HK1022497B - Method for immortalizing cells and immortalized cells thereof - Google Patents

Description

Method for immortalizing cell and immortalized cell

Technical Field

The present invention relates to the field of immortalization of cells and the use of cells as a repository for viral propagation and recombinant protein expression. In particular, the present invention relates to the use of the protein p53 under the control of an inducible promoter for the production of immortalized cells.

Background

Many avian viruses used in the production of avian and animal vaccines are propagated in embryonated chicken eggs or primary chicken fibroblast cell cultures. Examples of animal vaccines produced using these chicken substrates include Canine Distemper for dogs, Marek's disease vaccine for turkeys, reovirus, fowlpox (Fowl Pox) and infectious Bursal disease vaccine for poultry. Primary cell cultures can be diverse and present a risk of contamination with endogenous viruses, mycoplasma, and the like. The source of animal tissue from which primary cell cultures are obtained is often limited and expensive due to the need to maintain animal stocks in a pathogen-free state.

There is a need to develop a reproducible method to produce virus-free immortalized avian cell substrates suitable for use in the production of animal vaccine products. It is well studied that the availability of characterized immortalized (i.e., passaged) cell lines has the benefit of limiting or reducing dependence on primary animal tissue cultures, which are not easily controllable from a quality standpoint. Regulatory requirements in the vaccine industry regarding product safety, consistency and potency are driving companies to seek cell lines as the best alternative to the use of egg-based and primary cell vaccines as substrates in current practice. Vaccine production companies must first specify a consistent cell line for virus production to meet regulatory requirements that allow the company to spread its vaccine. Manufacturers of human and animal vaccine products must demonstrate that their vaccine substrates are non-contaminating, both in the united states and abroad. The advent of reproducible methods for generating passaged animal cell lines obtained from primary tissues will allow bioproduct manufacturers to better control the production process and improve product safety and consistency, while ultimately reducing costs.

Summary of The Invention

The present invention relates to methods for transforming cells and cells produced by introducing a nucleic acid encoding p53 under the control of a metallothionein promoter into said cells and by selecting cells having an immortalization profile.

In one aspect of the invention, a method of transforming a primary non-rodent cell is disclosed, comprising the steps of: locating a nucleic acid encoding p53 under the control of the metallothionein promoter in a genetic vector capable of directing expression of p 53; introducing the gene vector into a primary non-rodent cell; and screening for foci having a population doubling time of about 0.6 to 1.5 times per day, wherein the cells are reverse transcriptase negative and non-tumorigenic. In one embodiment, the primary non-rodent cell is of avian origin, while in another embodiment the primary non-rodent cell is of human origin. The primary cells used in the present invention may be derived from a variety of tissues, and in preferred embodiments the cells are obtained from skin tissue, breast muscle tissue and/or cardiac muscle tissue.

In another aspect of the invention, a cell is disclosed. These cells are immortalized fibroblasts containing a gene vector capable of expressing p53 under the control of the metallothionein promoter. In one embodiment, the cell is avian-derived.

The present invention also relates to a method of propagating a virus comprising the steps of: contacting at least one infectious viral particle with at least one cell of an immortalized cell culture, wherein the cells of said culture contain a gene vector capable of directing the expression of p53 under the control of a metallothionein promoter; and collecting the virus produced by the cells. In one embodiment, the virus is a reovirus, in another embodiment, the virus is HVT, and in a third embodiment, the virus is a fowlpox virus.

In another aspect of the invention, a method of propagating a virus is disclosed, comprising the steps of: contacting at least one infectious viral particle with a primary cell; in cell culture, primary cells are cultured with immortalized cells containing a gene vector expressing p53 under the control of a metallothionein promoter; and collecting the virus from the cell culture.

The invention also relates to immortalized, non-transformed cells containing p53 under the control of a metallothionein promoter and at least one vector capable of directing the expression of the recombinant protein in the cell. In one embodiment, the cell expresses a recombinant protein, while in another embodiment, the vector encodes at least a portion of a virus. In yet another embodiment, the vector is a retroviral vector.

The present invention relates to the following aspects.

1. A method of transforming primary chicken cells comprising the steps of:

positioning a nucleic acid encoding p53 under the control of a metallothionein promoter in a genetic vector capable of directing expression of p 53;

introducing the gene vector into primary chicken cells; and

the population doubling time was selected to be 0.6 to 1.5 population doubling foci per day, wherein the cells were reverse transcriptase negative and non-tumorigenic.

2. The method of 1 above, wherein the cell is a dermal cell.

3. The method of 1 above, wherein the cell is a pectoral muscle cell.

4. The method of 1 above, wherein the cell is a cardiomyocyte.

5. The method of 1 above, wherein the cell is a fibroblast.

6. A method of propagating a virus comprising the steps of:

contacting at least one infectious viral particle with at least one cell of a culture of immortalized chicken cells, wherein said cultured cells contain a genetic vector that expresses p53 under the control of a metal-sulfur protein promoter; and

the produced virus is collected by the cells.

7. The method of 6 above, wherein the virus is a reovirus.

8. The method of 6 above, wherein the virus is herpes virus of turkeys.

9. The method of 6 above, wherein the virus is fowlpox virus.

10. A method of propagating a virus comprising the steps of:

contacting at least one infectious viral particle with a primary cell;

in cell culture, primary cells are cultured with immortalized chicken cells containing a gene vector for p53 under the control of a metallothionein promoter; and

collecting the produced virus from the cell culture.

11. An immortalized, non-transformed chicken cell comprising p53 under the control of a metallothionein promoter and at least one vector capable of directing the expression of a recombinant protein in said cell.

12. The cell of the above 11, which expresses a recombinant protein.

13. The cell of above 12, wherein the vector encodes at least a portion of a recombinant virus.

14. The cell of above 11, wherein the vector is a retroviral vector.

Brief Description of Drawings

FIG. 1 is a schematic representation of the vector pJFNII used in a preferred embodiment of the present invention.

FIG. 2 is a schematic representation of the vector pJFNIIcMID used in a preferred embodiment of the present invention.

