Establishment of cell lines from mouse embryos with early embryonic lethality

Abstract

It is often difficult to determine molecular mechanisms leading to early embryonic lethality of genetically modified mice due to lack of cells for further analyses. The authors describe here establishment of mouse embryonic fibroblast (MEF) cell lines from gastrulation stage embryos. In this example, using a combination of in vivo and in vitro techniques, the authors successfully generated MEF cell lines that lack both fibronectin (FN) and focal adhesion kinase (FAK).

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/nl exogenously added FN for 2 days. FN matrix organization is assessed by immunostaining of fixed and permeabilized cells.

Figure 2. Generating FAK^−/−FN^−/−p53^−/− cell lines

(a) PCR verification of knocking out fak in vitro. Lane 1: the 100 bp Marker; lanes 2 and 3: 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. (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. (d) Western blot analysis of Pyk2 expression and (auto)phosphorylation on Y402. FAK^+/+, control cell line that express FAK; FAK^−/−, FAK^−/− is negative control line obtained directly from FAK^−/− embryos. Actin was used as a loading control.