Fig. 1

Functional silencing of the Grsf1 gene [C57BL/6J Grsf1tm1a(EUCOMM)Wtsi]. An artificial knockout exon was cloned between two FRT recognition sites (flipase recognition target) and this construct was used for transfection of mouse embryonic stem cells. This exon involves a splice acceptor side (En2 SA), an internal ribosomal insertion site (IRES), an open reading frame encoding for the beta-galactosidase gene (LacZ) and a polyadenylation sequence (PA). This sequence is followed by a loxP sequence and a neomycin resistance cassette consisting of the human beta-actin (hBactP) promoter and a downstream polyadenylation signal (PA). Immediately downstream of the PA signal second FRT sequence was included. Finally, the construct involves two unidirectional loxP sequences flanking exons 4 and 5. Stem cells that have incorporated this construct by homologous recombination were used for blastocyst injection and chimeric mice were bred. Crossing individuals containing this additional exon with FLT delete mice removes the sequences between the FRT signals but maintains the loxP flanked exons 4 and 5 (Grsf1-floxed mice). When these Grsf1-floxed mice were crossed with Cre-deleter mice, in which expression of the Cre recombinase is governed by unspecific or tissue specific promoters, tissue specific inactivation of the Grsf1 gene can be achieved. To induce systemic Grsf1 knockout in all cells and tissues we employed CMV-Cre mice (B6.C-Tg(CMV-cre)1Cgn/J) and this strategy truncated the Grsf1 gene in all cells and tissues removing exons E4 and E5