Detailed Description

There is a need for a method of reproducibly immortalizing primary cells and producing a continuous cell line. The development of techniques to generate well-characterized cell lines that support viral replication would allow companies to eliminate the time to repeatedly produce primary cells and avoid the problems associated with contamination and inconsistencies between batches of eggs. Immortalized cell lines defined for virus propagation reduce the costs associated with quality control testing of the final product.

Immortalization of primary non-rodent cells, in particular avian and human cells, using recombinant p53 under the control of an inducible metallothionein promoter is disclosed. The cells are useful for culturing virus stocks, for expressing viral proteins and as packaging cell lines for the production of recombinant viruses.

Primary cell cultures are most often obtained from newly isolated cells from intact tissues. These cells are often a good source of virus-free material and are well-suited as host cells for virus replication. Primary cells are not always effective at viral replication, and primary animal cells exhibit a limited lifespan in culture, eventually becoming senescent. Upon senescence, the cells stop dividing and die within a certain time. The ability of cells in culture to divide depends on several parameters, including the species from which the cells are derived and the time the tissue is in culture. Cells undergoing senescence cannot be maintained in culture for long periods of time and are therefore non-regenerative hosts for virus stock propagation. Primary cells typically undergo 23-26 passages before senescence is reached. Cells of the invention are preferably transfected with p53 between about passage 2 and about passage 4.

Immortalized cells as used throughout the present invention refers to cells that are capable of growing more than 25 passages in culture. Immortalized cells differ from transformed cells in that, unlike transformed cells, immortalized cells exhibit density-dependent growth constraints and maintain a normal morphology. In contrast to immortalized cells, transformed cells can grow on soft agar and often form tumors when injected into experimental animals.

The cells of the invention are immortalized by introducing p53 under the control of an inducible metallothionein promoter into a preferred primary cell. Partly due to the fact that mutations in the p53 gene somehow lead to a 50% increase in all human cancers, the nuclear oncogene p53 is one of the most well studied tumor suppressor genes (Levine et al, Nature 351: 453-456, 1991). p53 is a cellular phosphoprotein and often exhibits elevated levels in transformed cells (De Leo, A.B. et al, Pro.Natl.Acad.Sci 76: 2420-2424, 1979). Wild-type p53 appears to play a role in growth inhibition (Michalovisy et al, cell 62: 671-680, 1990) and p53 allows cells to remain at the checkpoint of the cell cycle in response to DNA damage (Kastan et al, cancer research 51: 6304-6311, 1991). This checkpoint function is achieved by the accumulation of p53 and subsequent induction of the GADD45 (an important splice repair protein), WAF1(p21), and MDM2 (which forms a stable complex with p 53) genes. (Kastan, et al, cell 71: 587-597, 1992). This theory is supported by the observation that mutations in p53 can lead to cell immortalization.

Several reports have linked the inhibition of cell division by the wild-type p53 molecule via a ubiquitous cyclin kinase inhibitor (i.e., Cipl, E1-Deiry et al, cell 75: 817-825, 1993: WAFl reported by Harper et al at cell 75: 805-816, 1993). The experiment shows that the p53 protein stimulates the production of another protein, p21, which is associated with a quaternary complex in normal cells, including cyclin-dependent kinases, cyclins, Proliferating Cell Nuclear Antigen (PCNA) and p 21. In transformed cells, the loss of p53 function appears to be due to mutations that result in the loss of p21 and PCNA from the multiprotein complex, resulting in uncontrolled growth.

When correlating mutations in p53 with the regulation of cell proliferation, there are other theories that infer other pathways by which p53 regulates cell proliferation. (Milner, et al, cell biol. int. Rep.4: 663-667, 1980, Milner et al, 112: 785-788, 1981, Mercer et al, Proc. Natl. Acad. Sci.79: 6309-6312, 1982 and Mercer, et al, molecular cell biology 4: 276-281, 1984). For example, mutations in the p53 gene may be associated with damage that occurs when cells fail to repair critical points in the cell cycle due to physiological and physical factors, such as stress, aging, ionizing radiation or exposure to carcinogens.

P53 is known to those skilled in the art to regulate cell proliferation. There was some evidence that non-mutated rat p53 could immortalize rodent cells. Jenkins et al demonstrated that rat chondrocytes were immortalized only when constitutive promoters from oncogenic viruses such as Rous Sarcoma Virus (RSV) and monkey virus 40(SV40) were used (Jenkins et al Nature 317: 816-818, 1985). Eliyahu et al (Nature 312: 646-649, 1984) and Parada et al (Nature 312: 649-. When these cells have the characteristics of transformed cells, the cells undergo senescence and stop proliferating within a relatively short time after transformation.

Unlike Jenkins, Eliyahu, and Parada (all above), Rovinski and Benchimol (oncogene 2: 445-Asn 452, 1988) demonstrated immortalization of rat embryonic fibroblasts with rat p53 under the control of its endogenous promoter. The results of the Rovinski publication can be explained by other studies that suggest that rat cells are known to be susceptible to immortalization and transformation. The cells are thought to be primed in such a way that they are particularly sensitive to immortalization and transformation stimuli. Studies have repeatedly demonstrated that rodent fibroblasts spontaneously undergo immortalization at high frequency (Ponten, J.Virol. monogr.8: 1, 1971; Ponten J.8, Biochim, Biophys.acta 458: 397, 1976; Todaro and Green J.Cell.Biol.17: 299-284, 1963; Meek et al, exp. cell Res.107: 277-284, 1977 and Curatolo, et al, In Vitro 20: 597-601, 1984). Indeed, Rovinski and Bechimol (page 446 above) noted that their rat embryonic fibroblast controls were transfected with an expression marker alone and had a spontaneous immortalization frequency of about 10%. In contrast, a review and other studies in science show that there is no report of spontaneous immortalization of human or chicken fibroblasts from normal donors (Smith et al science 273: 63-67, 1996, Hayflick, exp. cell Res.37: 614-. Moreover, rat cells carry a variety of endogenous viruses, particularly retroviruses that render them incompatible with commercial vaccine production.

