Conditional Inactivation of Transcription Factor AP-2 By Using the Cre/loxP Recombination System Uwe Werling, 1,2 and Hubert Schorle 2 * 1 Forschungszentrum Karlsruhe, Institute for Toxicology and Genetics, Eggenstein-Leopoldshafen, Germany 2 Institute for Pathology, Department of Developmental Pathology, University of Bonn Medical School, Bonn, Germany Received 14 December 2001; Accepted 2 January 2002 Published online 13 February 2002 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; Wil- liamson 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 me- diated 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 clo- sure (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 ex- traembryonic 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 electropo- ration 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 cas- sette using Cre-mediated excision. To check for the functional integrity of the loxP sites, a second transient transfection with a Cre-expressing plasmid was per- formed. 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 Sori- ano, 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 homozy- gous 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 time- and tissue-specific manner. ACKNOWLEDGMENTS We thank Peter Mombaerts for the pLTNL selection cassette, Pascal Dolle ´ for the AP-2 genomic clone, and Ivan Horak and Klaus Peter Knobeloch for the KPA-ES cells. We thank Andrea Jacob, Norma Howells, and Yvonne Petersen for brilliant technical assistance and maintenance of the animal colony. This work was sup- ported by a grant of the Deutsche Forschungsgemein- schaft to H.S. (DFG # 503-2). LITERATURE CITED Bosher JM, Totty NF, Hsuan JJ, Williams T, Hurst HC. 1996. A family of AP-2 proteins regulates c-erbB-2 expression in mammary carci- noma. Oncogene 13:1701–1707. * Correspondence to: Dr. Hubert Schorle, Institute for Pathology, Depart- ment of Developmental Pathology, University of Bonn, Sigmund-Freud Strasse 25, 53127 Bonn, Germany. E-mail: Hubert.Schorle@ukb.uni-bonn.de © 2002 Wiley-Liss, Inc. genesis 32:127–129 (2002) DOI 10.1002/gene.10057