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WO2000062604A1 - Systeme genetique destine a reguler l'expression en arriere-plan de produits transgeniques - Google Patents

Systeme genetique destine a reguler l'expression en arriere-plan de produits transgeniques Download PDF

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WO2000062604A1
WO2000062604A1 PCT/US2000/009837 US0009837W WO0062604A1 WO 2000062604 A1 WO2000062604 A1 WO 2000062604A1 US 0009837 W US0009837 W US 0009837W WO 0062604 A1 WO0062604 A1 WO 0062604A1
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transgene
promoter
gene
tetracycline
antisense
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Charles G. Orosz
Dongyuan Xia
Gayle M. Gordillo
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Ohio State University Research Foundation
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Ohio State University Research Foundation
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    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
    • A01K67/00—Rearing or breeding animals, not otherwise provided for; New or modified breeds of animals
    • A01K67/027—New or modified breeds of vertebrates
    • A01K67/0275—Genetically modified vertebrates, e.g. transgenic
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/85—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells
    • C12N15/8509—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells for producing genetically modified animals, e.g. transgenic
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
    • A01K2217/00—Genetically modified animals
    • A01K2217/05—Animals comprising random inserted nucleic acids (transgenic)
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
    • A01K2217/00—Genetically modified animals
    • A01K2217/20—Animal model comprising regulated expression system
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
    • A01K2227/00—Animals characterised by species
    • A01K2227/10—Mammal
    • A01K2227/105—Murine
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
    • A01K2267/00—Animals characterised by purpose
    • A01K2267/03—Animal model, e.g. for test or diseases
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2830/00—Vector systems having a special element relevant for transcription
    • C12N2830/008—Vector systems having a special element relevant for transcription cell type or tissue specific enhancer/promoter combination

Definitions

  • transgenic animals that serve as experimental models of disease.
  • transgenic animals that serve as experimental models of disease.
  • transgenes that had been operably linked to tetracycline-responsive promoters or to rapamycin-responsive promoters.
  • the first tetracycline-controlled promoter was constructed by fusing the operator sequence of the E. coli tetracycline-resistance gene (tetO) to the minimal promoter sequence of the human cytomegalovirus immediate-early gene (hCMV IE).
  • tetO E. coli tetracycline-resistance gene
  • hCMV IE human cytomegalovirus immediate-early gene
  • this promoter (tetO/hCMN) is activated when it binds to a fusion protein constructed from the tetracycline-controlled transactivator (tTA), which contains a tetO binding protein tetracycline repressor (tetR), plus a transcription activator, virion protein 16 (VP 16), from herpes simplex virus.
  • tTA tetracycline-controlled transactivator
  • tetR tetO binding protein tetracycline repressor
  • VP 16 virion protein 16
  • tetracycline In the presence of tetracycline, exogene transcription is blocked because tetracycline binds to the transactivator (tTA) and interferes with its binding to the tetO/hCMN promoter. Since tetracycline down-regulates transgene expression, this is called the "tet-Off ' promoter system.
  • tet-Off promoter system is associated with leaky gene expression which complicates the use of this system for basic research or pharmaceutical application.
  • Furth and colleagues first used the tet-Off tetracycline-controlled promoter in transgenic mice that expressed the reporter transgene, luciferase, under control of the tetO/hCMN promoter. When tetracycline was absent, luciferase expression was observed in numerous tissues. When tetracycline was provided subcutaneously, the luciferase activity was significantly reduced to low, but still detectable, background levels. This leaky transgene expression was observed with two different plasmid delivery systems, the paired plasmid system of Furth (Furth, P. A..).
  • leaky transgene expression has also been observed in transgenic models that utilized the tet-Off system linked to a tissue specific promoter, the cardiac-specific, ⁇ -myosin heavy chain promoter ( ⁇ -mhc) (Yu, Z., Redfern, C.S., arid Fishman, G.I., Conditional transgene expression in the heart. Circ.Res. 79(4), 691-697, 1998).
  • ⁇ -mhc ⁇ -myosin heavy chain promoter
  • the tetO/hCMN genetic system has also been modified to allow tetracycline to induce, rather then inhibit, transgene expression.
  • the modified system employs the reverse tetracycline-controlled transactivator (rtTA), comprised of a mutated tetO binding protein, rtetR (tetracycline repressor) linked to NP16 (Gossen, M., Freundlich, S., Bender, G., Muller, G., Hillen, W., and Bujard, H., Transcriptional activation by tetracyclines in mammalian cells. Science 268(5218), 1766-1769, 1995).
  • rtTA reverse tetracycline-controlled transactivator
  • the rtTA When tetracycline is absent, the rtTA cannot bind to the tetO in the tetracycline-controlled promoter. When tetracycline is present, it binds to the rtTA which allows the rtTA to bind to the tetO in the promoter and up-regulate transcription of the exogene. Since tetracycline induces gene expression, this is called the "tet-On" promoter system. Leaky gene expression is less prevalent in transgenic mice in which the transgene is under control of the tet-On promoter as compared to the mice in which the transgene is under control of the tet-Off promoter.
