Targeting of Pygo1 and Pygo2
Pygo1 and Pygo2 genes were each targeted by flanking the critical coding regions of the third exon containing the PHD motif with LoxP sequences. Targeting constructs were made using the Flp/Cre vector previously described [34], which carries a neomycin-resistance gene flanked by FRT sequences, and three unique restriction sites for subcloning the two blocks of homology for driving recombination, and the critical region to be flanked by LoxP sequences. For each construct, the three genomic segments required for subcloning were made by PCR from RI ES cell DNA. The resulting targeting constructs were confirmed by sequencing.
For Pygo1, the genomic sequences used for PCR were: 5' forward, GTGAAGGAGAGATGGATAAGTATG; 5' back, TAGACCCTAACCACCTACAAG; exon forward, GGTTAGGGTCTATGTGCTGG; exon back, TCACCAAATCTCTGTTCTACAC; 3' forward, TGTGTAGAACAGAGATTTGGTG; 3' back, CAGTGAAGAAAGAGGGTCAG. For Pygo2, the genomic sequences used for PCR were: GCCTGGGTTGCTTGTCTTCTG and CCACCTTACTTGTGTGTGAGGATACATAC, CCAAGTCCCAGCATCTCTTAC and CCAGTCATACCAGCAACAAG, and exon sequences TGGGTGCTGGGAACAGAAC and CAACAACAACAGAAGACAAGC.
Linearized constructs were electroporated into RI ES cells, and resulting targeted ES cells used for C57/Bl6 blastocyst injections according to standard protocols. Resulting chimeras were mated with Swiss Black mice, and the targeted stocks maintained on a mixed 129/Swiss Black background.
Germline null alleles of both Pygo1 and Pygo2 were generated by mating heterozygous floxed mice with the CMV-Cre mice [15]. The sequences of the primers used for genotyping PCR were: Pygo2 null allele, forward (F) CCTGGATTCTTGTTGCTGGTATG; reverse (R) AAGGTATTTGGTGCTCCGAGGG; Pygo2 WT or floxed allele, F TGTCTTGATGACAGCGTTTAGCC, R AGATTCAGTAAGCTGAGCCTGGTG; Pygo1 null allele, F AGTTTGAAATAGCGACGAGTTTGAG, R 5'-CACTTCTGCCCCTCTCTTTGC; Pygo1 WT or floxed allele, F AAGCGTGCCTCATCTCCATCCCTAAG, R GCCCTCCCCGACGTTTATATTG.
The noon of the day that vaginal plugs were observed was designated E0.5.