We have used large-insert cloning to obtain yeast and bacterial artificial chromosome clones (YACs and BACs) containing two genes of importance in human reproduction: PLAC1, expressed uniquely in one of the three layers of the placenta, and FOXL2, expressed only in developing eyelids and in follicular cells of the ovarian follicles. [Deficiency in FOXL2 leads to Premature Ovarian Failure (POF) in some women.] The goal is to determine the basis for the extraordinarily selective tissue-specific expression of these genes. In contrast to other instances such as liver-specific genes, the tissue specificity is not reproduced by segments of putative promoter DNA up to 10 Kb 5' of PLAC1 or FOXL2. This indicates that longer-range regulation is operative. Consistent with that notion, a translocation that disrupts transcription of FOXL2 lies very far (about 168 Kb away) from the gene sequence. In the next phase of these studies we are focusing on both human and mouse Plac1 genes. Using recombination-based methods in bacteria, we have introduced a Flag tag into the C-terminal end of the PLAC1, retrofitted the BAC with mammalian selectable drug marker, blasticidin, and made deletions of successive blocks of 10Kb, starting at the 5' most in the 65Kb sequence upstream of Exon 1. These constructs are now being transfected into cell-lines to study their expression, followed by probing at the protein level with anti-FLAG antibody. Recombineering methods were also used to recover 8.3Kb fragment containing Plac1 exon3 and flanking sequences. Exon3 was replaced with mammalian selectable PGKNeo marker and the knockout construct is currently being transfected into mouse ES cells to derive a Plac1 knockout mouse, in order to analyze its function in relation to its specific expression. In a complementary approach, experiments with rabbit antibodies raised against selected epitopes from both human and mouse proteins suggest that the PLAC1 protein exists in vivo as a dimer, undergoes substantial protein modification, and is localized in the cytoplasm. Experiments are in progress to track the human PLAC1 protein with the FLAG epitope, and to look for interacting partners by immunoprecipitation with anti-FLAG antibodies. The FLAG epitope distinguishes the BAC-encoded PLAC1 from endogenously expressed protein.
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