  • transgenic mice whose transgenes are responsive to rapamycin have also been shown to express detectable background levels of transgene transcription in the absence of rapamycin. Such "leaky" transgene transcription seriously compromises studies with these transgenic mice.
  • transgenic animals particularly transgenic mice, which have little to no background transgene transcription, particularly in specific tissues.
  • the present invention provides a method and system for controlling the expression of transgene products in specific tissues in a transgenic animal, particularly a transgenic mouse, while eliminating background expression of the transgene products.
  • the method comprises: a) providing a transgenic parent animal referred to hereinafter as a "P2" animal, whose genome comprises a transgene, hereinafter the "F” transgene, comprising an exogenous gene operatively linked to a promoter, hereinafter the "F” promoter; wherein the F promoter is upregulated by a transactivator protein; and wherein interaction of the transactivator protein with the F promoter occurs in the presence of a transactivator regulator; b) providing another transgenic parent animal, referred to hereinafter as a "PI” animal, whose genome comprises i) a second transgene, hereinafter the "S” transgene, comprising a second promoter, hereinafter the "S” promoter, operatively linked to an antisense gene; where
  • a transactivator regulator such as for example tetracycline
  • administration of a transactivator regulator results in enhanced expression of the exogenous gene and reduced expression of the antisense gene product in target tissues.
  • the FI mouse has reduced levels of the exogenous gene product in the absence of the transactivator regulator,
  • the FI mouse exhibits tissue specific expression of the exogenous gene product following administration of the transactivator regulator and little to no background expression of the exogenous gene product in the absence of the transactivator regulator. Accordingly, the FI mouse is a research tool useful for studying the impact of the transgene product on the biology of the mouse under defined conditions of expression.
  • the system for controlling expression of a desired transgene product in specific tissues of a transgenic mouse while eliminating background expression of the desired transgene product comprises a PI animal, preferably a PI mouse, and a vector or a DNA construct for producing the P2 animal, which is also preferably a mouse.
  • the S transgene comprises a CMN promoter, a tetracycline operator sequence, and a sequence encoding an antisense R ⁇ A which binds to the 5' untranslated sequence (5' UTR), and more preferably the first intron, of the human CMN immediate-early gene; and the T transgene comprises a tissue specific promoter operatively linked to a reverse tetracycline transactivator gene, hereinafter the "rtTA" gene.
  • the vector or D ⁇ A construct for producing the P2 animal comprises a tetracycline controlled promoter, hereinafter the "tetO/hCMN" promoter, and multiple restriction sites for subcloning a desired exogene.
  • the tetO/hCMN promoter comprises the operator sequence of the E. coli tetracycline-resistance gene fused to the minimal promoter sequence of the human cytomegalovirus immediate-early gene.
  • the vector or D ⁇ A construct further comprises the desired exogene operatively linked to the tetO/hCMN promoter.
  • the present invention also relates to a D ⁇ A construct comprising the S transgene.
  • the antisense gene of the S transgene comprises a sequence which binds to the F transgene transcript to inhibit or reduce translation of the F transgene transcript or splicing of the F transgene transcript.
  • the D ⁇ A construct is useful for preparing the PI animal.
  • the present invention also relates to a PI mouse.
  • the PI mouse comprises a S transgene comprising CMN promoter, a tetracycline operator sequence, and a sequence encoding an antisense R ⁇ A which binds to the 5' untranslated sequence and the intron of the human CMN immediate-early gene; and a T transgene comprising a tissue specific promoter operatively linked to the rtTA gene.
  • Figure 1 depicts the standard Tet-Off gene expression system.
  • Figure 2 depicts the standard Tet-On gene expression system.
  • Figure 3 depicts a tetracycline double controlled tissue specific gene expression system
  • Figure 4 depicts the tetracycline-controlled hCMN antisense gene expression system
  • Figure 5 depicts the design of the gene expression plasmids described in the Examples.
  • Figure 6 is a schematic showing construction of pNRBtA.
  • Figure 7 is a schematic showing construction of pTo 1012.
  • Figure 8 is a schematic showing construction of pToLd.
  • Figure 9 is a schematic showing construction of pTol412 and pToluci plasmids
  • Figure 10 is a schematic showing construction of Mhc-tetON plasmid.
  • Figure 11 is a map of pClone 22.
  • Figure 12 is a map of pUHD10-3.
  • the plasmid consists out of three main fragments: pBR322-sequences including colEl -origin of replication, ⁇ -lactamase-resistance-gene with the Pbla/p3 of Tn2661 (HincII-site and Pstl-site removed); the regulatory region with hCMN minimal promoter (-53 relative to start site) with heptomerized upstream tet-operators as described in P ⁇ AS 89, 5547-51 (1992); multiple cloning site, MCS, containing SacII, EcoRI and Xbal recognition sequences; and SN40 polyadenylation downstream of the MCS.
