The separation MK-2206 of hepatic parenchymal and nonparenchymal cells was performed essentially as previously described.24 The procedure is described in detail in the supporting information. A rat hepatic SEC line (NP31)25 was cultured on type I collagen–coated dishes (Iwaki, Chiba, Japan) in Dulbecco’s modified Eagle’s medium with 10% fetal bovine serum, penicillin (100 U/mL), and streptomycin (100 μg/mL) at 37°C with 5% CO2. A retrovirus vector (pMxIG)26 and a retrovirus packaging cell line (Plat-E)27 were used to
generate recombinant retroviruses. Hemagglutinin (HA)-tagged complementary DNAs (cDNAs) of full-length Cas (Cas FL) and a Cas mutant lacking the SH3 domain (Cas ΔSH3)28 were subcloned into pMxIG, and ecotropic retroviruses were produced by the transient transfection of Plat-E cells with viral vectors using FuGENE (Roche, Basel, Switzerland). Infection was performed in the presence of 8 μg/mL Polybrene (Sigma, St Louis, MO). After fixation Daporinad supplier in 4% paraformaldehyde in phosphate-buffered saline for 10 minutes, cells were permeabilized with 0.5% Triton X-100 in phosphate-buffered saline for 5 minutes at room temperature and incubated with Alexa 594–conjugated phalloidin (1:40; Invitrogen, Carlsbad, CA) in 1% bovine serum albumin in phosphate-buffered saline for 30 minutes at 37°C. Cells were mounted
with Vectashield MCE and observed on an Axioplan2 microscope with AxioCam MRm controlled by Axiovision software (Carl Zeiss, Germany). NP31 cells, cultured on glass cover
slips, were fixed in 2% glutaraldehyde buffered with a 1 M cacodylate buffer (pH 7.4) for 12 hours at 4°C and then with 1% osmium tetroxide in a cacodylate buffer (pH 7.4) for 1 hour at 4°C. After dehydration in a graded series of ethanol solutions, cells were dried to a critical point and sputter-coated with osmium. Cell surfaces were examined with an S-4300 scanning microscope (Hitachi, Tokyo, Japan) at a 30-kV accelerating voltage. To create a reduction-of-function Cas allele by gene targeting, we deleted exon 2 of the Cas gene, which encodes the entire SH3 and the N-terminal part of the SD domain containing one YLVP motif and four YQxP motifs. To this end, we constructed a targeting vector containing Cas exon 2 flanked by two locus of X-over P1 (loxP) sequences and followed by the Frt-flanked neomycin resistance (Neo) gene (Fig. 1A). When the floxed Cas exon 2 was correctly excised, exon 1 joined in frame to exon 3, and this resulted in a Cas transcript devoid of the exon 2–derived segment. Correctly targeted embryonic stem cells, identified by Southern blotting and genomic polymerase chain reaction (PCR; Fig. 1B, left and middle panels), were selected and used for the generation of heterozygous mice (Cas+/floxNeo).