The linearised targeting vector was electroporated into TT2 embryonic stem (ES) cells [40]. Homologous recombinants were selected by PCR using the following primers, AGN1: 5'-TCGTGCTTTACGGTATCGCCGCTCCCGATT-3' in the PGKneo cassette, and SGN033: 5'-GGACCCGGAAGACATTTGCACGGT-3' outside the targeting vector. Homologous recombined DNA was efficiently amplified by 35 cycles of the following amplification steps (94°C-30 sec and 68°C-5 min). Correctly targeted ES clones were injected into fertilised ICR mouse eggs at the eight-cell stage. The resulting male chimeras were mated with C57BL/6NCrj females (Charles River Japan, Tokyo, Japan), and resulting heterozygous (+/-) male and female mice were interbred to generate homozygous (-/-) mice.