Abstract
In man, deficiency of ADA activity is associated with an autosomal recessive form of severe combined immunodeficiency (SCID), a disease with profound defects of both cellular and humoral immunity. Current treatments of ADA deficient patients include bone marrow transplantation, enzyme replancement and somatic gene therapy. The mechanism of the selective immune cell pathogenesis in ADA-SCIDS is, however, still poorly understood. Thus, the generation of an ADA deficient mouse model will be of considerable benefit to understand better the pathophysiology of the disorder and to improve the gene therapy treatments.
We have disrupted the adenosime deaminase (ADA) gene in embryonic stem cells using a new efficient promoter trap gene-targeting approach. To this end, a dicistronic targeting construct containing a promoterless IRES β geo cassette was used. This cassette allows, via the internal ribosomal entry site (IRES), the direct cap-independent translation of the β geo reporter gene which encodes a protein with both β-galactosidase and neomycin activities. After indentification of targeted clones by Southern blot, successful inactivation of the ADA gene was first confirmed by producing, from our heterozygote clones, an homozygote cell line. This line shows no ADA activity as judged by zymogram analysis. Second, we have been able to detect in the targeted clones, a specific β galactosidase activity using a sensitive fluorogenic assay. The targeted ES cell clones are currently being injected into blastocysts to create an ADA deficient mouse model.
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References
Abbot, C.M., Skidmore, C.J., Searle, A.G. and Peters, J. (1986) Deficiency of adenosine deaminase in the wasted mouse.Proc. Natl Acad. Sci. USA 83, 693–5.
Al-Ubaidi, M.R., Ramamurthy, V., Maa, M.C., Ingolia, D.E., Chinksy, J.M., Martin, B.D. and Kellems, R.E. (1990) Structural and functional analysis of the murine adenosinedeaminase gene.Genomics 7, 476–85.
Anderson, W.F. (1992) Human gene therapy.Science 256, 808–13.
Aronow, B.J., Silbiger, R.N., Dusing, M.R., Stock, J.L., Yager, K.L., Potter, S.S., Hutton, J.J. and Wiginton, D.A. (1992) Functional analysis of the human adenosine deaminase gene thymic regulatory region and its ability to generate position-independent transgene expression.Mol. Cell. Biol. 12, 4170–85.
Bonnerot, C. and Nicholas, J.F. (1993) Application oflacZ gene fusions to postimplantation development. In Wassarman, P.M. and De Pamphilis, M.L. (eds),Guide to Techniques in Mouse Development: Methods in Enzymology. 451–69.
Boris-Lawrie, K.A. and Temin, H.M. (1993) Recent advances in retrovirus vector technology.Cur. Opinion Gen. Dev. 3, 102–9.
Bosma, G.C., Custer, R.P. and Bosma, M.J. (1983) A severe combined immunodeficiency mutation in the mouse.Nature 301, 527–30.
Capecchi, M.R. (1989) Altering the genome by homologous recombination.Science 244, 1288–92.
Chang, Z., Nygaard, P., Chinault, A.C. and Kellems, R.E. (1991) Deduced amino acid sequence ofEscherichia coli adenosine deaminase reveals evolutionarily conserved amino acid residues: Implications for catalytic function.Biochem. 30, 2273–80.
Chen, Z., Harless, M.L., Wright, D.A. and Kellems, R.E. (1990) Identification and characterization of transcriptional arrest sites in Exon-1 of the human adenosine-deaminase gene.Mol. Cell. Biol. 10, 4555–64.
Chinsky, J.M., Maa, M.C., Ramamurthy, V. and Kellems, R.E. (1989) Adenosine deaminase gene expression: tissuedependent regulation of transcriptional elongation.J. Biol. Chem. 264, 14561–5.
Cournoyer, D. and Caskey, C.T. (1993) Gene therapy of the immune system.Annu. Rev. Immunol. 11, 297–329.