A large number of p53 genes have been isolated from various mammals. For example, the sequence of chicken p53 (SEQ ID NO: 1) is available from GenBank under accession number X13057 and from Soussi et al (nucleic acids Res. 16 (23): 11383, 1988). The sequence of normal human p53 is available from GenBank under accession numbers W88747 and HSP53G and is publicly available by the college of pharmacy, Washington university. The human p53 gene is also provided as exon 1-11 under GenBank accession numbers M22881-M22884, M22887-M22888M 22894-M33898 and published by Buchman et al (Gene 70 (2): 245-. Horse p53 is available from GenBank under accession number U37120, while African green monkey p53 is available from GenBank under accession number X16384. Roman p53 is available from GenBank under accession number L20442, Bos domestica p53 from X81704 (see also Dequiedt F et al, DNA sequence 5 (4): 261-64, 1995), and Cat p53 from GenBank under accession number D26608(Okuda M. et al int.J. cancer 58 (4): 602-7, 1994). Other sequences and publications referencing these sequences are available from numerous gene databases such as GenBank and the like. Those skilled in the art will be readily able to find such literature or gene databases to obtain the p53 gene sequences well known in the art.

The metallothionein promoter has multiple metal binding regions, and gene expression regulated by the metallothionein promoter can be regulated by Cd⁺⁺，Cu⁺⁺And Zn⁺⁺And (4) inducing. The promoter contains multiple potential binding sites for metal regulatory factors and transcription factors, including SPI and MLTF. Metal response elements within the promoter region and other recognition regions in the promoter are discussed in detail by Mueller et al (Gene and development 4: 412-426, 1988) and Lee et al (Nature 325: 368-372, 1987).

Similar to the gene encoding p53, there are many metallothionein genes known in the art from various species. For example, the chicken metallothionein promoter sequence (SEQ ID NO: 2) is available from Fernando et al (Gene 8: 177-183, 1989). The promoter regions can be identified from the metallothionein gene sequences of many published metallothionein gene sequences available in the art based on the characteristics of the metallothionein promoter disclosed (Mueller et al, supra and Lee et al, supra). The sequence of the human metallothionein promoter gene is available from GenBank W68639, X65607, V00594 (see also Stennard et al Biochim. Biophys. acta1218 (3): 357-365, 1994; Richards et al, cell 37 (1): 263-272, 1984 and Karin et al, nucleic acids Res.10 (10): 3165-3173, 1982). The ovine metallothionein promoter is available from GenBank under accession number X04626 (see Peterson et al, J. Eur. Biochem., 160 (3): 579-585, 1986). Other sequences and publications referencing the use of the sequences are available from numerous gene databases such as GenBank, GenEMBL, and the like. Those skilled in the art will be able to readily find the literature or gene databases to obtain the gene sequences for metallothionein promoters well known in the art.

The cells of the invention are immortalized by introducing into the cell a nucleic acid encoding p53, wherein the nucleic acid encoding p53 is under the control of a metallothionein promoter, i.e., the metallothionein promoter is operably linked to the nucleic acid encoding p 53. In a preferred embodiment of the invention, p53 under the control of the metallothionein promoter is introduced into the cells from an expression vector. There are a variety of commercially available expression vectors available, and one skilled in the art will readily recognize that a wide variety of vectors may be used to express p53 in non-rodent cells. Expression vectors can also include a variety of other features that facilitate replication of the vector in prokaryotic cells, selection of the vector, integration of other gene sequences, or facilitate expression of the gene by addition of other regulatory sequences. Examples of such features include, but are not limited to, the inclusion of bacterial origins of replication, genes conferring antibiotic resistance, other selectable markers including, but not limited to, beta galactosidase, luciferase, or the like, enhancer sequences, multiple cloning sites, and the like.

Example 1 details the identification of the metallothionein promoter and the p53 gene and the insertion of the promoter with p53 into an expression vector. The metallothionein promoter and the p53 gene are preferably from the same species. The p53 construct under the control of the metallothionein promoter was introduced into the cells. The cells are preferably primary cells, and as used in the present disclosure, primary cells are preferably cells that have been cultured for 1-10 passages and/or for no more than 2 months.

Primary cells are generally characterized as cells that have a limited ability to grow in culture. Primary cells are those cells that are isolated from intact tissue and placed in culture. The cells can be obtained from a number of tissues of various species of mammals. Human biopsies, tissue samples from dogs, horses, pigs, chickens, cows, embryonic tissue and the like can be minced, trypsinized and isolated as single cells in suspension or used for transfection as a monolayer culture or small (but intact) tissue sample. Isolated cells suitable for transfection include fibroblasts, muscle cells, epithelial cells, endothelial cells and other cells. Those skilled in the art will recognize that there are many well known methods for obtaining and isolating various cells from various tissue samples, and that these methods can be performed without undue experimentation. Example 2 discloses a method for isolating cells from chick embryo tissue, and also includes methods for isolating cells from skin, cardiac muscle, and pectoral muscle.

There are many methods known in the art for introducing vectors capable of directing the expression of nucleic acid sequences or for introducing nucleic acid fragments into cells. These methods include, but are not limited to, calcium phosphate precipitation, electroporation, lipofection, polyamine transfection and viral particle transfection. Example 3 provides methods for introducing p 53/metallothionein nucleic acid fragments of the invention into eukaryotic cells using lipofectamine. Various commercially available kits are available that use various methods to incorporate nucleic acid fragments into eukaryotic cells. Thus, methods of introducing a nucleic acid encoding p53 under the control of the metallothionein promoter should not detract from the scope of the invention.

After transfection, the cells of the invention are cultured and expanded under selective growth pressure, and clones containing the transfected nucleic acid fragments are identified, if necessary. Cells are treated with cations necessary to promote expression of the metallothionein promoter. Example 3 Zinc sulfate was used to induce the metallothionein promoter. Foci were picked and grown in culture. The term "foci" refers to a population of cells having different characteristics than the surrounding cells. In the present invention, cells immortalized by a nucleic acid fragment encoding p53 under the control of the metallothionein promoter grew faster than cells not immortalized in their surroundings. Immortalized cells grow faster and form colonies on monolayers of primary cultures. The colonies can be isolated using cloning rings and allowed to expand in culture.

