Abstract
Transgenic mouse lines were engineered to express stably antisense mRNA or antisense mRNA containing catalytic ribozyme (rbz) structures complementary to bacterial chloramphenicol acetyltransferase (CAT) gene transcripts. One transgenic line expressed antisense mRNA that specifically targeted full-length CAT coding sequences (ACAT). Another transgenic line expressed full-length antisense CAT mRNA which was modified by mutagensis to include four rbz cassettes (rbz-ACAT) in order to compare antisense versus antisense-rbz function in vivo. Preliminary data were also collected from a transgenic mouse line expressing antisense mRNA targeting 72% of the 5′ region of CAT coding sequences (5′ ACAT). All constructs contained similar control elements in their design. Promoter elements were derived from the bovine αs1-casein gene, while the small t intron and 3′ control sequences were derived from SV40. The ability of these various constructs to down-regulate CAT p rotein levels was compared by analysis of CAT protein production in lactating double-hemizygous transgenic female mice. Every double-hemizygous mouse analysed expressed mRNA from the αs1-casein-CAT construct (Clarke et al., 1994) and equivalent levels of mRNA from one of the three antisense constructs. Transgenic mouse lines expressing both ACAT and CAT mRNA down-regulated CAT protein levels by 90% of that found in the CAT only transgenic population. Similarly, double-hemizygous transgenic lines expressing both rbz-ACAT and CAT mRNA regulated CAT protein levels by 87%. Preliminary data suggests that expression of mRNA from 5′ ACAT/CAT double-hemizygote mice allowed approximately 67% down-regulation of normal CAT protein levels. We conclude that incorporation of multiple ribozymes within the full-length antisense CAT construct does not enhance the effectiveness of antisense mRNA in the down-regulation of CAT protein production in our system
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Allshire, R., Javerzat, J., Redhead, N. and Cranston, G. (1994) Position effect variegation at fission yeast centromeres. Cell 76, 157-69.
Archibold, A., McClenaghan, M., Hornsey, V., Simons, J. and Clark, A. (1990) High level expression of biologically active human alpha 1-antitrypsin in the milk of transgenic mice. Proc. Natl Acad. Sci. USA 87, 5178-82.
Atkins, D., Young, M., Uzzell, S., Kelly, L., Filatti, J. and Gerlach, W. (1995) The expression of antisense and ribozyme genes targeting citrus exocortis viroid in transgenic plants. J. Gen. Virol. 76, 1781-90.
Ausubel, F., Brent, R., Kingston, R., Moore, D., Seidman, J., Smith, J. and Struhl, K. (1993) Current Protocols in Molecular Biology. Current Protocols, USA.
Aviv, H. and Leder, P. (1972) Purification of biologically active globin messenger RNA by chromatography on oligothymidylic acid cellulose. Proc. Natl Acad. Sci. USA 69, 1408-12.
Barash, I., Nathan, M., Kari, R., Neta, I., Shani, M. and Hurwitz, D. (1996) Elements within beta-lactoglobin gene inhibit expression of human serum albumin cDNA and minigenes in transfected cells but rescue their expression in the mammary gland of transgenic mice. Nucl. Acids Res. 24, 602-10.
Cameron, F. and Jennings, P. (1989) Specific gene suppression by engineered ribozymes in monkey cells. Proc. Natl Acad. Sci. USA 86, 9139-43.
Celander, D. and Cech, T. (1991) Visualizing the higher order folding of a catalytic RNA molecule. Science 251, 401-7.
Chen, C., Banerjea, A., Harmison, G., Hagtlaund, K. and Schubert, M. (1992) Multitarget-ribozyme directed to cleave at up to nine highly conserved HIV-1 env RNA regions inhibits HIV-1 replication-potential effectiveness against most presently sequenced HIV-1 isolates. Nucl. Acids Res. 20, 4581-89.
Chomczynski, P. and Sacchi, N. (1987) Single-step method of RNA isolation by acid guanidinium thiocyanate-phenol-chloroform extraction. Analytical Biochem. 162, 156-9.