Evans, M.J. (1989) Potential for genetic manipulation of mammals.Mol. Biol. Med. 6, 557–65.
Fox, I.H. and Kelley, W.N. (1978) The role of adenosine and 2′-deoxyadenosine in mammalian cells.Annu. Rev. Biochem. 47, 655–86.
Friedrich, G. and Soriano, P. (1991) Promoter traps in embryonic stem cells: a genetic screen to identify and mutate developmental genes in mice.Genes Dev. 5, 1513–23.
Hasty, P., Rivera-Perez, J. and Bradley, A. (1991) The role and fate of DNA ends for homologous recombination in embryonic stem cells.Mol Cell. Biol. 12, 2464–74.
Hershfield, M.S., Chaffee, S. and Sorensen, R.U. (1993) Enzyme replacement therapy with polyethylene clycoladenosine deaminase in adenosine deaminase deficiency: overreview and case reports of three patients, including two now receiving gene therapy.Pedaitr. Res. 33 (1 Suppl), S42–7.
Hirschhorn, R. (1993) Overview of the biochemical abnormalities and molecular genetics of adenosine deaminase deficiency.Pediatr. Res. 33 (1 Suppl), S35–41.
Innis, J.W., Moore, D.J., Kash, S.F., Ramamurthy, V., Sawadogo, M. and Kellems, R.E. (1991) The murine adenosine-deaminase promoter requires an atypical TATA box which binds transcription factor-IID and transcriptional activity is stimulated by multiple upsteam SP1 binding sites.J. Biol. Chem. 266, 21765–72.
Jeannotte, L., Ruiz, J.C. and E.J., R. (1991) Low level of Hox1.3 gene expression does not preclude the use of promoterless vectors to generate a targeted gene disruption.Mol. Cell. Biol. 11, 5578–85.
Kameoka, J., Tanaka, T., Nojima, Y., Schlossman, S. and Morimoto, C. (1993) Direct association of adenosine deaminase with a T cell activation antigen, CD26.Science 261, 466–9.
Kash, S., Innis, J., Jackson, A. and Kellem, R. (1993) Functional analysis of a stable transcription arrest site in the first intron of the murine adenosine deaminase gene.Mol. Cell. Biol. 13, 2718–29.
Kim, D.G., Kang, H.M., Jang, S.K. and Shin, H.S. (1992) Construction of a bifunctional mRNA in the mouse by using the internal ribosomal entry site of the encephalomyocarditis virus.Mol. Cell. Biol. 12, 3636–43.
Kredich, N.M. and Hershfield, M.S. (1989) In Scriver, C.R., Beaudet, A.L., Sly, W.S. and Valle, D.M. (eds), Immunodeficiency diseases caused by adenosine deaminase deficiency and purine nucleoside phosphorylase deficiency. The Metabolic Basis of Inherited Disease. pp. 1045–1075. New York, McGraw-Hill.
Lattier, D.L., States, J.C., Hutton, J.J. and Wiginton, D.A. (1989) Cell type-specific transcriptional regulation of the human adenosine deaminase gene.Nucleic Acids Res. 17, 1061–76.
Meera Khan, P., Rijken, H., Wijnen, J.T., Wijnen, L.M.M. and DeBoer, L.E.M. (1982) Red cell enzyme variation in the orangutan; electrophoretic characterization of 45 enzyme systems in Cellogel. Biology and Conservation of the Orangutan. pp. 61–108. The Hague, Junk.
Miller, A.D. (1992) Human gene therapy comes of age.Nature 357, 455–60.
Mombaerts, P., Iacomini, J., Johnson, R.S., Herrup, K., Tonegawa, S. and Papaioannou, V.E. (1992) RAG-1-deficient mice have no mature B and T lymphocytes.Cell 68, 869–77.