Cells are considered to be immortalized when the p 53/metallothionein-containing construct is introduced into the cells approximately over 25 tissue cultures and when the cells are population doubled by at least about 0.6 fold per day and preferably by about 0.6 to 1.5 fold per day. The cells maintain a normal morphology and exhibit density-dependent and/or contact-inhibited growth. The immortalizing capacity of cells from three tissues of chicken embryos was analyzed by the method of the invention. Numerous clones were identified from cardiomyocytes, pectoral myocytes and skin cells as provided in example 5.

The immortalized cells of the invention should be tested for viral contaminants as well as other tissue culture contaminants, such as low levels of bacterial contaminants, mycoplasma and the like. The cells can be tested for evidence of retroviral infection, avian influenza virus (type a), avian reovirus, avian adenovirus (group I-III), avian encephalomyelitis virus, fowlpox virus, newcastle disease virus, paramyxovirus (type 2), as well as mycoplasma, salmonella and other contaminants (e.g., contaminants listed in 9c.f.r. § 113 and from a list of its orders of finesse).

The cells are tested for a wide range of viral contaminants using the polymerase chain reaction to identify contaminating nucleic acid fragments. There are various commercially available test kits for various viruses, which can be used to determine whether the cells of the invention contain contaminating viruses. Also, there are commercially available assays for detecting viral antigens, where the antigens are from a variety of different viruses. These assays include ELISA assays, immunofluorescence assays, and the like. All of these assays are well known and involve routine experimental techniques. Example 4 provides a method for determining whether a cell of the invention is reverse transcriptase negative. Evidence of reverse transcriptase activity in immortalized cells containing p53 under the control of a metallothionein promoter is evidence of retroviral contamination.

The cells are also tested for their tumorigenic potential. The cells of the invention are preferably non-tumorigenic. Tests to determine whether a cell population is tumorigenic are known in the art. Example 6 provides a method for assessing the growth of the immortalized cells of the invention in soft agar, while example 7 provides a method for introducing the cells of the invention into an animal of a species preferred for the cells of the invention, in order to determine whether the cells of the invention are capable of inducing a tumor in the recipient animal. To test the tumorigenic potential of human cells containing a nucleic acid encoding p53 under the control of the metallothionein promoter, the cells can be tested for growth in soft agar and for growth in nude mice or in mice or other experimental animals with a reconstituted human immune system.

In one aspect of the invention, the cells can be used to propagate viruses. As demonstrated in example 8, the cells of the invention support reovirus infection, Herpes Virus of Turkeys (HVT) and marek's disease virus. The cells of the invention obtained from chicken tissue can also be used as hosts for infectious Bursal disease Virus, infectious bronchitis Virus, Newcastle disease Virus, infectious laryngotracheitis Virus, various adenoviruses including adenovirus type III, Circodnaviridae, Chicken HSV, fowlpox Virus and other viruses. Many human and animal viruses grow in embryonated eggs, including, but not limited to, hosts for rabies virus, canine parvovirus, feline leukopenia virus, calicivirus, hepatitis virus, influenza virus, varicella zoster virus, and other viruses. The ability of these viruses to grow in immortalized cells of the invention can also be tested.

For production of virus stocks, the cells of the invention may be inoculated into tissue culture flasks, roller bottles, spinner cultures or hollow fiber reactors. For virus propagation in roller bottles, about 2-5X 10⁴Cells were seeded per square centimeter of surface area. The multiplicity of infection (the ratio of infectious viral particles to cells) that initiates growth of the virus stock will vary depending on the strain. Those familiar with the field of virology and with the growth and strain of particular viruses will be able to maximize the yield of virus stocks by standard control of multiplicity of infection, temperature, changes in culture medium, etc. without undue experimentation.

The method of harvesting the virus after infection to obtain an infectious virus stock also varies from virus strain to virus strain. Enveloped viruses are released into the medium more slowly than non-enveloped viruses. The virus stock may be obtained from the medium alone or from cell lysates pooled with conditioned medium. For splitting viruses (which are sufficient to lyse one cell during virus release), it is sufficient to harvest the conditioned medium (e.g., the remaining medium containing the virus) after a gentle centrifugation step to remove cell debris. Moreover, methods for harvesting and preserving viruses from a wide range of viral strains are well known in the art.

Various methods of quantifying viral growth from cell cultures are also well known in the art. For example, the titer of virus stocks against members of the family herpesviridae and against various viruses that produce cytopathologically foci on the surface of cell monolayers was determined by plaque assay (e.g., plaque forming units/ml culture or dose quantified for viruses such as plaque forming units/vaccination) or at tissue culture infectious dose-50 (TCID)₅₀) And is easily quantified. By TCID₅₀Better quantification of fast lytic viruses, in which TCID₅₀Is the dose or dilution of a viral stock that can infect 50% of the culture over a period of time. Methods for culturing and quantifying viruses are well known in the art, and sources of information directing methods for quantifying viruses are in Fields et al (eds.) basic virology 1991, Raven Press, New York or in the principles and practice of infectious diseases in Mandell et al (eds.), 1985, John Wiley&Sons, new york.

The cells of the invention may also be used to produce recombinant proteins, including viral proteins and the like. Methods for inserting nucleic acids encoding recombinant proteins into nucleic acid vectors under the control of regulatory elements capable of directing the expression of the proteins in eukaryotic cells, such as immortalized cells of the invention, are well known in the art. Expression vectors are replicable nucleic acid fragments capable of directing the expression of a recombinant protein. Many expression vectors, including retroviral vectors, are well known in the art through journal publications and suppliers. Replicable expression vector components generally include, but are not limited to, one or more of the following: an origin of replication, one or more marker genes, an enhancer element, a promoter element, optionally a signal sequence and a transcription termination sequence. The selection or marker gene encodes a protein that functions to recognize a transformed or transfected cell population. Typical selection genes encode proteins that provide resistance to antibiotics or other toxins, complement auxotrophs, or provide key nutrients not present in complex media.

