MXPA98009700A - Methods for creating transgenic animals - Google Patents

Abstract

The present invention provides improved methods and compositions for the generation of transgenic non-human animals. The present invention permits the introduction of exogenous nucleic acid sequences into the genome of unfertilized eggs (e.g., pre-maturation oocytes and pre-fertilization oocytes) by microinjection of infectious retrovirus into the perivitelline space of the egg. The methods of the present invention provide an increased efficiency of production of transgenic animals with a reduced rate of generating animals which are mosaic for the presence of the transgene.

Description

METHODS TO CREATE TRANSGENIC ANIMALS.

DESCRIPTION.

This invention was developed with the support of the US Government. granted by the U.S. Department of Agriculture, Hatch Project No.3, 669. The U.S. Government has certain rights in the invention.

FIELD OF THE INVENTION.

The present invention relates to improved methods for the generation of transgenic non-human animals. In particular, the present invention relates to the introduction of retroviral particles into the perivitelline space of gametes, zygotes and embryos in the early stage, to allow the insertion of genetic material within the a-gene of the recipient gamete or embryo.

BACKGROUND OF THE INVENTION.

The ability to alter the genetic formation of animals, such as domesticated mammals, for example cows, pigs and sheep, allows a certain number of commercial applications. These applications include the production of animals that express large amounts of exogenous proteins in an easily harvestable form (for example, expression in milk), the production of animals that are resistant to infection by specific microorganisms and the production of animals that have high-risk regimens. improved development or better reproductive functioning. Animals that contain exogenous DNA sequences in their genome are referred to as transgenic animals.

The most widely used method for the production of transgenic animals is the microinjection of DNA into the pronucleus of fertilized embryos. This method is efficient for the production of transgenic mice but is much less efficient for the production of transgenic animals using large mammals such as cows and sheep. For example, it has been reported that 1,000 to 2,000 bovine embryos in the pronuclear stage should be injected to produce a single transgenic cow at an estimated cost of more than $ 500,000 [Wall et al. (1992) J. Cell. Biochem. 49: 113].

In addition, microinjection of pronuclei is more difficult when embryos of live domestic animals (eg, cattle, sheep or pigs) such as the pronucleus are used, often obscured by yolk material. Although techniques for the visualization of the pronuclei are known (ie, centrifugation of the embryo to sediment the yolk), the injection of pronuclei is an invasive technique that requires a high degree of skill of the operator.

Alternative methods for production include infection of embryos with retroviruses or with retroviral vectors. The infection of both mouse embryos pre- and post-plantation with either wild type or recombinant retroviruses has already been reported [Janenich (1976) Proc. Nati Acad. Sci. USA 73: 1260-1264; Janenich et al. (1981) Cell 24: 519; Stuhlmann et al) 1984) Proc. Nati Acad. Sci. USA 81: 7151; Jahner et al (1985) Proc. Nati Acad. Sci. USA 82: 6927-6931); Van der Putten, et al. (1985) Proc. Nati Acad. Sci. USA 82: 6148-6152; Steward, et al. (1987) EMBO J. 6: 383-388]. The resulting transgenic animals are typically mosaics for the transgene since incorporation occurs only in a subset of cells that make up the transgenic animal. The consequences of the incorporation of the mosaic of retroviral sequences (ie the transgene) include lack of transmission of the transgene to the progenere due to failures of the retrovirus to integrate within the germ line, it is difficult to detect the presence of viral sequences in the mice founders in those cases when the infected cell contributes only a small part of the fetus and makes it difficult to assess the effect of the genes transported by the retrovirus.

In addition, to produce mosaic founder animals, embryo infection with retroviruses (which is typically done using embryos in cell stage 8 or later) often results in the production of founder animals that contain multiple copies of retroviral provirus in different positions in the genome, that in general will segregate in the progeneie. The embryo infection of early mice by co-culturing early embryos with cells that produce retroviruses requires enzymatic treatment to remove the zona pellucida [Hogan et al. (1994) in Manipulating the Mouse Embryo: A Laboratory Manual, 2nd Ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, pp 251-252]. In contrast to mouse embryos, bovine embryos dissociate when removed from the zona pellucida. Therefore, infection protocols that remove the zona pellucida for the production of transgenic cattle or other animals whose embryos dissociate or suffer a decrease in significant viability under removal of the zona pellucida (for example, sheep embryos) can not be used. .

An alternative means to infect embryos with retroviruses is the injection of viruses or cells that produce viruses in the blastocels of embryos of mice [Jahner, D. Et al (1982) Nature 298: 623-628]. As is the case for infection of embryos from stage 8 of the cell, most of the founders produced by injection into the blastocoel will be mosaic. The introduction of transgenes into the mouse germ line has been reported using intrauterine retroviral infection of the mouse embryo at mid pregnancy [Jahner, D. et al. (1982) supra]. This technique suffers from a low efficiency of generation of transgenic animals and also produces animals that are mosaic for the transgene. T The infection of bovine and ovine embryos with retroviruses or retroviral vectors has been reported to create transgenic animals. These protocols comprise the micro-injection of retroviral particles or cells with repressed development (e.g., C-treated mitomycin) of cells that eject retroviral particles into the perivitelline space of fertilized eggs or early embryos [PCT International Application WO 90/08832 (1990 ) and Haskell and Bo in (1995) Mol. Reprod. Dev. 40: 386]. PCT International Application WO 90/08832 describes the injection of wild type feline leukemia virus B into the perivitelline space of sheep embryos in stage 2 to 8 of the cell. The fetuses derived from injected embryos showed to contain multiple integration sites. The production efficiency of the trsngénica sheep was low (efficiency is defined as the number of transgenic produced compared to the number of embryos handled); only 4.2% of the embryos injected were found to be transgenic.

Haskell and Bowen (supra) describe the micro-injection of C-treated mitomycin cells that produce retroviruses in the perivitelline space of bovine embryos in cells 1 a. The use of cells that produce viruses prevents "" the release of a controlled amount of viral particles per embryo. The resulting fetuses contained between 2 to 12 proviruses and proved to be mosaic for proviral integration sites, the presence of proviruses or both. The production efficiency of transgenic bovine embryos was low; only 7% of the embryos injected were found to be transgenic.

The technique needs improved methods for the production of transgenic animals, in particular for the production of transgenics using large domestic live animals. The ideal method will be simple in its implementation and less invasive than pronuclear, efficient injection, will produce mosaics of transgenic founder animals at a low frequency and will result in the integration of a defined number of copies of the sequences introduced into the transgenic animal's gene.

BRIEF DESCRIPTION OF THE INVENTION The present invention provides improved methods and compositions for the production of transgenic non-human animals. In one embodiment, the present invention provides a composition comprising an infectious non-human oocyte comprising a heterologous oligonucleotide (i.e., a heterologous polynucleotide) integrated into the oocyte gene. In a preferred embodiment, the infertile oocyte is a pre-mature oocyte. In another preferred embodiment the infertile oocyte is a pre-fertilization oocyte. The present invention is not limited by the nature of the heterologous oligonucleotide contained within the oocyte gene. In a preferred embodiment, the heterologous oligonucleotide is in the proviral form of a retroviral vector.

The invention is not limited by the nature of the retroviral vector employed. Retroviral vectors containing a variety of genes can be used. For example, the retroviral vector may contain sequences encoding proteins that modify the rate of development, size and / or carcass composition (e.g., bovine development hormone or other developmental hormones) or foreign proteins of commercial value that are expressed in, and collected from, a particular tissue component (e.g., blood or milk). The retroviral vector may contain genes that confer disease resistance to viruses or other microorganisms, including DNA sequences that are transcribed into RNA sequences that catalytically separate specific RNAs (ie, ribozymes) such as viral sequences, RNAs and DNA that are transcribed in RNA anti-direction of an essential gene of a pathogenic microorganism. The genes that encode protein and previous DNA sequences are examples of "genes of interest".

The compositions of the present invention are not limited by the nature of the non-human animal used to provide oocytes. In a preferred embodiment, the non-human animal is a mammal (e.g., cows, pigs, sheep, goats, rabbits, rats, mice, etc.). In a particularly preferred embodiment, the non-human animal is a cow.

The present invention further provides a method for introducing a heterologous polynucleotide into the geneome of an infertile non-human oocyte, comprising: a) providing: i) a non-human infertile egg comprising an oocyte having a plasma membrane and a zona pellucida , with the plasma membrane and the zona pellucida defining a perivitelline space; ii) an aqueous solution comprising a heterologous polynucleotide; and b) introducing the solution comprising the heterologous polynucleotide into the perivitelline space under conditions that allow the introduction of the heterologous polynucleotide into the oocyte gene. The method of the present invention is not limited by the nature of the heterologous polynucleotide employed. In a preferred embodiment, the heterologous polynucleotide encodes a protein of interest. In a particularly preferred embodiment, the heterologous polynucleotide is contained within the genome of a recombinant retrovirus.

The method of the present invention can be practiced using unfertilized eggs comprising a pre-mature oocyte. Alternatively, the method of the present invention can employ pre-fertilization oocytes such as the unfertilized egg.

When a recombinant retrovirus is employed, infectious retroviral particles comprising the heterologous polynucleotide are preferably used. The method of the present invention is not limited by the nature of the infectious retrovirus used to deliver nucleic acid sequences to an oocyte. Any retrovirus capable of infecting the oocyte species that is injected can be used. In a preferred embodiment, the infectious retrovirus comprises a protein associated with a heterologous membrane. In a preferred embodiment, the heterologous membrane associated protein is a G glycoprotein selected from a virus of the Rhabdoviridae family. In another preferred embodiment, the protein associated with heterologous membrane is selected from the group consisting of a vesicular stomatitis virus glycoprotein, Piry virus, Chandipura virus, Spring viraemia of carp virus and Mokola virus. In a particularly preferred embodiment, the protein associated with heterologous membrane is the G glycoprotein of vesicular stomatitis virus.

The method of the present invention is not limited by the nature of the non-human animal used to provide the oocytes. In a preferred embodiment, the non-human animal is a mammal (e.g., cows, pigs, sheep, goats, rabbits, rats, mice, etc.). In a particularly preferred embodiment, the non-human animal is a cow.

The present invention further provides a method for producing a transgenic non-human animal, comprising: a) providing: i) an unfertilized egg comprising an oocyte having a plasma membrane and a zona pellucida, the plasma membrane and the zone pellucid define a perivitelline space; ii) an aqueous solution containing infectious retrovirus; b) introducing the solution containing infectious retroviruses into the perivitelline space under conditions that allow infection of the oocyte; and c) contacting the infected oocyte with sperm under conditions that allow fertilization of the infected oocyte to produce an embryo. In a preferred embodiment, the method of the present invention further comprises, after fertilization of the infected oocyte, the step of transferring the embryo into an aminal hormonally synchronized non-human vessel (i.e., a hormonally synchronized female animal to stimulate pregnancy). precocious). In another preferred embodiment, the method comprises the step of allowing the transferred embryo to develop thermally. In still another preferred embodiment, at least one transgenic progene is identified from a progene that was allowed to develop at term.

The method of the present invention can be practiced using unfertilized eggs comprising a pre-mature oocyte. Alternatively, the method of the present invention may employ pre-fertilization oocytes such as the infertile egg.

When pre-mature oocytes are employed in the method of the present invention, the method may further comprise, after introducing the solution containing infectious retrovirus into the pre-mature oocyte, the additional step of culturing the pre-mature oocyte. - infected maturation under conditions that allow the maturation of the pre-mature oocyte. The technique is well aware of culture conditions that allow the in vitro maturation of pre-mature oocytes from a variety of malarial species.

The method of the present invention is not limited by the nature of the infectious retrovirus used to deliver the nucleic acid sequences to an oocyte. Any retrovirus capable of infecting the oocyte species to be injected can be used. In a preferred embodiment, the infectious retrovirus comprises a heterologous associated membrane protein. In a preferred embodiment, the heterologous associated membrane prstein is a G glycoprotein selected from a virus of the R family abdoviridae. In another preferred embodiment, the heterologous associated membrane protein is selected from the group consisting of a G glycoprotein from vesicular stomatitis virus, Piry virus, Chandipura virus, Spring viraemia from carp virus and Mokola virus. In a particularly preferred embodiment of the invention, the heterologous associated membrane protein is the glycoprotein G of a vesicular stomatitis virus.

The method of the present invention is not limited by the nature of the non-human animal employed to provide oocytes. In a preferred embodiment, the non-human animal is a mammal (e.g., cows, pigs, sheep, goats, rabbits, rats, mice, etc.). In a particularly preferred embodiment, the non-human animal is a cow.

DESCRIPTION OF THE DRAWINGS Figure 1 provides a scheme showing the production of pre-mature oocytes, prefertilization oocytes and fertilized oocytes (zygotes).

Figure 2 shows an autoradiogram of a Southern blot of genomic DNA isolated from the hide (A) and blood (B) of calves derived from preferentialized oocytes and zygotes which are injected with pseudo-typed LSRNL retroviruses.

Figure 3 shows an agarose gel stained with ethidium bromide containing electrophoresis PCR products that were amplified using primary neogenes (A) or primary HBsAg (B) blood and calf leather derived from pre-fertilization oocytes and zygotes injected with pseudo-typed LSRNL retroviruses.

DEFINITIONS To facilitate the understanding of the invention, in the following, a certain number of terms is defined.

As used herein, the term "egg" when used with reference to the egg of a mammal, means an oocyte, surrounded by a zona pellucida and a mass of cumulus cells (follicular cells) with its associated proteoglycan. The term "egg" is used with reference to eggs recovered from antral follicles in an ovary (These eggs comprise pre-mature oocytes) as well as "eggs" that have been released from an antral follicle (a broken follicle).

As used herein, the term "oocyte" refers to a female gamete cell, and includes primary oocytes, secondary oocytes and mature unfertilized egg. An oocyte is a large cell that has a large nucleus, (for example, the germinal vesicle) surrounded by ooplasm. Ooplasm contains non-nuclear cytoplasmic material that includes mRNA, ribosomes, itochondria, yolk proteins, etc. Here, the oocyte membrane, is referred to as the "plasma membrane".

The term "pre-maturation oocyte" as used here, refers to a female gamete cell that follows the oogony stage (ie, mitotic proliferation has occurred) that is isolated from an ovary (e.g., by aspiration) but that has not been exposed to an in vitro maturation medium . Those skilled in the art know that the aspiration process causes the oocytes to initiate the maturation process but that, upon completion of the maturation process, (ie, the formation of a secondary oocyte that has extruded the first polar body) in vitro, requires exposure of the oocytes aspirated to the maturation medium. Premature oocytes in general will be repressed in the first anaphase of eosis.

The term "prefertilization oocyte" as used herein, refers to a female gamete cell such as a pre-maturation oocyte that follows exposure of the maturation medium in vitro, but prior to exposure to sperm (i.e., matured but not fertilized). The prefertilization oocyte has completed the first meiotic division, has released the first polar body and lacks a nuclear membrane (the nuclear membrane will not reform until fertilization occurs, after fertilization, the second meiotic division occurs along with the extrusion of a second polar body and the formation of male and female pronuclei). The prefertilization oocytes can also refer to oocytes matured in metaphase II of the second meiosis.

