Generation and characterization of CPEB4 null mice.

<p>(<b>A</b>) Schematic illustration of the targeting strategy. The <i>cpeb4</i> gene consists of 10 exons (numbered boxes) and spans a region of approximately 60-kb (WT, wild-type). The targeting vector containing the flip recombinase target (FRT)-flanked neomycin-resistance gene (Neo) and loxP-flanked exon 2 cassette was inserted into the <i>cpeb4</i> gene by homologous recombination. The correctly targeted locus was examined by Southern blotting using DNA digested with BamHI (B1) or BglII (B2) and probed with a 5′-end or 3′-end probe, respectively. The mouse line carrying the floxed allele of CPEB4 (fCPEB4) was then crossed with a transgenic male mouse expressing Cre recombinase in the sperm (protamine-Cre) to produce the knockout (KO) allele. (<b>B</b>) The WT and KO alleles were distinguished on a Southern blot using DNA digested with HindIII (H3). The brain RNAs isolated from male offspring carrying WT (+/+), heterozygous (+/−) or KO (−/−) alleles were analyzed by (<b>C</b>) Northern blotting using a probe against exon 1 of CPEB4 RNA or GAPDH RNA (loading control). (<b>D</b>) RT-qPCR analysis. The brain RNAs were reverse transcribed and PCR-amplified for CPEB4 and β-actin. The CPEB4 RNA level after normalization with the β-actin signal was expressed as a relative ratio to the WT which was arbitrarily set to 100%. (<b>E</b>) RT-PCR confirmed the presence of exon 2-deleted CPEB4 transcripts in the KO brain. The doublet bands amplified from the WT or KO cDNA were resulted from the presence of two alternatively spliced isoforms (4a and 4b). PTC, premature termination codon.