Construction of an Adam22 gene-targeting vector
The 18-kb genomic DNA fragments covering exons 5, 6, 7, 8 and 9 of the Adam22 gene were amplified from C57BL/6 genomic DNA by PCR using Expand Hi-Fidelity enzyme mix (Roche Diagnostics) and primers designed for each exon. To generate a mutation in the mouse Adam22 gene, we inserted the PGKneo cassette into exon 8 of the Adam22 gene. The final targeting construct consisted of a 10.5-kb genomic DNA fragment that was interrupted by the insertion of the PGKneo cassette and contained a MC1/TK cassette as a negative selection marker against random integration [5].