  • Figure 13 is a map of pL d .444.
  • Figure 14 is a map of ICAM2/Ul/tTA
  • Figure 15 depicts the tetracycline triple controlled transgenic system.
  • Figure 16 is a schematic showing construction of pToGFP and pToRFP plasmids.
  • the present invention provides a method for controlling the expression of transgene products in specific tissues in a transgenic animal while eliminating background expression of the transgene products in tissues of the animal.
  • the method comprises providing a PI animal and a P2 animal and breeding the PI animal with the P2 animal to provide an FI animal.
  • the PI, P2, and FI animals are non-human mammals such as, for example, pigs.
  • the PI animal, the P2 animal, and the FI animal are mice.
  • the P2 animal is a transgenic parent animal whose genome comprises an F transgene which comprises an exogenous gene operatively linked to an F promoter that is upregulated by a transactivator protein. Interaction of the transactivator protein with the F promoter occurs in the presence of a transactivator regulator.
  • the P2 animal can comprise any desired exogenous gene.
  • an exogenous gene is a gene that is not normally present in the genome of the P2 animal.
  • the exogenous gene can be a reporter gene such as for example a gene which encodes luciferase or beta-galactosidase, green fluorescence protein (GFP) or red fluorescence protein (RFP). Clones comprising such reporter genes are commercially available.
  • the exogenous gene encodes a biologically active molecule.
  • An example of such gene is the H- 2L d gene which encodes the H-2L d protein.
  • the P2 animal is homozygous for the F transgene.
  • the F promoter is a tetracycline controlled promoter.
  • the tetracycline controlled promoter comprises the operator sequence of the E. coli tetracycline- resistance gene fused to the minimal promoter sequence of the human cytomegalovirus immediate-early gene.
  • the tetracycline controlled promoter further comprises the first intron of the human CMN immediate early gene.
  • the P2 animal ftirther comprises a transgene comprising a constitutive promoter operatively linked to an antisense gene which encodes a transcript that blocks processing of the exogene transcript.
  • the genome of the PI animal comprises an S transgene comprising an S promoter operatively linked to an antisense gene which encodes a transcript that blocks processing of the exogene transcript, i.e., the F transgene transcript, and a T transgene comprising a tissue specific promoter operatively linked to a gene encoding the transactivator protein.
  • the S promoter is downregulated by the transactivator protein which up-regulates the S promoter of the S transgene. Interaction of the transactivator with the S promoter occurs in the presence of the transactivator regulator.
  • the S antisense gene encodes a sequence which blocks the mR ⁇ A splicing of the F transgene transcript.
  • the PI animal expresses the transactivator protein in target tissues, i.e., those tissue corresponding to the tissue specific promoter. In the absence of the transactivator regulator the PI animal also expresses the antisense gene product. Administration of the transactivator regulator to the PI animal results in reduced expression of the antisense gene product.
  • the transactivator regulator is tetracycline and the transactivator protein is the rtTA protein.
  • the genome of the FI animal comprises the F transgene, the S transgene, and the T transgene.
  • the FI animal expresses the antisense gene product in all tissues and the transactivator protein in target tissues.
  • Administration of the transactivator regulator to the FI animal results in enhanced expression of the exogenous gene and reduced expression of the antisense gene product in target tissues.
  • the FI animal particularly an FI mouse, is a research tool useful for studying the impact of the transgene product function on the biology of the animal under defined conditions of expression.
  • the FI animal As compared to the P2 animal, the FI animal has reduced levels of the exogenous gene product in the absence of the transactivator regulator, Accordingly, the FI animal exhibits tissue specific expression of the exogenous gene product following administration of the transactivator regulator and little to no background expression of the exogenous gene product in the absence of the transactivator regulator.
  • the system for controlling expression of a desired exogene product in specific tissues of a transgenic mouse while eliminating background expression of the desired exogene product comprises the PI animal, which is preferably a PI mouse, and a vector or a DNA construct for producing the P2 animal, which, preferably, is also a mouse.
  • the S transgene comprises a CMN promoter, a tetracycline operator sequence, and a sequence encoding an antisense R ⁇ A which binds to the 5' untranslated sequence (5' UTR), and more preferably, the first intron of the human CMN immediate-early gene,; and the T transgene comprises a tissue specific promoter operatively linked to a reverse tetracycline transactivator gene, i.e., the "rtTA" gene.
  • tissue specific promoter is suitable for use in the present invention.
  • the tissue specific promoter is selected to be an endothelial cell specific promoter, such as for example an ICAM-2 promoter.
  • the tissue specific promoter is selected to be a cardiac myocyte specific promoter, such as for example the alpha-myosin heavy chain promoter.
  • the present system can easily be modified to accommodate different exogenes and different tissue specific promoters.
  • the vector or D ⁇ A construct for producing the P2 animal comprises a tetracycline controlled promoter and multiple restriction sites for subcloning a desired exogene.