The term "unfertilized egg" or "unfertilized oocyte" as used herein refers to any female gamete cell that has not been fertilized and these terms encompass both oocytes of pre-maturation and prefertilization.

The term "perivitelline space" refers to the space located between the zona pellucida and the plasma membrane of an oocyte or mammalian egg.

The term "infectious retrovirus" refers to a retroviral particle that is capable of penetrating a cell (i.e., the particle contains an associated membrane protein such as a coated protein or a viral glycoprotein G that can bind to a surface of host cell and facilitate the entry of the viral particle into the cytoplasm of the host cell) and integrate the retroviral geneome (as a double-stranded provirus) into the host cell's genome.

Retroviral vectors can be used to transfer genes efficiently into the host cells by exploiting the viral infectious process. Cloned foreign or heterologous genes (ie, inserted using molecular biology techniques) into the retroviral gene can be efficiently delivered to host cells that are susceptible to infection by the retrovirus. By means of well-known genetic manipulations, the replicative capacity of the retroviral geneome can be destroyed. The resulting replication-defective vectors can be used to induce new genetic material to a cell but are unable to replicate. A Helper virus or packaging cell line can be used to allow the vector particle to attach and exit the cell.

The term "vector particle" or "retroviral particle" refers to similar viral particles that are capable of introducing nucleic acid into a cell through a similar viral entry mechanism.

The host range of a retroviral vector (that is, the range of cells these vectors can infect) can be altered by including a coat protein from another closely related virus.

The term "membrane-associated protein" refers to a protein (e.g., a viral envelope glycoprotein or the G proteins of the Rhabdoviridae family such as VSV, Piry, Chandipura and Mokola) that are associated with the surrounding membrane a viral particle; these proteins with associated membrane mediate the entrance of the viral particle inside the host cell. The associated membrane protein can bind to specific cell surface protein receptors, as is the case for retroviral coated proteins or the associated membrane protein, can interact with a phospholipid component of the plasma membrane of the host cell, as is the case for G proteins derived from members of the Rhabdoviridae family.

The term "heterologous associated membrane protein" refers to an associated membrane protein that is derived from a virus that is not a member of the same viral class or family as that from which the cleocapsid n protein is derived. of the vector particle. "Family or viral class" refers to a class or family taxonomic rank, as assigned by the International Committee on Taxonomy of Viruses.

The term "Rhabdoviridae" refers to a family of covered RNA viruses that infect animals, including humans and plants. The Rhabdoviridae family encompasses the genus Vesiculovirus that includes vesicular stomatitis virus (VSV), Cocal virus, Piry virus, Chandipura virus and Spring viremia of carp viruses (sequences encoding the Spring viraemia of carp virus are available under access number U18101 in GeneBank). The G proteins of the viruses in the genera Vesiculovirus are virally encoded membrane-integrated proteins that form glycoprotein complexes of homotrimeric spikes that project externally, which are required for receptor binding and fusion of the membrane. in genres Vesicifloviruses have one half (Ci6) of palmitic acid covalently bound. The amino acid sequences of the G proteins of Vesiculoviruses are conserved perfectly well. For example, the G protein of the Piry virus shares 38% identity and approximately 55% similarity with the G VSV proteins (several strains of VSV are known, for example, the strains Indiana, New Jersey, Orsay, San Juan, etc.). , and its G proteins are very homologous). The G protein of the Chandipura virus and the G. VSV proteins share approximately 37% identity and 52% similarity. Given the high degree of conservation (amino acid sequence) and the related functional characteristics (for example, virus binding to the host cell and membrane fusion, including the formation "syncytia") of the G proteins of the Vesiculoviruses, the G Non-VSV Vesiculovirus proteins can be used in place of the VSV protein G for the pseudotyping of viral particles. The G proteins of the Lyssa viruses (another genus within the Rhabdoviridae family) also share a high degree of conservation with the VSV G proteins and function in a similar way (for example mediate fusion of membranes) and can therefore be used instead of the VSV protein G for the pseudotyping of viral particles. Lyssa viruses include Mokola virus and Rabies virus (Several strains of Rabies virus are known and their G proteins have been cloned and sequenced). The G protein of the Mokola virus shares homology elongations (particularly on the extracellular and transmembrane domains) with the G proteins of the VSV showing approximately 31% identity and 48% similarity with the G proteins of the VSV. The preferred G proteins share at least 25% identity, preferably at least 30% identity and more preferably at least 35% identity with the G proteins of the VSV. The VSV protein G of which the New Jersey strain is used (the sequence of this G protein is "provided with the access numbers M27165 and M21557 of the GeneBank) as the G protein of the reference VSV.

The term "conditions that allow maturation of a pre-maturation oocyte" refers to in vitro cell culture conditions that allow maturation of a pre-mature oocyte to a mature egg (eg, a pre-fertilization oocyte). These culture conditions allow and induce the events that are associated with the maturation of the pre-maturation oocyte including the stimulation of the first and second eiodic divisions. The conditions of in vitro culture, * - "that allow the maturation of the oocytes of A 'pre-maturation of a variety of mammalian species (e.g., cattle, hamsters, pigs and goats) are well known in the art [see, for example, Parrish et al. (1985) Theriogeneology 24: 537; Resonarais ad First (1994) J. Ani. Sci. 72: 434; Bavister ad Yanagimachi (1977) Biol.

Reprod. 16: 228; Bavister et al. (1983) Biol. Reprod. 28: 235; Leibfried ad Bavister (1982) J. Reprod. Fert. 66:87; Keskintepe et al. (1994) Zygote 2:97; Funahashi et al. (1994) J. Reprod. Fert. 101: 159 ad Funahashi et al. (1994) Biol Reprod. 50: 1072].

DETAILED DESCRIPTION OF THE INVENTION.

The present invention provides improved methods for the production of transgenic animals. The methods of the present invention provide, for the first time, the production of transgenic animals by introducing exogenous DNA into mature and pre-mature oocytes, unfertilized oocytes (that is, pre-fertilization oocytes) using retroviral vectors that transduce the division of cells [e.g., vectors derived from murine leukemia virus (MLV)].

The description of the invention is divided into the following sections: I) retroviruses and retroviral vectors; II) integration of retroviral DNA; III) introduction of retroviral vectors in gametes before the last meiotic division; and IV) detection of retroviruses, after injection into oocytes and embryos.

I) RETROVIRUS AND RETROVIRAL VECTORS.

The retroviruses (family Retroviridae) are divided into three groups: the foam viruses (for example, (the human foamy viruses), the lentiviruses (for example the human immunodeficiency virus and the sheep virus "visna"), and the oncoviruses (for example, MLV, Rous sarcoma virus).

Retroviruses are single-stranded RNA viruses covered (ie, they are surrounded by a membrane of two layers of lipids derived from host cells) that infect animal cells. When a retrovirus infects a cell, its RNA geneome is converted into a double-stranded linear DNA form (ie it is reverse transcribed). The DNA form of the virus is then integrated into the host cell's genome as a provirus. The provirus serves as a model for the production of additional viral genomes and viral RNAs. The mature viral particles contain two copies of outbreaks of gene-derived RNA from the surface of the infected cell. The viral particle comprises the gene RNA, reverse transciptase and other products of the "pol" gene within the viral capsid (which contains the products of the viral "gag" gene) that is surrounded by a membrane of two layers of lipids derived from the cell host containing the viral envelope glycoproteins (also referred to as proteins with associated membrane).

The organization of the genomes of numerous retroviruses is well known in the art and this has allowed the adaptation of the retroviral geneome to produce retroviral vectors.

The production of a recombinant retroviral vector carrying a gene of interest is typically achieved in two stages. First, the gene of interest is inserted into a retroviral vector that contains the sequences necessary for the efficient expression of the gene of interest [including promoter elements and / or enhancers that can be provided by the viral long terminal repeats (LTRs) or by an element internal promoter / enhancer and relevant dividing signals], sequences required for the efficient packaging of ~ ~ * viral RNA in infectious virions [eg, the packaging signal (Psi), the tRNA base binding site (-PBS), the sequences 3 'regulators required for reverse transcription (+ PBS)) and viral LTRs]. The LTRs contain the sequences required for the association of viral gene RNA, reverse transcriptase and integrase functions and sequences that involve directing the expression of the gene RNA that is to be packaged in viral particles. For safety reasons, many retroviral vectors. recombinants lack functional copies of genes that are essential for viral replication (these essential genes are either deleted or disabled); it is said that the resulting virus is a defective replication.

Second, after construction of the recombinant vector, the vector DNA is introduced into a packaging cell line. Packing cell lines provide viral proteins required in trans for packaging of the viral gene in viral particles having the desired host range, (ie, the viral encoded gag, pol and env proteins). The range of lodging is controlled, in part, by the type of cover gene product expressed on the viral particle surface.

Packing cell lines can express ecotrophic, amphotropic or geneotropic cover gene products. Alternatively, the packaging cell line may lack sequences encoding a viral envelope (env) protein. In this case, the packaging cell line will pack the viral genome, into particles that lack an associated membrane protein (eg env protein). In order to produce viral particles that contain an associated membrane protein, which will allow the entry of the virus into a cell, the packaging cell line containing the retroviral sequences is transfected with sequences that encode an associated membrane protein [ example, the G vesicular stomatitis virus (VSV) protein]. The transfected packaging cell will then produce viral particles containing the associated membrane protein, expressed by the line of transfected packaging cells; you are viral particles that contains viral gene RNA derived from a virus encapsidated by the envelope proteins of another virus are said to be pseudotyped virus particles.

Viral vectors, including recombinant vectors, provide a more efficient means for transferring genes within cells compared to other techniques such as calcium phosphate DNA coprecipitation or DEAE-dextran-mediated transfection, electroporation or nucleic acid microinjection. The efficiency of viral transfer is due in part to the fact that the nucleic acid transfer is a mediated receptor process (ie, the virus binds to a specific receptor protein on the surface of the cell that is going to infect). In addition, virally transferred nucleic acid once inside a cell is integrated in a controlled manner in contrast to the integration of nucleic acids that are not virally transferred; nucleic acids transferred by other means such as coprecipitation of DNA with calcium phosphate are subjected to re-arrangements and degradation.

JliOS Most commonly used recombinant retroviral vectors are derived from murine amphotropic murine leukemia virus (MoMLV) [Miller and Balti ore (1986) Mol. Cell. Biol. 6: 2895]. The system "MoMLV has several advantages: 1) this specific retrovirus can infect many different types of cells, 2) established packaging cell lines are available for the production of recombinant MoMLV viral particles, and 3) the transferred transcripts are permanently integrated into the chromosome of the target cell The established MoMLV vector systems comprise a DNA vector that contains a small portion of the retroviral sequence (the repeated long viral terminal or "LTR" and the packaging or "psi" signal) and a line of packaging cells The gene to be transferred is inserted into the DNA vector The viral sequences present in the DNA vector provide the necessary signals for the insertion or packaging of the vector RNA in the viral particle and for the expression of the gene inserted The packaging cell line provides the viral proteins required for the coupling of particles [M Arkowitz et al (1988) J. Virol. 62: 1120].

Despite these advantages, existing retroviral vectors based on MoMLV are limited by several intrinsic problems: 1) they do not infect undivided cells [Miller et al (1990) Mol. Cell. Biol. 10: 4239]; 2) produce low titers of the recombinant virus [Miller and Rosman (1989) BioTecniques 7: 980 and Miller (1992) Natura 357: 455]; and 3) they infect certain cell types (eg, human lymphositos) with low efficiency [Adams et i (1992) Proc. Nati Acad. Sci. USA 89: 8981]. The low titers associated with the MoMLV-based vectors have been attributed, at least in part, to the instability of the protein coated with coded virus. Concentrations of retroviruses provided by physical means (e.g., by ultracentrifugation and ultrafiltration) lead to severe loss of infectious virus.

The low titre and inefficient infection of certain cell types by means of MoMLV-based vectors has been overcome by the use of pseudotyped retroviral vectors containing the VSV protein G as the associated membrane protein. Unlike retroviral-coated proteins that bind to a specific cell surface protein receptor to gain entry into the cell, the VSV protein G interacts with a phospholipid component of the plasma membrane [Mastro arino et al ( 1977) J. Gene. Virol.68: 2359]. Because the entry of the VSV into a cell does not depend on the presence of specific protein receptors, the VSV has an extremely broad host range. The pseudotyped retroviral vectors carrying the VSV protein G have an altered VSV host range characteristic (ie, they can infect almost all vertebrate, invertebrate, and insect5 cell species). Importantly, retroviral vectors G seu ofcified of VSV can be concentrated 200 times or more by ultracentrifugation without significant loss of infectivity [Burns et al (1993) Proc. Nat. Acad. Sci.

USA 90: 8033].

The VSV protein G has also been used for pseudotyped retroviral vectors based on human immunodeficiency virus (HIV) [Naldini et al (1996) Science 272: 263]. Therefore the VSV protein G can be used to generate a variety of pseudotyped retroviral vectors and is not limited to MoMLV based vectors.

The present invention is not limited to the use of the VSV protein G when a viral protein G is used as the heterologous membrane associated protein within a viral particle. The G proteins of the viruses of the genus Vesiculovirus other than VSV, such as the viruses Piry and Chandipura, which are highly homologous to the VSV protein G and, like the VSV protein G, contain covalently bound palmitic acid [Brun et al. al (1995) Intervirol. 38: 274 and Masters et al (1990) Virol. 171: 285]; therefore, the protein G of the viruses Piry and Chandipura can be used instead of the G protein of the VSV for the pseudotyping of viral particles. In addition, the G protein of the VSV of the viruses that are within the genus of Lyssa virus such as the Rabies and Mokola viruses show a high degree of conservation (amino acid sequence as well as functional conservation with the G proteins of the VSV. , the G protein of the Mokola virus has been shown to function in a manner similar to the G protein of the VSV (this is for mediated membrane fusion) and therefore can be used in place of the VSV protein G for the pseudotyping of particles Viral proteins [Mebatsion et al (1995) J. Virol 69: 1444]. The sequence of nucleotides encoding the G protein of Piry is provided in SEQ ID (identification sequence), NO: 5 and the amino acid sequence of G Piry protein is provided in SEQ ID NO: 6. The sequence of nucleotides encoding the G protein of Chandipura is provided in SEQ ID NO: 7 and the amino acid sequence of the G protein of Chandipura is provided in SEQ ID NO. 8. The sequence of nucleotides encoding the G protein of Mokola is provided in SEQ ID NO: 9 and the amino acid sequence of the G protein Mokola is provided in SEQ ID NO: 10. Viral particles can be pseudotyped using either the G protein of Piry, Chandipura or Mokola as described in example 2 with the exception that a plasmid containing sequences encoding either the G protein of Piry, Chandipura or Mokola under the transcriptional control of a promoter elements suitable [eg, the first CMV intermediate promoter; numerous expression vectors containing the CMV IE promoter are available, such as the vectors of pcDNA3.1 (Invitrogene)], are used in place of pHCMV-G. Sequences encoding other G proteins derived from other members of the Rhadbdoviridae family may be used; Sequences that encode numerous G rhabdoviral proteins are available in the GeneBanK database.