An expression vector containing a nucleic acid encoding a recombinant protein is transfected into the cells and used to direct expression of the recombinant protein in the immortalized cells of the invention. The vector preferably can encode any recombinant protein capable of being expressed in chicken embryo fibroblasts, including but not limited to viral proteins, including reverse transcriptase and/or viral structural proteins. Examples of vectors for producing recombinant proteins in cells include vectors producing tumor suppressor proteins or viral structural proteins [ oncogenes 11(12) such as Givol: 2609-2618, 1995, Givol et al cell growth and differentiation 5 (4): 419-429, 1994, Akiyama et al virology 203 (2): 211-220, 1994 and Boyer, et al oncogene 20: 457-66, 1993.

The cells of the invention can be used as a medium for expression of recombinant viruses, including but not limited to recombinant retroviruses. The cells of the present invention can be used as a packaging cell line for genetically engineered viruses useful for gene therapy and the like. Constructs and methods involved in using particular cell lines as packaging cell lines are well known in the art. For example, Boerkoel et al (virology 195 (2): 669-79, 1993) disclose the use of primary chicken embryo fibroblasts for packaging viruses for packaging cell lines. Those same methods can be used to package viruses in the immortalized cells of the invention.

Most avian cell lines and all transformed avian cells as well as almost all rodent transformed cell lines contain viral contaminants (e.g. endogenous viruses) or produce viral proteins, which are not suitable for the production of human or animal vaccines. The cells cannot be used to produce recombinant proteins because the endogenous contaminants would contaminate the purified recombinant protein preparation. As an advantage, the cells of the invention provide a suitable alternative to these problems.

The cells of the invention may also be used as substrates to support the growth of viruses from other cells. These other cells include primary cells or cultured cells that exhibit improved growth or prolonged life in culture in the presence of other cells or in the presence of extracellular matrix proteins (e.g., collagen, laminin, etc.). In one embodiment, the cells are mixed with the virus and then with the cells of the invention, preferably in the following ratios: the ratio of cells to cells of the invention is between about 1: 5 cells to about 1: 20 cells, and more preferably about 1: 10(1 cell to about 10 cells of the invention). The mixed cells are then placed in culture. In a second embodiment, the cells are mixed with virus and placed on the surface of immortalized cells of the invention that have been attached to a tissue culture surface. The cells of the invention serve as supports for other cells and (without intending to limit the scope of the invention) the cells of the invention may provide growth factors and the like as well as extracellular matrix components and the like to support other cells when they produce viruses. Example 9 provides an example of the use of the cells of the invention as a cell substrate.

All references cited herein are expressly incorporated into this disclosure by reference. Specific embodiments of the invention will be discussed in detail and possible variations have been mentioned within the scope of the invention. There are various alternative techniques and methods available to those skilled in the art that will also enable the practitioner to successfully practice the intended invention.

Example 1

Preparation of an exemplary p53 expression vector

Plasmid pJFNII (5436bp) was constructed by starting with pBluescriptSK vector (Stratagene, LaJolla, Calif.). The multiple cloning site and lacZ gene were removed with PvuII. The 2513bp vector fragment was treated with calf intestinal alkaline phosphatase. The fragments were then treated with EDTA, incubated at 65 ℃ and extracted with phenol/chloroform followed by ethanol precipitation. Plasmid pRSVneo was digested with BamH1 and NdeI (Gorman, C., et al, science 221: 551-. The fragment was treated with Klenow fragment of DNA polymerase (Stratagene, LaJolla, Calif.) and blunt-ended ligated with a 2513bp vector fragment. After isolation of the clone, the vector was digested with EcoRI, treated with Klenow fragment and religated. The digestion removed the EcoRI site from the promoter region. This plasmid was designated pJFNII (see FIG. 1).

pJFNII was cut with BamHI at a site approximately 1000bp downstream from the polyadenylation signal of SV40 used to encode the neomycin resistance gene in the vector. The chicken metallothionein promoter was obtained by PCR. The following primers were used in Polymerase Chain Reaction (PCR) to obtain the metallothionein promoter:

left primer:

5’CGAAGATCTCTCAGCACGGCCCCACGCT3’(SEQ ID NO：3)

right primer:

5’CGAAGATCTTTATTCTCGAGATATCGAATTCTCGGGTGGGCTCGTAGCAGT3’(SEQ ID NO：4)

in these experiments, the template for the PCR reaction was plasmid pCBcMTlacZ. The chicken metallothionein-inducible promoter in plasmid pCBcMtlacZ (Fernando and Andres, Gene 81: 177-183, 1989) was obtained from Dr. Ann Gibbins (university of Guelph, Guelf, Onta, Canada). Those skilled in the art will recognize that the promoter may also be identified from gene expression libraries to obtain promoters for other species, including chickens. The primers were purchased from IDT (Coralville, LA). Both the left and right primers were designed to include a BglII site. Amplification with these primers yielded a sequence identical to SEQ ID NO: 2 the metallothionein promoter contained within the corresponding nucleic acid fragment.

Preferred chicken metallothionein promoter sequences (SEQ ID NO: 2) are:

CT CTCAGCACGG CCCCACGCTG TGCGCACCGC CTCGCAGCGC

GGCCCGGGGG GGTGGCGGGG GTGGGAGCAG CAGTGGCGCA

ATGACCCCTC CGGGTCACAT TCCCGCAACC GAGCGCAGAG

TGCGTGGCCG GGAAATTCCC CCCCCCCAAT TCGCCTTTCG

GCAGCCAAAG CGGGAGGGGG GGAGTGAGGA GGGTCAGGCA

CGTTGGGGTC CGTGCCGTGT TCTGGCAAAG TGTCGTGTTT

GGGGGGGGGG GGGAGCAAGG AAGGGAGGCG AGGGGTGAGG

ACACAAAGCA AAAGCGCCCT AAATCTGTTG GCACACATGG

CCATCCCACA GCTGTATCCC CCTGCTTTGG GGGAACCCCA

ACACCAGGGC TGGCCCCGCG GTGAGGCTCC CCCCAGGCAG

GGGGCACGGC CGTGACCCCG CTGAGCACGG CACGGCGCTG

CCCCGCCCCG CTGAGCACGG CACGGCACGG CACGGCACGG

CCCCCCGAGC ACGGCTCAGC ACGGCACGGC GCTCAGCACG

GCACGGATCG GCACCGCCCC GCCGTGCGCT GCGCGCAGCA

CCACCCCGGC CCTATAAATA CAGGGCGGGC AGCGGGACTC

GGGACTGCTA CGAGCCCACC CGAG 3’