Chowrira, B., Berzal-Herranz, A. and Burke, J. (1993) Ionic requirements for RNA binding, cleavage, and ligation by the hairpin ribozyme. Biochem. 32, 1088-95.
Clarke, R. (1992) Ph.D. Thesis, University of New South Wales, New South Wales, Australia.
Clarke, R., Sokol, D., Rigby, N., Ward, K., Murray, J. and Mackinlay, A. (1994) Mammary gland-specific expression of bovine αS1-casein derived transgenes in transgenic mice. Transgenics 1, 313-9.
Cotten, M. and Birnstiel, M. (1989) Ribozyme mediated destruction of RNA in vivo. EMBO J. 8, 3861-66.
Dale, T., Krnacik, M., Schmidhausen, C., Yang, C., Bissell, M. and Rosen, J. (1992) High level expression of the rat whey acidic protein gene is mediated by elements in the promoter and 3′ untranslated region. Mol. Cell. Biol. 12, 905-14.
de Feyter, R., Young, M., Schroeder, K., Dennis, E. and Gerlach, W. (1996) A ribozyme gene and an antisense gene are equally effective in confering resistance to tobacco mosaic virus in transgenic tobacco. Mol. Gen. Genetics 250, 329-38.
Devinoy, E., Thepot, D., Stinnnakre, M., Fontaine, M., Grabowski, H., Puissant, C., Pavirani, A. and Houdebine, L. (1994) High level production of the human growth hormone in the milk of transgenic mice: The upstream region of the rabbit whey acidic protein (WAP) gene targets transgene expression to the mammary gland. Transgenic Res. 3, 79-89.
Dobie, K., Lee, M., Fantes, J., Graham, E., Clark, A., Springbett, A., Lathe, R. and McClenaghan, M. (1996) Variegated transgene expression in mouse mammary gland is determined by the transgene integration locus. Proc. Natl Acad. Sci. USA 93, 6659-64.
Dorer, D. and Heinkoff, S. (1994) Expansions of transgene repeats cause heterochromatin formation and gene silencing in Drosophila. Cell 77, 993-1002.
Efrat, S., Leiser, M., Wu, Y-J., Fusco-Demanc., D., EmRan, O., Surana, M., Jetton, T., Magnuson, M., Weir, G. and Fleischer, N. (1994) Ribozyme-mediated attenuation of pancreatic β-cell glucokinase expression in transgenic mice results in impaired glucose-induced insulin secretion. Proc. Natl Acad. Sci. USA 91, 2051-5.
Fraser, P., Pruzina, S., Antoniou, M. and Grosveld, F. (1993) Each hypersensitive site of the human beta globin locus control region confers a different developmental pattern of expression on the globin genes. Genes Dev. 7, 106-113.
Gampel, A., Nishikimi, M. and Tzagoloff, A. (1989) Binding of the CBPC protein to a yeast mitochondrial group 1 intron requires the catalytic core of the RNA. Mol. Cell. Biol. 9, 5424-33.
Gibson, I. (1994) Antisense DNA and RNA strategies: new approaches to therapy (review). J. Royal Coll. Physicians London 28, 507-11.
Gottschling, D., Aparicio, B., Billington, B. and Zakian, V. (1990) Position effects at S. cerevisiae telomeres: reversible repression of polymerase II transcription. Cell 63, 751-62.
Green, P., Pines, O. and Inouye, M. (1986) The role of antisense RNA in gene regulation. Annu. Rev. Biochem. 55, 569-97.
Grosshans, C. and Cech, T. (1989) Metal ion requirements for sequence-specific endoribonuclease activity of the tetrahymena ribozyme. Biochem. 28, 6888-94.
Haseloff, J. and Gerlach, W. (1988) Simple RNA enzymes with new and highly specific endoribonuclease activities. Nature 334, 585-91.
Heinrich, J., Tabler, M. and Louis, C. (1995) Influence of chromosomal position and copy number of a white directed ribozyme gene on the suppression of eye pigmentation in Drosophila melanogaster. Antisense Res. Dev. 5, 155-60.