II. INTEGRATION OF RETROVIRAL DNA.

Most retroviruses can transfer or integrate a linear double-stranded form of the virus (the provirus) into the recipient cell's genome only if the recipient cell is cyclized (ie, divided) at the time of infection. Retroviruses that have been shown to infect cells that divide exclusively, or more efficiently, include MLV, splenic necrosis virus, Rous sarcoma virus, and human immunodeficiency virus (HIV), while HIV infects cells that divide more efficiently, HIV can infect cells that do not divide ^.

It has been shown that the integration of MLV virus DNA depends on the progression of the host cells through mitosis and it has been postulated that the dependence on mitosis reflects a requirement for the breaking of the nuclear envelope in order that the complex viral integration gain entry to the core [Roe et al. (1993) EMBO J: 12: 2099]. However, since integration in cells retained in metaphase does not occur, the breaking of the envelope of the nucleus alone may not be sufficient to allow viral integration; there may be additional requirements such as the state of condensation of the geneomic DNA (Roe et al., supra).

III. INTRODUCTION OF RETROVIRAL VECTORS IN GAMETOS BEFORE THE LAST MEIOTIC DIVISION.

The envelope of the nucleus of a cell breaks during meiosis as well as during mitosis. Meiosis occurs only during the final stages of gametogenesis. The methods of the present invention exploit the breakdown of the nuclear envelope during meiosis to allow the integration of the recombinant retroviral DNA and allow for the first time the use of unfertilized oocytes (ie pre-fertilization and pre-mature oocytes). as the recipient cell for the transfer of retroviral genes for the production of transgenic animals. Because the infection of unfertilized oocytes, allows the integration of the proviruses. Before the division of the cell embryo, all the cells in the embryo will contain the proviral sequences.

Oocytes that have not undergone the final stages of gametogenesis are infected with the retroviral vector. Then the injected oocytes are allowed to complete maturation with the accompanying eiodic divisions. The rupture of the nucleus envelope during meiosis allows the integration of the proviral form of the retrovirus vector into the oocyte gene. When pre-mature oocytes have been used, the injected oocytes are then cultured in vitro under conditions that allow oocyte maturation before in vitro fertilization.

Oocyte maturation conditions from a number of mammalian species are well known in the art (eg, cattle, sheep, swine, murine, caprine). In general, the base medium used here for the in vitro maturation of bovine oocytes, medium TC-M199, can be used for the in vitro maturation of other mammalian oocytes. The TC-M199 medium is supplemented with hormones (e.g., luteinizing hormone and estradiol) from the appropriate mammalian species. The amount of time a pre-maturation oocyte should be exposed to the maturation medium will allow varied maturations between mammalian species, as is well known in the art. For example, an exposure of approximately 24 hours is sufficient to allow the maturation of bovine oocytes whereas the oocytes of portions require approximately 44-48 hours.

The oocytes may mature in vivo and be used in place of oocytes matured in vitro, in the practice of the present invention. For example, when porcine oocytes are to be employed in the methods of the present invention, pre-breeding oocytes matured directly from pigs that are induced to superovulation can be harvested, as is known in the art. Briefly, on day 15 or 16 of estrus, the sow (s) is injected with approximately 1000 units of pregnant mare serum (PMS); available from Sigma and Calbiochem). Approximately 48 hours later, the sows are injected with approximately 1000 units of human chorionic gonadotropin (hCG; Sigma) and 24-48 hours later the mature oocytes are collected from the oviduct. These prefertilization oocytes matured in vivo are then injected with the desired retroviral preparation as already described herein. Methods for superovulation and collection of matured oocytes in vivo (ie, oocytes in the metaphase 2 stage) are known for a variety of mammals [eg, for mouse super-ovulation, see Hogan et al. (1994), supra at pp. 130-133; for superovulation of pigs and in vitro fertilization of pig oocytes see Cheng, W. (1995) Doctoral Dissertation, Cambridge University, Cambridge, United Kingdom].

Retroviral vectors capable of infecting the desired species of non-human animals that can be grown and concentrated to very high titres (eg,> 1 x 108 cfu / ml), are preferably used. The use of high titer virus materials allows the introduction of a defined number of viral particles into the perivitelline space of each injected oocyte. The perivitelline space of most mammalian oocytes can accommodate approximately 10 picolitres of fluid (ie, those in the known technique, which in volume can be injected into the periviral space of a mammalian or zygote oocyte). , they vary a little between species when the volume of an oocyte is smaller than that of a zygote and therefore, it can accommodate a little less oocytes than zygotes).

The vector used may contain one or more genes that encode a protein of interest; alternatively, the vector may contain sequences that produce s-sequences of anti-di rectional RNA or ribozymes. Infectious virus is microinjected into the priviral space of oocytes (including pre-mature oocytes or cell stage zygotes »L, microinjection in the perivitelline space is much less invasive than nucleic acid microinjection within the rate of an embryo and results in lesser embryo viability.In addition, a higher level of operator experience is required to perform a prenuclear injection compared to the perivitic injection.No pronucleus visualization is required when the virus is injected into the embryo. the perivitelline space (in contrast to injection into the pronucleus); therefore, injection into the perivi-tel space IBT obviates the difficulties associated with the visualization of pronuclei, especially such as cattle, pigs and pigs .

The virus material can be titrated and diluted before the microinjection in the space perivite inp - - ^ so that the number of proviruses integrated in • * • is controlled? the resulting transgenic animal. The use of a viral material (or a dilution thereof) having a titer of 1 x 108 cfu / l allows the delivery of a single viral particle per oocyte. The use of pre-mature oocytes or mature fertilized oocytes as the container of the viruses, minimizes the production of animals that are mosaics for the provirus, when the virus is integrated into the oocyte's gene, before the occurrence of division ( cleavage) of the cell.

In order to supply, on average, a single infectious particle per oocyte, the micropipettes used for the injection are calibrated as follows. Small volumes (eg, about 5-10 pl) of undiluted high titer viral material (eg, a titer of approximately 1 x 10 8 cfu / ml) are delivered to the cavities of a microtiter plate by pressing the micromanipulator. The titer of the virus delivered for a given number of pulses is determined by diluting the viral material in each well and determining the titer using a suitable cell line (e.g., cell line 208F) as described in example 2. The number of pulses that supply, on average, a volume of virus containing an infectious viral particle (ie, gives an MOI of 1 when titrated on 208F cells), is used for injection of the viruses into the oocytes.

Prior to microinjection of the titrated and diluted virus material (if required), the accumulated cell layer opens to provide access to the perivitelline space. The layer of accumulated cells does not need to be completely removed from the oocyte and actually for certain species of animals (for example, cows, sheep, pigs, mice) a portion of the layer of accumulated cells must remain in contact with the oocyte to allow the development appropriate and post-injection of fertilization. The injection of viral particles into the perivitelline space allows the vector RNA (i.e., the viral genome) to penetrate the cell through the plasma membrane, thereby allowing proper reverse transcription of the viral RNA.

IV. DETECTION OF THE RETROVIRUS AFTER INJECTION WITHIN THE OOCYTES OR EMBRYOS.

The presence of the retroviral genome in cells (e.g., oocytes or embryos) infected with pseudotyped retrovirus can be detected using a variety of means. The expression of the product (s) of the gene (s) encoded by the retrovirus can be detected by detection of the mRNA corresponding to the gene products encoded with vector using techniques well known in the art (eg, Northern blot). , spot spot, in situ hybridization and RT-PCR analysis). The direct detection of the gene encoded vector product is used when the gene product is a protein that either has an enzymatic activity (for example, beta-galactosidase) or when an antibody capable of reacting with the vector-encoded protein is available.

Alternatively, the presence of the integrated viral gene can be detected using Southern blot or PCR analysis. For example, the presence of the genomes LZRNL or LSRNL, can be detected following infection of oocytes or embryos using PCR as follows. Geneomic DNA is extracted from the infected oocytes or embryos (the DNA can be extracted from the entire embryo or alternatively several tissues of the embryo can be examined) using techniques well known in the art. The LZRNL and LSRNL viruses containing the neo gene and the base pair that follows can be used to amplify a 349-bp segment of the neo gene: the upstream base: 5'-GCATTGCATCAGCCATGATG-3 '(SEQ ID NO.l) and the current base aft jo: 5'-GATGGATTGCACGCAGGTTC-3 '(SEQ ID NO.2). PCR is carried out using well-known phenols [for example, using a GeneA p kit according to the manufacturer's instructions (Perkin-Elmer)) .- The DNA present in the reaction is denatured by incubation at 94 ° C for 3 minutes followed by 40 cycles of 94 ° C for 1 minute, 60 ° for 40 seconds and 72 ° C for 40 seconds followed by a final extension at 72 ° C for 5 minutes. The PCR products can be analyzed by electrophoresis of 10 to 20% of the total reaction on a 2% agarose gel; the 349-bp product can be visualized by dyeing the gel with ethidium bromide and exposing the gel dyed to ultraviolet light. If the expected PCR product can not be detected visually, the DNA can be transferred to a solid support (eg, a nylon membrane) and hybridized with a labeled neo 32P probe.

Southern blot analysis of the gene-derived DNA extracted from the infected oocytes and / or the resulting embryos, progene and tissues derived therefrom, is used when information is desired related to the integration of the viral DNA into the host geneome. To examine the number of integration sites present in the host gene, the extracted genomic DNA is typically digested with a restriction enzyme that is cut at least once within the sequences of the vectors. If the selected enzyme cuts twice within the vector sequences, a band of known (ie, predictable) size is generated in addition to two fragments of new length that can be detected using appropriate probes.

EXPERIMENTATION The following examples serve to illustrate certain preferred embodiments and aspects of the present invention and are not constructed as limiting the scope thereof.

In the experimental description that follows, the following abbreviations apply: M (molar); mM (millimolar); μM (micromolar), nM (nanomolar); mol (moles); mmol (millimoles), μ ol (micromoles); nmol (nanomoles); gm (grams); mg (milligrams); μg (micrograms); pg (picogramos); L (liters); ml (milliliters); μl (microliters); cm (centimeters), mm (millimeters); μm (micrometers); nm (nanometers), ° C (degrees centigrade); AMP (5 '- adenosine monophosphate); BSA (bovine serum albumin); CDNA (complementary or copy DNA); CS (serum • calf); DNA (deoxyribonucleic acid); SsDNA (DNA from single cord); DsDNA (double cord DNA); dNTP (deoxyribonucleotide triphosphate); LH (luteinizing hormone); NIH (National Institute of Health of Besthesda, MD); RNA (ribonucleic acid); PBS (salt buffered with phosphate); g (severity); OD (optical density 10); HEPES (N- [2-hydroxyethyl] piperazine-N- (2- (ethanesulfonic acid)); HBS (buffered saline HEPES); PBS (salt buffered with phosphate); SDS (sodium dodecyl sulfate); Tris-HCl (tris [hydroxymethyl] aminomethane hydrochloride); Klenow (large fragment (Klenow) of DNA polymerase I); rpm (revolutions per minute); EGTA (N, N, N ', N'-tetraacetic acid of bis (beta-aminoethyl ether) -ethylene glycol); EDTA ethylene diamine tetra acetic acid); bla (beta-lactamase or ampicillin-resistant gene); ORÍ (origin of replication of the plasmid); lacl (lac repressor); X-gal (5-bromo-4-chloro-3-indolyl-beta-D-galactoside); ATCC (American Type Culture Collection, Rockville, MD); GIBCO / BRL (GIBCO / BRL, Grand Island NY); Perkin-Elmer (Perkin-Elmer, Norwalk, CT); and Sigma (Sigma Chemical Company, St. Louis, MO). 25 EXAMPLE 1.

Generation of stable cell lines expressing the G proteinsag and pol of MoMLV.

The expression of the f G protein fusogenic on the surface of the cells, results in syncytium formation and cell death. Therefore, in order to produce retroviral particles containing the VSV protein G as the associated membrane protein, a three-step solution was made. First, stable cell lines expressing the G proteinsag and pol of MoMLV were generated at high levels (eg, 293GP cells, example 1). These stable cell lines were then infected using the desired retroviral vector, which is derived from an amphotrophic packaging cell (e.g., PA317 cells transfected with the desired retroviral vector, example 2a). The line of stable, infected cells expressing G proteins and pol produce non-infectious viral particles lacking an associated membrane protein (e.g., a coat protein). Third, these infected cell lines are then transiently transfected with a plasmid capable of directing the high level expression of the VSV protein G (example 2b). The transiently transfected cells produce pseudotyped VS retroviral G vectors that can be harvested from the cells over a period of 3 to 4 days before cell death occurs as a result of syncytium formation.

The first step in the production of pseudotyped retroviral G VSV vectors, the generation of stable cell lines expressing the G proteinsag and pol of MoMLV, is described below.

Line 293 (ATCC CRL 1573) of cells of the embryonic kidney 5. transformed from human adenovirus, was cotransfected with the plasmids pCMVgag-pol and pFR400 using a ratio of 10: 1 (pCMVgag-pol and pFR400) .- pCMVgag-pol contains the genes gag and pol of MoMLV under the control of the CMV promoter (gag -pol pCMV is available from ATCC). pFR400 encodes a mutant dihydrofolated reductase that has a reduced affinity for methotrexate [Simonsen et. al., Proc. Nati Acad. Sci. 80: 2495 (1983)].

Plasmid DNA was introduced into 293 cells using co-precipitation with calcium phosphate [Graha and Van der Eb, Virol, 52: 456 (1973)].

Approximately 5 x 105 293 cells were placed in a 100 mm tissue culture plate the day before the coprecipitate was added. A total of 20 μg of plasmid DNA (18 μg gag-pol from pCMV and 2 μg from pFR400) were added as a calcium coprecipitate DNA to each 100 mm plate. Stable transformants were selected by development in a high glucose DMEM medium containing 10% JCS, 0.5 μM methotrexate and 5 μM dipyridimole (selective medium). The colonies that developed on the selective medium were screened for extracellular reverse transcriptase activity [Goff et al., J. Virol. 38: 239 (1981)] and intracellular p30gag expression. The p30gag expression was determined by Western blotting using a goat anticurepo anti p30 (NCI antiserum 77S000087). A clone that exhibited stable expression of the retroviral genes was selected in the absence of continued methotrexate selection. This clone was named 293GP (293 gag-pol). The 293GP cell line, a derivative of line 293 of enbrionic kidney cells transformed from human Ad, was grown in a high glucose DMEM medium containing 10% FCS. The 293GP cell line is commercially available from Viagene, Inc., San Diego, CA.

EXAMPLE 2 Preparatof pseudotyped retroviral vectors carrying the VSV protein G.

In order to produce pseudotyped retroviruses of VSV protein G, the following steps were followed. First, the 293GP cell line was infected with virus derived from the PA317 line of amphotrophic packaging cells. The infected cells packed the retroviral RNA into viral particles lacking an associated membrane protein (because the 293GP cell line lacks a gene env or another gene encoding an associated membrane protein). The infected 293GP cells were then tansitorily transfected with a plasmid encoding the VSV protein G to produce pseudotyped viral particles bearing the VSV protein G.

