Generation of <i>Cul4b</i> flox mice.

Abstract

<p>(A) Strategy for generation of <i>Cul4b</i> floxed targeting vector. On the top was shown the wild-type allele of <i>Cul4b</i> gene. The targeting vector and targeted allele were shown in the middle and at the bottom, respectively. <i>BamHI</i> (labeled B) and <i>XbaI</i> (labeled X) sites are indicated. Black bars indicating the positions of the probes used in Southern blots are also indicated. (B) Southern blot analysis of genomic DNA isolated from wild-type ES cells and selected ES cell clones. <i>BamHI</i> digested DNA was hybridized with 5′ probe and <i>XbaI</i> digested DNA was hybridized with 3′ probe. WT, wild-type allele; F, flox allele. (C) cDNA sequencing of <i>Cul4b</i> gene from the brain tissue of brain-specific knockout mice (<i>Cul4b</i>^flox/Y;<i>Nesin-Cre</i>). As predicted, exon 2 was spliced onto exon 6 after the excision of exons 3–5. (D) Real-time RT-PCR analysis of <i>Cul4b</i> mRNA isolated from brain tissues of wild-type males (<i>Cul4b</i>^+/Y;<i>Nestin-Cre</i>^+/−), wild-type females (<i>Cul4b^+/+</i>;<i>Nestin-Cre</i>^+/−), heterozygous females (<i>Cul4b</i>^+/flox;<i>Nestin-Cre</i>^+/−), and conditional knock-out males (<i>Cul4b</i>^flox/Y;<i>Nestin-Cre</i>^+/−). (E) Western blot analysis of Cul4b protein isolated from brain tissues of wild-type, heterozygous and conditional knockout mice using an anti-Cul4b antibody. Gapdh was used as a loading control.</p