Generation of THOC5/FMIP (flox/flox) and Mx-cre THOC5/FMIP (flox/flox) mice. (A) The genomic structure of the 33,523 kb THOC5/FMIP locus is depicted. THOC5/FMIP deficient mice were generated using a genomic DNA fragment containing exons II, III, IV, and V isolated from 129/ola genetic background. The targeting vector was constructed from a 3.9 kb PCR fragment and an adjacent 2.2 kb fragment harboring exons IV/V. Fragments were introduced into the pPNTloxPneo vector via NotI, XhoI restriction sites and KpnI, respectively. An additional loxP site was inserted into the SacI site downstream of exon V: (B) After transfection, ES cell clones carrying the inserted floxed neo cassette and floxed Fmip exons IV/V were identified by the presence of an 1.9 kb BamHI restriction fragment in Southern Blot analysis with external 3'probe ((*) THOC5/fmip neo flox/flox). (C) Mx-cre THOC5/FMIP (flox/flox) were sacrificed before (zero day) and after two (one × poly (I:C) injection), four (two × poly (I:C) injection) and seven (three × poly (I:C) injection) days after the first poly (I:C) injection and genomic DNAs were extracted from liver, spleen, and bone marrow. Genomic DNAs were supplied for the deletion of exons IV/V determined by PCR using 5'-TGCTGGCATTGAACTGTG-3' and 5'-CAGCACTGGAGCGGGAGATGT-3'. PCR product: wild type allel: 1110 bp; THOC5/FMIP del allele: 355 bp.
Mancini et al. BMC Biology 2010 8:1 doi:10.1186/1741-7007-8-1