A) Cell lines and plasmids The line of amphotropic packaging cells, PA317 (ATCC CRL 9078) was developed in a DMEM-high glucose medium containing 10% FCS. The 293GP cell line was grown in a DMEM-high glucose medium containing 10% FCS. The pseudotyped virus titer can be determined using either 208F cells (Quade (1979) Virol. 98: 461] or NIH / 3T3 cells (ATCC CRL 1658); 208F and NIH / 3T3 cells were grown in a high glucose DMEM medium containing 10% CS.

Plasmid pLZRNL [Xu et al. (1989) Virol. 171: 331] contains the gene encoding E. coli beta-galactosidase (LacZ) under the transcriptl control of the murine murine sarcoma virus of Moloney (MSV) followed by the gene encoding neomycin phosphotransferase (Neo) under the transcriptl control of the Rous sarcoma virus promoter (RSV). The pLSRNL of the plasmid contains the gene encoding the hepatitis B surface antigenic gene (HBsAg) under the transcriptl control of the MSV LTR followed by the Neo gene under the control of the RSV promoter (US Patent Number 5,512,421, the discussof which is incorporated herein) as reference) . The pHCMV-G of the plasmid contains the VSV G gene under the transcriptl control of the intermediate-early promoter of the human cytomegalovirus [Ye et al. (1994) Meth. Cell Biol. 43:99].

B) Productand titratof the pseudotyped LZRNL virus.

PLZRNL DNA was so affected in a PA317 amphotropic packaging line to produce LZRNL virus. The resulting LZRNL virus was then used to infect 293GP cells to produce pseudotyped LZRNL viruses bearing the VSV protein G (after transient transfectof 293GP cells infected with a plasmid encoding the VSV protein G). The procedure for producing pseudotyped LZRNL virus was performed as described [Ye et al-, (1994) Meth. Cell Biol. 43:99], Briefly, on the first day, approximately 5 x 10 5 PA317 cells were placed in a 100 mm tissue culture plate. On the next day (the second day), the PA317 cells were transformed with 20mug of plasmid DNA pLZRNL (the plasmid DNA was purified using CsCl gradients) using the normal calcium phosphate coprecipitatprocedure [Graham and Van der Eb ( 1973) Virol. 52: 456). A range of 10 to 40 mug of plasmid DNA can be used. Because 293GP cells can take more than 24 hours to bind tightly to tissue culture plates, 293GP cells can be placed on 100-mm plates 48 hours before transfect Transfected PA317 cells provide an amphotropic LZRNL virus.

On the third day, approximately 1 x 105 293GP cells were placed in a 100 mm culture dish 24 hours prior to collectof the amphotropic virus from the transfected PA317 cells. On the fourth day, the culture medium of the transfected PA317 cells was harvested 48 hours after the applicatof pLZRNL DNA. The culture medium was filtered through a 0.45 micron filter and polybrene was added to a final concentratof 8 μg / ml. A solutof the polybrene material was prepared by dissolving 0.4 grams of hexadimethrine bromide (polybrene; Sigma) in 100 ml of sterile water; the solution of the material was stored at 4 ° C. The culture medium containing LZRNL virus (containing polybrene) was used to infect the 293GP cells as follows. The culture medium was removed from the 293GP cells and replaced with the LZNRL virus containing the culture medium. The virus containing the culture medium was allowed to remain on the 293GP cells for 16 hours. After 16 hours of the infection period (on the fifth day), the medium was removed from the 293GP cells and replaced with a new medium containing 400 μg / ml of G418 (GIBCO / BRL). The medium was changed every three days until resistant colonies of G418 appeared two weeks later. Care was taken not to disturb the resistant colonies of G418 when the medium was changed as 293GP cells rather loosely attached to the tissue culture plates.

The resistant colonies 293 of G418 were collected using an automatic pipeton and transferred directly to 24-well plates (ie, the colonies were not removed from the plates using trypsin). The resistant colonies 293 of G418 (named as "293GP / LZRNL" cells) were selected for expression of the LacZ gene in order to identify clones that produce high titers of LZRNL pseudotypificadis virus. The clones from the 24-well plates were transferred to 100 mm tissue culture plates and allowed to develop to confluence. Protein extracts were prepared from confluent plates by washing the cells once with 10 ml of PBS (137 mM NaCl, 2.6 mM KC1, 8.1 mM Na2HP04, 1.5 mM KH2P04). Two ml of 250 mM tris-HCl, pH 7.8 were added, and the cells were scraped off the dish using a rubber-tipped glass rod. The cells were then harvested by centrifugation at room temperature and resuspended in 100 microliters, 250 mM tris-HCl, pH 7.8. The cells were subjected to four rapid freeze / thaw cycles followed by centrifugation at room temperature to remove cell debris. The beta-galactosidase activity present in the resulting protein extracts was determined as follows. Five microliters of protein extract were mixed with 500 μl of beta-gal buffer (50 mM tris-HCl, pH 7.5, 100 mM NaCl, 10 mM MgCl2) containing 0.75 of ONPG (Sigma). The mixtures were incubated at 37 ° C until a yellow color appeared. The reactions were stopped by the addition of 500 μl 10 mM EDTA and the optical density of the reactions was determined at 420 nm.

The 293GP / LZRNL clone that generated the highest amount of beta-galactosidase was then expanded and subsequently used for the production of the pseudotyped LZNRL virus. Approximately 1 x 106 293GP / LZRNL cells were placed in a 100 mm tissue culture plate. 24 hours later, the cells were transfected with 20 micrograms of plasmid DNA pHCMV-G, using co-precipitation with calcium phosphate. 6 to 8 hours after the precipitate of calcium DNA was applied to the cells, the DNA solution was replaced with a new culture medium (lacking G418). It has been found that longer transfection times (overnight), result in the separation of most of the 293GP / LZRNL cells from the plate and, therefore, these times are avoided. Transfected 293GP / LZRNL cells produce pseudotyped LZRNL viruses.

The pseudotyped LZRNL viruses generated from the transfected 293GP / LZRNL cells can be harvested at least once a day between 24 and 96 hours after transfection. The highest virus titer was generated approximately 48 to 72 hours after the transfection of initial pHCMV-G. Although the syncytium formation became visible approximately 48 hours after transfection in most of the transfected cells, the cells continued to generate pseudotyped viruses for at least an additional 48 hours as long as the cells remained attached to the culture plate. tissue. The collected culture medium containing the pseudotyped VSV LZRNL G virus was pooled, filtered through a 0.45 micron filter and stored at -70 ° C.

The title of the pseudotyped LZRNL G virus of the VSV was then determined as follows. 5 x 105 rat 208F fibroblasts or NIH 3T3 cells were placed in a 100 mm culture dish. 24 hours after they were placed on the plate, the cells were infected with serial dilutions of the LZRNL virus containing the culture medium in the presence of 8 μg / ml polybrene. Sixteen hours after infection with the virus, the medium was replaced with a new medium containing 400 μg / ml of G18 and the selection was continued for 14 days until resistant colonies of G418 became visible. Viral titers were typically from about 0.5 to 5.0 x 106 colony forming units (cfu) / ml. The virus titer can be concentrated to a titer greater than 109 cfu / ml as described in the following.

EXAMPLE 3 Concentration of pseudotyped retroviral vectors.

The pseudotyped VSV LZRNL G virus was concentrated to a high titer by two cycles of ultracentrifugation. The frozen culture medium, harvested as described in Example 2 containing the pseudotyped LZRNL virus, was thawed in a 37 ° C water bath and then transferred to ultra-clear centrifuge tubes (14 x 89 mm; Beckman, Palo Alto, CA) that had been previously sterilized by exposing the tubes to ultraviolet light in a laminar flow cap overnight. The viruses were pelleted in a SW41 (Beckman) rotor at 50,000 x g (25,000 rpm) at 4 ° C for 90 minutes. Then the culture medium was removed from the tubes in a laminar flow cap and the tubes were well drained. The virus pellets were resuspended from 0.5 to 1% of the original volume of the culture medium in either TNE (50 M Tris-HCl, pH 7.8, 130 mM NaCl, 1 mM EDTA) or in a balanced salt solution of Hank 0.1X [the balanced salt solution of Hank IX contains 1.3 mM CaCl2, 5 mM KC1, 03 M H2P04, 0.5 mM MgCl2-6H20), 0.4 mM MgSO4-7H20, 138 mM NaCl, 4 mM NaHC03, 0.3 M NaH2P04-H20, 0.1X Hank is made by mixing 1 part of Hank IX with 9 parts of PBS]. The resuspended virus pellets were incubated overnight at 4 ° C without stirring. The virus pellet could be dispersed with a gentle pipette movement after incubation overnight without significant loss of infectious virus. The titer of the viral material was routinely increased 100 to 300 times after one round of ultracentrifugation. The efficiency of recovery of infectious virus varied between 30% and 100%.

The viral material was then subjected to low speed centrifugation in a microfuge for 5 minutes at 4 ° C to remove any visible cellular debris or added virions that were not resuspended under the above conditions (if the virus material is not to be used) for injection into oocytes or embryos, this centrifugation stage can be omitted).

The viral material was then subjected to another round of ultracentrifugation to concentrate the virus material even more. The resuspended virus from the first round of centrifugation was joined and tabletted by a second round of ultracentrifugation which was performed as described above. Viral titers were increased approximately 200 times after the second round of ultracentrifugation (titers of the pseudotyped LZRNL virus were typically greater than or equal to 1 x 109 cfu / ml after the second round of ultracentrifugation).

The titres of the pre and post-centrifuged fluids were determined by infection of 208F (NIH 3T3 or Mac-T cells can also be used) followed by selection of resistant colonies of G18 as described above in example 2. Concentrated viral material was stable (ie, did not lose infectivity) when stored at 4 ° C for several weeks.

EXAMPLE 4 PREPARATION OF PSEUDOTIPED RETROVIRUS BY INFECTION OF OOCYTES AND EMBRYOS.

Concentrated pseudotyped retroviruses were resuspended in 0.1X HBS (2.5 mM HEPES, pH 7.12, 14 mM NaCl, 75 μM Na2HP04-H20) and 18 μl aliquots were placed in 0.5 ml vials (Eppendorf) and stored at -80 ° C until they were used. The titer of the concentrated vector was determined by diluting 1 μl of the concentrated virus 10"7 or 10" 8 times with HBS 0.1X. The diluted virus solution was then used to infect 208F and Mac-T cells and viral titers were determined as described in example 2.

Before infection of oocytes or embryos (by microinjection), 1 μl of polybrene [25 ng / μl; the working solution of polybrene was generated by diluting a solution of the material having a concentration of 1 mg / ml (in sterile water) in 0.1 of HBS, pH 7.12) was mixed with 4 μl of concentrated virus to produce a solution that contains 103-104 cfu / μl and 8 μg / ml polybrene. This solution was loaded on an injection needle (the tip with an internal diameter of approximately 2-4 micras) for injection into the perivitelline space of gametes (pre-mature oocytes, mature oocytes) or a zygote of the cell stage (stage embryo). precocious). An Eppendorf Transjector 5246 was used for all micro-injections.

EXAMPLE 5 PREPARATION AND MICROINJECTION OF GAMETOS AND ZÍGOTOS. The gametes (oocytes of pre-maturation and pre-fertilization) and zygotes (fertilized oocytes) were prepared and micro-injected with retroviral materials as described in the following, a) Solutions Tirodes-lactato with HEPES (TL-HEPES); 114 mM NaCl, 3.2 mM KC1, 2.0 mM NaHCO3, 0.4 mM Na2H2P04 * H20, 10 mM Na-lactate, 2 mM CaCl2-2H20, 0.5 mM MgCl2'6H20, lOmM HEPES, 100 IU / ml penicillin, 50 μg / ml red phenol, 1 mg / ml of fraction V of BSA, 0.2 mM of pyruvate and 25 μg / ml of genetamicin.

Maturing medium: TC-199 medium (GIBCO) containing 10% FCS, 0.2 mM pyruvate, 5 μg / ml NIH or LH (NIH), 25 μg / ml of genetamycin and 1 μg / ml of estradiol-17beta.

Sperm-thyroid-lactate (sperm-TL): 100mM NaCl, 3.2mM KC1, 25mM NaHCO3, 0.29mM Na2H2P0 -H20, 21.6mM Na-lactate, 2.1mM CaCl2-2H20, 0.4mM MgCl2-6H2 ?, 10 mM HEPES, 50 μg / ml red phenol, 6 mg / ml fraction V of BSA, 1.0 mM pyruvate and 25 μg / ml of genetamycin.

Fertilization medium: 114 mM NaCl, 3.2 mM KC1, mM NaHC03, 0.4 mM Na2H2P04 -H20, 10 M Na-lactate, 2 mM CaCl2-2H20, 0.5 mM MgCl2-6H20, 100 IU / ml penicillin, 50 μg / ml red phenol, 6 mg / ml BSA without fatty acid, 0.2 mM pyruvate and 25 μg / ml of genetamycin.

PHE: 1 mM hypotaurine, 2 mM penicillamine and 250 μM epinephrine.

Embryo incubation + amino acids (EIAA): 114 μM NaCl, 3.2 μM KC1, 25 μM NaHCO- ,, 1.6 μg / ml L (+) -lactate, 10.7 μg / ml L-glutamine, 300 μg / ml BSA without acid fatty, • 0.275 μg / ml of pyruvate, 25 μg / ml of genetamycin, 10 μl of 5 amino acid MEM 100X (M7145, Sigma) per ml and 20 μl of BME 50X amino acid material (B6766, Sigma) per ml, HBS 0.1X: 2.5 mM HEPES (pH 7.12), 14 M NaCl and 75 £ 10 μM Na2HP04-H-0. b) Preparation, injection, maturation and fertilization of pre-mature oocytes. 15 Oocytes were aspirated from small antral ovarian follicles of dairy cows obtained from a trail. Freshly aspirated oocytes were selected in the stage with germinal vesicle (GV), meiosis stopped, 20 with the accumulated mass together (ie pre-mature oocytes). The oocytes were then washed twice in freshly prepared TL-HEPES and transferred to a 100 microliter drop of TL-HEPES for microinjection.

After the injection, the pre-mature oocytes were washed twice in new TL-HEPES and transferred to the maturation medium (10 oocytes in 50-microliters). The pre-maturation oocytes were then incubated in a maturing medium for 24 hours at 37 ° C, which allowed the oocytes to mature to the metaphase II stage. Then the mature oocytes were washed twice in Sperma-TL and 10 oocytes were then transferred to 44 μl of fertilization medium. Mature cysts (10 oocytes / 44 μl of fertilization medium) were then fertilized by the addition of 2 μl of sperm at a concentration of 2.5 x 107 / ml, 2 μl of PHE and 2 μl of heparin (fertilization mixture). Sperm was prepared by the discontinuous separation of percolating gradient of frozen-thawed semen as described [Kim et al. (1993) Mol. Reprod. Develop. 35: 105], Briefly, percolation gradients were formed by placing 2 ml of each 90% percolator and 45% in a 15 ml conical tube. The frozen-thawed semen was layered on top of the gradient and the tubes were centrifuged for 10 minutes at 700xg. The motile sperm was collected from the bottom of the tube.

