A model for the mechanism of precise integration of a microinjected transgene
- 52 Downloads
A unique transgenic mouse line has undergone transgene integration in a very precise fashion. The phenotype displayed by mice of the line followed the predicted inheritance patterns for X-linked transgene insertion which has been confirmed. In order to investigate the mechanism of integration the DNA sequence of the transgene and cellular junctions have been determined. A comparison between wild type and transgenic mutant sequences at the site of insertion revealed that there was no loss or rearrangement of cellular DNA upon integration of the transgene. The cellular sequences at the transgene 5′ and 3′ joins are contiguous in the wild type. The integrant exists as a head to tail tandem dimer with minimal loss of sequence compared with the injected monomer. Analysis of the site of insertion has revealed a 5 bp homology between the 5′ end of the transgene and the cellular sequences. In addition, adjacent to the site of insertion within the cellular sequences, there are several sequence motifs implicated in recombination events including a clustering of strong consensus sites for DNA topoisomerase type I and a region of homology to the human minisatellite consensus core sequence, theEscherichia coli Chi site and the meiotic recombination hotspot within the Eβ gene of the murine major histocompatibility complex. This clustering of features is likely to have been factorial in the integrity of the insertion event. A model depicting the mechanism of this precise integration is proposed.
Keywordsrecombination hotspot Chi site minisatellite topoisomerase I transgenic mice integration site
Unable to display preview. Download preview PDF.
- Macleod, D., Lovell-Badge, R., Jones, S. and Jackson, I. (1990) A promoter trap in embryonic stem (ES) cells selects for integration of DNA into CpG islands.Nucl. Acids Res. 19, 17–23.Google Scholar
- Ninomiya, T., Hoshi, M. and Yuki, A. (1988) Structures of integrated DNA containing human adenovirus Ela genes in transgenic mice.Agric. Biol. Chem. 52, 2537–46.Google Scholar
- Radding, C.M. (1988) In Kucherlapati, R. and Smith, G.R. eds, Homologous pairing and strand exchange promoted byEscherichia coli RecA protein inGenetic Recombination, pp. 193–229. Washington: Am. Soc. for Microbiol.Google Scholar
- Taylor, A.F. and Smith, G.R. (1992) RecBCD enzyme is altered upon cutting DNA at a Chi recombination hotspot.Proc. Natl. Sci. USA 89, 5226–30.Google Scholar
- Wilson, J.B., Ferguson, M.W.J., Jenkins, N.A., Lock, L.F., Copeland, N.G. and Levine, A.J. (1993) Transgenic mouse model of X-linked cleft palate.Cell Gr. Different. 4, 67–76.Google Scholar