Figure 1

Generation of ES cells with various Rex1 genotypes. A. Strategy for generation of Rex1-KO ES cells. The schematic maps of the Rex1 allele (top), the KO vector carrying the floxed pacEGFP-pA cassette (middle), and the KO allele generated by homologous recombination (bottom) were shown in scale. The EcoRI sites (E) provide the polymorphism between the wild-type and mutant alleles, 8.0 kb and 2.4 kb, respectively, on southern blot analysis using the indicated 3' external probe. B. Southern blot hybridization of wild-type (+/+), Rex1 heterozygous (+/-) and homozygous (-/-) ES cells using the EcoRI digestion and the 3' external probe. The expected sizes of wild-type (wt) and mutant (mut) bands were detected. The 5.6 kb fragment corresponds to the polymorphism of the Rex1 pseudogene on chromosome 15 reported previously as well as found in the mouse genome data. C. Northern blot analysis of Rex1 expression in wild-type (+/+), Rex1 heterozygous (+/-) and homozygous (-/-) ES cells. The Rex1 cDNA probe detects 1.8 kb mRNA from the wild-type allele and 3.5 kb mRNA, which is generated by inefficient function of the polyA addition signal in the pacEGFP-pA cassette, from the mutant allele. The Rex1 KO ES cells lack the wild-type transcript. D. Northern and western blot analysis of wild-type (+/+) and Rex1 KO (-/-) ES cells with the Rex1 transgene (Tg:+) or the empty vector (Tg:-). The 2.7 kb transcripts from the transgene were detected with or without the 2.2 kb endogenous transcripts in Northern blot with the Rex1 cDNA probe (top), in which equal loading of total RNA was confirmed by ethidium bromide staining of 28S and 18S ribosomal RNAs (middle). Western blot using anti-Rex1 antisera detects ~38 kd band in wild-type, wild-type+Tg and Rex1 KO+Tg lanes but not Rex1 KO lane (bottom), confirming the proper production of Rex1 protein from Tg. E. QPCR analysis of Rex1 expression in undifferentiated (+LIF) and differentiated (-LIF and EB) ES cells with various Rex1 genotypes. Three independent clones with each genotypes were cultured with or without LIF for 4 days or for formation of EBs for 5 days, analyzed separately with normalization by the amount of Gapdh, and plotted with standard deviation against the expression level in undifferentiated wild-type ES cells (wt) cultured with LIF, set as 1.0. The primer pair was set in the region deleted in the KO allele.