The oocytes were incubated for 16 to 24 hours at 37 ° C in the fertilization mixture. After fertilization, the accumulated cells were removed by a swirling movement of the cells ((zygotes of the cell stage, pronucleus stage) for 3 minutes to produce oocyte "knots." The oocyte nodes were then washed twice in a medium of embryo culture (EIAA) and 20 to 25 zigotes were then cultured in 50 microliter drops of EIAA (without serum until the fourth day at which time the zygotes were placed in EIAA containing 10% serum) until the desired development stage: approximately 48 hours or two days (day 0 is the day when the mature oocytes were cocultivated with sperm) for the morula stage (stage 8 of the cell) or day 6 or 7 for the blastocyst stage. embryos in the morula stage were analyzed for .beta-galactosidase expression as described in example 6. Embryos derived from injected pre-oocyte oocytes were also analyzed for beta-galactosi expression dasa in cell 2, cell 4 and blastocyst stage and all the developed stages examined were positive. c) Preparation, injection and fertilization of prefertilization oocytes.

Pre-re oocytes were harvested, washed twice with TL-HEPES as described above. The oocytes were then cultured in a ring medium (10 oocytes per 50 microliters of the medium) for 16 to 20 hours to produce prefertilized oocytes (metaphase II stage). Then the prefertilization or re oocytes were swirled for 3 minutes to remove the accumulated cells to produce oocyte nodes. The knotted oocytes were washed twice in TL-HEPES and then transferred to a 100 microliter drop of TL-HEPES for microinjection. The microinjection was performed as described here before.

After the microinjection, the prefertilization oocytes were washed twice with TL-HEPES and then placed in a ration medium until fertilization. Fertilization was conducted as described above. After fertilization, the zygotes were then washed twice in EIAA and 20 to 35 zigotes were then cultured by drop of 50 microliters of EIAA until the desired stage of development was reached.

Next, the embryos were examined for beta-galactosidase expression (example 6) or transferred to recipient cows (example 7). d) Preparation and injection of stage zigotes of a cell.

As described above, re oocytes were generated (metaphase stage II). The re oocytes were then co-cultured in the presence of sperm for 16 to 20 hours as described above to generate zygotes in the pronucleus stage. . The zygotes in the pronucleus stage were swirled for 3 minutes to remove the layer of accumulated cells before microinjection. The microvirus microinjection was conducted as described above. After the microinjection, the zygotes were washed four times in EIAA and then placed in a drop (25 zigotes per drop of 50 microliters of EIAA). The zygotes were cultivated in EIAA (20 to 25 zigotes per drop of 50 microliters of EIAA) until the desired stage of development was reached. The embryos were then examined for beta-galactosidase expression (example 6) or transferidos to recipient cows (example 7).

EXAMPLE 6 Injection of pseudotyped retrovirus into the perivitelline space of bovine ration oocytes, results in the efficient transfer of vector sequences, Single cell zygote oocytes that have been microinjected with pseudotyped LZRNL virus and cultured in vitro were examined for expression of vector sequences by staining for beta-galactosidase activity when the embryos reached the morula stage. The activity of beta-galactosidase was examined as follows. The embryos were washed twice in PBS and then fixed in 0.5% glutaraldehyde in PBS containing 2mM magnesium chloride for 40 minutes at 4 ° C. The fixed embryos were then washed three times with PBS containing 2 mM magnesium chloride and then incubated at 37 ° C overnight in an X-gal solution (20 mM K3Fe (CN) 6, 20 mM K4Fe (CN) 6.N20, 2mM magnesium chloride and 1 mg / ml X-gal). The presence of the blue precipitate indicates expression of beta-galactosidase activity. The results are shown below in the following Table 1.

TABLE ^ Positive number / number injected.

From the results of Table 1, it is clear that infection of pre-fertilization and zygote oocytes using the methods of the present invention results in the transfer and expression of retrovirally encoded nucleic acid. Although the present invention is not limited to any particular theory, it is commonly believed that only half of the daughter cells of an initial founder cell infected with a retrovirus will contain the provirus because the retroviral provirus is integrated into the host post-replication DNA [ Hajihosseini et al. (1993) EMBO J. 12: 4969], Therefore, the finding that 47% of the prefertilization oocytes injected are positive for β-galloctosidase expression suggests that 100% of these injected oocytes were infected with the retrovirus recombinant. Therefore, the methods of the present invention provide a generation efficiency of transgenic embryos that is superior to existing methods.

EXAMPLE 7 Generation of transgenic cows that contain integrated retroviral nucleic acid sequences.

Embryos derived from infected prefertilization oocytes and early zygotes were transferred to recipient cows that were allowed to reach term as described below. a) Treatment of embryos derived from infected zygotes and oocytes. Preferrtilization oocytes (infected approximately 17 hours after exposure to the maturation medium) and early stage zygotes (= cell stage 8) were prepared and infected as described in example 5 with the exception that: VSV pseudotyped viruses G used were the LSRNL viruses that were prepared as described for the LZRNL virus in Example 2; and 2) on the fourth post-fertilization day embryos derived from injected pre-fertilized zygotes and oocytes were placed in a freshly prepared EIAA medium containing 10% FCS and allowed to develop in vitro until transfer to recipient cows. The embryos on the seventh day were transferred to female recipients that were prepared as described below. b) Preparation of container cows and embryo transfer.

Cows were synchronized by injecting 100 μg of gonadotropin releasing hormone (GnRH, Sanofi Winthrop Pharmaceutical Inc., New York, NY) (day zero). Seven days later, the recipients were injected with 25 mg PGF2o (Upjohn Co., Kala azoo, MI). Thirty to 48 hours after the injection of PGF2a, a second injection of 100 μg of GnRH was applied. Ovulation occurs approximately 24 to 32 hours after the injection. Seven days after ovulation occurred, embryos derived from infected zygotes and oocytes (seven day old embryos) were then transferred without surgery to the uteri of the recipient cows. Two embryos were transferred to each container (it is expected that only one calf will be born from the transfer of the two embryos in a single container).

A total of twenty embryos were transferred into the containers in three separate days. On the first day, 8 transfer embryos derived from infected prefertilization oocytes were transferred in four vessels. Four calves were born from these containers and it was found that all 4 were positive for the presence of proviral vector DNA (ie, 100% were transgenic). On the second day, 8 transfer embryos derived from prefertilization oocytes were transferred in 4 containers; 2 calves were born to those containers and it was found that one of these animals is transgenic (in the second transfer, the product was lost in the first month of a pregnant woman and another pregnant lost twins in the eighth month, no embryo with 8 months of pregnancy he was transgenic). On the third day, 4 transfer embryos derived from infected zygotes (infected in stage 4-8 of the cells) were transferred to 2 recipients; 3 calves were born to these containers and none were transgenic.

The nine calves appeared healthy at birth and continued to appear healthy at the age of 6 months. After the birth of the offspring derived from the injected zygotes and oocytes, the offspring were examined by Southern blot and PCR analysis to determine if they contained the retroviral transgenes and if they exhibited somatic cell mosaicism. The skin tissue and white blood cells (buffy coat) were collected from the calves. Genomic DNA is extracted using common techniques. Briefly, tissue samples were digested with 50 μg / ml proteinase K (GIBCO) at 55 ° C. The samples were then extracted in sequence 2 times with an equal volume of phenol, once with phenol: chloroform (1: 1) and again with chloroform. Then the DNA present in the aqueous layer was precipitated by the addition of two volumes of isopropanol. The DNA was collected by centrifugation and the DNA pellets were resuspended in a buffered TE (lOmM Tris-Cl, 1 M EDTA, pH 8.0) and the concentration was determined spectrophotometrically. The DNA was then analyzed by Southern blot and PCR analysis. The results are shown in figures 2 and 3.

Figure 2 shows an autoradiograph of a Southern blot of genomic DNA isolated from the hide (Figure 2A) and blood (Figure 2B) of 6 calves derived from either prefertilization oocytes infected with a pseudo-typed LSRNL virus VSV about 17 hours after exposure to the maturation medium (calves numbered 17, 18, 20 and 21) or a few zygotes of infected cells approximately 12 hours after postfertilization (calves numbered 15 and 16). The DNA of the calves was digested with HindTII which cuts the pLSRNL vector to generate a 1.6 kb fragment (Figure 2C). DNA digested with HindTIT from blood (line marked * 12 derived from a random non-transgenic calf), ovary and semen from non-transgenic cattle (derived from random adult females and males), were also included. Lines marked "3989 M and F" represent DNA derived from two late-term embryos that were born one month prematurely (these calves were generated from injected fertilized eggs and both are not transgenic). Lines marked "LSRNL pDNA" contain plasmid DNA of pLSRNL digested with HindIII and provide controls for the quantification of copy number of the progenies integrated in the progeny (DNA equivalent to 5, 10 or 25 copies of LSRNL, were applied in these lines ).

Approximately 10 μg of the digested DNA with HindIII were electrophoresed in 0.8% agarose gels and stained on a nylon membrane. The membrane was hybridized with a 32P labeled probe that hybridizes to the HBsAg gene present in the pLZRNL vector (Figure 2C). The HBsAG probe was generated by PCR amplification of the pLSRNL plasmid DNA using the upstream base S-1 [5'-GGCTATCGCTGGATGTGTCT-3 '(SEQ ID NO: 3)] and the downstream base S-3 [5' -ACTGAACAAATGGCACTAGT-3 '(SEQ ID NO: 4)]. The generated PCR probe (334 bp) was signaled using a Rediprime kit (Amersham, Arlington Heights, IL) according to the manufacturer's instructions. The autoradiographs shown in Figure 2 were generated by exposing the spots to X-ray film for 3 weeks at -80 ° C.

The results shown in Figure 2 show that calves 16, 17, 18, 20 and 21 contained Retroviral vector DNA in both the leather (Figure 2A) and the blood (Figure 2B). Like the blood cells (buffy coat) are derived from the cells of the mesoderm and the cells of the leather, are derived from the hectoderm, these results show that the transgenic animals do not exhibit somatic cell mosaicism. Southern blot analysis has shown that the majority (this is, 7/9) of the transgenic calves contain a simple copy of the proviral sequence; a few animals (ie 2/9) appear to contain two copies of the integrated proviral sequence. These results further demonstrate that the retroviral infection, both of pre-fertilization oocytes and early stage zygotes, was successful in integrating the viral sequences into the genome of the resulting transgenic animals.

In order to confirm the presence of retroviral sequences integrated into the genome of the somatic cells of transgenic animals, PCR analysis was performed (figure 3) using genomic DNA isolated from the 5 transgenic calves that was determined by Southern blot analysis, be transgenic by the retroviral sequences. Figure 3 shows the result of the PCR analysis after the amplification of two different regions (ie, the neo gene and the HBsAg gene) of the retroviral genome of the LZRNL that was injected into the oocytes. The genomic DNA of the leather and blood of each of the five transgenic calves was amplified using the bases upstream and downstream (SEQ ID NOS: 1 and 2 and NOS: 3 and 4 described supra) for the neo genes (Figure 3A) and HBsAg (Figure 3B), respectively. The PCRs analyzes were conducted using the following thermocyclic conditions: 94 ° C (4 minutes); [94 ° C (2 minutes); 50 ° C (2 minutes); 72 ° C (2 minutes)] 30 C? Ios; 72 ° C (10 minutes). The amplification produced the expected dimensions of the amplified sequence with the bases neo (349 bp) and HBsAg (334 bp) in both the blood and the leather of each of the five transgenic calves. The genomic DNA isolated from the blood of the non-transgenic calves, as well as the semen and the ovary of non-transgenic cattle were used as negative controls in the PCRs. PLSRNL DNA was used as the positive control.

These data demonstrated that infection of prefertilization oocytes results in the efficient transfer of retroviral vector DNA (100% of birth or of 4 transgenic calves / 4 calves of embryos derived from infected prefertilization oocytes). In addition, means for efficiently generating transgenic animals are provided. The methods of the present invention provide means for generating transgenic animals that do not exhibit mosaisis or somatic cells. In addition, these methods allow the production of transgenic animals that contain a single copy of the transgene.

In order to confirm the transmission of the germ line of the integrated viral sequences, the transgenic progeny are generated with non-transgenic cattle and the presence of the viral sequences (ie, the transgene) is determined using a Southern blot analysis or an amplification. PCR as described above. Animals that are heterozygotes or homozygotes for the transgene are produced using methods well known in the art (e.g. integergendering of animals heterozygous for the transgene).

From the foregoing it is evident that the invention provides improved methods and compositions for the preparation of non-human transgenic animals. The methods of the present invention provide the production of non-human transgenic animals with improved efficiency and a reduced incidence of generation animals that are mosaics for the presence of the transgene.

All publications and patents mentioned in the above specification are incorporated herein by reference. Various modifications and variations of the described methods and systems of the invention will be apparent to those skilled in the art without departing from the scope and spirit of the invention.

Although the invention has been described in connection with specific preferred embodiments, it will be understood that the invention as claimed will not be unduly limited to those specific embodiments. Indeed, various modifications of the modes described to carry out the invention that are obvious to those skilled in molecular biology or related fields are intended to be within the scope of the clauses indicated below.

