International Mammalian Genome Society

The 16th International Mouse Genome Conference (2002)


M Wentland
Baylor College of Medicine

2) Wang X, 3) Bradley A
1,2) Baylor College of Medicine, 3) The Wellcome Trust Sanger Institute

We have devised a method to isolate gene trap mutations on a chromosome of interest in mouse embryonic stem (ES) cells using Cre/loxP technology. Specifically, we used ES cells with a neo selection cassette, a loxP site, and the 5 part of the Hprt mini-gene targeted to the Hsd17b1 locus on the distal part of mouse chromosome 11. We infected these cells with a gene trapping retrovirus that contains a 3 gene trap construct, a loxP site, and the 3 part of the Hprt mini-gene. If a proviral insertion and gene trap occurred on chromosome 11, we isolated it by selecting for a Cre mediated inversion between the targeted locus and the gene trap locus. The inversion event reconstitutes a functional Hprt gene and is selected in HAT containing media. By selecting for inversions, we isolated gene traps proximally and distally to the targeted locus on chromosome 11. To date, we have isolated 21 gene traps on chromosome 11. We were able to select gene traps within a 117 Mb region; however, most (60%) of the gene traps were concentrated within 5 Mb proximal and 5 Mb distal to the Hsd17b1 locus. So far, we have confirmed one of these inversion events using FISH. Ultimately, we hope to decrease the time required to make and maintain homozygous mutant mice by crossing chimera transmitting gene trap alleles on chromosome 11 to a mouse harboring a balancer in the same region.

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