WO1994021111A1 - Mammifere transgenique utilise en tant que modele de maladie - Google Patents

Mammifere transgenique utilise en tant que modele de maladie Download PDF

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WO1994021111A1
WO1994021111A1 PCT/GB1994/000569 GB9400569W WO9421111A1 WO 1994021111 A1 WO1994021111 A1 WO 1994021111A1 GB 9400569 W GB9400569 W GB 9400569W WO 9421111 A1 WO9421111 A1 WO 9421111A1
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cells
transgenic
disease
nef
transgenic mammal
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David John Abraham
Elaine Anne Dzierzak
Hugh Joseph Martin Brady
Colin Graham Miles
Daniel John Pennington
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Medical Research Council
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/005—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from viruses
    • 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
    • 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
    • A01K67/0278—Knock-in vertebrates, e.g. humanised vertebrates
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    • 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
    • A01K2207/00—Modified animals
    • A01K2207/15—Humanized animals
    • 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
    • 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
    • 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
    • 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
    • A01K2267/0337—Animal models for infectious 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
    • C12N2740/00—Reverse transcribing RNA viruses
    • C12N2740/00011—Details
    • C12N2740/10011—Retroviridae
    • C12N2740/16011—Human Immunodeficiency Virus, HIV
    • C12N2740/16311—Human Immunodeficiency Virus, HIV concerning HIV regulatory proteins
    • C12N2740/16322—New viral proteins or individual genes, new structural or functional aspects of known viral proteins or genes
    • C—CHEMISTRY; METALLURGY
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    • 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

