Journal: PLoS ONE
Article Title: In Vivo Functional Requirement of the Mouse Ifitm1 Gene for Germ Cell Development, Interferon Mediated Immune Response and Somitogenesis
Figure Lengend Snippet: Schematic view of the Ifitm1 targeting strategy by homologous recombination in mouse ES cells. ( A ) Shows the scheme of the linearized targeting vector used for electroporation in mouse ES cells. The coding sequence of the lacZ reporter gene (lacZ) was inserted in frame into the first codon of the Ifitm1 gene. The three Ifitm1 exons are indicated as I, II, and III. The lacZ gene is followed by a poly-adenylation signal (bGHpA) and a neomycin/kanamycin selection cassette (neo/kan) that was flanked by loxP sites (L) for subsequent deletion of the cassette. The backbone of the vector (vb) included the coding sequence for the diphtheria toxin fragment A (DTA) to support the selection of clones carrying the homologous recombination of the targeting vector. The location of the probe used for Southern blotting (sp) and PvuII restriction sites (P) are also indicated. The PvuII restriction sites that are relevant for the diagnostic restriction fragments in Southern blots in combination with DNA probe sp are marked with an underline ( P ). ( B ) Shows a scheme of the wildtype Ifitm1 locus on mouse chromosome 7. The three Ifitm1 exons (I, II, and III) are indicated as boxes. Black shading in the boxes indicates the coding sequence of Ifitm1 . The genomic region that was amplified by PCR to genotype the Ifitm1 wt allele is indicated (wt-PCR). ( C ) Shows the Ifitm1 locus following homologous recombination in mouse ES cells. As result of recombination, the complete Ifitm1 coding sequence is replaced by the lacZ reporter gene and the selection cassette (lacZ-bGHpA-Lneo/kanL). The genomic region that is covered by the homologous sequence of the targeting vector (tv) is designated. The genomic region that was amplified by PCR to genotype the targeted Ifitm locus in the electroporated ES cells following G418 selection is indicated (ES-PCR). ( D ) Shows a scheme of the Ifitm1 tm1IEG loss-of-function allele that was generated by expression of the Cre recombinase and subsequent deletion of the neo/kan selection cassette from the targeted Ifitm1 allele shown in ( C ). As result of this excision, a single loxP site (L) is left behind as indicated. The genomic region that was amplified by PCR to genotype the targeted Ifitm tm1IEG loss-of-function allele in mice obtained from matings between chimeric mice and Cre expressing mice is indicated (flox-ko-PCR). The size bar (bottom left) indicates 1 kb length.
Article Snippet: DNA was isolated from ES cells (and later from mouse tails) by standard procedures and 15 µg of genomic DNA were digested with PvuII restriction enzyme (MBI Fermentas, Germany) over night.
Techniques: Homologous Recombination, Plasmid Preparation, Electroporation, Sequencing, Selection, Clone Assay, Southern Blot, Diagnostic Assay, Amplification, Polymerase Chain Reaction, Generated, Expressing, Mouse Assay