LIST OF SEQUENCES (1) GENERAL INFORMATION (i) APPLICANT: Bremel, Robert D. Chan, Anthony W.S. Burns, Jane C. (ii) TITLE OF THE INVENTION: Methods for creating transgenic animals. (iii) SEQUENCE NUMBER: 10 (iv) CORRESPONDENCE ADDRESS: (A) ADDRESSES: Medien & Carroll, LLP (B) STREET: 220 Mantgomery Street, Suite 2200 (C) CITY: San Francisco (D) STATE. California (E) COUNTRY. E. ü. A. (F) ZIF: 94104 (v) READABLE FORM OF THE COMPUTER: (A) MIDDLE TYPE: Flexible disk. (B) COMPUTER: IBM PC compatible. (C) OPERATING SYSTEM: PC-DOS / MS-DOS. (D) SOFTWARE: PATENT RELEASE # 1.0, version # 1.30 (vi) NORMAL APPLICATION DATA (A) APPLICATION NUMBER: US (B) APPLICATION DATE: (C) CLASSIFICATION: (viii) INFORMATION FROM THE LAWYER / AGENT: (A) NAME: INGOLIA, DIANE E. (B) REGISTRATION NUMBER: 40,027. (C) REFERENCE NUMBER / FILE: WARF-02184. (ix) TELEPHONE INFORMATION: (A) TELEPHONE: (415) 705-84-10 (B) TELEFAX. (415) 397-8338 (2) INFORMATION FOR SEQ ID NO: l: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 20 base pairs (B) TYPE: Nucleic acid (C) BRAIDED: Simple (D) TOPOLOGY : Linear (ii) TYPE OF MOLECULE: different nucleic acid (A) DESCRIPTION: / desc = "DNA" (xi) SEQUENCE DECRIPTION: SEQ ID NO: l: GCATTGCATC AGCCATGATG (2) INFORMATION FOR SEQ ID NO: 2: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 20 base pairs (B) TYPE: Nucleic acid (C) BRAIDED: Simple (D) TOPOLOGY: Linear (ii) TYPE OF MOLECULE: different nucleic acid (A) DESCRIPTION: / desc = "DNA" (xi) SEQUENCE DECRIPTION: SEQ ID NO: 2: GATGGATTGC ACGCAGGTTC (2) INFORMATION FOR SEQ ID NO: 3: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 20 base pairs (B) TYPE: Nucleic acid (C) BRAIDED: Simple (D) TOPOLOGY: Linear (ii) TYPE OF MOLECULE: different nucleic acid (A) DESCRIPTION: / desc = "DNA" (xi) DECRIPTION OF. SEQUENCE: SEQ ID NO: 3: GGCTATCGCT GGATGTGTCT (2) INFORMATION FOR SEQ ID NO: 4: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 20 base pairs (B) TYPE: Nucleic acid (C) BRAIDED: Simple (D) TOPOLOGY: Linear (ii) TYPE OF MOLECULE: different from nucleic acid (A) DESCRIPTION: / desc = "DNA" (xi) SEQUENCE DECRIPTION: SEQ ID NO:: ACTGAACAAA TGGCACTAGT (2) INFORMATION FOR SEQ ID NO: 5: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 1590 base pairs (B) TYPE: Nucleic acid (C) BRAIDED: double (D) TOPOLOGY: Linear (ii) TYPE OF MOLECULE: different nucleic acid (A) DESCRIPTION: / desc = "DNA" (ix) CHARACTERISTICS: (A) NAME / KEY: CDS (B) LOCATION: 1: 1587 (xi) SEQUENCE DECRIPTION: SEQ ID NO: 5: ATG G? T CTC TTT CCC ATT TTG GTC GTG GTG CTC ATG ACA GAT ACT GTC 48 Met Aap Leu Phe Pro lie Leu Val Val Leu Met Thr Asp Thr Val 1 5 10 15 TTA GGG AAG TTT CAA ATT GTC TTC CCG GAT CAG A? T GAA CTG GAO TGG 96 Leu Gly Lyß Phß Gln lie Val Phe Pro Aap CJln Aßn Glu Leu Glu Trp 20 25 30 AGA CCA GTT GTG GGT GAC TCT CGG CAT TGC CCA CAG TCA TCA GAA ATG 144 Arg Pro Val Val Gly Aap Ser Arg His Cys Pro Gln Ser Ser Glu Met 35 40 45 CAA TTC GAT GGA AGC A.JA TCC CAG ACC ATA CTG ACT GGG AAA GCT CCC 192 Gln Phe Asp Gly Ser Arg be Gln Thr He Leu Thr aly Lyß Ala P «50 55 60 GTG GGG ATC ACG CCC TCT AAA TCA GAT GGA TTT ATC TGC CAT CCC GCA Í40 Val Gly He Thr Pro Ser Lys Ser Asp aly Phe He -Cyß His Ala Ala 65 70 75 80 AAA TGG GTG ACÁ ACÁ TGT GAT TTC AGG TGG TAT GGG CCG AAA TAC ATC 288 Lys Trp Val Thr Thr Cys Asp Phe Arg Trp Tyr Gly Pro Lys Tyr He 85 90 95 ACT CAT TCA ATA CAT C.YT CTG ^ -3A CCG ACÁ ACÁ TCA GAC TGT GAG ACÁ 336 Thr His Ser He His His Leu Arg Pro Thr Thr Ser Asp Cys Glu Thr 100 105 110 GCT CTC CAG AGG TAT AAA GAT GGG AGC TTA ATC AAT CTT GGA TTC CCC 384 Wing Leu Gln Arg Tyr Lys Asp Gly Ser Leu He Asn Leu Gly Phe Pro 115 120 125 CCA GAA TCC TGC GGT TAT GCA ACÁ GTC ACÁ GAT TCT GAG GCA ATG TTG 432 Pro Glu Ser Cys Gly Tyr Ala Thr Val Thr Asp Ser Glu Wing Met Leu 130 135 140 GTC CAG GTG ACT CCC CAC GTG GTG GTG GAT TAT AGA GGT CAC 480 Val Gln Val Thr Pro His His Val Gly Val Asp Asp Tyr Arg Gly His 145 150 155 160 TGG ATC GAC CCA CTA TTT CCA GGA GGA GAA TGC TCC ACC AAT TTT TGT 528 Trp He Aßp Pro Leu Phß Pro ßly Gly Glu Cyß Ser Thr Asn Phß Cys 165 170 175 GAT ACÁ GTC CAC AAT TCA TCG GTG TGG ATC CCC AAG AGT CAÁ AAG ACT 576 Asp Thr Val His Asn Ser Ser Val Trp He Pro Lys Ser Gln Lye Thr 180 185 190 GAC ATC TGT GCC CAG TCT TTC AAA AAT ATC AAG ATG ACC GCA TCT TAC 624 Aßp He Cys Wing Gln Ser Phß Lys Asn He Lys Met Thr Wing Ser Tyr 195 200 205 CCC TCA GAA GGA GG TTG GTG AGT GAC AGA TTT GCC TTC CAC AGT GCA 672 Pro Ser Glu Gly Wing Leu Val Ser Asp Arg Phe Wing Phe His Ser Wing 210 215 220 TAT CAT CCA AAT ATG CCG GGG TCA ACT GTT TGC ATA ATG GAC TTT TGC 720 Tyr His Pro Asn Met Pro Gly Ser Thr Val Cys He Met ASD Phe Cys 225 230 235 * 2 0 GAA CAA AAG GGG TTG AGA TTC ACA AAT GGA GAG TGG ATG GGT CTC AAT 768 Glu Gln Lys Gly Leu Arg Phe Thr Asn Gly Glu Trp Met Gly Leu Asn 245 25o 255 GTG GAG CA TCC ATC CGA GAG AAG AAG ATA AGT GCC ATC TTC CCA AAT 816 val Glu Gln Ser He Arg Glu Lyß Lys He Ser Ala He Phe Pro Asn 260 265 270 TGT GTT GCA GGG ACT GAA ATC CGA GCC ACA CTA GAA TCA GAA GGG GCA 864 Cys Val Wing Gly Thr Glu He Arg Wing Thr Leu Glu Ser Glu Gly Wing 275 280 285 AGA ACT TTG ACG TGG GAG ACT CA AGA ATG CTA GAT TAC TCT TTG TGT 912 Arg Thr Leu Thr Trp Glu Thr Gln Arg Met Leu Asp Tyr Ser Leu Cys 290 295 300 CAG AAC ACC TGG GAC AAA GTT TCC AGG AAA GAA CCT CTC AGT CCG CTT 960 Gln Asn Thr T rp Asp Lys Val Ser Arg Lys Glu Pro Leu Ser Pro Leu 305 310 315 320 GAC TTG AGC TAT CTG TCA CCA AGG GCT CCA GGG AAA GGC ATG GCC TAT 1008 Asp Leu Ser Tyr Leu Ser Pro Arg Wing Pro Gly Lys Gly Met Wing Tyr 325 330 335 ACC GTC ATA AAC GGA ACC CTG CAT TCG GCT CAT GCT AAA TAC ATT AGA 1056 Thr Val He Asn Gly Thr Leu His Ser Wing His Wing Lys Tyr He Arg 340 345 350 ACC TGG ATT GAT TAT GGA GAA ATG AAG GAA ATT AAA .GGT GGA CGT GGA 1104 Thr Trp He Asp Tyr Gly Glu Met Lys Glu He Lys Gly Gly Arg Gly 355 360 365 GAA TAT TCC AAG GCT CCT GAG CTC CTC TGG TCC CAG TGG TTC GAT TTT 1152 Glu Tyr Ser Lys ^ ~ -a - Glu Leu Leu Trp Ser Gln Trp Phe Asp Phe 370 375 380 GGA CCG TTC AAA ATT GGA CCG AAT GGA CTC CTG CAC ACA GGG AAA ACC 1200 Gly Pro Phe Lyß He Gly Pro Asn Gly Leu Leu Hxs Thr Gly Lys Thr 385 390 395 400 TTT AAA TTC CCT CTT TAT TTG ATC GGA GCA GGC ATA ATT GAC GAA GAT 1248 Phe Lys Phe Pro Leu Tyr Leu He Gly Wing Gly He He Asp Glu Asp 405 410 415 CTG CAT GAA CTA GAT GAG GCT GCT CCC ATT GAT CAC CCA CAG ATG CCT 1296 Leu His Glu Leu Aßp Glu Ala Wing Pro He Asp Hiß Pro Gln Met Pro 420 425 430 GAC GCG AAA AGC GTT CTT CCA GAA GAT GAA GAG ATA TTC GTC GCA 1344 Asp Ala Lys Ser Val Leu Pro Glu Asp Glu Glu He Phe Phe Gly Asp 435 440 44S ACÁ GGT GTA TCC AAA AAC CCT ATC GAG TTG ATT CA? GGA TGG TTC TCA 1392 Thr Gly Val Ser Lys Asn Pro He Glu Leu He Gln Gly Trp Phe Ser 450 455 460 AAT TGG AGA GAG AGT GTA ATG GCA ATA GTC GGA ATT GTT CTA CTC ATC 1 440 Asn Trp Arg Glu Ser Val Met Wing He Val Vally Val Leu Le He 465 470 475 480 GTT GTG ACA TTT CTG GCG ATC AAG ACG GTC CGG GTG CTT AAT TGT CTC 1 8 or Val Val Thr Phe Leu Wing He Lyß Thr Val Arg Val Leu Asn Cyß Leu 485 490 495 TGG AGA CCC AGA AAG AAA AGA ATC GTC AGA CAA GAA GTA GAT GTT GAA? 536 Trp Arg Pro Arg Lyß Lys Arg He Val Arg Gln Glu Val Asp Val Glu 500 505 *? O TCC CGA CTA AAC CAT TTT GAG ATG AGA GGC TTT CCT GAA TAT GTT AAG 1534 Ser Arg Leu Asn His Phe Glu Met Arg Gly Phe Pro Glu Tyr Val Lys 515 520 525 AGA TAA 1590 Arg (2) INFORMATION FOR SEQ ID NO: 6: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 529 amino acids (B) TYPE: amino acids (D) TOPOLOGY: Linear (ii) TYPE OF MOLECULE: protein (xi) SEQUENCE DECRIPTION: SEQ ID NO: 6: Mßt Asp Leu Phe Pro He Leu Val Val Leu Met Thr Asp Thr Val 1 5 10 15 Leu Gly Lys Phe Gln He Val Phe Pro Asp Gln A3n Glu Leu Glu Trp 20 25 30 Arg Pro Val Val Gly Asp Ser Arg His C s Pro Gln Ser Ser Glu Met 35 40 45 Gln Phe Asp Gly Ser Arg Ser Gln Thr He Leu Thr Gly Ly3 Wing Pro 50 55 60 Val Gly He Thr Pro Ser Lys Ser Asp Gly Phe He Cys His Wing Wing 65 70 75 80 Lys Trp Val Thr Thr Cys Asp Phe Arg Trp Tyr Gly Pro Lys Tyr He U 90 95 Thr His Ser Hi Hi His Leu Arg Pro Thr Thr Ser Asp Cys Glu Thr 100 ios no Wing Leu Gln Arg Tyr Lys Asp Gly Ser Leu He Asn Leu Gly Phe Pro 115 120 125 Pro Glu Ser Cys Gly Tyr Wing Thr Val Thr Asp Ser Glu Wing Met Leu 130 135 140 Val Gln Val Thr Pro His His Val Gly Val Asp Asp Tyr Arg Gly His 145 150 155 160 Trp He Asp Pro Leu Phe Pro Gly Gly Glu Cys Ser Thr Asn Phe Cys 165 170 175 Asp Thr Val His Asn Ser Ser Val Trp He Pro Lys Ser Gln Lys Thr 180 185 190 Asp He Cys Wing Gln Ser Phe Lys Asn He Lys Met Thr Wing Ser Tyr 195 200 205 Pro Ser Glu Gly Ala Leu Val Ser Asp Arg I- I have Ala Phe His Ser Ala 210 215 220 Tyr His Pro Asn Met Pro Gly Ser Thr Val Cys He Met Asp Phe Cys 225 230 235 240 Glu Gln Lys Gly Leu Arg Phe Thr Asn Gly Glu Trp Met Gly Leu Asn 245 250 255 Val Glu Gln Ser He Arg Glu Lys Lys He Be Wing He Phe Pro Asn 260 265 270 Cyß Val Wing Gly Thr Glu He Arg Wing Thr Leu Glu Ser Glu Gly Wing 275 280 285 Arg Thr Leu Thr Trp Glu Thr Gln Arg Met Leu Asp Tyr Ser Leu Cys 290 295 300 Gln Asn Thr Trp Asp Lys Val Ser Arg Lys Glu Pro Leu Ser Pro Leu 305 310 315 320 Asp Leu Ser Tyr Leu Ser Pro Arg Ala Pro Gly Lys Gly Met Ala Tyr 325 330 335 Thr Val He Asn Gly Thr Leu His Ser Wing His Wing Lys Tyr He Arg 340 345 350 Thr Trp He Asp Tyr Gly Glu Met Lys Glu He Lys Gly Gly Arg Gly 355 360 365 Glu Tyr Ser Lys Wing Pro Glu Leu Trp Ser Gln Trp Phe Asp Phe 370 375 380 Gly Pro Phe Lyß He Gly Pro Aßn Gly Leu Leu His Thr Gly Lys Thr 385 390 395 400 Phe Lys Phe Pro Leu Tyr Leu He Gly Wing Gly He He Asp Glu Asp 405 410 415 Leu His Glu Leu Asp Glu Ala Ala Pro He Asp His Pro Gln Met Pro 420 425 430 Asp Ala Lys Ser Val Leu Pro Glu Asp Glu Glu He Phe Phe Gly Asp 435 440 445 Thr Gly Val Ser Lyß Even Pro He Glu Leu He G n Gly Trp Phe Ser 450 455 460 Asn Trp Arg Glu Ser Val Met Wing He Val Gly He Val Leu Leu He 465 470 475 480 Val Val Thr Phe Leu Wing He Lys Thr Val Arg Val Leu Asn Cys Leu 485 490 495 Trp Arg Pro Arg Lys Lys Arg He Val Arg Gln Glu Val Asp Val Glu 500 505 510 Ser Arg Leu Asn His Phe Glu Met Arg Gly Phe Pro Glu Tyr Val Lys 515 520 525 Arg (2) INFORMATION FOR SEQ ID NO: 7: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 1590 base pairs (B) TYPE: Nucleic acid (C) BRAIDED: double (D) ) TOPOLOGY: Linear (ii) TYPE OF MOLECULE: different nucleic acid (A) DESCRIPTION: / desc = "DNA" (ix) CHARACTERISTICS: (A) NAME / KEY: CDS (B) LOCATION: 1: 1587 (xi) SEQUENCE DECRIPTION: S? Q ID NO: 7: ATG GAT CTC TTT CCC ATT TTG GTC GTG GTG CTC ATG ACA GAT ACT GTC 48 Met Asp Leu Phe Pro He Leu Val Val Val Leu Met Thr Aap Thr Val 1 5 10 15 TTA GGG AAG TTT CAA ATT GTC TTC CCG GAT CAG AAT GAA CTG GAG TGG 96 Leu Gly Lys Phe Gln He Val Phe Pro Asp Gln Asn ülu Leu Glu Trp 20 25 30 AGA CCA GTT GTG GGT GAC TCT CGG CAT TGC CCA CAG TCA TCA GAA ATG 144 Arg Pro Val Val Gly Asp Ser Arg His Cys Pro Gln Ser Ser Glu Met 35 40 45 CAA TTC GAT GGA AGC AGA TCC CAG ACC ATA CTG ACT GGG AAA GCT CCC 192 Gln Phe Asp Gly Ser Arg Ser Gln Thr He Leu Thr ßly Lys Wing Pro 50 55 60 GTG GGG ATC ACG CCC TCT AAA TCA GAT GGA TTT ATC TGC CAT GCC GCA 240 Val Gly He Thr Pro Ser Lys Ser Asp Gly Phe He Cys His Ala Ala 65 70 75 80 AAA TGG GTG ACÁ ACÁ TGT GAT TTC AGG TGG TAT GGG CCG AAA TAC ATC 286 Lys Trp Val Thr Thr Cys Asp Phe Arg Trp Tyr Gly Pro Lys Tyr He 85 90 95 ACT CAT TCA ATA CAT CAT CTG AGA CCG ACÁ ACÁ TCA GAC TGT GAG ACÁ 336 Thr His Ser He His His Leu Arg Pro Thr Thr Ser Asp Cys Glu Thr 100 105 110 GCT CTC CAG AGG TAT AAA GAT GGG AGC TTA ATC AAT CTT GGA TTC CCC 384 Ala Leu Gln Arg Tyr Lys Asp Gly Ser Leu He Asn Leu Gly Phe Pro 115 120 -125 CCA GAA TCC TGC GGT TAT GCA ACA GTC ACA GAT TCT GAG GCA ATG TTG 432 Pro Glu Ser Cys Gly Tyr Ala Thr Val Thr Asp Ser Glu Ala Met Leu 130 135 1 40 GTC CA GTG ACT CCC CAC CTC GTT GGG GTG GAT GAT TAT AGA GGT CAC 480 val Gln Val Thr Pro His His Val Gly Val Aep Asp Tyr Arg Gly His 145 150 155 160 TGG ATC GAC CCA CTA TTT CCA GGA GGA GAA TGC TCC ACC AAT TTT TGT 528 Trp He Asp Pro Leu Phe Pro Gly Gly Glu Cys Ser Thr Asn Phe Cys 165"O 175 GAT ACA GTC CAC AAT TCA TCG GTG TGG ATC CCC AAG AGT CAA AAG ACT 576 Asp Thr Val His Asn Ser Ser Val Trp He Pro Lys Ser ßln Lys rhr 180 185 »0 G GAACC AATTCC TTGGTT GGCCCC CCAAGG TTCCTT TTTTCQJ AAAAAA AAAKT_ A? TIC < _ A? AG» A? TG «A" .CC- G-CA ~ "TCC? T * . TT-VAn-C 624 mi. *. M * £ p He CCyyss Wing 01 »Ser Phe Lys Asn He Lys Met Thr Wing Ser Tyr 195 - 200 205 CCC TCA GAA GGA GCA TTG GTG AGT GAC AGA TTT GCC TTC CAC AGT OCA 672 Pro Ser Glu Gly Ala Leu Val Ser Asp Arg Phß Wing Phe Hiß Ser Wing 210 215 220 TAT CAT CCA AAT ATG CCG GGG TCA ACT GTT TGC ATA ATO GAC TTT TGC 720 Tyr His Pro Asn Met Pro Gly Ser Thr Val Cys He Met Asp Phe Cyß 225 230 235 240 GAA CAÁ AAG GGG TTG AGA TTC ACA AAT GGA GAG TGG ATG GGT CTC AAT 768 Glu Gln Lys Gly Leu Arg Phe Thr Asn Gly Glu Trp Met Gly Leu Asn 245 250 255 GTG GAG CA CA TCC ATC CGA GAG AAG AAG ATA AGT GCC ATC TTC CCA AAT 816 Val Glu Gln Ser He Arg Glu Lys Lys He Be Wing He Phe Pro Asn 260 