Establishment of KRIT1-Null and KRIT1-Expressing MEF Cells

KRIT1−/− and KRIT1+/+ mouse embryonic fibroblasts (MEFs) were obtained by Dr. Luca Goitre in the laboratory of Cell Biology, under the supervision of Prof. Saverio Francesco Retta, at the Department of Clinical and Biological Sciences of the University of Torino. Specifically, KRIT1−/− and KRIT1+/+ mouse embryonic fibroblast (MEF) isogenic cell lines were established from KRIT1−/− and KRIT1+/+ E8.5 mouse embryos, respectively, using the 3T3 protocol [45 (link)], and were previously described [17 (link),42 (link)]. The KRIT1 gene was inactivated by homologous recombination, using a vector obtained by Dr. Luca Goitre, deleting a 631 bp genomic region encompassing 77 nucleotides of the first exon, including the ATG codon, and 554 bp of the upstream 59 untranslated sequence, and replacing this region with a pMC1-Neo-Poly(A) cassette. KRIT1+/+ cells were derived from KRIT1−/− MEFs by lentiviral re-expression of KRIT1 in order to obtain KRIT1-null and KRIT1-expressing MEF cells with uniform genetic backgrounds to be used for comparative molecular and cellular biology studies [17 (link),42 (link)]. Specifically, KRIT1−/− MEF cells were infected with a lentiviral vector encoding KRIT1 (pCCLsin.PPT.PGK.KRIT1.Wpre, obtained by Dr. Luca Goitre) to restore KRIT1 expression. Cells were cultured in DMEM (Gibco) containing 10% v/v heat-inactivated fetal bovine serum (FBS; Gibco), 2 mM glutamine (Gibco), 10 U/mL penicillin (Invitrogen), and 10 µg/mL streptomycin (Invitrogen), at 37 °C and 5% CO₂.