Generation of GASP-1 knockout mice.

<p><b>A</b>. Targeting vector design for generating GASP-1 KO mice. A cassette expressing the G418 resistance gene flanked by lox P sites was inserted into the intron upstream of the GASP-1 open reading frame (ORF) (intron 4) and a third loxP site was inserted in the intron downstream of the GASP ORF (intron 5). ES cells from C57/Bl6 mice were transfected with this vector. Properly targeted clones (see B, C) were transfected with Cre-recombinase and blastocysts from clones in which the GASP-1 ORF was disrupted were implanted into C57/BL6 females. B, C. Southern blotting analysis identified homologous recombination and single insertion using <b>B.</b> 5′ and <b>C.</b> Neo probes. <b>D.</b> Immuno-detection of GASP-1 in KO mice. Whole brain lysates from WT, HET and KO mice were prepared, separated by SDS-PAGE, transferred to nitrocellulose and immunoblotted for GASP-1 using a GASP specific antibody (upper panel) or β-Actin (lower panel) to control for protein loading.