Targeted disruption of the mouse ESG1 gene
To study the function of ESG1, we deleted the gene by homologous recombination in mouse ES cells. We replaced the three exons with either a fusion of the neomycin-resistance and β-galactosidase genes (β-geo) or the hygromycin resistant gene (HygR) using two targeting vectors (Figure 4A) introduced into RF8 ES cells by electroporation. We obtained eight ES cell clones with correct homologous recombination of the β-geo targeting vector, which was confirmed by Southern blot analysis (Figure 4B). We obtained only one clone with correct homologous recombination of the HygR targeting vector.
Figure 4  Targeted disruption of the mouse ESG1 gene. A) Targeting strategy. Homologous regions are indicated by thick lines. Recognition sites of PstI (P) and SpeI (S), which were used for Southern blot analyses, are shown. The gene encoding diphtheria toxin A (DTA) was inserted at the 3' end of the targeting vectors to facilitate negative selection. B) Southern blot analyses confirming homologous recombination. WT, wild-type ES cells; β, β-geo +/- ES cells; H, HygR +/- ES cells; -/-, ESG1-null ES cells. Numbers indicate clone numbers. C) Northern blot (upper) and western blot (lower) analyses of wild-type ES cells (WT), ESG1-null ES cells (-/-, three clones) and heterozygous ES cells (+/-). Northern blot was performed as previously described [20]. To confirm the loading of equal amounts of RNA, ethidium bromide staining of ribosomal RNA is also shown (middle). To obtain homozygous mutant ES cells, we introduced the β-geo vector into HygR heterozygous ES cells. Of 105 G418-resistant colonies tested, 49 were homozygous for ESG1 deletion. Northern blot and western blot analyses confirmed the absence of ESG1 in these cells (Figure 4C). In 29 clones, the β-geo vector had replaced the HygR vector, such that the cells remained heterozygous. In the remaining 27 clones, the β-geo vector was integrated at non-homologous sites.
ESG1-/- ES cells exhibited normal morphology (Figure 5A). These cells also proliferated at a speed comparable to that of the control (heterozygous and wild-type) cells (Figure 5B). ESG1-/- cells differentiated normally after the removal of leukemia inhibitory factor (Figure 5B) or retinoic acid treatment (not shown). When transplanted into hind flanks of nude mice, these cells produced teratomas, tumors containing components of all three germ layers (Figure 5C). These results indicate that ESG1 is dispensable for the self-renewal properties and pluripotency of ES cells.
Figure 5  Analyses of ESG1-null ES cells. A) The morphology of ESG1-null ES cell colonies grown on STO feeder cells. B) Growth curve of wild-type (WT), ESG1-null (-/-) and heterozygous (+/-) ES cells. Each clone (1 × 104 cells/well) was plated in 24-well plates. Cell numbers were determined with a Coulter counter at 2, 4, and 6 days. Data of +/- and -/- cells are shown as averages and standard deviations of three independent clones. C) A section of teratoma derived from ESG1-null ES cells (hematoxylin & eosin staining). We examined the gene expression profiles of ESG1-/- ES cells using oligonucleotide-based DNA microarrays representing ~20,000 genes. In comparison to control ES cells, ESG1 was identified as the gene reduced to the greatest extent in ESG1-/- ES cells (Figure 6A). The expression of ES cell marker genes, such as Nanog and Oct3/4, was normal in ESG1-/- ES cells. We confirmed normal Oct3/4 expression at protein levels by Western blot (Figure 6B). The overall gene expression profiles were similar between control ES cells and ESG1-/- ES cells. Several genes exhibited a greater than two-fold reduction in ESG1-/- cells, including Krt1-8, Pem, Ctgf, Ptgs2, Igf2 and Inhba. These genes might be regulated directly or indirectly by ESG1. Since ESG1 contains a KH-type RNA-binding domain, it may stabilize mRNA of these genes. Further studied are required to clarify this possibility.
Figure 6  Gene expression analyses of ESG1-null ES cells. A) DNA microarray analyses. Total RNA from wild-type ES cells and ESG1-null ES cells were labeled with Cy3 and Cy5, respectively. Samples were hybridized to Agilent Mouse Developmental Microarrays. The averages of two independent clones are shown. B) Western blot analyses. Cell lysates from ESG1+/- and ESG1-/- cells were examined for expression of ESG1, Oct3/4 and CDK4 with immunoblotting. To generate ESG1-knockout mice, we injected β-geo-ESG1+/- ES cell clones into the blastocysts of C57BL6 mice. We obtained germline transmission from three clones. We obtained ESG1-/- mice at the Mendelian ratios (36 wild-type, 69 ESG1+/-, and 45 ESG1-/-) from intercrosses of ESG1+/- mice. These animals exhibited normal development, gross appearance, and fertility (not shown). Histological examination of testis and ovary could not identify any abnormalities (not shown). These data demonstrated that ESG1 is dispensable for both mouse development and germ cell formation.
We also generated ES cells from blastocysts obtained by intercrosses of ESG1+/- males and ESG1-/- females. Of the eight ES cell lines established, two clones were ESG1-/-. These ESG1-null ES cells demonstrated normal morphology, proliferation, and differentiation (not shown), confirming that ESG1 is dispensable in ES cells.