DEV01877 Supplementary Material

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  Fig. S1. Gene targeting of the Msx2 locus with the nlacZ gene. (A) Restriction map of the Msx2 endogenous locus as established from the initial P1 phage DNA. (B) The recombination vector contains 3.1 kb and 5.6 kb of Msx2 sequences at the 5¢ and the 3¢, respectively. Msx2 coding and intron sequences are replaced by an nlacZ-neo cassette (Bonnerot et al., 1987), bracketed with the I-SceI sites (Choulika et al., 1995) such that the b-galactosidase is produced from the Msx2 ATG. An HSV-tk counter-selection gene (Thomas and Capecchi, 1990) is further inserted in a unique SalI site outside the regions of homology. New restriction sites created in the vector are in italics. This construct was transfected into HM1 ES cells (129 genetic background) (Magin et al., 1992) (a kind gift from Dr D. Melton) as previously described (Houzelsteinet et al., 1997). (C) Structure of the modified allele. A total of 560 G418:gancyclovir resistant clones were analysed. For Southern blot identification, ES cell or mouse DNA was restricted with BglII and NcoI and diagnostic bands revealed with a 5¢ BglII-BamHI probe (1), or digested with EagI and EcoRI and revealed with a 3¢ BamHI-EcoRI probe (2). Cells from four positive clones were injected into E3.5 blastocysts. Ten chimeras were produced; one transmitted the mutant allele. Scale bar: 1 kb. B, BamHI; Bg, BglII; E, EcoRI; S, SalI; Xb, XbaI.

  References

  Bonnerot, C., Rocancourt, D., Briand, P., Grimber, G. and Nicolas, J. F. (1987). A beta-galactosidase hybrid protein targeted to nuclei as a marker for developmental studies. Proc. Natl. Acad. Sci. USA 84, 6795-6799.

  Choulika, A., Perrin, A., Dujon, B. and Nicolas, J. F. (1995). Induction of homologous recombination in mammalian chromosomes by using the I-SceI system of Saccharomyces cerevisiae. Mol. Cell. Biol. 15, 1968-1973.

  Thomas, K. R. and Capecchi, M. R. (1990). Targeted disruption of the murine int-1 proto-oncogene resulting in severe abnormalities in midbrain and cerebellar development. Nature 346, 847-850.