  • the present invention relates to a transgenic mammalian model for a disease.
  • the present invention relates to a transgenic mouse which expresses HIV- derived peptides in a tissue-specific manner.
  • mice as model systems, although overcoming many of the above disadvantages, is limited by the existence of an animal equivalent of the human disease in question.
  • the most common animal models, rodents do not share many diseases with humans.
  • the use of animals such as primates is objectionable on ethical as well as financial grounds. It has therefore been sought, in a few cases, to adapt rodents in order to reproduce human diseases therein, so that the disease may be studied. Examples range from the irradiation of mice to produce immunocompromised animals, through the use of specific mutants such as non-obese diabetic (NOD) mice, to the recent use of mice rendered highly susceptible to tumorigenesis (the Harvard Oncomouse; see European Patent Application 0162672).
  • NOD non-obese diabetic
  • NEF is encoded by an open reading frame overlapping the 3' HIV LTR (Guy et al ., 1987) and displays a high degree of polymorphism between HIV isolates (Ratner et al . , 1985). Up to 80% of the early, multiply spliced class of viral transcripts encode NEF (Robert-Guroff et al . , 1990), yet its function is unclear. The 27 Kd myristilated protein is expressed at a very high level early in the HIV life cycle (Haseltine, 1991) and is found in the cytoplasm (Franchini et al ., 1986). While NEF shows some sequence homology to G proteins (Guy et al .
  • NEF may provide an important function in the life cycle of the virus, it may have an adverse effect on host, cell function.
  • the NEF protein in the absence of other HIV sequences, has been shown to downregulate the levels of cell surface CD4 on human T cell lines (Garcia and Miller, 1991; Garcia et al . , 1993).
  • a post-translational mechanism has been postulated since downregulation has been observed not at the level of mRNA but in surface CD4 protein.
  • CD4 was found to be localized in the cytoplasm (Garcia and Miller, 1991).
  • NEF has also been found to downregulate mouse and simian cell surface CD4 suggesting a common mechanism of action (Garcia et al . , 1993). Since NEF has such effects on established mature human CD4+ T cell lines, the relevance of such downregulation should be established in vivo .
  • Transgenic animals such as the Harvard Oncomouse are fundamentally incompatible with the study of most diseases with the exception of certain tumours. This is because the Oncomouse expresses oncogenes under the control of a viral LTR promoter/enhancer. Such control sequences essentially lead to widespread yet unpredictable expression of the oncogene, as evidenced by the experimental data referred to in the examples of EP 0162672.
  • the oncomouse is incapable of producing tissue-specific expression of oncogenes on a repeatable basis.
  • a transgenic mammal comprising a gene encoding an effector associated with a disease, the gene comprising at least one control sequence effective to direct expression of the effector substantially exclusively to cells where the effector is expressed in the normal course of the disease.
  • effector it is intended to denote that the heterologous gene encodes an agent which promotes a biological effect in the course of the disease.
  • agents may be polypeptides or nucleic acids.
  • the effector is a polypeptide.
  • polypeptide effectors include the products of oncogenes such as ras, myc and fos, the aberrant proteins such as tau protein which are implicated in Alzheimer's disease, HIV-specific peptides such as TAT, NEF, REV, VPU, VPR and VIF, bacterial toxins, self-antigens and any other peptides which cause or have a role in disease.
  • the disease is a human disease. Any disease may be studied using the transgenic mammal of the invention, provided that the disease involves the expression of an effector in particular cell types of the host. Examples of such diseases include, but are not limited to, Alzheimer's disease, cancer, autoimmune diseases, cystic fibrosis and infectious diseases of all kinds.
  • the invention be applied to viral diseases, especially AIDS.
  • the invention allows the study of a particular aspect of the pathology of the disease to be studied in isolation, or in connection with any other preselected aspects of the disease.
  • the effect of virally-encoded proteins on the immune system may be studied, without the risk of pathological immunodeficiency arising from viral infection colouring the results.
  • the effect of the interaction of two or more virally encoded proteins on the immune system may be studied.
  • control sequences used in the invention may be any sequences capable of directing tissue-specific expression in a host animal.
  • combinations of promoters, enhancers and tissue-specific responsive elements may be used. Such combinations have been studied in cell lines and transgenic animals in the prior art and the selection of appropriate combinations will be within the capabilities of a person skilled in the art.
  • control sequences may comprise Locus Control Regions (LCRs).
  • LCRs are position-independent, copy number-dependent activators of gene transcription which display strong tissue specificity.
