Generation of Adam22 deficient mice
The linearised targeting vector was electroporated into TT2 embryonic stem (ES) cells [31]. Homologous recombinants were selected by PCR. The extracted genomic DNA from each clone was amplified using the forward primer AGN2: 5'-GCCTGCTTGCCGAATATCATGGTGGAAAAT-3' in the PGKneo cassette, and the reverse primer MFP065R: 5'-ACTATTTCTGTGATGAGGGCACAGCATC-3' outside the targeting vector. Homologous recombined DNA was efficiently amplified by 35 cycles of the following amplification steps (94°C-30 s and 68°C-5 min). The targeting efficiency of this construct was about 4%. Correctly targeted ES clones were injected into fertilized ICR mouse eggs at the eight-cell stage. The resulting male chimeras were mated with C57BL/6N females, and heterozygous male and female mice were interbred to generate homozygous mice. The ataxic phenotypes of homozygous mice were observed in two independent lines.