Generation of Rgnef−/− mice.

<p>(A) Rgnef schematic. Depicted are the N-terminal leucine-rich (Leu), zinc finger-like (Zn), Dbl- and pleckstrin-homology (DH-PH), FAK binding region, and C-terminal coiled-coil (coil) domains. (B) Genetic targeting strategy to insert LoxP sites and a neomycin selectable marker (Neo) around <i>Rgnef</i> exon 24 by homologous recombination (Rgnef ^flox). Primer binding sites used for PCR screening are indicated (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0037830#pone-0037830-t003" target="_blank">Table 3</a>). Rgnef ^flox/flox mice were crossed with transgenic (Tg) cytomegalorvirus-driven Cre (CMV-Cre) recombinase expressing mice to inactivate Rgnef expression during early development (Rgnef ^flox, Cre+). (C) Representative PCR genotyping of progeny from Rgnef ^WT/flox (Cre+) mouse crosses: Rgnef ^WT/WT (582 bp), Rgnef ^WT/flox (582 and 125 bp), and Rgnef ^flox/flox (125 bp). Cre PCR genotyping (500 bp) is also shown. (D) No truncated Rgnef mRNA transcript detected in Rgnef ^flox/flox (Cre+) mice. Embryos were isolated at e13.5 from Rgnef ^WT/flox Cre+ mouse crosses, total RNA isolated, and analyzed by RT-PCR using primers 5′ to deleted exon 24 (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0037830#pone-0037830-t003" target="_blank">Table 3</a>) to Rgnef. GAPDH amplification is a positive control. (E) No Rgnef protein expressed in Rgnef ^flox/flox (Cre+) mice. Embryo lysates were immunoblotted with antibodies to Rgnef, FAK, and actin as a control. (F) Representative image of male littermates showing reduced size of Rgnef ^flox/flox (Cre+) mice at 4 weeks.