Mbd3^tm2Bh

This allele, in which exon 1 is flanked by loxP sites, was generated in embryonic stem (ES) cells heterozygous for Mbd3tm1Bh using a negative selection approach. First, a loxP-flanked hygromycin/thymidine kinase (hyg/tk) cassette was introduced into the wild-type allele upstream of exon 1. The ES cells were re-targeted with a vector having loxP sites flanking exon 1; two rounds of exposure to 6-thioguanine selected for cells in which the hyg/tk-tagged exon 1 had been replaced by the floxed exon. Immunoblot analysis of nuclear extracts of ES cells heterozygous for this floxed allele and the null Mbd3tm1Bh allele using antibody to MBD3 demonstrated expression of both normal protein isoforms, one of which (referred to as Mbda) contains the entire methyl-CpG-binding domain (MBD) and the other (Mbdb) lacking the N-terminal half of the MBD, encoded by exon 1. Following Cre recombinase-mediated excision of exon 1, neither isoform is detected. (J:106974, J:167984)