Beyond the Identification of Transcribed Sequences: Functional and Expression Analysis

9th Annual Workshop, October 28-31, 1999

Co-sponsored by the U.S. Department of Energy


Disruption of a gene induced in early mouse development results in severe emphysema and Adenocarcinoma

Irmgard S. Thorey, Anja Sterner-Kock, Jürgen Otte, and Harald von Melchner

Laboratory for Molecular Hematology, University of Frankfurt Medical School Frankfurt, Germany

Several strategies involving gene traps have been developed to identify genes that are regulated during mouse development.  However, none specifically selects for mutations into genes that are only transiently ex-pressed.  Since these genes are key regulators of many important biological processes and are generally difficult to isolate by cDNA based methodology, we have developed a strategy based on gene trap mutagenesis and site specific recombination (Cre/loxP) to isolate short lived transcripts during early mouse development.  Five ES cell clones isolated by this method were passaged to the germ line and mice heterozygous for the transgene were mated to obtain null mutations.  One clone (3C7) generated an overt phenotype in F2 homozygous offspring.  By the age of 2-3 weeks, mice with a prevalent C57BL6 background (4 backcrossings) develop a rectal prolapse associated with invasive adenocarcinoma, severe pulmonary emphysema and die around 6 months of age.  Molecular analysis revealed that the gene trap integration disrupted an exon of a yet unknown gene just several nucleotides downstream of a 3' splice consensus sequence. When hybridized to mRNA from wild type mice, the cloned exon sequences identified a 4.4 kb transcript which was missing from the mRNA of mutant mice. This indicated that the phenotypic abnormalities in mice homozygous for the gene trap integration are caused by a null mutation of a cellular gene. Using a combination of 5' and 3' RACE, we are presently cloning the full length cDNA of the disrupted cellular gene.


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