Establishment of Cell Lines from Mouse Embryos with Early Embryonic Lethality

Figures & data

Figure 1.  Generating FAK^loxP/loxPFN^−/ −p53^−/ − cell lines. (a) Genotyping of embryos from FAK^loxP/loxPFN^+/ −p53^−/ − crosses, by PCR for FN. Embryos #1, #4, and #10 were wild-type, #5 and #8 were FN^−/ − mutants, whereas others were FN^+/ −. Genomic DNA isolated from primary wild-type MEF and FN^−/ − cell line was used as a positive and negative control, respectively. (b) Outgrowth of fibroblast-like cells from E8.0–8.5 mouse embryo cultured in a drop of Matrigel for 3, 5, or 7 days. (c) Organization of FN matrix. Cells were cultured in the presence of 20 µg/ml exogenously added FN (Sigma) for 2 days. FN matrix organization is assessed by immunostaining of fixed and permeabilized cells. Rabbit polyclonal anti-FN antibody was from Sigma. FITC-conjugated donkey anti-rabbit antibody was from Jackson Immunoresearch.

Figure 2.  Generating FAK^−/ −FN^−/ −p53^−/ − cell lines. (a) PCR verification of knocking out FAK in vitro. Marker lane (Ma): the 100-bp marker; lanes 1 and 2: FAK^+/ + and FAK^−/ − control cell lines, respectively; lane 3: parental FAK^loxP/loxPFN^−/ −p53^−/ − line #5; lane 4: line #5 transduced with Cre-expressing adenovirus for 48 h; lane 5: single clone #5.41; lane 6: parental FAK^loxP/loxPFN^−/ −p53^−/ − line #8; lane 7: line #8 transduced with Cre-expressing adenovirus for 48 h; lane #8: single clone #8.36. PCR product size: FN⁺, 900 bp; FN⁻ 1060 bp; FAK⁺ 290 bp; FAK^loxP, 400 bp; Cre, 419 bp. The same pair of primers is used to detect both FAK⁺ (290 bp) and FAK loxP (400 bp) alleles. (b) Western blot analyses of FAK knockout in vitro. Lane 1, FAK wild-type control cell line; lane 2, FAK-null control cell line; lane 3, cell line 5 (FAK^loxP/loxPFN^−/ −p53^−/ −); lane 4, cell line 5 (FAK^loxP/loxPFN^−/ −p53^−/ −) plus Cre; lane 5, cell line 5.41 (FAK^−/ −FN^−/ −p53^−/ −) 48 h after adding Cre; lane 6, cell line 8 (FAK^loxP/loxPFN^−/ −p53^−/ −); lane 7, cell line 8 (FAK^loxP/loxPFN^−/ −p53^−/ −) plus Cre; lane 8, cell line 8.36 (FAK^−/ −FN^−/ −p53^−/ −) 48 h after adding Cre. Arrow indicates size of FRNK, independently expressed C-terminal region of FAK. GFP and Cre expression are detected 48 h upon infection with adenovirus and they are gone in single cell clones. Paxillin used as a loading control. (c) Western blot analysis of FAK expression in clones derived from cell lines #5 and #8 after in vitro deletion of floxed region of FAK with adnovirus-delivered Cre. FAK^+/ +, control cell line that express FAK; FAK^−/ −, negative control line obtained directly from FAK^−/ − embryos (Furuta et al. 1995). (d) Western blot analysis of Pyk2 expression and (auto)phosphorylation on Y402. FAK^+/ +, control cell line that express FAK; FAK^−/ −, negative control line obtained directly from FAK^−/ − embryos (Ilic et al. 1995). Actin was used as a loading control. Anti-FAK antibodies were purchased from BD Transduction Laboratories and from Santa Cruz Biotechnology. Anti-Pyk2 antibody was from from BD Transduction Laboratories, anti-paxillin and ant-GFP from Zymed, anti-phosphoY402 Pyk2 from BioSource, anti-Cre from Covance. All secondary Abs were from Jackson Immunoresearch.

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