Morgan, G., Levinsky, R.J., Hugh-Jones, K., Fairbanks, L.D., Morris, G.S. and Simmonds, H.A. (1987) Heterogeneity of biochemical, clinical and immunological parameters in severe combined immunodeficiency due to adenosine deaminase deficiency.Clin. Exp. Immunol. 70, 491–9.
Mortensen, R.M., Conner, D.A., Chao, S., Geisterfer-Lowrance, A.A.T. and Seidman, J.G. (1992) Production of homozygous mutant ES cells with a single targeting construct.Mol. Cell. Biol. 12, 2391–5.
Mountford, P., Zevnik, B., Duwel, A., Nichols, J., Li, M., Dani, C., Robertson, M., Chambers, I. and Smith, A. (1994) Dicistronic targeting constructs: reporters and modifiers of mammalian gene expression.Proc. Natl Acad. Sci. USA 91, 4303–7.
Namikawa, R., Kaneshima, H., Lieberman, M., Weissman, L.L. and McCune, J.M. (1988) Infection of the SCID-hu mouse by HIV-1.Science 242, 1684–6.
Orkin, S.H., Daddona, P.E., Shewach, D.S., Markham, A.F., Bruns, G.A., Goff, S.C. and Kelly, W.N. (1983) Molecular cloning of human adenosine deaminase gene sequences.J. Biol. Chem. 258, 753–6.
Pantelouris, E.M. (1968) Absence of thymus in a mouse mutant.Nature 217, 370.
Robertson, E.J. (1987) Embryo-derived stem cells lines. Teratocarcinomas and Embryonic Stem Cells: a Practical Approach. Oxford, IRL Press.
Santisteban, E., Arredondo-Vega, F.X., Kelly, S., Mary, A., Fischer, A., Hummell, D., Lawton, A., Sorensen, R.U., Stiehm, E.R., Uribe, L., Weinberg, K. and Hershfield, M.S. (1993) Novel splicing, missense and deletion mutations in seven adenosine deaminase-deficient patients with late/delayed onset of combined immunodeficiency disease.J. Clin. Invest. 92, 2291–302.
Shinkai, Y., Rathbun, G., Lam, K.P., Oltz, E.M., Stewart, V., Mendelsohn, M., Charron, J., Datta, M., Young, F., Stall, A.M. and Alt, F.W. (1992) RAG-2-deficient mice lack mature lymphocytes owing to inability to initiate V(D)J rearrangement.Cell 68, 855–67.
Tolstoshev, P. (1993) Gene therapy, concepts, current trials and future directions.Annu. Rev. Pharmacol. Toxicol. 33, 573–96.
Valerio, D., Duyvesteyn, M.G.C., Dekker, B.M.M., Weeda, G., Berkvens, T.M., Voorn, L. van der, Ormondt, H. Van and Eb, A.B. Van Der (1985) Adenosine deaminase: characterization and expression of a gene with a remarkable promoter.EMBO J. 4, 437–43.
Valerio, D., Putten, H. van der, Botteri, F.M. and Hoogerbrugge, P.M. (1988) Activity of the adenosine deaminase promoter in transgenic mice.Nucleic Acids Res. 16, 10083–96.
Wiginton, D.A., Kaplan, D.J., States, J.C., Adeson, A.L., Perme, C.M., Bilyk, I.J., A.J., V., Lattier, D.L. and Hutton, J.J. (1986) Complete sequence and structure of the gene for human adenosine deaminase.Biochem. 25, 8234–44.
Wilson, D.K., Rudolph, F.B. and Quiocho F.A. (1991) Atomic structure of adenosine deaminase complxed with a transition-state analog: understanding catalysis and immunodeficiency mutations.Science 252, 1278–84.
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Vaulont, S., Daines, S. & Evans, M. Disruption of the adenosine deaminase (ADA) gene using a dicistronic promoterless construct: Production of an ADA-deficient homozygote ES cell line. Transgenic Research 4, 247–255 (1995). https://doi.org/10.1007/BF01969118
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DOI: https://doi.org/10.1007/BF01969118