265 270 TGT GTT GCA GGG ACT GAA ATC CGA GCC ACA CTA GAA TCA GAA GGG GCA 864 Cys Val Ala Gly Thr Glu He Arg Ala Thr Leu Glu Ser Glu Gly Wing 275 280 285 AGA ACT TTG ACG TGG GAG ACT CAA AGA ATG CTA GAT TAC TCT TTG TGT 912 Arg Thr Leu Thr Trp Glu Thr Gln Arg Met Leu Asp Tyr Ser Leu Cys 290 295 300 CAG AAC ACC TGG GAC AAA GTT TCC A GG AAA GAA CCT CTC AGT CCG CTT 960 Gln Aßn Thr Trp Asp Lys Val Ser Arg Lys Glu Pro Leu Ser Pro Leu 30S 310 315 320 GAC TTG AGC TAT CTG TCA CCA AGG GCT CCA GGG AAA GGC ATG GCC TAT 1008 Asp Leu Ser Tyr Leu Ser Pro Arg Wing Pro Gly Lys Gly Met Wing Tyr 325 330 335 ACC GTC ATA AAC GGA ACC CTG CAT TCG GCT CAT GCT AAA TAC ATT AGA 1056 Thr Val He Asn Gly Thr Leu His Ser Wing His Wing Lys Tyr He Arg 340 345 350 ACC TGG ATT GAT TAT GGA GAA ATG AAG GAA ATT AAA GGT GGA CGT GGA 1104 Thr Trp He Aßp Tyr Gly Glu Met Lyß Glu He Lyß Gly Gly Arg aly 355 360 365 GAA TAT TCC AAG GCT CCT GAG CTC CTC TGG TCC CAG TGG TTC GAT TTT 1152 Glu Tyr Ser Lys Wing Pro Glu Leu Leu Trp Ser Gln Trp Phe Asp Phe 370 375 380 GGA CCG TTC AAA ATT GGA CCG AAT GGA CTC CTG CAC ACA GGG AAA ACC 1200 Gly Pro Phe Lys He Gly Pro Asn Gly Leu Leu His Thr Gly Lys Thr 385 390 395 400 TTT AAA TTC CCT CTT TAT TTG ATC GGA GCA GGC ATA ATT GAC GAA GAT 1248 Phe Lys Phe Pro Leu Tyr Leu He Gly Wing Gly He He Asp Glu Asp 405 410 415 CTG CAT GAA CTA GAT GAG GCT GCT CCC ATT GAT CAC CCA CAA ATG CCT 1296 Leu His Glu Leu Asp Glu Ala Wing Pro He Asp His Pro Gln Met Pro 420 425 430 GAC GCG AAA AGC GTT CTT CCA GAA GAT GAA GAG ATA TTC TTC GGA GAC 1344 Asp Ala Lys Ser Val Leu Pro Glu Asp Glu Glu He Phe Phe Gly Asp 435 440 445 ACÁ GGT GTA TCC AAA AAC CCT ATC GAG TTG ATT CAA GGA TGG TTC TCA 1392 Thr Gly Val Ser Lys Asn Pro He Glu Leu He Gln Gly Trp Phe Ser 450 455 460 AAT TGG AGA GAG AGT GTA ATG GCA ATA GTC GGA ATT GTT CTA CTC ATC 1440 Asn Trp Arg Glu Ser Val Met Wing He Val Vally Val Leu Leu He 465 470 475 480 GTT GTG ACA TTT CTG GCG ATC AAG ACG GTC CGG GTG CTT AAT TGT CTC 1488 Val Val Thr Phe Leu Ala He Lys Thr Val Arg Val Leu Asn Cys Leu 485 490 495 TGG AGA CCC AGA AAG AAA AGA ATC GTC AGA CAA GAA GTA GAT GTT GAA 1536 Trp Arg Pro Arg Lys Lys Arg He Val Arg Gln Glu Val Asp Val Glu 500 505 510 TCC CGA CTA AAC CAT TTT GAG ATG AGA GGC TTT CCT GAA TAT GTT AAG 1584 Ser Arg Leu Asn His Phe Glu Met Arg Gly Phe Pro Glu Tyr Val Lys 515 520 525 AGA TAA 1S90 Arg (2) INFORMATION FOR SEQ ID NO: 8: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 529 amino acids (B) TYPE: amino acids (D) TOPOLOGY: Linear (ii) TYPE OF MOLECULE: protein (xi) SEQUENCE DECRIPTION: SEQ ID NO: 8: Met Asp Leu Phe Pro He Leu Val Val Leu Met Thr Asp Thr Val 1 5 10 15 Leu Gly Lys Phe Gln He Val Phe Pro Asp Gln Asn Glu Leu Glu Trp 20 25 30 Arg Pro Val Val Gly Asp Ser Arg His Cys Pro Gln Ser Ser Glu Met 35 40 45 Gln Phe Asp Gly Ser Arg Ser Gln Thr He Leu Thr Gly Lyß Wing Pro 50 55 60 Val Gly He Thr Pro Ser Lya Ser Asp Gly Phe He Cyß Hiß Ala Ala 65 70 75 80 Lyß Trp Val Thr Thr Cys Asp Phe Arg Trp Tyr Gly Pro Lyß Tyr He 85 ^ 3S Thr Hiß Ser He Hiß His Leu Arg Pro Thr Thr Ser Asp Cyß Clu Thr 100 105 110 Ala Leu ßln Arg Tyr Lyß Asp Gly Ser Leu He Asn Leu Gly Phe Pro 115 12 ° '125 Pro Glu Ser Cys Gly Tyr Wing Thr Val Thr Asp Ser Glu Wing Met Leu 130 1 5 140 Val Gln Val Thr Pro His His Val Gly Val Asp Aap Tyr Arg Gly His 145 150 155 160 Trp He Asp Pro Leu Phe Pro Gly Gly Glu Cys Ser Thr Asn Phe Cys 165 170 175 Asp Thr Val His Asn Being Ser Val Trp He Pro Lys Ser Gln Lys Thr 180 185 190 Aßp He Cys Wing n Ser Phß Lyß Aßn He Lyß Met Thr Ala Ser Tyr 195 200 205 Pro Ser Glu Gly Ala Leu Val Ser Asp Arg Phe Wing Phe His Ser Wing 210 215 220 Tyr His Pro Asn Met Pro Gly Ser Thr Val Cys He Met Asp Phe Cys 225 230 235 240 Glu Gln Lys Gly Leu Arg Phe Thr Asn Gly Glu Trp Mßt Gly Leu Asn 245 250 255 Val Glu Gln Ser He Arg Glu Lys Lys He Ser Wing He Phe Pro Asn 260 265 270 Cys Val Wing Gly Thr Glu He Arg Wing Thr Leu Glu Ser Glu Gly Wing 275 280 285 Arg Thr Leu Thr Trp Glu Thr Gln Arg Met Leu Asp Tyr Ser Leu Cys 290 295 300 Gln Asn Thr Trp Asp Lys Val Ser Arg Lys Glu Pro Leu Ser Pro Leu 305 310 315 320 Asp Leu Ser Tyr Leu Ser Pro Arg Ala Pro Gly Lys Gly Met Ala Tyr 325 330 335 Thr Val He Asn Gly Thr Leu His Ser Wing His Wing Lys Tyr He Arg 340 345 350 Thr Trp He Asp Tyr Gly Glu Met Lvs Glu He Lys Gly Gly Arg aly 355 360 * 365 Glu Tyr Ser Lys Wing Pro Glu Leu Leu Trp Ser Gln Trp Phe Asp Phe 370 375 380 Gly Pro Phe Lys He Gly Pro Asn Gly Leu Leu His Thr Gly Lys Thr 385 390 395 400 Phe Lya Phe Pro Leu Tyr Leu He Gly Wing Gly He He Asp Glu Asp 405 410 415 Leu His Glu Leu Aßp Glu Ala Ala Pro He Asp His Pro Gln Met Pro 420 425 430 Asp Ala Lys Ser Val Leu Pro Glu Asp c-iu Glu He Phe Phe Gly Asp 435 440 445 Thr Gly Val Ser Lys Asn Pro He Glu Leu He Gln Gly Trp Phe Ser 450 455 460 Asn Trp Arg Glu Ser Val Met Wing He Val Val Gly He Val Leu Leu He 465 470 475 480 Val Val Thr Phe Leu Ala He Lys Thr Val Arg Val Leu Aßn Cys Leu 485 490 495 Trp Arg Pro Arg Lyß Lyß Arg He Val Arg Gln Glu Val Aßp Val Glu 500 505 510 Being Arg Leu Aßn Hiß Phe Glu Met Arg Gly Ph * Pro Glu Tyr Val Lyß 515 520 525 Arg (2) SEQ IP INFORMATION N0: 9 :: (i) SEQUENCE CHARACTERISTICS (A) LENGTH: 1569 base pairs (B) TYPE: nucleic acid (C) BRAIDED; double (D) TOPOLOGY: linear (ii) TYPE OF MOLECULE: nucleic acid (A) DESCRIPTION: / desc = "DNA" (ix) CHARACTERISTICS: (A) NAME / CLAY ?: CDS (B) (LOCALIZATION 1: 1566 (Xi) DESCRIPTION OF SEQUENCE: SEQ ID NQ: 9; ATG AAT ATA CCT TGC TTT GCT GTG ATC CTC AGC TTA GCT ACT ACA CAT 48 Met Asn He Pro Cys Phe Wing Val He Leu Ser Leu Wing Thr Thr Hiß 1 5 10 15 TCT CTG GGA GAA TTC CCC TTG TAT ACG ATT CCC GAG AAA ATA GAG AAA 96 Ser Leu Gly Glu Phe Pro Leu Tyr Thr He Pro Glu Lys He Glu Lyß 20 25 30 TGG ACC CCC ATA GAC ATG ATC CAT CTT AGT TGC CCT AAT AAC ATG CTG 144 r Trp Thr Pro He Aβp Met He Hiβ Leu Ser Cys Pro Aßn Asn Met Leu ° 35 40 45 TCT GAG GAA GAA GGT TGC AAT ACÁ GAG TCT CCT TTC ACC TAC TTC GAO 192 Ser Glu Glu Glu Gly Cys Asn Thr Glu Ser Pro Phe Thr Tyr Phe Glu 50 55 60 CTC AAG AGT GGT TAC CTA OCC CAT CAG AAG OTC CCA GGA TTT ACA TGC 240 Leu Lyß Ser Gly Tyr Leu Ala Hiß Gln Lys Val Pro Gly Phß Thr Cyß 65 70 75 80 ACT GGG GTT GTG AAT GAO GCA GAG ACÁ TAC AC AAC TTT GTC GGA TAT 288 Thr Gly Val Val Asn Glu Ala Glu Thr Tyr Thr Aßn Phß Val Gly Tyr 10 85 90 95 GTC ACC ACC ACC TTC AAA AGG AAG CAC TTT AAA CCT ACÁ GTG GCT GCT 336 Val Thr Thr Thr Phe Lys Arg Lys His Phe Lys Pro Thr Val Ala Ala 100 105 110 TGT CGT GAT GCC TAC AAC TGG AAA GTA TCA GGG GAC CCC CGA TAT GAA 384 Cys Arg Asp Ala Tyr Asn Trp Lys Val Ser Gly Aßp Pro Arg Tyr Glu 115 120 125 GAA TCT CTA CAC ACC CCG TAT CCC GAC AGC AGG TGG TTA AGG ACT GTG 432 Glu Ser Leu His Thr Pro Tyr Pro Asp Ser Ser Trp Leu Arg Thr Val 15 130 135 140 ACC ACA ACC AAA GAA GCC CTT CTT ATA ATA TCG CCA AGC ATT GTA GAO 480 Thr Thr Thr Lyß Glu Wing Leu Leu He Ii Being Pro Pro Being He Val Glu 145 150 155 160 ATG GAC ATA TAT GGC AGG ACC CTT CAC TCT CCC ATO TTC CCT TCG GGG 528 Met Asp He Tyr Gly Arg Thr Leu His Ser Pro Met Phe Pro Ser ßly 165 170 175 AAA TGT TCC AAG CTC TAT CCT TCT GTC CCC TCT TGT ACA ACC AAC CAT 576 Lys Cys Ser Lys Leu Tyr Pro Ser Val Pro Ser Cys Thr Thr ZJB? I His 20 180 185 190 GAT TAC AC TTG TGG TTG CCA GAA GAT TCT AGT CTG AGT TTQ ATT TGC 624 Asp Tyr Thr Leu Trp Leu Pro Glu Asp Ser Ser Leu Ser Leu He CyS 195 200 205 GAC ATC TTC ACT TCC AGC AGT GGA CAG AAG GCC ATG AAT GGG TCT CGC 672 Aßp He Phe Thr Ser Ser Gly Gln Lyß Ala Met Aßn Gly Ser Arg 210 215 220 ATC TGC GGA TTC AAG GAT GAA AGG GGA TTT TAC AGA TCC TTG AAG GGA 720 He Cys Gly Phe Lys Asp Glu Arg Gly Phe Tyr Arg Ser Leu Lys Gly 25 225 230 235 240 TCC TGT AAG CTG ACA TTG TGC GGG AAA CCT GGA ATT AGG CTG TTC GAC 768 Ser Cys Lys Leu Thr Leu Cys Gly Lys Pro Gly He Arg Leu Phe Asp 245 250 255 GGA ACT TGG GTC TCT TTT ACA AAG CCG GAC GTT CAT GTG TGG TGC ACT 816 Gly Thr Trp Val Ser Phe Thr Lys Pro Asp Val His Val Trp Cys Thr 260 265 270 CCC AAC CAG TTA GTC AAT ATA CAT AAC GAC AGA CTA GAT GAG GTT GAA 864 Pro Asn Gln Leu Val Asn He His Asn Asp Arg Leu Asp Glu Val Glu 275 280 285 CAT CTG ATC GTG GAC GAT ATC ATC AAG AAG AGA GAG GAG TGT TTA GAC 912 His Leu Hep Asp Asp He He Lys Lys Arg Glu Glu Cys Leu Asp 290 295 300 ACG CTG GAA ACT ATA CTT ATG TCT CAA TCA GTT AGT TTT AGA CGG TTG 960 Thr Leu Glu Thr He Leu Met Ser Gln Ser Val Ser Phe Arg Arg Leu 305 310 315 320 AGC CAT TTC AGA AAG TTA GTT CCA GGA TAT GGA AAA GCT TAC ACT ATT 1008 be Hxs Phe Arg Lyß Leu Val Pro Gly Tyr Gly Lyß Ala Tyr Thr He 325 330 335 lo TTG AAC GGC AGC TTA ATG GAA ACÁ AAT GTC TAC TAC AAA AGA GTT GAC 1056 Leu Asn. Gly Ser Leu Met Glu Thr Asn Val Tyr Tyr Lyß Arg Val Aßp 340 345 350 AGG TGG GCG GAC ATT TTG CCT TCG AGG GGA TGT CTG AAA GTC GGA CAA 1104 Arg Trp Wing Asp He Leu Pro Ser Arg Gly Cys Leu Lys Val Gly Gln 355 360 365 CAG TGC ATO GAC CCT GTC AAA GGG GTC CTC TTC AAC GGA ATT ATC AAG 1152 G n Cys Met Aßp Pro Val Lyß Gly Val Leu Phe Asn Gly He He Lyß 370 375 380 GGT CCG QAT GGA CAA ATA TTG ATT CCA GAG ATG CAG TCA GAG CAG CTC 1200 Ib G Pro Aap Gly Gln He Leu He Pro Glu Mßt Gln Ser Glu G n Leu 385 390 395 400 AAA CAG CAT ATO GAT CTG TTG AAA GCA GCT ATO TTT CCT CTC CGT CAT 1248 Lyß sln Hiß Mee Asp Leu Leu Lyß Wing Wing Met Phß Pro Leu Arg Kiß 405 410 415 CCT TTA ATC AAC AGA GAG GCA GTC TTC AAG GAT GAT AAT GCC GAT 1296 Pro Leu He Asn Arg Glu Wing Val Phe Lyß Lys Asp Gly Asn Wing Asp 420 425 430 GAT TTT GTT GAT CTC CAT ATO CCT GAT GTT CAA AAA TCT GTG TCG GAT 1344 0 Asp Phe Val Aso Leu Hiß Met Pro Asp Val Gl.u Lys Ser Val Ser Asp 435 ~ 440 445 GTC GAC CTG GGC CTG CCT CAT TGG GGG TTC TGG TTG TTA GTC GGG GCA 1392 Val Asp Leu Gly Leu Pro His Trp Gly Phe Trp Leu Leu Val Gly Wing 450 455 460 9 ACA GTA GTA GCC TTT GTG GTC TTG GCG TGC TTG CTC CGT TGT TGT TGT 1440 Thr Val Val Wing Phe Val Val Leu Wing Cys Leu Leu Arg Val Cyß Cyß * 65 470 475 480 AGG AGA ATO AG AGG AGA AGG TCA CTG CGT GCC ACT CAG GAT ATC CCC 1488 Arg Arg Arg Arg Arg Arg Leu Le Arg Ar Thr Gln Asp He Pro 485 490 495 CTC AGC GTT GCC CCT GCC CCT GTC CCT CGT GCC AAA GTG GTG TCA TCA 1536 Leu Ser Val Ala Pro Wing Pro Val Pro Arg Ala Lys Val Val Ser Ser 500 505 510 TGG GAG TCT TCT AAA GGG CTC CCA GGT ACT TGA 1569 Trp Glu Be Ser Lys Gly Leu Pro Gly Thr 515 520 (2) INFORMATION FOR SEQ ID NO: 10: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 522 amino acids 0 (B) TYPE: amino acids (D) TOPOLOGY: Linear (ii) MOLECULE IPO: protein (xi) SEQUENCE DECRIPTION: SEQ ID NO: 10: Met Asn He Pro Cys Phe Wing Val He Leu Ser Leu Wing Thr Thr His 1 5 10 15 Ser Leu Gly Glu Phe Pro Leu Tyr Thr He Pro Glu Lys He Glu Lyß 20 25 30 0 Trp Thr Pro He Asp Met He His Leu Ser Cye Pro Asn Aan Met Leu 35 40 45 Ser Glu Glu Glu Gly Cyß Aßn Thr Glu Ser Pro Phe Thr Tyr Phe Glu 50 55 60 Leu Lys Ser Gly Tyr Leu Ala His Gln Lys Val Pro Gly Phe Thr Cy 65 65 75 75 Thr Thr Gly Val Val Asn Glu Ala Glu Thr Tyr Thr Aßn Phe Val Gly Tyr 85 90 95? Val Thr Thr Thr Phe Lys Arg Lys His Phe Lys Pro Thr Val Ala Wing 5 100 105 no Cys Arg Asp Ala Tyr Asn Trp Lys Val Ser Gly Asp Pro Arg Tyr Glu 115 120 125 Glu Ser Leu His Thr Pro Tyr Pro Asp Ser Ser Trp Leu Arg Thr Val 130 135 140 Thr Thr Thr Lys Glu Wing Leu Leu He He Ser Pro Pro He Val Glu 145 150 155 160 Met Asp He Tyr Gly Arg Thr Leu Hiß Pro Pro Met Phe Pro Ser Gly 165 170 175 Lyß Cyß Ser Lyß Leu Tyr Pro Ser Val Pro Ser Cys Thr Thr Asn His 180 185 190 Aßp Tyr Thr Leu Trp Leu Pro Glu Asp Ser Ser Leu Ser Leu He Cys 195 200"205 Asp He Phe Thr Ser Ser Ser Gly Gln Lys Ala Met Asn Gly Ser Arg 210 215 220 He Cys Gly Phe Lys Asp Glu Arg Gly Phe Tyr Arg Ser Leu Lys aly 225 230 235 240 Be Cys Lys Leu Thr Leu Cys Gly Lys Pro Gly He Arg Leu Phe Asp 245 250 255 Gly Thr Trp Val Ser Phe Thr Lys Pro Asp Val His Val Trp Cys Thr 260 265 270 Pro Asn Gln Leu Val Asn He His Asn Asp Arg Leu Asp Glu Val Glu 275 280 285 Hiß Leu He Val Asp Asp He He Lys Lys Arg Glu ßlu Cyß Leu Aßp 290 295 300 Thr Leu Glu Thr He Leu Met Ser Gln Ser Val Ser Phe Aro Arg Leu 305 310 315 ~ 320 Ser Hiß Phß Arg Lyß Leu Val Pro Gly Tyr Gly Lyß Ala Tyr Thx He 325 330 335 Leu Asn Gly Ser Leu Met Glu Thr Aßn Val Tyr Tyr Lyß Arg Val Asp 340 345 350 Arg Trp Wing Asp He Leu Pro Ser Arg Gly Cyß Leu Lyß Val ß and G n 355 360 365 'Gln Cyß Met Asp Pro Val Lyß Gly Val Leu Phe Asn Gly He He Lys 370 375 380 Gly Pro Asp Gly Gln He Leu He Prc Glu Met Gln Ser Glu Gln * ßu 385 390 395 400 Lys Gln His Met Asp Leu Leu Lys Wing Wing Met Phe Pro Leu Arg His 405 410 41: »Pro Leu He Asn Arg Glu Wing Val Phe Lys Lys Asp Gly Asn Wing Asp 420 425 430 Asp Phe Val Asp Leu His Met Pro Asp Val Gln Lys Ser Val Ser Asp 435 440 445 Val Asp Leu Gly Leu Pro His Trp Gly Phe Trp Leu Leu Val Gly Wing 450 455 460 Thr Val Val Wing Phe Val Val Leu Wing Cys Leu Leu Arg Val Cys Cys 465 470 475 480 Arg Arg Met Arg Arg Arg Arg Ser Leu Arg Ala Thr Gln Asp He Pro 485 490 495 Leu Ser Val Wing Pro Wing Pro Pro Arg Wing Lys Val Val Ser Ser 500 505 510 Trp Glu Ser Ser Lys Gly Leu Pro Gly Thr 515 520