  • LCRs other than those for globin genes has now been described.
  • Particularly preferred for use in the present invention are the CD2 LCR (Greaves et al . , 1989) which is specific for T-lymphocytes, the macrophage-specific lysozyme LCR (Bonifer et al . , 1990) and the Class II LCR specific for dendritic cells and macrophages (Carson et al . , 1993).
  • the gene used in the invention comprises an LCR together with an appropriate promoter/enhancer to drive transcription of the gene in the intended cell type.
  • an appropriate promoter/enhancer to drive transcription of the gene in the intended cell type.
  • greater tissue-specificity can be achieved by the use, in combination, of an LCR and a tissue-specific promoter and/or enhancer.
  • promoter elements may be used to introduce developmental regulation as well as tissue-specific regulation of the transgene. For example, when considering T-cell specific expression, use of an IL-2 promoter and CD2 LCR will ensure that the transgene is expressed only late in the T-cell development cycle, in peripheral T-cells, rather than in the thymus. This mirrors an HIV infection, which would occur initially in peripheral cells.
  • a greater degree of control over the gene can be provided by the use of a regulatable promoter and/or enhancer, which may be susceptible to regulation by, for example, transcription factors (Hu and Davidson, 1987; Kakidani and Ptashne, 1988), hormones, such as glucocorticoids (Picard et al . , 1988), oestrogen (Boehmelt et al . , 1992) or orally administrable non-toxic small molecules, such as tetracycline (Gatz and Quail, 1988; Gossen and Bujard, 1992).
  • transcription factors Hu and Davidson, 1987; Kakidani and Ptashne, 1988
  • hormones such as glucocorticoids (Picard et al . , 1988), oestrogen (Boehmelt et al . , 1992) or orally administrable non-toxic small molecules, such as tetracycline (Gatz and Quail, 1988; Gossen and
  • the transgenic mammal of the present invention may be any non-human mammal. However, rodents, especially mice, are preferred.
  • Transgenic mammals may be generated by any technique known in the art.
  • transgenic it is intended to infer that the mammal in question comprises at least one active copy of a heterologous gene in a substantial proportion of the cells of interest.
  • the heterologous gene may be inserted by conventional techniques, such as microinjection of embryos, such that the gene is present in substantially all the cells of the mammal.
  • it may be delivered in a targeted or non-targeted manner to mature animals, using, for example, virus vectors or liposome-based vectors according to techniques known in the art.
  • the heterologous gene encoding the effector may be present in an episomal state. However, it is preferred that the gene be integrated into the genome of the transgenic mammal.
  • a transgenic mouse comprising the coding sequence of an HIV peptide under the control of the CD2 LCR.
  • the HIV peptide may be the HIV nef gene product.
  • transgenic mammals of the invention are useful for the study of diseases in general and particularly for the study of therapy intended for diseases.
  • VIF and VPR may be assayed in transgenic animals according to the invention. It will be apparent that regulators derived from viruses other than HIV may be studied in a similar manner.
  • the invention provides a method for studying a potential therapeutic agent for a disease comprising administering the agent to a transgenic mammal according to the invention.
  • FIGURE 1 is a schematic representation of the transgene used for the generation of CD2-nef transgenic mice
  • FIGURE 2A shows a southern blot of DNA isolated from four transgenic mouse lines
  • FIGURE 2B shows a slot blot analysis of RNA from the same lines
  • FIGURE 2C shows western blot analysis of spleen tissue extract in the four lines
  • FIGURE 3 shows representative FACS analysis for transgenic and non-transgenic littermates performed on cells from the thymus and peripheral lymphoid organs, the spleen and lymph nodes;
  • FIGURE 4 shows a FACS histogram analysis of surface levels of CD4 on thymocytes from nef transgenic mice
  • FIGURE 5 shows the results of an anti-CD3 ⁇ mediated activation assay
  • FIGURE 6 compares the results of analysis of normal and transgenic thymocytes of the presence of CD4 by direct immunofluorescence
  • FIGURE 7 shows a similar experiment to Figure 6, except that the cells have been double-stained for CD4 and subcellular compartments;
  • FIGURE 8 shows the construction of a CD2 LCR - tat transgene
  • FIGURE 9 shows: A. Slot blot identification of transgenic mice carrying the CD2 LCR - tat transgene
  • FIGURE 10 shows a FACS analysis of CD4 and CD8 T-cell subsets in tat transgenic mice
  • FIGURE 11 shows a northern blot analysis of RNA derived from CD2 - tat transgenic mice, probed with a number of cytokine-specific probes
  • FIGURE 12 shows the observed increase in TNF- ⁇ transcription in tat transgenic mice.
  • the 800 bp BamHI-Smal fragment from either pTG1147 or pTG1191 was blunted and ligated into a unique blunted EcoRl site in the first exon of the p2629 CD2 expression plasmid (gift from D.Kioussis) to give either p2629N47 or p2629N91.
  • a 4.5Kb BamHI-NotI fragment containing the 3' CD2 LCR from p2694 (gift from D.Kioussis) was then ligated into the unique BamHI-NotI sites in P2629N47 or p2629N91, resulting in either pCD2nef 1147 or pCD2nef 1191.
