Generation of <i>Pank1</i>^−/− mice and liver PanK activity.

<p>(<b>A</b>) Diagram of the gene targeting strategy. A selection cassette containing cDNAs encoding thymidine kinase (TK) and neomycin resistance (NEO) was inserted into the intron between exons 2 (Ex2) and 3 (Ex3) of the <i>Pank1</i> gene. The cassette was flanked by loxP (▸) sites. A third loxP site was inserted between exons 3 (Ex3) and 4 (Ex4). Cre-recombinase-mediated deletion of the DNA between the outermost loxP sites resulted in a knockout (KO) allele which lacked Ex3 and the selection cassette. Locations of the primers (arrows) for PCR analysis and genotyping are shown. PCR products and sizes are indicated (bars). Locations of the loxP sites are indicated with triangles. A PCR product of 274 bp using primers F and R1 indicated a wild-type (WT) allele, and a PCR product of 220 bp using primers F and R2 indicated a deleted (KO) allele. Heterozygous (Hz) mice had both alleles. (<b>B</b>) Total PanK activity (pmoles/min) was determined in liver homogenates from wild-type (WT; •) or <i>Pank1</i> knockout (KO; ◯) mice as a function of protein concentration. The data are the average of duplicate measurements obtained from 4 control and 5 knockout mice. (<b>C</b>) Inhibition of total PanK activity by acetyl-CoA (AcCoA) in liver homogenates from wild-type (WT; •) or <i>Pank1</i> knockout (KO; ◯) mice. For WT, the 100% value corresponds to 33±3 pmoles/min/mg protein, and 17±2 pmoles/min/mg protein for KO. The data are the average of duplicate measurements obtained with 3 mice of each genotype. Statistically significant differences between wild-type and knockout are indicated by * (p<0.05).