Figure 1.

Generation of the chimeric Sec24c^c-d allele. (A) Schematic representation of dRMCE to generate the Sec24c^c-d allele. The replacement vector pUC19-Sec24c-d contains the Sec24c intron 2 splice acceptor (yellow), the Sec24d coding sequence beginning with G¹²⁰ (gray), and a stop codon followed by a poly A signal sequence (red). Arrows represent primers used for genotyping, long-range PCR, and RT-PCR (for sequences, see Table S2). (B) The SEC24C-D fusion protein encoded by the dRMCE generated Sec24c^c-d allele, which contains the first 57 amino acids of SEC24C followed by the SEC24D sequence corresponding to the remaining ~ 95% of the SEC24C protein. Val58/41 (Val58 in SEC24C, Val41 in SEC24D) indicates the junction point for this chimeric protein. SEC24C^Met1-Val58 and SEC24D^Met1-Val41 have 34% protein sequence identity. (C) PCR results for dRMCE subclone 12275. Primer combinations are indicated at the top of each lane. Correct targeting was observed for the 5′ recombination (5′ rec) site (lanes 1 and 2) and the 3′ recombination (3′ rec) site (lanes 3 and 4). Additionally, the presence of the Sec24d cDNA (“any ins.”) (lanes 5,6), and the loxP and FRT sites (103 bp and 106 bp products in lanes 7,8, respectively) was confirmed. The signal in lane 9 is due to the Sec24c⁺ allele, confirming that ESC clone 12275 is heterozygous for the Sec24c^c-d allele. Clone 12275 does not carry any random insertions of pCAGGS-iCre (lane 10) or pCAGGS-Flpo (lane 11). (D) A genotyping PCR assay on mouse genomic DNA from tail clip to distinguish between the wild type and Sec24c^c-d allele using primers E, F, and G. (E) Long range PCR confirms correct targeting. Primers GF4 + U were used to amplify the 5′ arm resulting in a 7.6 kb product, and primers F and GR4 were used to amplify the 3′ arm to yield a 5.8 kb product. Primers were located outside the homology arms (GF4 and GR4) and within the Sec24d cDNA (F and U). Neither set of primers yields a band from the Sec24c^wt allele.