Claims (23)

1. A composition comprising an unfertilized non-human oocyte comprising an oligonucleotide 5 heterologous integrated into the oocyte genome.

2. The composition of claim 1, wherein the unfertilized oocyte is a pre-mature oocyte. 10 flp

3. The composition of claim 1, wherein the unfertilized oocyte is a prefertilization oocyte.

4. The composition of claim 1, wherein the oligonucleotide is in the proviral form of a reroviral vector.

5. The composition of claim 1, wherein the non-human animal is a mammal.

6. The composition of claim 1, wherein the mammal is a cow.

7. A method for introducing a heterologous polynucleotide into the genome of an unfertilized non-human oocyte, comprising: a) provide: i) an unfertilized nonhuman egg comprising an oocyte having a plasma membrane and a zona pellucida, this plasma membrane and this zona pellucida define a perivitelline space; ii) an aqueous solution comprising a heterologous polynucleotide; Y b) introducing the solution comprising the heterologous polynucleotide into the perivitelline space under conditions that allow the introduction of the heterologous polynucleotide into the oocyte genome.

8. The method of claim 7, wherein the heterologous polynucleotide encodes a protein of interest.

9. The method of claim 7, wherein the unfertilized eye is a pre-mature oocyte.

10. The method of claim 7, wherein the unfertilized oocyte is a prefertilization oocyte.

11. The method of claim 1, wherein the heterologous polynucleotide is contained within the genome of a recombinant retrovirus.

12. A method for production of a non-human transgenic animal, comprising: a) provide: i) an unfertilized egg comprising an oocyte having a plasma membrane and a zona pellucida, this plasma membrane and this zona pellucida define a perivitelline space; ii) an aqueous solution containing an infectious retrovirus; b) introducing the solution containing the infectious retrovirus into the perivitelline space under conditions that allow oocyte infection; and c) contacting the infected oocyte with sperm under conditions that allow feritilization of the infected oocyte to produce an embryo.

13. The method of claim 12, further comprising, after fertilization of the infected oocyte, the step of transferring the embryo into a hormonally synchronized non-human recipient animal.

14. The method of claim 13, further comprising the step of allowing the embryo to develop at term.

15. The method of claim 14, further comprising identifying at least one transgenic progeny.

16. The method of claim 12, wherein the unfertilized egg comprises a pre-mature oocyte.

17. The method of claim 12, wherein the unfertilized egg comprises a prefertilization oocyte.

18. The method of claim 16, further comprising, after introducing the solution containing the infectious retrovirus into the pre-maturation oocyte, the further step of culturing the infected pre-maturation oocyte under conditions that allow maturation of the pre-maturation oocyte.

19. The method of claim 12, wherein the infectious retrovirus comprises a heterologous associated membrane protein.

20. The method of claim 19, wherein the heterologous membrane-associated protein is a G-glycoprotein selected from a virus of the Rhabdoviridae family.

21. The method of claim 20, wherein the G-glycoprotein is selected from the group consisting of G-glycoprotein from vesicular stomatitis virus, Piry virus, Chandipura virus, Spring viraemia of the carp virus and Mokola virus.

22. The method of claim 12, wherein the non-human animal is a mammal.

23. The method of claim 22 wherein the mammal is a cow. SUMMARY . The present invention provides improved method and compositions for the generation of non-human transgenic animals. The present invention allows the introduction of exogenous nucleic acid sequences into the genome of unfertilized eggs (e.g., pre-maturation oocytes and pre-fertilization oocytes) by infectious retrovirus microinjection into the perivitelline space of the egg. The methods of the present invention provide increased production efficiency of transgenic animals with reduced rate of generation of animals that are mosaic for the present transgene.