International Mammalian Genome Society

The 14th International Mouse Genome Conference (2000)

I3. Identification and Characterization of the Gene Underlying the Mouse Germ Cell Deficient Mutation (gcd)

Zhu Q1, Agoulnik AI1, Ty MT1, Arango NA2, Chada K3 and Bishop CE1,4
1Baylor College of Medicine, Department of Obstetrics and Gynecology, Houston TX
2University of Texas MD Anderson Cancer Center, Houston TX
3University of Medicine and Dentistry of New Jersey, Piscataway, NJ, USA.
4Baylor College of Medicine, Department of Molecular and Human Genetics, Houston TX

In human populations infertility is a common problem affecting 10-15% of all individuals. Defects in migration and/or proliferation of the primordial germ cells during embryonic development can lead to both Sertoli Cell Only syndrome (SCOS) in males and Premature Ovarian Failure (POF) in females (0.3% of all young women are affected). Identification of genes that play a role in the early stages of primordial germ cell (PGC) development are key to the better understanding of such conditions. In these studies the non-pleiotropic, transgene-insertional, germ cell deficient mouse mutant, (gcd) has been used as a model system. Analysis of this mutant clearly shows that disruption at a single locus, can drastically reduce the number of PGCs in the embryonic gonad by 11.5 dpc, giving rise to male and female infertility in the adult. The male phenotype of a vacuolated testis, with only a few functional tubules, is very similar to the human Sertoli Cell Only syndrome (SCOS) seen in infertile males. The female phenotype of rapid follicular depletion closely parallels that of Premature Ovarian Failure

Using the inserted transgene DNA as a probe we cloned the genomic fragments adjacent to the insertion site and the corresponding genomic region from normal DNA. We mapped the mutation to a genetic interval of less than 1cM using interspecific backcross analysis and established that the transgene insertion caused a deletion in gcd genomic DNA. A BAC contig of the critical region was then constructed. A single 200 kb BAC was identified which fully spans the gcd critical deletion interval. Sample sequencing of the BAC in combination with BLAST analysis of the publicly available mouse and human genomic sequence databases was then performed. We have been able to construct a gene map of the mouse gcd region and also the homologous region of the human (chromosome 2p15-p16). Two candidate genes within this critical area for were identified. The 3' exons and UTR regions of both genes were deleted in the gcd mutant. One of the genes is a known member of a conserved family of Serine/Threonine kinases termed Vrk2 (vaccinia related kinase 2). The other gene is novel having no sequence homologies in Genbank or to any other known proteins. We have termed it Can1 (candidate 1).

Using transgenic technology, Vrk2 was correctly expressed in homozygous gcd/gcd males and females. There was no rescue of the sterility and the gonadal histology remained typical of the gcd mutant.This indicated that deletion of Vrk2 was not responsible for the gcd phenotype and that Can1 may well underlie the mutation. Using gene targeting we have now shown that ablation of Can1 leads to the typical gcd phenotype of reduced numbers of PGC's, and adult male and female gonads severely depleted for germ cells. Data on the characterization and spatio-temporal expression pattern of this gene will be presented

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