  • the 12Kb Sall-NotI fragment from these plasmids was prepared for microinjection into (CBAxC57BL/10) fertilised mouse oocytes as previously described (Grosveld et al . , 1987). Positive founder animals were bred with CBAxC57BL/10) mice and lines were maintained as heterozygotes.
  • RNA was prepared using the lithium chloride/urea method (Fraser et al . , 1990).
  • For Northern blot analysis (Sambrook et al . , 1989) 10 ⁇ g of RNA was run on a 1% formaldehyde gel, blotted onto nitrocellulose and probed with an 800 bp BamHI-Smal nef fragment from pTG1147.
  • For RNA slot blots (Sambrook et al . , 1989) 5 ⁇ g of RNA was blotted onto nitrocellulose and probed as above.
  • RNA from the NEF producing CRIP L producer cell line (Schwartz et al . , 1992) was used as a positive control.
  • FACS analysis was used to detect cell surface markers on lymphocytes from transgenic mice.
  • the antibodies used were: a PE-conjugated rat monoclonal antibody (mAb) against murine CD4; a FITC-conjugated rat mAb against murine CD8 (both Becton Dickinson, San Jose, CA); a FITC-conjugated hamster mAb against murine CD3e (Pharmingen, San Diego, CA) and a FITC-conjugated rat mAb against murine Thy-1.2 (Sigma Chemical Co., St. Louis, MO).
  • the thymus, spleen and lymph nodes were removed and homogenised to single cell suspensions in FACS medium ( ⁇ MEM, 5% FCS, lO ⁇ g/ml Na azide) on ice. Accurate cell counts were obtained and 10 cells were washed in 5 ml FACS media, pelleted and the supernatant removed. Antibodies were added at a dilution of 1:200 in FACS medium and incubated for 30 min on ice. Cells were washed once with 5 ml of cold FACS medium, once with 5 ml of cold PBS, fixed in 1% formaldehyde/PBS and filtered through fine gauze. Stained cells were analysed with a Becton Dickinson FACSCAN cell sorter and the LYSIS II software package.
  • the extracts were cleared by centrifugation (14,000 rpm) for 5 min at 4°C and incubated with 50 ⁇ l of normal rabbit serum for 1 hr at 4°C and for 30 min with 100 ⁇ l of a 10% suspension of protein A- sepharose in lysis buffer followed by 15 min centrifugation at 4°C.
  • Anti-NEF antibodies HV-1 HXB3 NEF antisera
  • Hammes et al . , 1989 were then added at a 1:250 dilution to each cell lysate and incubated overnight on ice. Following this the extracts were incubated again with 100 ⁇ l of 10% suspension of protein A-sepharose beads for 1 hr.
  • the beads were collected by centrifugation for 15 min at 4°C and washed three times in lysis buffer. Pellets were resuspended in reducing sample buffer, heated at 100°C for 5 min and the supernatants recovered. The supernatants were resolved on 15% SDS-polyacrylamide gels and transferred to nitrocellulose filters by electroblotting. Filters were blocked in 5% low fat dried milk dissolved in phosphate buffered saline with 0.1% Tween-20 (PBS-T) at 4°C overnight. After extensive washing in PBS-T the filters were incubated for 1 hr at room temperature with a mAb to NEF (AE6) diluted at 1:1000 in PBS.
  • AE6 mAb to NEF
  • Thymocytes and erythrocyte depleted splenocytes were cultured in 200 ⁇ l of ⁇ MEM, 10% FCS, 2 mM glutamine, 10 U/ml Penicillin, 100 ⁇ g/ml Streptomycin and 50 ⁇ M ⁇ - mercaptoethanol. Cells were cultured in microtiter wells from a density of 8 x 10 per well for splenocytes.
  • Thymocytes were stimulated with anti-CD3e (145-2C11) mAb (0.36 ⁇ g/well) and 5 ng/ml PMA (Sigma) or with 5 ng/ml PMA and 500 ng/ml ionomycin (Sigma) or 5 ng/ml PMA alone as a control. Splenocytes were stimulated as above except no PMA was added with anti-CD3e antibodies. Controls contained no PMA. 48 hours after stimulation cells were labelled for 16 hours with 1 ⁇ Ci/well of 3 H thymidine (Amersham) before harvest. The incorporated radioactivity was precipitated on glassfibre filter paper and subsequently counted by liquid scintillation.
  • Indirect immunofluorescence was used to detect the intracellular and surface distribution of CD4 on lymphocytes from transgenic mice.
  • the antibodies used were a rat mAb against murine CD4 (Pharmingen, San Diego, CA) detected via a FITC-conjugated goat anti-rat IgG (Calbiochem., La Jolla, CA); a golgi-specific rabbit anti- ⁇ mannosidase II antibody (gift from Dr. K.
  • the coverslips were first overlaid with 5 ⁇ l of block solution (0.8% BSA, 0.1% gelatin in PBS) for 15 min. Diluted antibodies in the same block solution were then applied (10 ⁇ l) in the following sequence; (1) anti-CD4 (1:200); (2) FITC anti-rat IgG (1:100); then for double staining either; (3) anti-golgi-marker (1:1000), or anti-ER marker (1:20), or anti-p56 lck (1:50); (4) Texas Red anti-rabbit IgG (1:100). The incubation period for each antibody was 30 min at room temperature in a humidified chamber with three 5 min washes with 0.05% Tween-20/PBS between each application.
  • Coverslips were mounted with a drop of Univert (BDH, Poole, UK) containing 100 mg/ml of DABCO (Sigma Chemical Co., St. Louis, MO) as anti-fading agent, and cells were viewed under a Zeiss Axiophot fluorescence microscope.
  • CD2 nef transgenic mice express NEF in thymocytes and peripheral T cells
