Specifically, significantly regulated genes were placed at the top or bottom of the list and ordered by descending or ascending fold changes, respectively. Less significantly regulated Wnt inhibitor manufacturer genes were placed in the middle of the list and ordered by ascending or descending p values. GO terms with the top ten Normalized Enrichment Scores were selleck chemical selected and combined from both the upregulated and downregulated GO terms in each time point, compared to the 0 min treatment condition. 2 function in the gplots package in R U0126 clinical with default parameters. In the present study, we used two germ line competent haploid mouse ESC lines, H129 2 and HAP 1. After the enrichment of haploid cells by flow sorting, H129 2 and HAP 1 ESCs were treated with 0. 25 and 0. 20 mgml of ENU, respectively. For the purpose of further increasing the mutation rate, pretreatment with the alkyltransferase inhibitor O6 benzylguanine was also included in the experimental design. Most cells were killed by these treatments, and the re sidual surviving cells were treated with 1 nM toxin to select for GPI anchor pathway mutants. A total of 114 resistant clones were separately isolated and subjected to the previously described functional complementation assay involving transfection with various combinations of plasmids encoding the 26 candidate genes. As a reporter, we co transfected ESCs with a plasmid encoding green fluorescent protein fused with a GPI anchor attachment motif. Only when the mutated gene were exogen ously restored did the cells express GFP at their surface, enabling mutants to be identified by their fluorescence. Representatively, four ESC clones were also analyzed by Sanger sequencing and their causative mutations were detected in the corresponding GPI pathway genes. Eventually, all isolated 114 clones harbored at least one mutant allele in any of 20 known genes involved in the GPI anchor biosynthetic pathway. Among the 114 mutant ESC clones, clone B502 exhibited fluorescence when transfected with a mixture of cDNA expressing vectors for all known GPI anchor pathway genes, not when transfected with a single gene plasmid. A series of step wise reductions in the repertoire of cDNA expressing vectors revealed that both Dpm1 and Pigv cDNAs were essential to restore the fluorescence. Sanger sequencing of genomic DNA from the B502 ESC clone identified causative point mutations of both genes. Thus, the appear ance of this double mutant in our screening suggests that the mutagenicity of ENU is of a sufficiently high level for saturation mutagenesis. Haploid ESCs have an inherent tendency to become diploid during culture.
This process, autodiploidiza tion, resulted in undesirable but inevitable contamination of the diploidized ESCs. In this study, 86. 0% of ESCs remained haploid at the point of ENU treatment and the rest were diploid. In the diploidized ESCs, because either of the duplicated X chro mosomes could undergo X inactivation or chromosomal loss, an ENU induced mutation on the other allele of the X linked gene would immediately lead to a complete loss of function. This meant that, in the mixture of hap loid and diploid ESCs, X linked mutants would be more frequently obtained than autosomal recessive mutants.