  • the tetracycline controlled promoter or tetO/hCMN promoter comprises the operator sequence of the E.
  • the construct comprises both the 5' UTR and the first intron of the human cytomegalovirus (CMN) immediate early gene.
  • the vector or D ⁇ A construct further comprises an exogenous gene such as for example a reporter gene or a coding sequence for a biologically active molecule. The preferred system allows for tetracycline controlled, tissue specific expression of any exogene, and eliminates unwanted background expression of the product of such exogene.
  • the present invention also provides a D ⁇ A construct comprising the S transgene.
  • the construct comprises a sequence encoding an antisense R ⁇ A which binds to a sequence selected from the group consisting of the 5' untranslated sequence human CMN immediate-early gene, the first intron of human CMN immediate early gene, or a combination thereof.
  • the preferred S transgene further comprises a CMN promoter, and a tetracycline operator sequence operably linked to the antisense R ⁇ A coding sequence. As shown in Figure 3, the tetracycline operator sequence is located at the 3' end of the CMN promoter and the 5' end of the antisense R ⁇ A encoding sequence.
  • the D ⁇ A construct is useful for preparing the PI animal.
  • the present invention also relates to a PI mouse.
  • the PI mouse comprises a S transgene comprising a sequence encoding an antisense RNA which binds to the 5' untranslated sequence and the intron of the human CMN immediate-early gene, a CMN promoter, and a tetracycline operator sequence; and a T transgene comprising a tissue specific promoter operatively linked to the rtTA gene.
  • the PI mouse is homozygous for the S transgene and the T transgene.
  • the tetracycline-controlled promoter (tetO/hMHC) was obtained from the plasmid PNR1250 (Vical, Inc. San Diego, CA) with Fsp I and Sst II double cut and replaced the hCMV promoter of the high expression vector pVR1012 (Vical Inc.).
  • This modified vector, pTol012 contains the tetracycline-controlled promoter, multiple subclone sites, and the Bovine Growth Hormone transcription terminated sequence (Fig. 5a and Fig. 7).
  • the pTol012 plasmid can be used to build any exogene expression construct.
  • Vector function was tested in vitro by inserting a luciferase gene (pToLuci, Fig. 5b and Fig. 9), followed by transfection into COS 1 cells (Cowan, P.J., Shinkel, T.A., Witort, W.J., Barlow, H., Pearse, M.J., and d'Apice, A.J.F., Targeting gene expression to endothelial cells in transgenic mice using the human intercellular adhesion molecule 2 promoter.
  • pToLuci Fig. 5b and Fig. 9
  • the mouse H-2Ld cDNA from the pLd.444 plasmid was digested with Bam HI and inserted into the Bam HI site of the pTol012 subclone site.
  • the new plasmid pToLd (Figs. 5e and 8) contains the murine H-2Ld gene driven by the tetracycline-controlled hCMV promoter.
  • C. Tetracycline-controlled ⁇ -galactosidase expression plasmid The ⁇ -galactosidase gene from the pVR1412 plasmid (Fig. 5d and 9), a gift from Vical Inc.) was double digested with
  • the pTol412 plasmid (Figs. 5e and 9) contains the ⁇ -galactosidase gene driven by the tetracycline-controlled promoter. Function of the pTol412 was tested by co-transfection of
  • pVRBtA A tetracycline-controlled antisense expression plasmid, pVRBtA was generated as shown in Fig. 6.
  • the plasmid pVRBtA expresses a 0.9kb antisense RNA which is highly specific, and binds only to the 5 'untranslated sequence and the intron of the hCMV immediate-early gene. This antisense blocks the splicing of any mRNA which is transcripted from the hCMV promoter.
  • an antisense expression plasmid was constructed by reverse ligation of the 0.9kb Xma III fragment which contains the 5' UTR and the intron of the hCMV IE1 gene back into a pVR1012 vector.
  • the CMV promoter was modified into a tetracycline-controlled promoter by inserting a tetracycline operator (tetO) sequence at the Sac I site that is right at the 3' end of the CMV promoter (Fig. 4f).
  • tetO tetracycline operator
  • COS1 cells were transfected with 1.5 ⁇ g of pVRBtA using LipofectAMINE (Life Technologies, BRL, Gaithersburg, MD). After 48 hr culturing, the total RNA was isolated with the neutral-phenol extraction method. Plasmid DNA contamination was removed by DNase digestion at 37°C for 2 hours. The total cDNA was obtained by reverse-transcription.
  • the primer pairs for the antisense RNA detection (sense: 5'-AGA AGA CAC CGG GAC CGA TC -3', and antisense: 5'-ATG TAA TCG CAG CCG TCG TAG -3') were designed to produce a DNA fragment of 243bp. For PCR, 30 cycles were used (94°C/30 sec, 60°C/20 sec, and 72°C/1 min.). The results showed that the antisense RNA was detectable in the transfected COS cells via RT-PCR.