  • mice Four transgenic lines of mice were produced with a construct containing the human CD2 promoter and LCR element and a 621 bp nef fragment (Figure 1). Two different alleles of the HIV-1 nef gene were used to examine the effects of NEF in vivo , 1147 with threonine at amino acid position 15 and 1191 with an alanine at position 15. In vitro studies show CD4 downregulation with both alleles (Guy et al . , 1990) but only the 1147 allele is phosphorylated at position 15. DNA from the four lines A (1147), F (1147), B (1191) and D (1191) was analyzed by Southern blot analysis and copy numbers were determined to be 6, 25, 48 and 26 respectively (Figure 2A).
  • Thymocytes and peripheral T cells populations are altered in nef transgenic mice
  • FIG. 1 shows representative FACS analysis for transgenic and non- transgenic littermates. In all lines we observed a decrease in the percentage of CD4 single positive (SP) thymocytes and a concomitant increase in the percentage of CD4/CD8 double positive (DP) cells.
  • SP single positive
  • DP double positive
  • the total number of CD8 SP cells was decreased 12 fold while the number of DN cells were not significantly changed.
  • the reduction in total number of thymocytes was less than line F, again a significant decrease was observed in the CD4 SP subset.
  • NEF expression results in a downregulation of CD4 on the surface of developing T cells
  • CD8 levels were found to be slightly decreased and the normally high CD3 expressing population of thymocytes was found to be greatly reduced in line F and less so in the other three lines. This loss of CD3 high cells correlates well with the loss of CD4 SP cells in the thymus. Thy-1 levels did not change, thus demonstrating the specific effects of NEF.
  • T cell activation is decreased in nef transgenic mice
  • Mitogen induced or anti-CD3e mediated activation assays were performed to examine whether downregulation of CD4 or loss of CD4+ cells ha ⁇ negative effects on thymocyte or peripheral T cell activation.
  • Proliferation of thymocytes as measured by H thymidine incorporation after activation via the calcium ionophore (ionomycin) and phorbol ester (PMA) revealed small differences between nef transgenic and non-transgenic cells of both alleles demonstrating that the total response of transgenic thymocytes to mitogen is not impaired.
  • NEF has been shown to have no effect on the steady-state levels of CD4 mRNA or CD4 protein and the surface downregulation was found to be a consequence of intracellular localization of CD4 (Garcia and Miller, 1991).
  • thymocytes from nef transgenic mice for the presence of intracellular CD4.
  • Indirect immunofluorescence was performed on permeablized thymocytes with anti-CD4 antibody.
  • Figure 6A non- transgenic permeablized cells have normal cell surface expression of CD4 whereas transgenic thymocytes ( Figure 6B) express only low levels. Instead, CD4 was observed as a singular brightly staining area within the cytoplasm of nef transgenic thymocytes.
  • CD4 was localized to any particular subcellular compartment.
  • thymocytes with antibodies specific for the endoplasmic reticulum (ER) and the ⁇ -mannosidase II protein of the golgi apparatus.
  • ER endoplasmic reticulum
  • ⁇ -mannosidase II protein of the golgi apparatus Double staining with CD4 and compartment specific antibodies revealed that CD4 was sequestered within the specific region stained by the anti- golgi (figure 7, A and B) but not the anti-ER antibodies ( Figure 7, C and D).
  • CD4 colocalises with the golgi marker due to the coalescence of these organelles in the perinuclear region we cannot rule out an endosomal localization for CD4.
  • NEF expression was found throughout the cytoplasm of transgenic thymocytes as previously described for HIV infected cells (Franchini et al . , 1986; Ovod et al . , 1992). Since the cytoplasmic domain of CD4 is known to interact directly with the tyrosine kinase p56 lck (Shaw et al . , 1989; Veillette et al . , 1988), double staining with CD4 and p56 lck specific antibodies was performed to determine whether the NEF mediated downregulation of CD4 also affected the cellular location of p56 lck.
  • HIV-1 has an effect on the CD4 subsets of thymocytes which extends to the peripheral T cells in transgenic mice. Due to the CD2 gene regulatory elements, nef begins expression in the transgenic mice very early in T cell differentiation while the cells are in the CD4/CD8 DN stage in the thymus and continues in the DP and SP thymocytes as well as in the peripheral T cells (Kamoun et al . , 1981; Lang et al . , 1988; Owen et al . , 1988). We observed a dramatic decrease in the levels of cell surface CD4 on the DP subset of thymocytes and a significant reduction in the number of CD4 SP thymocytes in all four mouse lines.
  • CD4 SP cell number could be due to a NEF specific cytopathic effect, as has been indicated previously (Luria et al . , 1991; Skowronski et al . , 1993).
  • NEF mediated depletion of CD4 SP cells may be a consequence of aberrant positive selection in the thymus.
  • the effect of NEF is most likely to occur early in the T cell differentiation pathway at the DP stage, initiated by the downregulation of CD4.
