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. 1994 Jul 5;91(14):6298–6302. doi: 10.1073/pnas.91.14.6298

Partial rescue of a lethal phenotype of fragile bones in transgenic mice with a chimeric antisense gene directed against a mutated collagen gene.

J S Khillan 1, S W Li 1, D J Prockop 1
PMCID: PMC44188  PMID: 8022775

Abstract

Previously, transgenic mice were prepared that developed a lethal phenotype of fragile bones because they expressed an internally deleted mini-gene for the pro alpha 1(I) chain of human type I procollagen. The shortened pro alpha 1(I) chains synthesized from the human transgene bound to and produced degradation of normal pro alpha 1(I) chains synthesized from the normal mouse alleles. Here we assembled an antisense gene that was similar to the internally deleted COL1A1 minigene but the 3' half of the gene was inverted so as to code for an antisense RNA. Transgenic mice expressing the antisense gene had a normal phenotype, apparently because the antisense gene contained human sequences instead of mouse sequences. Two lines of mice expressing the antisense gene were bred to two lines of transgenic mice expressing the mini-gene. In mice that inherited both genes, the incidence of the lethal fragile bone phenotype was reduced from 92% to 27%. The effects of the antisense gene were directly demonstrated by an increase in the ratio of normal mouse pro alpha 1(I) chains to human mini-pro alpha 1(I) chains in tissues from mice that inherited both genes and had a normal phenotype. The results raise the possibility that chimeric gene constructs that contain intron sequences and in which only the second half of a gene is inverted may be particularly effective as antisense genes.