  • the blocking function of the antisense construct was tested by co-transfection of COS 1 cells with the luciferase reporter plasmid, pToLuci, and either the hCMV antisense plasmid, pVRBtA or the control plasmid, pVR1012.
  • the in vitro blocking results showed that the antisense plasmid is able to block 40 to 45% activity of the reporter gene. This is significant blocking by antisense, considering that the inhibition in this system can be observed only when the COS 1 cells are co-transfected.
  • EXAMPLE 3. Tissue Specific Expression Systems A Endothelial specific expression system.
  • the ICAM-2 promoter is a tissue specific transcription promoter which drives gene expression only in endothelial cells (Volloch, V., Schweitzer, B., and Rits, S., Inhibition of pre-mRNA splicing by antisense RNA in vitro: effect of RNA containing sequences complementary to introns. Biochem Biophys Res Commun 179(3), 1600-1605, 1991).
  • the hCMV promoter in the pTet-On plasmid was replaced with the ICAM-2 promoter (obtained from Dr. Peter Cowan at the St. Vincent's Hospital). This plasmid construct, pICAM/rtTA (Figs.
  • rtTA reverse tetracycline-controlled transactivator
  • MAE cells a murine endothelial cell line derived from aortic endothelial cells (obtained from Dr. Robert Auerbach at the University of Wisconsin).
  • the MAE cells were co-transfected with the pICAM/rtTA plasmid and pVR1250 luciferase reporter plasmid, which contains a luciferase gene driven by a tetracycline-controlled promoter.
  • pICAM/rtTA plasmid a murine endothelial cell line derived from aortic endothelial cells (obtained from Dr. Robert Auerbach at the University of Wisconsin).
  • the MAE cells were co-transfected with the pICAM/rtTA plasmid and pVR1250 luciferase reporter plasmid, which contains a luciferase gene driven by a tetracycline-controlled
  • the cardiac myocytes specific promoter a-myosin heavy chain promoter (Mhc) is another promoter that can be used for generating tissue specific expression systems.
  • the hCMV promoter in the Tet-On plasmid was removed (Sal I /blunted and the Barn HI double cutting) and replaced by the ⁇ -myosin heavy chain promoter, 5.5kb Eco RI (blunt) and Bam HI fragment (Figs. 5h and 10).
  • the PIE line a transgenic mouse line that displays endothelial-specific rtTA expression, was generated by micro injection of C57BL/6 embryo with the I-CAM-1-rtTA and the pVRBtA plasmids.
  • the pICAM-1-rtTA plasmid contains the rtTA gene driven by the endothelial specific ICAM-2 promoter in a cassette that can be isolated by digestion with Pvu II restriction endonuclease.
  • the plasmid, pVRBtA contains the hCMV antisense gene driven by the tetracycline down regulated promoter in a cassette that can be isolated with Xmn I/Msc I restriction endonucleases.
  • These two gene expression cassettes were isolated and purified by gel electrophoreses. Three pico-litres of these two purified DNA constructs, in a concentration of 3ng/ ⁇ l, were co-injected into the C57BL/6 embryo by micro-injection.
  • these PIE mice should express the genes of the reverse tetracycline-controlled transactivator (rtTA) protein only in endothelial cells, whereas they should expression the hCMV-antisense gene in multiple tissues (in the absence of tetracycline).
  • rtTA reverse tetracycline-controlled transactivator
  • Putative PIE transgenic mice were screened by PCR for detection of rtTA gene and the antisense gene sequence.
  • Tail tissues (3mm) from transgenic mice were digested with 25 ⁇ l 0.1% collagenase at 37°C for 2hr. After a phenol and chloroform extraction to obtain DNA, 5 ⁇ l of DNA was analyzed by PCR.
  • the PCR primer pair for rtTA transgene detection (sense: rtTA-5, 5'-AGA TCA AGA GCA TCA AGT CG -3' and antisense: rtTA-3, 5'-AGT CGG CCA TAT CCA GAG -3') was designed to produce a DNA fragment of 512bp (Fig. 18a).
  • PCR used 30 cycles for analysis with these primer pairs, (94°C/60 sec, 57°C/60 sec, and 72°C/60/cycle).
  • the primer pair for hCMV antisense gene detection (sense: 5' -AGA AGA CAC CGG GAC CGA TC -3' and antisense: S'-GAA GAA GAT GCA GGC AGC TGA G -3')was designed to produce a DNA fragment of 243bp (Fig.l2c),which is not present in the normal B6 genome.
  • 30 cycles was used (94°C/60sec, 61°C/60 sec, and 72°C/60 sec/cycle).
  • rtTA and laCMV antisense gene products in PIE transgenic mice.
  • the rtTA gene product was detected by Western blot with a specific anti-Tet monoclonal antibody (CLONTECH Laboratories, Inc.). Since the rtTA gene is controlled by the ICAM-2 promoter, the rtTA mRNA and protein is transcribed and translated in endothelial cells. Since tail and pinnae are endothelial cell-rich tissues, either may b used for detection of the rtTA.
