Figure 1

Targeting vector for creating Fam20A -mutant mice and genotyping strategy. (a) The mouse Fam20A gene contains 11 exons (green boxes). Intron 4 is the largest among the 10 introns. In the targeting vector, an IRES-lacZ-Neo cassette flanked by two flippase recognition target (FRT) sites (red ovals) was inserted into intron 4, and one LoxP site (yellow triangle) was placed into intron 8, which is also relatively large. Recombination after Flp recombinase scission removed the IRES-lacZ-Neo cassette from the Fam20A^lacZ-flox allele and produced the conditional allele Fam20A^flox, in which the region of exons 5 through 8 was flanked by two LoxP sites (yellow triangles). (b) We used the primer set of a and b to distinguish the Fam20A^lacZ-flox allele from the WT allele; PCR with these primers produced a 273-bp fragment for the mutant allele and 216-bp fragment for WT allele. (c) The primer set of c and d was used to identify the Fam20A^flox allele after the IRES-lacZ-Neo cassette was removed by Flp recombinase. Note that the use of primers c and d was not expected to generate any PCR products for the Fam20A^lacZ-flox allele. (d) The primer set of c and b was used to identify the Fam20A-ablated (Fam20^Δ) allele in the K14-Cre;Fam20A^flox/+ or the K14-Cre;Fam20A^flox/flox mice; PCR with these primers did not produce any fragment for the Fam20A-floxed (Fam20A^flox/+ and Fam20A^flox/flox) or WT alleles. PCR, polymerase chain reaction; WT, wild type.