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Selected References

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  1. Bird C. R., Ray J. A. Manipulation of plant gene expression by antisense RNA. Biotechnol Genet Eng Rev. 1991;9:207–227. [PubMed] [Google Scholar]
  2. Chomczynski P., Sacchi N. Single-step method of RNA isolation by acid guanidinium thiocyanate-phenol-chloroform extraction. Anal Biochem. 1987 Apr;162(1):156–159. doi: 10.1006/abio.1987.9999. [DOI] [PubMed] [Google Scholar]
  3. Colige A., Sokolov B. P., Nugent P., Baserga R., Prockop D. J. Use of an antisense oligonucleotide to inhibit expression of a mutated human procollagen gene (COL1A1) in transfected mouse 3T3 cells. Biochemistry. 1993 Jan 12;32(1):7–11. doi: 10.1021/bi00052a002. [DOI] [PubMed] [Google Scholar]
  4. D'Alessio M., Bernard M., Pretorius P. J., de Wet W., Ramirez F., Pretorious P. J. Complete nucleotide sequence of the region encompassing the first twenty-five exons of the human pro alpha 1(I) collagen gene (COL1A1) Gene. 1988 Jul 15;67(1):105–115. doi: 10.1016/0378-1119(88)90013-3. [DOI] [PubMed] [Google Scholar]
  5. Gogarten J. P., Fichmann J., Braun Y., Morgan L., Styles P., Taiz S. L., DeLapp K., Taiz L. The use of antisense mRNA to inhibit the tonoplast H+ ATPase in carrot. Plant Cell. 1992 Jul;4(7):851–864. doi: 10.1105/tpc.4.7.851. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Gray J., Picton S., Shabbeer J., Schuch W., Grierson D. Molecular biology of fruit ripening and its manipulation with antisense genes. Plant Mol Biol. 1992 May;19(1):69–87. doi: 10.1007/BF00015607. [DOI] [PubMed] [Google Scholar]
  7. Izant J. G., Weintraub H. Constitutive and conditional suppression of exogenous and endogenous genes by anti-sense RNA. Science. 1985 Jul 26;229(4711):345–352. doi: 10.1126/science.2990048. [DOI] [PubMed] [Google Scholar]
  8. Izant J. G., Weintraub H. Inhibition of thymidine kinase gene expression by anti-sense RNA: a molecular approach to genetic analysis. Cell. 1984 Apr;36(4):1007–1015. doi: 10.1016/0092-8674(84)90050-3. [DOI] [PubMed] [Google Scholar]
  9. Katsuki M., Sato M., Kimura M., Yokoyama M., Kobayashi K., Nomura T. Conversion of normal behavior to shiverer by myelin basic protein antisense cDNA in transgenic mice. Science. 1988 Jul 29;241(4865):593–595. doi: 10.1126/science.2456614. [DOI] [PubMed] [Google Scholar]
  10. Khillan J. S., Olsen A. S., Kontusaari S., Sokolov B., Prockop D. J. Transgenic mice that express a mini-gene version of the human gene for type I procollagen (COL1A1) develop a phenotype resembling a lethal form of osteogenesis imperfecta. J Biol Chem. 1991 Dec 5;266(34):23373–23379. [PubMed] [Google Scholar]
  11. Kuivaniemi H., Tromp G., Prockop D. J. Mutations in collagen genes: causes of rare and some common diseases in humans. FASEB J. 1991 Apr;5(7):2052–2060. doi: 10.1096/fasebj.5.7.2010058. [DOI] [PubMed] [Google Scholar]
  12. Melton D. A. Injected anti-sense RNAs specifically block messenger RNA translation in vivo. Proc Natl Acad Sci U S A. 1985 Jan;82(1):144–148. doi: 10.1073/pnas.82.1.144. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Mizuno T., Chou M. Y., Inouye M. A unique mechanism regulating gene expression: translational inhibition by a complementary RNA transcript (micRNA). Proc Natl Acad Sci U S A. 1984 Apr;81(7):1966–1970. doi: 10.1073/pnas.81.7.1966. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Olsen A. S., Geddis A. E., Prockop D. J. High levels of expression of a minigene version of the human pro alpha 1 (I) collagen gene in stably transfected mouse fibroblasts. Effects of deleting putative regulatory sequences in the first intron. J Biol Chem. 1991 Jan 15;266(2):1117–1121. [PubMed] [Google Scholar]
  15. Pepin M. C., Pothier F., Barden N. Impaired type II glucocorticoid-receptor function in mice bearing antisense RNA transgene. Nature. 1992 Feb 20;355(6362):725–728. doi: 10.1038/355725a0. [DOI] [PubMed] [Google Scholar]
  16. Pereira R., Khillan J. S., Helminen H. J., Hume E. L., Prockop D. J. Transgenic mice expressing a partially deleted gene for type I procollagen (COL1A1). A breeding line with a phenotype of spontaneous fractures and decreased bone collagen and mineral. J Clin Invest. 1993 Feb;91(2):709–716. doi: 10.1172/JCI116252. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Pestka S., Daugherty B. L., Jung V., Hotta K., Pestka R. K. Anti-mRNA: specific inhibition of translation of single mRNA molecules. Proc Natl Acad Sci U S A. 1984 Dec;81(23):7525–7528. doi: 10.1073/pnas.81.23.7525. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Prockop D. J. Mutations that alter the primary structure of type I collagen. The perils of a system for generating large structures by the principle of nucleated growth. J Biol Chem. 1990 Sep 15;265(26):15349–15352. [PubMed] [Google Scholar]
  19. Rosenberg U. B., Preiss A., Seifert E., Jäckle H., Knipple D. C. Production of phenocopies by Krüppel antisense RNA injection into Drosophila embryos. Nature. 1985 Feb 21;313(6004):703–706. doi: 10.1038/313703a0. [DOI] [PubMed] [Google Scholar]
  20. Saijo Y., Perlaky L., Valdez B. C., Busch R. K., Henning D., Zhang W. W., Busch H. The effect of antisense p120 construct on p120 expression and cell proliferation in human breast cancer MCF-7 cells. Cancer Lett. 1993 Feb;68(2-3):95–104. doi: 10.1016/0304-3835(93)90134-u. [DOI] [PubMed] [Google Scholar]
  21. Schuch W. Using antisense RNA to study gene function. Symp Soc Exp Biol. 1991;45:117–127. [PubMed] [Google Scholar]
  22. Sheehy R. E., Kramer M., Hiatt W. R. Reduction of polygalacturonase activity in tomato fruit by antisense RNA. Proc Natl Acad Sci U S A. 1988 Dec;85(23):8805–8809. doi: 10.1073/pnas.85.23.8805. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Sokolov B. P., Mays P. K., Khillan J. S., Prockop D. J. Tissue- and development-specific expression in transgenic mice of a type I procollagen (COL1A1) minigene construct with 2.3 kb of the promoter region and 2 kb of the 3'-flanking region. Specificity is independent of the putative regulatory sequences in the first intron. Biochemistry. 1993 Sep 7;32(35):9242–9249. doi: 10.1021/bi00086a033. [DOI] [PubMed] [Google Scholar]
  24. Szyf M., Rouleau J., Theberge J., Bozovic V. Induction of myogenic differentiation by an expression vector encoding the DNA methyltransferase cDNA sequence in the antisense orientation. J Biol Chem. 1992 Jun 25;267(18):12831–12836. [PubMed] [Google Scholar]
  25. Tanaka Y., Tang X., Hou D. X., Gao H., Kitabayashi I., Gachelin G., Yokoyama K. Phenotypic conversion of SV40-immortalized human diploid fibroblasts to senescing cells by introduction of an antisense gene for SV40-T antigen. Cell Struct Funct. 1992 Dec;17(6):351–362. doi: 10.1247/csf.17.351. [DOI] [PubMed] [Google Scholar]
  26. Westerhausen A., Constantinou C. D., Pack M., Peng M. Z., Hanning C., Olsen A. S., Prockop D. J. Completion of the last half of the structure of the human gene for the Pro alpha 1 (I) chain of type I procollagen (COL1A1). Matrix. 1991 Dec;11(6):375–379. doi: 10.1016/s0934-8832(11)80191-5. [DOI] [PubMed] [Google Scholar]

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