  • Tissues from PIE transgenic mice found to be DNA-positive by gene screening for both rtTA and hCMV antisense, were homogenized and treated with SDS PAGE sample buffer at 100°C for 5 minutes. The total proteins were separated by PAGE and blotted into a PVDF membrane. A 37 kDa of rtTA protein is specifically detected with the mouse anti-TetR monoclonal antibody, which binds to the TetR domain in the tTA or rtTA fusion proteins (CLONTECH Laboratories, Inc., Palo Alto, CA).
  • the hCMV antisense RNA product is detected by the RT-PCR technique. Since it is expected that the CMV promoter for the hCMV antisense gene will be expressed in multiple tissues, tail or pinna tissues can also be used for the screening of the hCMV antisense RNA transcription.
  • Endothelial specific rtTA expression in the PIE mice is evaluated by immunohistochemistry. Ear tissues from the PIE transgenic mice (rtTA gene expression positive in Western blot and antisense RNA positive in RT-PCR) is embedded with Tissue Tek OCT (Miles, inc., Elkhart, IN) then sectioned in 5 ⁇ m. The biotin-labeled mouse anti-TetR mAb and streptavidin-Horseradish Peroxidase is used for the rtTA protein staining. Additional tissues, such as liver, heart, kidney, muscle, and spleen are screened for endothelial-specific rtTA expression. The PIE transgenic mice with endothelial specific rtTA expression are used to prepare endothelial specific FI mice as shown in Fig. 15
  • the PIM line a transgenic mouse line that displays myocyte-specific rtTA expression
  • the plasmid, MHC/rtTA contains the rtTA gene driven by the cardiac myocyte specific alpha-Myosin Heavy Chain promoter in a cassette that can be isolated by digestion with Xho I/Hind III restriction endonucleases.
  • the antisense plasmid, pVRBtA was digested with Xmn I/Msc I restriction endonucleases as before.
  • a tissue specific rtTA expression mouse lines Cardiac myocyte-specific rtTA expression in the PIM mice is evaluated by immunohistochemistry. Heart, liver, kidney, skeletal muscle, and spleen tissues from the PIM transgenic mice, found to be positive for rtTA gene product and hCMV antisense RNA, is embedded with OCT and sectioned. The biotin-labeled mouse anti-TetR mAb and streptavidin-HRP is used for the rtTA protein staining. The rtTA should be detected only in myocytes, but not endothelial cells of the cardiac tissues. None of the other tissues should express rtTA. The littermates of these rtTA positive PIM transgenic mice are used to prepare FI mice expressing myocyte specific exogenes as shown in Figure 15.
  • P2Ld Tetracycline-controlled H-2 Ld transgenic mice
  • the P2Ld line a transgenic mouse line that displays tetracycline transactivator-controlled H2Ld expression in all tissues, is generated with the plasmid, pToLd, which contains the mouse H-2Ld gene driven by the tetracycline-controlled promoter in a cassette that can be isolated by digestion with Xmn III/Xho I restriction endonucleases. This gene expression cassette will be isolated., and purified by gel electrophoreses. Three pico-litre of these two purified DNA constructs, in a concentration of 3ng/ ⁇ l.
  • P2Ld mice should not express H-2L d protein in any tissue without the tetracycline-controlled transactivator and tetracycline present. However, the background or leaky expression of the H-2L d gene from the hCMV minimal promoter should be found in multiple tissues.
  • a. Gene Screening Putative P2Ld transgenic mice are screened by PCR analysis of DNA from tail tissues using the primer pair (sense: 5'-AGA AGA CAC CGG GAC CGA TC -3' and antisense: 5'-ATG TAA TCG CAG CCG TCG TAG -3').
  • This primer pair specifically targets small contiguous portions of the hCMV and H-2Ld genes, and produces a DNA fragment of 512bp (Fig. 12e).
  • the sense primer is directed at an hCMV intron sequence that is not present in the normal B6 genome. This eliminates the chance of inadvertent detection of other MHC class I genes that might occur if an H-21,d-specific PCR primer pair were used.
  • This primer pair uses 30 cycles for arrays (94°C/60 sec, 62°C/60 sec, and 72°°C/60 sec/cycle).
  • H-2Ld Gene product detection The H2Ld gene should not be expressed in any tissue without the tetracycline-controlled transactivator and tetracycline present.
  • RNA from liver, heart, kidney, muscle, and spleen tissues is isolated by the neutral-phenol extraction method. The contaminating DNA is removed by DNase digestion at 37°C for 2 hr. Then the RNA is reverse transcripted into cDNA with the reverse-transcriptase. The cDNA is tested by PCR with the same primers used above for the H-2Ld gene screening.
  • P2LacZ Tetracycline-controlled ⁇ -galactosidase transgenic mice
  • the P2LacZ line a transgenic mouse that displays tetracycline transactivator-controlled ⁇ -galactosidase expression in all tissues is/was generated with the plasmid pTol412 which contains the ⁇ -galactosidase gene driven by the tetracyline- controlled promoter in cassette that can be isolated by digestion with Xmn III/Xho I restriction endonucleases. This gene expression cassette is isolated, and purified by gel electrophoreses.