the promoter contains three major metal regulatory elements, a GC-box functional region and a TATA box. The metal regulatory element binds to the metal and induces the chicken metallothionein gene located downstream. The amplified metallothionein fragments inserted into the BglI termini from the left and right primers were ligated with BamHI to pJFNII. The ability to select for neomycin resistance provided by the vector clones identified from the ligation. The clone was named pJFNIIcMTD and was approximately 6088 bp. The vector includes multiple cloning sites including EcoRI, EcoRV and Xho I. The polyadenylation signal within the right primer allows for efficient expression of DNA sequences lacking this signal.

The chicken p53cDNA (SEQ ID NO: 1) (accession # X13057 to GenBank), as supplied by the EcoRI insert from T.Soussi, was inserted into the EcoRI endonuclease restriction site in the multiple cloning site of pJFNIIcMTD. The cDNA sequence (Soussi et al, nucleic acids Res. 16: 11383, 1988) contained a 64bp 5 'untranslated region (UTR), a 1101bp open reading frame encoding a p53 molecule of 367 amino acids, a 390' UTR and a small poly-A tail. Chicken p53 has about 47% homology with the human homologous fragment. 1555bp chicken p53cDNA containing EcoRI cohesive end was obtained and cMT/SK was digested with EcoRI⁺Constructs. The chicken p53cDNA insert was ligated into the EcoRI site. Clones containing the p53 insert in the forward (sense) orientation were identified. The constructs were transfected into competent E.coli XL-1 Blue cells (Stratagene) and cultured overnight in LB broth supplemented with ampicillin. The plasmid was isolated by the large (maxi) plasmid preparation method of Sambrook et al (molecular cloning laboratory Manual, 2 nd edition, Cold spring harbor, New York, 1989). Plasmid DNA was purified twice by cesium chloride centrifugation prior to transfection.

Example 2

Isolation of primary cells from intact tissue

Embryonic SPAFAS line chick embryos (HyVac, Abel, IA) were the primary cell source for these experiments. The eggs and chickens which produced these eggs were proven by the supplier to be negative for avian influenza virus (type a), avian reovirus, avian adenovirus (types I-III), avian encephalomyelitis virus, fowlpox virus, newcastle disease virus, paramyxovirus (type 2), mycoplasma, salmonella and other infection agents known to infect livestock. Fertilized eggs are cultured in sterile, isolated incubators and processed for 10-day-old and 19-day-old embryos to establish primary cultures.

Cells were obtained from heart, liver, skin and muscle (chest and thigh) using three 10-day-old SPAFAS embryos. 19-day-old embryos were also used to establish primary skin cultures. The embryonic tissue was dissociated with trypsin/EDTA solution and plated on DMEM medium (Gibco) containing 10% fetal bovine serum (Gibco), 1% antibiotic/antifungal (Gibco) and 2 mML-glutamine (Gibco). The dissociated cell suspension was collected in a 50 ml centrifuge tube containing 10% ml fetal calf serum to inactivate the trypsin and centrifuged at 700Xg for 10 minutes.

The cells were resuspended in 10 ml Dulbecco's modified Eagles's medium enriched with 36. mu.g/ml insulin (Sigma), 1.6. mu.g/ml transferrin (Sigma, St. Louis, Mo.), 2 mML-glutamine, 10% fetal bovine serum, 1% antibiotic/antifungal solution and at about 1X 10⁶Cells/plate cells were pipetted into 4-5 100 mm square culture dishes and incubated in 5% carbon dioxide, 95% air at 40.5 ℃. After 24 hours of culture, the medium was changed.

Cultures were grown to confluent plates (5 days) and removed from the plates with a trypsin/EDTA solution (0.05% trypsin and 0.02% ethylenediaminetetraacetic acid (EDTA) in PBS) and re-plated to give the second generation. The cell stock was frozen in liquid nitrogen.

Example 3

Transfection of the p 53/metallothionein promoter construct into cells

Primary chicken skin cells from ELL-O19-day-old embryos were transformed between first generation to second generation. The cells were plated at 5X 10 in 100 mm square plates⁵The cells were plated at high density on glucose DMEM supplemented with 10% certified fetal bovine serum and enriched with 1.6 microgram/ml transferrin (Sigma, st. louis, MO), 36 microgram/ml insulin (Sigma) and antibiotics. Cells were cultured overnight to stabilize the culture and 9 ml of additional medium was added the next morning and transfected with lipofectamine TransitII using package insert (PanVera, Madison, Wis.). 10 micrograms of purified p53 construct was used per transfection. Cells were transfected for 5 hours and the medium was replaced with 600 μ G/ml of the selective antibiotic G418(Sigma, st. Rotating shaftThe chemodifferentiated cells are passaged in selective medium until foci are formed (typically 4-10 days) and the resulting foci are clonally selected and propagated.

