Figure 1.

Generation of p150- and p60-conditional knockout DT40 cells. (A and B) Schematic representations of the chicken p150 and p60 genome loci (top), and two targeted alleles changed by different targeting vectors (middle and bottom). Solid boxes and horizontal lines indicate positions of exons and introns. White boxes indicate drug resistance cassettes: hisD, histidinol dehydrogenase; neo, neomycin; bsr, blasticidin S; Cre-ER, cre-recombinase fused with part of estrogen receptor. The 3′ outer probes are indicated by gray lines. B, BamHI site; H, HindIII site; E, EcoRI site; ER, EcoRV site. (C) Southern blot analysis of wild-type (+/+), heterozygous mutant (+/−), and homozygous mutant (−/−) clones. Genomic DNA digested by indicated enzymes was hybridized with 3′ probes shown in A and B. The sizes of DNA fragments are shown on the left. (D). Western blot analysis for p150 and p60 expression after the addition of tet. Equal amounts of whole cell extracts prepared from DT40 (top and bottom), p150- (top) and p60 (bottom)-conditional knockout cells treated with tet for indicated times were immunoblotted with polyclonal antibodies against either chicken p150 or p60. HA-p150, endogenous p150, HA-p60, and endogenous p60 are indicated on the left.