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Conditional inactivation of transcription factor AP‐2γ by using the Cre/loxP recombination system

The family of AP-2 transcription factors consists of four different genes: AP-2 , AP-2 , AP-2 , and AP-2 (Bosher et al., 1996; McPherson et al., 1997; Mitchell et al., 1987; Moser et al., 1995; Oulad-Abdelghani et al., 1996; Williamson et al., 1996; Zhao et al., 2001). All AP-2 family members share a characteristic protein structure with a unique helix-span-helix motif at the C-terminal region, which mediates protein dimerization, and, overlapping with a basic domain binding with varying affinity to GC-rich DNA elements. Transcriptional activation is mediated by an N-terminal proline/glutamine-rich region (Williams et al., 1988). Gene knockout experiments revealed that AP-2 and AP-2 proteins harbor distinct functions during mouse development. AP-2 is predominantly essential for craniofacial morphogenesis and ventral body wall closure (Schorle et al., 1996; Zhang et al., 1996), while the lack of AP-2 causes polycystic kidney disease (Moser et al., 1997). AP-2 expression is detected as early as day 3.5 of murine development in the trophoblast lineage and persists in all of its derivatives (Shi and Kellems, 1998). In the embryo proper, AP-2 expression starts at day 7.5 of development and can be detected in neural, neural crest, and epithelial cells (Chazaud et al., 1996). Using gene-knockout technologies, we demonstrated that AP-2 , located on mouse chromosome 2H3-4 and human chromosome 20q13.2 (Williamson et al., 1996), is essential for the maintenance of proliferation of extraembryonic tissue, derived from trophoblastic cells. As a consequence, the lack of AP-2 results in an embryonic lethal phenotype short after implantation (Werling and Schorle, submitted). To overcome this early defect and to address the roles of this transcription factor in the embryo proper, we generated a mouse line harboring a conditional AP-2 allele by using the site-specific Cre/ loxP recombination system. The targeting vector encompassed a 6-kb genomic fragment, spanning exons 2–7 of the AP-2 locus. To generate the conditional allele, a single loxP recognition site was inserted at the PstI site 5 of exon 5, and a selection cassette containing the pgk-neo and the HSV-tk genes, which are flanked by loxP sites, were introduced at the XbaI site 3 of exon 5 (Fig. 1A). After electroporation into ES cells and G418 selection clones that had undergone homologous recombination were identified by Southern blot analysis (Fig. 1B). The conditional AP-2 allele was generated by removing the neo/tk cassette using Cre-mediated excision. To check for the functional integrity of the loxP sites, a second transient transfection with a Cre-expressing plasmid was performed. As expected, this resulted in the generation of the AP-2 null allele (Fig. 1A). A PCR assay was used to verify the Cre-mediated loop-out reaction (Fig. 2A). To generate mice harboring the conditional AP-2 allele, cells were injected into C57Bl/6 blastocysts, resulting in several chimeric animals. The in vivo function of the conditional AP-2 allele was tested by mating the AP-2 line to Mox2 Cre transgenic animals (Tallquist and Soriano, 2000). PCR amplification of DNA from tail biopsies showed that the majority of the conditional allele had been converted to the null allele in the embryos carrying both the “floxed” AP-2 allele and the Mox2-driven Cre transgene (Fig. 2B, lanes 2 and 5). Mice heterozygous for the conditional AP-2 allele were mated and a homozygous line was established, which is being kept on the 129/ola x C57Bl/6 mixed background. These animals will provide a tool to overcome early lethality of AP-2 deficient embryos and to analyze AP-2 deficiency in a timeand tissue-specific manner.