Conditional knockout of Foxc2 gene in kidney: efficient generation of conditional alleles of single-exon gene by double-selection system
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Foxc2 is a single-exon gene and a key regulator in development of multiple organs, including kidney. To avoid embryonic lethality of conventional Foxc2 knockout mice, we conditionally deleted Foxc2 in kidneys. Conditional targeting of a single-exon gene involves the large floxed gene segment spanning from promoter region to coding region to avoid functional disruption of the gene by the insertion of a loxP site. Therefore, in ES cell clones surviving a conventional single-selection, e.g., neomycin-resistant gene (neo) alone, homologous recombination between the long floxed segment and target genome results in a high incidence of having only one loxP site adjacent to the selection marker. To avoid this limitation, we employed a double-selection system. We generated a Foxc2 targeting construct in which a floxed segment contained 4.6 kb mouse genome and two different selection marker genes, zeocin-resistant gene and neo, that were placed adjacent to each loxP site. After double-selection by zeocin and neomycin, 72 surviving clones were screened that yielded three correctly targeted clones. After floxed Foxc2 mice were generated by tetraploid complementation, we removed the two selection marker genes by a simultaneous-single microinjection of expression vectors for Dre and Flp recombinases into in vitro-fertilized eggs. To delete Foxc2 in mouse kidneys, floxed Foxc2 mice were mated with Pax2-Cre mice. Newborn Pax2-Cre; Foxc2loxP/loxP mice showed kidney hypoplasia and glomerular cysts. These results indicate the feasibility of generating floxed Foxc2 mice by double-selection system and simultaneous removal of selection markers with a single microinjection.
KeywordsSelection Marker loxP Site Proximal Convoluted Tubule Selection Marker Gene Conditional Allele
The mouse strain used for this research project, STOCK Tg (Pax2-cre) 1Akg/Mmnc, identification number 10569-UNC, was obtained from the Mutant Mouse Regional Resource Center, a NIH-funded strain repository, and was donated to the MMRRC by Andrew Groves, Ph.D., House Ear Institute. We thank Dr. Valentina Kon, Vanderbilt University, for helpful discussions during a preparation of this manuscript and Drs. Iekuni Ichikawa and Kiyoshi Ando, Tokai University, for technical and financial support. This study was supported by Grant-in Aid for Scientific Research 25461234 and 22590899 from the Ministry of Education, Culture, Sports, Science and Technology, Japan. We also thank Ms. Shiho Imai and Ms. Chie Sakurai for technical assistance.
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