Herschlag, D. and Cech, T. (1990) Catalysis of RNA cleavage by the Tetrahymena thermophila ribozyme. Kinetic description of the reaction of an RNA substrate complementary to the active site. Biochem. 29, 10159-71.
Hogan, B., Beddington, R., Costantini, F., and Lacy, E. (1994) Manipulating the Mouse Embryo. Cold Spring Harbor, NY, USA: Cold Spring Harbor Laboratory Press.
Homann, M., Tzortzakaki, S., Rittner, K., Sczakiel, G. and Tabler, M. (1993) Incorporation of the catalytic domain of a hammerhead ribozyme into antisense RNA enhances its inhibitory effect on the replication of human immunodeficiency virus type 1. Nucl. Acids Res. 21, 2809-14.
Huber, M., Bosch, F., Sippel, A. and Bonifer, C. (1994) Chromosomal position effects in chicken lysozyme gene transgenic mice are correlated with suppression of DNase I hypersensitive site formation. Nucl. Acids Res. 22, 4195-201.
Kazakov, S. and Altman, S. (1991) Site specific cleavage by metal ion cofactors and inhibitors of m1 RNA, the catalytic subunit of RNaseP from Escherichia Coli. Proc. Natl Acad. Sci. USA 88, 9193-7.
Larsson, S., Hotchkiss, G., Andang, M., Nyholm, T., Inzunza, J., Jansson, I. and Ahrlund-Richter, L. (1994) Reduced β2-microglobulin mRNA levels in transgenic mice expressing a designed hammerhead ribozyme. Nucl. Acids Res. 22, 2242-8.
L'Huillier, P., Davis, S. and Bellamy, A. (1992) Cytoplasmic delivery of ribozymes leads to efficient redfuction in α-lactalbumin mRNA levels in C1271 mouse cells. EMBO J. 11, 4411-8.
L'Huillier, P., Soulier, S., Stinnakre, M., Lepourry, L., Davis, S., Mercier, J. and Vilotte, J. (1996) Efficient and specific ribozyme-mediated reduction of bovine α-lactalbumin expression in double transgenic mice. Proc. Natl Acad. Sci. USA 93, 6698-703.
Lo, K., Biasolo, M., Dehni, G., Palu, G. and Haseltine, W. (1992) Inhibition of replication of HIV-1 by retroviral vectors expressing tat-antisense and anti-tat ribozyme RNA. Virol. 190, 176-83.
Maniatis, T., Fritsch, E. and Sambrook, J. (1982) Molecular Cloning: a Laboratory Manual. Cold Spring Harbor, NY, USA: Cold Spring Harbor Laboratory Press.
Manuelidis, L. (1991) Heterochromatic features of an 11-megabase transgene in brain cells. Proc. Natl Acad. Sci. USA 88, 1049-53.
Monstein, H., Geijer, T. and Bakalkin, G. (1992) Blotto-MF, an inexpensive and reliable hybridization solution in northern blot analysis using complementary probes. BioTechniques 13, 842-4.
Neckers, L., Rosolen, A. and Whitesell, L. (1992) Antisense inhibition of gene expression: a tool for studying the role of n-MYC in the growth and differentiation of neuroectoderm-derived cells. J. Immunotherapy 12, 162-6.
Ninomiya, T., Hirabayashi, M., Sagara, J. and Yuki, A. (1994) Function of milk protein gene 5′ flanking regions of human growth hormone gene. Mol. Reprod. Dev. 37, 276-83.
Ohkawa, J., Koguma, T., Kohda, T. and Taira, K. (1995) Ribozymes: from mechanistic studies to applications in vivo. J. Biochem. 118, 251-8.
Perriman, R., Graf, L. and Gerlach, W. (1993) A ribozyme which enhances gene suppression in tobacco protoplasts. Antisense Res. Dev. 3, 253-63.
Pyle, M. (1993) Ribozymes: a distinct class of metalloenzymes. Science 261, 709-14.
Saenger, W. (1984) in Principles of Nucleic Acid Structure: New York, NY, USA: Springer-Verlag Press.