  • DP cells In normal positive selection, DP cells interact with MHC class I and II molecules presented by the cortical thymic epithelium and receive a signal to expand (Berg et al . , 1989). Mice deficient in MHC Class II (Cosgrove et al . , 1991) and mice treated with anti-class II antibodies (Kruisbeek et al . , 1983) show defective development of their CD4+ T cells and clearly demonstrate that CD4 must interact with Class II for expansion to occur. Additionally, in mice homozygous for CD4 which have reduced levels of CD4 on the surface of DP thymocytes (Rahemtulla et al . , 1991), decreased numbers of CD4 SP cells have been found in the thymus.
  • thymocyte activation assays with anti-CD3e antibody result in proliferation of only SP cells (Havran et al . , 1987; Weiss et al . , 1987). Differences in thymocyte activation could be due to quantitative changes in the total number of SP cells or the level to which individual SP cells can be activated.
  • Our results concerning the effects of NEF on the in vivo immune system indicate that equal numbers of transgenic thymocytes are stimulated to a lesser degree than those from non- transgenic littermates. This is most likely due to the quantitative loss of the CD4 SP subset (which are CD3 high expressing cells).
  • the levels of activation in the transgenic thymocytes are corrected for the depleted SP cells, they become comparable to those from non-transgenic littermates. Further analysis is required to determine if the activation level of individual thymocytes is also perturbed. These data are in direct contrast to those of others (Skowronski et al . , 1992) where, despite large losses in CD4 SP cells, Nef transgenic thymocytes are hyperactivated through anti-CD3 stimulation. The nef transgene in these mice is controlled via murine CD3 gene regulatory elements which direct expression of nef much later in T cell ontogeny than CD2 elements (Yagita et al . , 1989). Thus developmental expression differences may account for the opposing data.
  • position effects on the transgene could play a role in variable NEF expression on T cells since, unlike human CD2 (Greaves et al . , 1989), no elements have been identified in the CD3 gene to confer position independent expression (Lacy et al . , 1983; Lee et al . , 1992).
  • the thymus probably acts as a site of T cell differentiation and maturation throughout life (Steinmann, 1986) and thus dysfunction of thymopoiesis may be a pathogenic mechanism for HIV as recently suggested in the SCID-hu model (Bonyhadi et al . , 1993; Aldrovandi et al . , 1993). HIV infection of adult thymus/liver implants affects DP and SP thymocyte percentages and numbers, with the DP population harbouring greater than 90% of the virus. Taken together, these studies may provide clues as to why HIV infected patients become depleted for CD4 cells over a long period of time (Fauci, 1986).
  • CD4 in the absence of gpl60, CD4 is downregulated specifically by NEF and that CD4 colocalises to the perinuclear region. Further to this, the interaction of CD4 with cytoplasmic tyrosine protein kinase p56 lck (Veilette et al . , 1988; Shaw et al . , 1989) has been examined. It has been shown that p56 lck and gpl60-CD4 form a ternary complex in the ER (Crise and Rose, 1992).
  • transgenic mice could serve as a useful model to further elucidate the mechanism of NEF mediated CD4 downregulation, to study effect of NEF on the host developing immune system and for the testing of NEF inhibitors that may have a therapeutic effect against HIV replication in vivo .
  • Transgenic mice expressing the HIV-2 tat gene were constructed, in order to study the effect of the TAT transactivator on T-cells.
  • the tat transgene was expressed under the control of the CD2 LCR and regulatory sequences in order to achieve T-cell specific expression.
  • Exon 1 (encoding aa 1-72) of the HIV-1 tat gene was inserted downstream of the transcriptional start site in the first exon of the human CD2 gene.
  • a stop codon was constructed in the sequence of human CD2 exon 2 so as to eliminate the production of CD2 protein.
  • the human CD2 LCR element was ligated to the 3' end of the construct.
  • a Sall- Notl fragment was injected into fertilized mouse eggs. At least three transgenic lines were created. Lice C (2 in Figure 9A) contains 70 copies and line E (4 in Figure 9A) contains 40 copies.
  • Thymocyte and peripheral T cell CD4 and CD8 subsets are not perturbed by expression of the CD2-tat transgene.
  • Cytokine gene expression is affected by the presence of HIV- TAT
  • RNA from CD2- tat transgenic mice was prepared from thymocytes of line C transgenic mice (C+1 and C+2) and a non-transgenic littermate (C-) and from thymocytes of line E transgenic mice (E+1 and E+2) and a non-transgenic littermate (E-). 10 ⁇ g of RNA was loaded per lane on a formaldehyde agarose gel. RNA was transferred onto a filter and hybridized with a ⁇ -actin probe as a RNA quantitation control and a tat probe for verification of transgene expression.
  • the filter was rehybridized several times with probes for cytokine genes TGF- ⁇ , IL-4R, TNF- ⁇ and TNF- ⁇ .
  • Autoradiagrams of the Northern blot demonstrate an increase in expression of TGF- ⁇ , IL-4R and TDF- ⁇ gene expression in the TAT transgenic mice.
  • hybridization signal with the TNF- ⁇ probe suggests no change or a decrease in TNF- ⁇ gene expression in the TAT positive mice.
  • TAT induced transcriptional upregulation of TNF- ⁇ leads to overproduction of functional TNF- ⁇ as measured by cytotoxicity.