Cells were cultured for 2-3 passages to generate foci (about 10. mu.g/ml) of G418 (600. mu.g/ml) resistant cells³-10⁴). At the beginning at 5 × 10 when needed⁵Cells were plated per 100 mm square plate to avoid the effect of plating density on the expression of p 53. Untransfected control cells were mock transfected with control plasmid. The control cells senesced and died by the time of 12 passages. Cells were cultured in 50mM zinc sulfate selection medium for 2 weeks until stably transfected cells began to proliferate. Several control plates were not supplied with zinc sulfate (no induction of metallothionein promoter was present). Two weeks after the re-addition (4 weeks post-transfection) phenotypically specific foci began to appear that grew faster than the surrounding older appearing cells. After further re-addition, one of the foci of the transformed skin cells appeared as rapidly growing cells. About 5X 10 removal from the culture plate with cloning Ring⁴Cells/foci. These cells were transferred to a six-well plate. Two dishes of nearly confluent cells grown in the presence of zinc at 2X 10⁵The cells/dish were divided into six dishes, and 50. mu.M of zinc was added to the dishes. In the presence of zinc at 1X 10⁵Cells/cm the skin cells were propagated every 3-4 days until 17 passages. At about 20-22 passages, the population doubling appeared to decline and later rose back to a population doubling number of about 0.7 to about 1.0 times per day. Foci were also obtained from transfected skin cells isolated from 10-day-old chicken fetuses. The results of the experiments are unexpected since p53 is well known in the literature as a tumor suppressor and as a growth regulator. Identical cultures of skin cells were transfected with the same construct containing the antisense p53 gene fragment. Cells transfected with this construct were unable to undergo immortalization.

For primary cardiac and thoracic muscle cells, the frozen cultures were thawed and passaged. In place 2, the sense p53 construct was combined with a polyamine compound, lipofectamine TransitII (PanVera, Madison,WI) transfected 8X 10 of each type of cells together⁶Cells (confluent 40-70%). Untransfected cells were maintained as a control for positive growth and as a control for negative cell death (culture with additional G418 added). For transfection, 2-12. mu.l/microgram DNA was added dropwise to 100. mu.l of serum-free medium (RPMI 1640, Life technologies). The mixture was gently mixed and incubated at room temperature for 5 minutes. 1-3. mu.g of DNA was diluted in Transit II reagent supplied by the manufacturer and incubated for 6 hours. Cells were washed and medium was added again. After a 24 hour recovery period, heart and thoracic cells were plated out into 4 dishes, each at 3X 10⁵Cells were plated under G418 selection and zinc induction. Identifying foci in the experimental culture. No foci were obtained from control dishes supplemented with 50. mu.M zinc.

Example 4

Detection of viral contaminants in cells containing the p 53-construct

Detecting reverse transcriptase activity of the cell. Isolation of 1X 10 from rapidly growing cultures⁶Cells were in 4 ml of medium. The medium was taken to lyse the cells by several freeze-thawing at-80 ℃. The medium containing lysed cells was plated onto a 10% glycerol gradient medium. The gradient media was spun at 40,000rpm for 60 minutes using a SW40 centrifuge (Beckman Instruments, Pal Alto, Calif.). Small pieces of virus particles, if any, are formed. The medium was discarded and the pellet was resuspended in 20. mu.l of Nonidet P-40(Sigma chemical Co., St. Louis, Mo.).

One microcentrifuge tube was heated at 41 ℃. A sample of 5. mu.l was added to 45. mu.l of a reverse transcriptase mixture containing 45mM Tris, pH7.8, 2mM 2-. beta.mercaptoethanol, 2mM manganese acetate, 0.1% Triton X-100, 10. mu.M dATP, dCTP, dGTP IN portions (Boehringer Mannheim Biochemical, Indianapolis, IN), 2.4. mu.g of poly A (Sigma), 60ng of primer dT12-19(Pharmacia), 0.4. mu.g of 3H thymidine triphosphate per reaction (15,000 to 28,000cpm/pmole activity, Amersham).

The reaction solution was incubated at 41 ℃ for 1 hour. For the negative control, 5. mu.l of double distilled water and 45. mu.l of the mixture were used. Two known positive controls were included in the assay. The assay was terminated by the addition of 1 ml of 10% trichloroacetic acid (TCA, Columbus chemical industries, Columbus, WI). The mixture was filtered through a Whatman GF/C glass 0.45 micron prefilter. Washed several times with 5% TCA. The filtrate was transferred to a scintillation vial containing 5 ml of scintillation counting solution. Samples were counted on a Beckman instrument scintillation counter with a 050 to 600 window setting. Amounts more than three times the background of the mixture (negative control) were considered positive.

The primary cells were tested negative as were the other cells used in the present invention. For further information on reverse transcriptase assays see (Crittenden et al, virology 57: 128-138, 1974).

Example 5

Identification of immortalized cells

After introducing the p 53/metallothionein-containing construct into cells, the cells are said to be immortalized when they have passed more than about 25 passages in tissue culture and multiplied by a population doubling of about 0.6 to about 1.5 per day. This rate was compared to a late untreated (i.e., cells not receiving the p 53/metallothionein promoter construct) control having a population doubling rate of about 0.1 to about 0.2 population doublings/day. Skin cells that received the p 53/metallothionein promoter are currently in the 70 th passage after the introduction of the gene construct into the cells. In the transfection method, more than 20 immortalized clones were identified. The cells are morphologically normal and contact-inhibited. The pectoral cells are currently at passage 52, and morphologically normal, exhibiting contact inhibited growth and a current population doubling rate of about 0.72. 13 clones were selected for analysis. The cardiac cells were currently at passage 20, morphologically normal, contact inhibited, and the average population doubling rate was 0.6. The population doubling rate for several clones was between about 0.8 and 1.1. More than 10 clones were selected for further study.

Example 6

Soft agar colony formation assay to assess tumorigenic potential of cells

To test for tumorigenic potential, cells transfected with the p 53/metallothionein promoter were tested for growth in soft agar. A soft agar matrix was prepared by mixing 12 ml of 2% agar solution (autoclaved and cooled to 56 ℃) in 21.6 ml of McCoy's 5A medium [ BRL/Gibco, 120 ml of fetal bovine serum (heat inactivated, 5 ml of sodium pyruvate (2.2% stock), 1 ml of L-serine (21 mg/ml stock), 5 ml of L-glutamine (200mM stock), 12.5 ml of Hepes (1M stock) ]. 7 ml of warm medium/agar was poured onto 100 mM square tissue culture dishes and allowed to solidify in tissue culture for 1 hour at room temperature.