  • ⁇ -galactosidase product detection Gene transcription is detected by the ⁇ -galactosidase activity analysis or ⁇ -galactosidase staining of histologic tissue specimens. Various tissues, such as liver, lung, heart, muscle, kidney, are harvested and fixed in 10% formalin for 3 hr.
  • transgenic mice Each of the four types of transgenic mice described in Examples 4-7, i.e. the transgenic mice designated PIE, PIM, P2Ld, and P2LacZ, are inbred 2 to 3 generations to produce homozygous transgenic lines. For each line, this requires the screening and selection protocol outlined below.
  • the transgenic founders that are proven to be transgene-positive are outbred with normal, syngenic C57BL/6 mice to generate additional offspring that express the transgene. These offspring are screened by PCR to identify the transgene carrier mice, some of which will be sacrificed to test for relative levels of transgene mRNA and transgene protein expression. The goal is to identify lines with the highest levels of transgene expression.
  • transgene-positive littermates of mice with high transgene expression which should express similar high levels of transgene function, are inbred for 2 to 3 generations to produce homozygous transgenic mouse lines. Routine PCR is used to identify and select transgenic mice during the process. When inbred lines have been obtained, Southern Blot is used to identify lines with the best transgene expression. These lines are tested for homozygosity by cross-breeding them with normal, non-transgenic mice. All litter mates from a homozygous transgene parent should be transgene positive, as detected by DNA screening. If some of the offspring are transgene negative, the line is not homozygous. In this way, four homozygous transgenic mouse lines, PIE, PIM, P2Ld and P2LacZ, are established.
  • P2 double transgenic mice that express red fluorescence protein under the control of the tretracycline-controlled promoter, as well as the anti-sense RNA for the first intron of the CMV immediate-early gene were prepared.
  • C57BL/6 embryos were micro-injected with plasmids pToRFP and pVRBtA, respectively (See Fig. 16).
  • Expression of the RFP gene product is detected by fluorescence histology of tissues from the transformed animals. Expression of the antisense RNA is assayed by RT-PCR as described above.
  • the endothelial-specific rtTA expression transgenic mice (PIE) is crossed with the ⁇ -galactosidase gene transgenic mice (P2LacZ mice) to produce the E-LacZ FI transgenic mice.
  • PIE endothelial-specific rtTA expression transgenic mice
  • P2LacZ mice ⁇ -galactosidase gene transgenic mice
  • one set of chromosomes carries the PIE-derived rtTA hCMV antisense genes and another set of chromosomes carrying the P2LacZ derived, transactivator-controlled LacZ gene.
  • tail tissues are collected and digested with 25 ⁇ l 0.1% collagenase at 37°C for 2hr. After a phenol and chloroform extraction to obtain DNA, 5 ⁇ l of DNA is analyzed by PCR with the rtTA hCMV antisense, and ⁇ -galactosidase primer pairs.
  • EXAMPLE 10 Transgenic Mice which exhibit tetracycline-controlled and endothelial specific H-2Ld expression (E-Ldl The endothelial-specific rtTA expression transgenic mice (PIE) are crossed with the
  • H-2Ld transgenic mice to produce the E-Ld FI transgenic mice.
  • one set of chromosomes carries the PIE-derived rtTA hCMV antisense genes, and the other set of chromosomes carries the P2Ld-derived transactivator-controlled H-2Ld gene.
  • tail tissues are collected, digested and tested by PCR with the rtTA, hCMV antisense, and CMV/H-2Ld primer pairs.
  • the cardiac myocyte-specific rtTA gene expression transgenic mice (P-IM) is crossed with the ⁇ -galactosidase gene transgenic mice (P2LacZ mice) to produce the M-LacZ transgenic FI mice.
  • P2LacZ mice ⁇ -galactosidase gene transgenic mice
  • one set of chromosomes carries the PIM-derived rtTA/hCMV antisense genes and the other set of chromosomes carries the P2LacZ-derived, transactivator-controlled LacZ gene.
  • the myocyte-specific rtTA expression transgenic mice are crossed with the H-2Ld transgenic mice (P2Ld ) to produce the M-Ld transgenic FI mice.
  • PIM myocyte-specific rtTA expression transgenic mice
  • P2Ld H-2Ld transgenic mice
  • one set of chromosomes carries the PIM-derived rtTA/hCMV antisense genes and the other set of chromosomes carries the P-2Ld-derived transactivator-controlled H2Ld gene.
  • Tail tissues are collected, digested and tested by PCR with the rtTA, hCMV antisense primers, and
  • the tetracycline-responsiveness and the tissue specificity of ⁇ -gal expression can also be easily tested by in situ tissue staining.
