International Mammalian Genome Society

The 15th International Mouse Genome Conference (2001)


POSTER 247 - REGION-SPECIFIC GENE TRAP MUTAGENESIS AND TRANSCRIPT MAPPING ON MOUSE CHROMOSOME 11

Ms Meredith Wentland
Baylor College of Medicine
Department of Molecular and Human Genetics
One Baylor Plaza, Rm. T928
Mail Stop BCM 225
Houston
TX 77030
USA

Co-Authors: 1)Wang, X  2)Bradley, A
Institutions: 1)Howard Hughes Medical Institute, Baylor College of Medicine, 2)The Sanger Centre

To identify and mutate genes in a candidate region, we designed a strategy to select gene trap mutations specifically in a region of interest in mouse embryonic stem cells.  We selected the distal part of chromosome 11 because loss of heterozygosity in the syntenic region of human chromosome 17 in several tumor types suggests the presence of tumor suppressor genes.  Also, initial transcript mapping of the human genome shows that this region is gene rich.

To isolate region-specific gene trap mutations, a locus on chromosome 11 is targeted with a selectable marker, a loxP site, and the 5 part of the HPRT minigene.  This locus is an anchor point around which mutations are selected.  Mutations are generated by insertion of a retrovirus containing a gene trap construct, a loxP site, and the 3 part of the HPRT minigene.   A mutation on chromosome 11 is selected in HAT-containing media after a Cre mediated inversion between the proviral integration site and the anchor point reconstitutes a functional HPRT gene.  In this selection scheme, only mutations in cis to the anchor locus are selected.  The trapped gene is identified by 3 Rapid Amplification of cDNA Ends(RACE).  The inversion also introduces a breakpoint in the trapped gene, increasing the chance that the gene is mutated.  Additionally, chimera transmitting gene trapped alleles can be mated to mice harboring deletions in the same region ina two-generation screen for recessive mutations.


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