Cells were removed from actively growing (approximately 40 to 70% confluent) cultures by trypsinization to obtain single cell suspensions in fresh DMEM medium containing 10% fetal bovine serum (with L-glutamine and antibiotic-antifungal agent). Adding about 1X 10⁶Cells were plated in 4.2 ml DMEM medium containing 10% fetal bovine serum, 0.75 ml 1% agar, and 50. mu.l 2 β -mercaptoethanol. Care was taken to ensure that the warm medium/agar was at 42 ℃ before the cells were added. Quickly, 5 ml of the above cell suspension was plated on the agar plate.

Cells were grown at 37 ℃ in a 5% carbon dioxide and 95% air incubator and observed for 35 days. Duplicate plates were stained with 3 p-nitrophenyl-5-phenyltetrazole chlorite (INT dye) and colony formation and growth was detected on days 0, 5, 10, 15, 20, 30, and 35. All stained colonies larger than 60 microns were considered positive.

All cells were negative. Further information on soft agar assays can be obtained from Hamburger et al (clinical biology research progress 48: pps43, 135, 179, 1980).

Example 7

Tumorigenicity of immortalized cells

Cells were injected into experimental animals to determine whether the cells were tumorigenic under the guidelines outlined in the animal utilization protocol of the university of minnesota (protocol No. 950300-1, 3 months 1995 to 12 months 1996). To test the tumorigenic potential of chicken p53 under the control of the chicken metallothionein promoter, the immortalized cells were injected into chickens.

Actively growing cells were removed from the cell culture plate and injected into 6 adult chickens of the SPAFAS strain. Injecting subcutaneously with 4X 10⁶Cells were placed in the web of the chicken wings. The injection sites were examined weekly over 3.5 months. For any transfected cells produced to date (skin, heart and muscle), no tumors were observed at the injection site and all animals remained healthy.

Example 8

Viral propagation in the p 53/metallothionein leukocyte line

The cells were tested for their ability to support virus production. With a titre of 8.2TCID₅₀Perml of the WSS-Reol733 strain of reovirus infected a 2.5X 10 vector containing the p 53/metallothionein promoter construct⁸A cell. Cells are infected at a multiplicity of infection of 0.005, 0.001 or 0.0005 infectious viral particles/cell. Infected cells were cultured in roller bottles and tested at 48, 64 and 72 hours post infection and proliferation of productive virus was determined.

In addition, the ratio of the total weight of the powder to the total weight of the powder in a roller bottle is 2.0 multiplied by 10⁴Cells were seeded per square centimeter. The roller bottles were incubated at 37 ℃ for 8 days at 0.4 to 0.5RPM on a roller frame apparatus. The cells were infected with herpes virus of turkeys (HVT virus, strain R2/23). When the rolling bottle is about 1.33 multiplied by 10⁷When cells were infected, the cells were infected at a multiplicity of infection of 0.001. The cells are harvested at about 90 to about 96 hours when about 40% of the monolayer cells have cytopathological symptoms associated with infection. In gamma-irradiated FBS with 10% Hyclone, 2.5g/L TPB, 2mM L-glutamine, 100U/ml penicillin, 0.10 mg/ml streptomycin,cells were cultured and maintained in DMEM at 0.25 μ g/ml amphotericin B, 1.6 mg/l insulin and 1.6 mg/l transferrin. Preliminary studies demonstrated that the cells produced approximately 1.4X 10⁴pfu/ml。

The cells can also support the replication of Marek's disease virus.

Example 9

Use of infected skin cells as cell substrates

The cells of the invention can be used as substrates to support viral replication in primary cells. In these experiments, p53 immortalized skin cells were mixed with primary cells. In one study, the primary cells were infected and mixed with immortalized cells and placed in culture, while in another study, primary cells were infected and placed on the immortalized cells, wherein the immortalized cells had been arranged into a lawn in the tissue culture flask. In one embodiment, the virus is an Egg Drop Syndrome virus and the primary cells are primary chick embryo hepatocytes. In a second embodiment, the primary cells are endothelial cells, preferably renal endothelial cells, and the virus is infectious bronchitis virus. Preferably, the ratio of primary cells to immortalized cells is from about 1: 5 to about 1: 20, and more preferably about 1: 10. The virus titer from primary cells grown in the mixed cell population is higher than the virus titer from primary cells in culture alone. The immortalized cells make the primary cells useful for virus propagation under commercial conditions.

While specific embodiments of the invention have been described in detail, those skilled in the art will recognize that these embodiments are by way of example, and not by way of limitation, and that the true scope of the invention is defined by the following claims.

Claims (14)

1. A method of transforming primary chicken cells comprising the steps of:

positioning a nucleic acid encoding p53 under the control of a metallothionein promoter in a genetic vector capable of directing expression of p 53;

introducing the gene vector into primary chicken cells; and

the population doubling time was selected to be 0.6 to 1.5 population doubling foci per day, wherein the cells were reverse transcriptase negative and non-tumorigenic.

2. The method of claim 1, wherein the cells are dermal cells.

3. The method of claim 1, wherein the cell is a pectoral muscle cell.

4. The method of claim 1, wherein the cell is a cardiomyocyte.

5. The method of claim 1, wherein the cell is a fibroblast.

6. A method of propagating a virus comprising the steps of:

contacting at least one infectious viral particle with at least one cell of a culture of immortalized chicken cells, wherein said cultured cells contain a genetic vector that expresses p53 under the control of a metal-sulfur protein promoter; and

the produced virus is collected by the cells.

7. The method of claim 6, wherein the virus is a reovirus.

8. The method of claim 6, wherein the virus is herpes virus of turkeys.

9. The method of claim 6, wherein the virus is a fowlpox virus.

10. A method of propagating a virus comprising the steps of:

contacting at least one infectious viral particle with a primary cell;

in cell culture, primary cells are cultured with immortalized chicken cells containing a gene vector for p53 under the control of a metallothionein promoter; and

collecting the produced virus from the cell culture.

11. An immortalized, non-transformed chicken cell comprising p53 under the control of a metallothionein promoter and at least one vector capable of directing the expression of a recombinant protein in said cell.

12. The cell of claim 11 which expresses a recombinant protein.

13. The cell of claim 12, wherein the vector encodes at least a portion of a recombinant virus.

14. The cell of claim 11, wherein the vector is a retroviral vector.