Rex1/Zfp42 is dispensable for pluripotency in mouse ES cells

We showed the function of Rex1 in mouse ES cells as well as in embryos using the conventional gene targeting strategy. Our results clearly indicated that Rex1 function is dispensable for both the maintenance of pluripotency in ES cells and the development of embryos. However, Rex1-/- ES cells showed the defect to induce a subset of the marker genes of visceral endoderm, when differentiated as embryoid body, as found in EC cells. Conclusion: Rex1 should be regarded just as a marker of pluripotency without functional significance like the activity of alkaline phosphatase. Keywords: genetic modification design,reference design,replicate design We established ES cell lines with four different genotypes for Rex1; ES cells carrying the wild-type Rex1 alleles and the empty CAG-IZ vector (wt), the wild-type Rex1 alleles and the Rex1 transgene (wt-Tg), the Rex1-/- alleles and the empty vector(KO), and the Rex1-/- alleles and the Rex1 transgene (KO-Tg). The genetically-engineered ES cell lines were generated to analyze the function of Rex1 in the maintenance of pluripotency and to analyze its gain- and loss-of function. For loss-of-function analysis, we disrupted the endogenous Rex1 allele by conventional gene targeting via homologous recombination in ES cells. The knock-out (KO) allele should be a functionally null allele because the first 100 bp of the open reading frame in the exon 4 including the start codon was replaced by the pacEGFP chimeric gene cassette containing the puromycin-resistant gene (pac) and the green fluorescent protein (Egfp) cDNA. Interestingly, all of the puromycin resistant clones obtained by transfection of this KO vector carried the correctly targeted alleles. One of the Rex1+/- ES cell line (RKPG9) was cultured with high-dose puromycin to obtain the Rex1-/- ES cell lines generated via spontaneous gene conversion. Multiple Rex1-/- ES cell lines were established with extremely high efficiency (4 of 4 clones obtained after the selection were homozygous for Rex1 KO allele). Correct targeting events were confirmed by the loss of the polymorphic signature of the wild-type allele on the southern blot analysis of the genomic DNA, in which the 5.6 kb fragment corresponds to the Rex1 pseudogene on chromosome 15 reported previously as well as found in the mouse genome data. Northern blot revealed the loss of the transcript derived from the wild-type allele in Rex1-/- ES cells, which express the large transcripts composed by the truncated Rex1 and pacEGFP. Rex1-/- ES cells were also established by introduction of the second knockout vector carrying the hygromycin-resistant gene as a selection marker. Rex1+/- ES cell line (RKPG9), Rex1-/- ES cells (HP3 and HP4), and one wild-type ES cells (EB5)