Characterization of the PB insertion site in Alpk1PB/PB mice. (A) Position of the PB insertion. PB integration site in Alpk1PB/PBmice was mapped to the first intron of Alpk1 gene on mouse chromosome 3, nucleotide 128212040 (ENSMUSG00000028028, NCBI m37). Diagram shows the genomic structure of Alpk1 gene with introns (lines) and exons (black boxes). Two putative start codons and kinase domain are indicated above; gene specific primers are indicated below. TTAA: the four nucleotides required for PB insertion. (B) Disruption of endogenous Alpk1 transcript in Alpk1PB/PB mice. Alpk1 5'-end transcript in wild type (WT), Alpk1PB/+ and Alpk1PB/PB mice were analyzed by RT-PCR using the indicated primers P1/P2. cDNAs from whole-brain were amplified as templates. GAPDH was used as an internal control. The different genotypes were determined by 3-primer PCR to detect WT (801 bp) and PB insertion alleles (631 bp). (C-H) Alteration of Alpk1 3'-end transcript levels in Alpk1PB/PBmice. The transcription level of Alpk1 3'-end transcript in skeletal muscle (C),thymus (D), spleen (E), lymph node (F), small intestine (G)and brain (H) were measured by real-time quantitative RT-PCR using the indicated primers P3/P4. Alpk1 mRNA expression levels were normalized to GAPDH. *P < 0.05, ***P < 0.001, n = 3 per group.