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Abstract

L'invention concerne un mammifère transgénique comprenant un gène hétérologue codant un effecteur associé à une maladie, le gène comprenant au moins une séquence de régulation efficace pour diriger l'expression de l'effecteur sensiblement exclusivement vers des cellules où l'effecteur est exprimé dans l'évolution normale de la maladie. Ledit mammifère est efficace dans des recherches effectuées sur des maladies, ainsi que dans la thérapie de maladies.
PCT/GB1994/000569 1993-03-19 1994-03-21 Mammifere transgenique utilise en tant que modele de maladie WO1994021111A1 (fr)

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Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1995008635A1 (fr) * 1993-09-24 1995-03-30 Therexsys Limited Expression de proteines leurres virales soumises a une region de regulation de locus et leurs utilisations
US5907080A (en) * 1995-11-30 1999-05-25 Nexia Biotechnologies, Inc. Method for development of transgenic dwarf goats

Citations (2)

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Publication number Priority date Publication date Assignee Title
WO1992020790A1 (fr) * 1991-05-15 1992-11-26 L'institut De Recherches Cliniques De Montreal Animal transgenique non humain portant un genome non infectieux de vih
WO1993002189A1 (fr) * 1991-07-18 1993-02-04 The Regents Of The University Of California Modeles animaux transgeniques pour l'etude de la maladie d'alzheimer

Patent Citations (2)

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Publication number Priority date Publication date Assignee Title
WO1992020790A1 (fr) * 1991-05-15 1992-11-26 L'institut De Recherches Cliniques De Montreal Animal transgenique non humain portant un genome non infectieux de vih
WO1993002189A1 (fr) * 1991-07-18 1993-02-04 The Regents Of The University Of California Modeles animaux transgeniques pour l'etude de la maladie d'alzheimer

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Dialog Information Services, File 154, Medline, Dialog accession no. 08445139, Medline accession no. 93155139, Shih DM et al: "A 5' control region of the human epsilon-globin gene is sufficient for embryonic specificity in transgenic mice". J Biol Chem (UNITED STATES) Feb 15 1993, 268 (5) p 3066-71 *
Dialog Information Services, File 154, Medline, Dialog accession no. 08636735, Medline accession no. 93346735, Probert L et al: "Wasting, ischemia, and lymphoid abnormalitites in mice expressing T cell-targeted human tumor necrosis factor transgenes". J Immunol (UNITED STATES) Aug 15 1993 151 (4) p 1894-906 *
STEVEN H. HINRICHS ET AL: "A Transgenic Mouse Model for Human Neurofibromatosis", SCIENCE, vol. 237, 1987 *

Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1995008635A1 (fr) * 1993-09-24 1995-03-30 Therexsys Limited Expression de proteines leurres virales soumises a une region de regulation de locus et leurs utilisations
AU697095B2 (en) * 1993-09-24 1998-09-24 Cobra Therapeutics Limited Expression of viral decoy proteins under the control of a locus control region and uses thereof
US5907080A (en) * 1995-11-30 1999-05-25 Nexia Biotechnologies, Inc. Method for development of transgenic dwarf goats

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