Transgenic Research

, Volume 4, Issue 1, pp 52–59 | Cite as

Frequent deletions and sequence aberrations at the transgene junctions of transgenic mice carrying the papillomavirus regulatory and the SV40 TAg gene sequences

  • Chuan-Mu Chen
  • Kong-Bung Choo
  • Winston T. K. Cheng
Papers

Abstract

Exogenous DNA microinjected into one-cell mouse zygotes either integrates into the host genome within a short time span, or is rapidly degraded. On integration, a transgene squence is frequently reiterated. In this report, we describe the enzymatic amplification analysis of transgene junctions of 12 transgenic mice carrying different copy numbers of the same transgene with dissimilar ends. The transgene was composed of the regulatory sequence of the type 18 human papillomavirus linked to the TAg gene of the SV40 virus. Nucleotide sequences of 36 of these junctions were also determined. Deletions were found in 33 (91.7%) of the junctions analysed. At the crossover regions, 55.6% contained short overlapping sequences of one to six nucleotides. Insertions of 2–6 extraneous nucleotides were also found in 8.3% of the transgene junctions. Within a 10-nucleotide sequence on both sides of the transgene junctions, topoisomerase I (topo I) cleavage sites, runs of homogeneous purines or pyrimidines, alternating purine-pyrimidine tracks and (A-T)-rich sequences were found frequently. Stringent control experiments were also performed to ascertain that the observations made were not artefacts resulting from the polymerase chain reaction. Our data therefore indicate that damage had occurred quite frequently and extensively in our transgene construct. Such transgene damage may also occur to various extents in mice carrying other transgenes. Primary structure of the nucleotide sequences of the injected DNA seems to influence the process of transgene reiteration and aberration.

Keywords

transgenic mice transgene junctions DNA integration sequence deletion 

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References

  1. Chong, K.Y., Chen, C.M. and Choo, K.B. (1993) Post-hybridization recovery of membrane filter-bound DNA for enzymatic DNA amplification.Bio Techniques 14, 575–7.Google Scholar
  2. Choo, K.B., Chong, K.Y., Liew, L.N., Hsu, H.C. and Cheng, W.T.K. (1992) Unregulated and basal transcription activities of the regulatory sequence of the type 18 human papillomavirus genome in transgenic mice.Virology 188, 378–83.Google Scholar
  3. Chou, Q. (1992) Minimizing deletion mutagenesis artifact duringTaq DNA polymerase PCR byE. coli SSB.Nucl. Acids Res. 20, 4371.Google Scholar
  4. Covarrubis, L., Nishida, Y. and Mintz, B. (1986) Early postimplantation embryo lethality due to DNA rearrangements in a transgenic mouse strain.Proc. Natl Acad. Sci. USA 83, 6020–4.Google Scholar
  5. Covarrubis, L., Nishida, Y., Terao, M., D'Eustachio, P. and Mintz, B. (1987) Cellular DNA rearrangements and early developmental arrest caused by DNA insertion in transgenic mouse embryos.Mol. Cell. Biol. 7, 2243–7.Google Scholar
  6. Hofman-Liebermann, B., Libermann, D., Troutt, A., Kedes, L.E. and Cohen, S. (1986) Human homologs of TU transposon sequences: polypurine/poly-pyrimidine sequence elements that can alter DNA conformationin vitro andin vivo.Mol. Cell. Biol. 6, 3632–42.Google Scholar
  7. Jefferys, A.J., Wilson, V. and Thein, S.L. (1985) Hypervariable ‘minisatellite’ regions in human DNA.Nature 314, 67–70.Google Scholar
  8. Konopka, A.K. (1988) Compilation of DNA strand exchange sites for non-homologous recombination in somatic cells.Nucl. Acids Res. 16, 1739–58.Google Scholar
  9. Lebkowski, J.S., DuBridge, R.B., Antell, E.A., Greisen, K.S. and Calos, M.P. (1984) Transfected DNA is mutated in monkey, mouse and human cells.Mol. Cell. Biol. 4, 1951–60.Google Scholar
  10. Lee, H.H., Lo, W.C. and Choo, K.B. (1992) Mutation analysis by a combined application of the multiple restriction fragment-single strand conformation polymorphism and the direct linear amplification DNA sequencing protocols.Anal. Biochem. 205, 289–93.Google Scholar
  11. Moser, H.E. and Dervan, P.B. (1987) Sequence-specific cleavage of double helical DNA by triple helix formation.Science 238, 645–50.Google Scholar
  12. Nakamura, Y., Leppert, M., O'Connell, P., Wolff, R., Holm, T., Culver, M., Martin, C., Fujimoto, E., Hoff, M., Kumlin, E. and White, R. (1987) Variable number of tandem repeat (VNTR) markers for human gene mapping.Science 235, 1616–22.Google Scholar
  13. Rohan, R.M., King, D. and Frels, W.I. (1990) Direct sequencing of PCR-amplified junction fragments from tandemly repeated transgenes.Nuc. Acids Res. 18, 6089–95.Google Scholar
  14. Roth, D.B., Porter, T.N. and Wilson, J.H. (1985) Mechanisms of nonhomologous recombination in mammalian cells.Mol. Cell. Biol. 5, 2599–607.Google Scholar
  15. Roth, D.B., Chang, X.B. and Wilson, J.H. (1989) Comparison of filler DNA at immune, nonimmune and oncogenic rearrangements suggests multiple mechanisms of formation.Mol. Cell. Biol. 9, 3049–57.Google Scholar
  16. Roth, D.B., Proctor, G.N., Stewart, L.K. and Wilson, J.H. (1991) Oligonucleotide capture during and joining in mammalian cells.Nucl. Acids Res. 19, 7201–5.Google Scholar
  17. Sambrook, J., Fritsch, E.F. and Maniatis, T. (1989)Molecular Cloning: a Laboratory Manual, second edition. Cold Spring Harbor, New York: Cold Spring Harbor Laboratory Press.Google Scholar
  18. Wake, C.T., Gudewicz, T., Porter, T., White, A. and Wilson, J.H. (1984) How damaged is the biologically active subpopulation of transfected DNA?Mol. Cell. Biol. 4, 387–98.Google Scholar
  19. Wilkie, T.M. and Palmiter, R.D. (1987) Analysis of the integrant in MyK-103 transgenic mice in which males fail to transmit the integrant.Mol. Cell. Biol. 7, 1646–55.Google Scholar

Copyright information

© Chapman & Hall 1995

Authors and Affiliations

  • Chuan-Mu Chen
    • 1
    • 2
  • Kong-Bung Choo
    • 1
    • 2
  • Winston T. K. Cheng
    • 2
  1. 1.Recombinant DNA Laboratory, Department of Medical ResearchVeterans General HospitalTaipeiTaiwan
  2. 2.Graduate Institute of Animal ScienceNational Taiwan UniversityTaipeiTaiwan

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