Sambrook, J., Fritsch, E., and Maniatis, T. (1989) Molecular Cloning, a Laboratory Manual. Cold Spring Harbor, NY, USA: Cold Spring Harbor Laboratory Press.
Sankar, T., Cheah, K. and Porter, A. (1989) Antisense oligonucleotide inhibition of encephalomyocarditis virus RNA translation. J. Biochem. 184, 39-45.
Sarver, N., Cantin, E., Chang, P., Zaia, J., Ladne, P., Stephens, D. and Rossi, J. (1990) Ribozymes as potential anti-HIV-1 therapeutic agents. Science 247, 1222-5.
Saxena, S. and Ackermann, E. (1990) Ribozymes correctly cleave a model substrate and endogenous RNA in vivo. J. Biol. Chem. 265, 17106-9.
Scanlon, K., Jiao, L., Funato, T., Wang, W., Tone, T., Rossik, J. and Kashani-Sabet, M. (1991) Ribozyme-mediated cleavage of c-fos mRNA reduces gene expression of DNA synthesis enzymes and metallothionine. Proc. Natl Acad. Sci. USA 88, 10591-5.
Sioud, M. and Drlica, K. (1991) Prevention of human immunodeficiency virus type 1 integrase expression in Escherichia coli by a ribozyme. Proc. Natl Acad. Sci. USA 88, 7303-7.
Sokol, D. and Murray, J. (1996) Antisense and ribozymes constructs in transgenic animals. Transgenic Res. 5, 363-71.
Steinecke, P., Herget, T. and Schreier, P. (1992) Expression of a chimeric ribozyme gene results in endonuleolytic cleavage of target mRNA and a concomitant reduction of gene expression in vivo. EMBO J. 11, 1525-30.
Tropsha, A., Kizer, J. and Chaiken, F. (1992) Making sense from antisense: a review of experimental data and developing ideas on sense-antisense peptide recognition. J. Mol. Recognition 5, 43-54.
Valera, A., Solanes, G., Fernandez-Alvarez, J., Pujol, A., Ferrer, J., Asins, G., Gomis, R. and Bosch, F. (1994) Expression of GLUT-2 antisense RNA in β cells of transgenic mice leads to diabetes. J. Biol. Chem. 269, 28543-6.
Wagoner, D., Steinecke, P., Herget, T., Petereit, I., Philipp, C. and Schreier, P. (1994) Expression of a reporter gene is reduced by a ribozyme in transgenic plants. Mol. Gen. Genetics 245, 465-70.
Wang, S. and Dolnick, B. (1993) Quantitative evaluation of intracellular sense: antisense RNA hybrid duplexes. Nucl. Acids Res. 21, 4383-91.
Wei, Y., Yarus, S., Greenberg, N., Whitsett, J. and Rosen, J. (1995) Production of human surfactant protein C in milk of transgenic mice. Transgenic Res. 4, 232-40.
Wilson, C., Bellen, H., and Gehring, W. (1990) Position effects on eukaryotic gene expression. Ann. Rev. Cell. Biol. 6, 679-714.
Woolf, T. (1995) To cleave or not to cleave: ribozymes and antisense. Antisense Res. Dev. 5, 227-232.
Zabaleta, E., Oropeza, A., Assad, N., Mandel, A., Salerno, G. and Herrera-Estrella, L. (1994) Antisense expression of chaperonin 60-beta in transgenic tobacco plants leads to abnormal phenotypes and altered distribution of photoassimilates. Plant J. 6, 425-432.
Zhao, J. and Pick, L. (1993) Generating loss of function phenotypes of the fushitarazu gene with a targeted ribozyme in Drosophila. Nature 365, 448-451.
Zyskind, J. and Bernstein, S. (1989) Recombinant DNA Laboratory Manual. San Diego, CA, USA: Academic Press, Inc.
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Sokol, D.L., Passey, R.J., Mackinlay, A.G. et al. Regulation of CAT protein by ribozyme and antisense mRNA in transgenic mice. Transgenic Res 7, 41–50 (1998). https://doi.org/10.1023/A:1008803905445
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DOI: https://doi.org/10.1023/A:1008803905445