PARENT SESSION
Sex Determination
160
TRANSGENIC MOUSE EXPRESSION MODEL FOR DMRT1 PROMOTER.
Boyer, Alexandre1, Dornan, Saffron2, Daneau, Isabelle1, Lussier, Jacques1, Silversides, David1, 1 2
ABSTRACT- Based on cross phyla homologies and sex reversal phenotypes in humans, DMRT1 was identified as a candidate gene for involvement in mammalian sex determination and differentiation. To facilitate comparative studies of DMRT1 in these processes, we have cloned the corresponding pig cDNA and 5-prime flanking genomic sequences. Pig cDNA was obtained in a two step cloning procedure, initiated by generating partial pig sequences via RT-PCR of adult pig testes RNA using heterologous (human, mouse) primers. These pig sequences were then used to screen an adult pig testicular library to obtain full length cDNA sequences. To obtain 5-prime flanking genomic sequences, a probe was generated based on exon 1 coding sequences and used to screen a pig genomic DNA library; 2.8 kb of 5-prime flanking DNA was subsequently generated. To pursue in vivo studies of DMRT1 regulation in a heterologous model system, a transgene was constructed incorporating green fluorescent protein (GFP) coding sequences placed under the control of the 2.8 kb of pig DMRT1 5-prime flanking DNA sequences. This was injected into single celled mouse embryos and transgenic mouse lines generated. One mouse line (#9) has shown appropriate expression of fluorescence within cells of the testicular cords at day e14.5, demonstrating that promoter elements for developmental expression of DMRT1 are found within the 2.8 kb fragment 5-prime flanking DNA, and that regulatory mechanisms for expression are conserved across mammalian species.
KEY WORDS: sex differentiation, genital ridge, DMRT1, transgenic mouse