Fig. 1

Generating conditional and conventional Fam20c^D446N knock-in mice by gene targeting. (A) Sequencing of the targeting vector showed that the gat encoding Aspartate (D) was mutated into aat encoding Asparagine (N). (B) In the targeting construct, the full DNA from exon 7 to 10 and a PGK-neo cassette flanked by Frt sites (orange ovals) were flanked by loxp sites (blue triangles). A cDNA of exon 7–10 carrying the D446N mutation was fused with a IRES-EGFP reporter cassette and followed the loxp flanked sequence. A MC1-HSV-TK cassette was downstream to the 3’-homologous arm (target construct). The PGK-neo cassette was removed from the targeted allele after correct targeting (Neo-removed Fam20c cKI allele). To ensure the correct targeting, a 5.4Kb segment at 5’ arm and a 4.1Kb segment at 3’ arm of the target construct were tested through PCR with the primers eGFP-ScF3 and Fam20c-ScrR2, and the primers Fam20c-ScrF2 and Lox-ScR2, respectively (Fam20c targeted allele). When the loxp-flanking exon 7–10 was removed by Cre, the Fam20c KI allele was formed by recombining the exon 1–7 with the cDNA of exon 7–10 carrying the mutation of D446N. (C) PCR screening for targeted ES clones showed the correct targeting took place in 13 ES clones. Two correctly targeted ES clones (5 A and 10 C) were selected for injection and both got germline transmission