  • the ⁇ -galactosidase should not be detected in any of tissues from either the E-LacZ or M-LacZ transgenic mice. This is tested by histochemistry in various tissues, such as liver, lung, heart, muscle, and kidney. To do this, the tissues is harvested from the transgenic FI mice and fixed in 10% formalin for 3 hr, embedded in Tissue Tek OCT (Miles, inc., Elkhart, IN), sectioned (14 ⁇ m), and stained with routine X-gal staining solution. Tissues from non-transgenic animals serve as controls.
  • ONPG is a chromogenic substrate for the enzyme ⁇ -galactosidase.
  • Total protein will be extracted from the various tissues with PBS buffer, incubated with the ONPG for 30 min. at 37°C.
  • ⁇ -galactosidase activity is quantitated by spectrophotometric absorption at 450nm.
  • X-gal staining of histologic sections is sensitive, and allows the localized detection of ( ⁇ -gal production within the tissues, but X-gal staining is essentially a qualitative test. X-gal staining is utilized to detect background levels of gene expression and for detection of tissue-specific ⁇ -gal production.
  • the ONPG colorimetric assay is quantitative, but does not permit analysis of tissue specific ⁇ -gal expression.
  • the ONPG assay is complicated by the fact that most tissues have background absorption at 450nm, so matched tissues from non-transgenic mice must be used to establish baseline absorption values. This technique is primarily utilized for the general quantitation of ⁇ -gal expression in murine tissues.
  • tissue specific expression of the ⁇ -galactosidase gene should be turned on when tetracycline is administrated to E-LacZ or MLacZ mice. To test this, tetracycline is given drinking water or via subcutaneous pellet. In initial studies, tetracycline or doxycycline (2mg/ml) is administrated via drinking water for 48 hours before the tissue harvesting. Thereafter, various tissues are collected and tested for ⁇ -galactosidase.
  • the FI mice are either treated with tetracycline or not via the drinking water or subcutaneously implanted pellets as described above.
  • the tissue specific expression of Ld protein is detected in different tissues by immunohistochemistry with the specific anti-H-2Ld antibody. Briefly, various tissues from the transgenic FI mice, are harvested, embedded and sectioned in 5 ⁇ m.
  • a biotin-labeled mouse anti-Ld monoclonal antibody 30-5-7 (American Type Culture Collection, Rockville, MD) is used as the primary antibody for the L d protein detection, and HRP conjugated aviden is used for the colorogenic reaction.

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Abstract

L'invention concerne un procédé et un système pour réguler l'expression de produits transgéniques dans des tissus déterminés d'un animal transgénique. Le procédé consiste en ce qui suit: a) fournir un premier animal parent transgénique dont le génome comprend un transgène F comportant un gène exogène lié de manière fonctionnelle à un promoteur régulé positivement par une protéine transactivatrice; b) fournir un deuxième animal parent transgénique dont le génome comprend i) un deuxième transgène comportant un deuxième promoteur qui est régulé négativement par la protéine transactivatrice et lié de manière fonctionnelle à un gène antisens qui code une séquence inhibant ou réduisant le traitement du transcrit du gène F; ii) un troisième transgène comprenant un promoteur spécifique au tissu lié de manière fonctionnelle à un gène codant la protéine transactivatrice; et c) accoupler le premier animal transgénique avec le deuxième animal transgénique pour obtenir une descendance transgénique dont le génome comprend les trois transgènes. La présente invention se rapporte aussi à une structure d'ADN qui comprend le deuxième transgène et à une deuxième souris parente transgénique utilisée dans ce procédé.
PCT/US2000/009837 1999-04-16 2000-04-12 Systeme genetique destine a reguler l'expression en arriere-plan de produits transgeniques Ceased WO2000062604A1 (fr)

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Citations (2)

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US5650298A (en) * 1993-06-14 1997-07-22 Basf Aktiengesellschaft Tight control of gene expression in eucaryotic cells by tetracycline-responsive promoters
US6004941A (en) * 1993-06-14 1999-12-21 Basf Aktiengesellschaft Methods for regulating gene expression

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* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5650298A (en) * 1993-06-14 1997-07-22 Basf Aktiengesellschaft Tight control of gene expression in eucaryotic cells by tetracycline-responsive promoters
US6004941A (en) * 1993-06-14 1999-12-21 Basf Aktiengesellschaft Methods for regulating gene expression

Non-Patent Citations (3)

* Cited by examiner, † Cited by third party
Title
BOYD, A. L.: "Review: Molecular Biology of Transgenic Animals", J. ANIM. SCI., vol. 71, no. SUPP.3, 1993, pages 1 - 9, XP002929986 *
KAPPEL, C. A.: "Regulating Gen Expression in Transgenic Animals", CURRENT OPINION IN BIOTECHNOLOGY, vol. 3, 1992, pages 548 - 553, XP002929987 *
KUHN, R.: "Inducible Gene Targeting in Mice", SCIENCE, vol. 269, September 1995 (1995-09-01), pages 1427 - 1429, XP002081198 *

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