Journal: Nucleic Acids Research

Article Title: An efficient method for generation of bi-allelic null mutant mouse embryonic stem cells and its application for investigating epigenetic modifiers

doi: 10.1093/nar/gkx811

Figure Lengend Snippet: Schematic of allele structures in second allele targeted and revertant ES cells and cell line validation. ( A ) Insertion-type targeting vector pI_hygGFP for inactivation of the WT allele in ES cells heterozygous for a standard IKMC knockout-first allele ( tm1a ). ( B ) Structure of the bi-allelic locus after targeting the second allele. The tm2 allele contains the pI_hygGFP targeting cassette and duplicated homology region, where exon 2 is re-generated by gap repair. ( C ) Western blots of Cbx1 and Jarid2 parental IKMC heterozygous ES cells (B01 and E08 lines), and examples of cell lines following pI_hygGFP electroporation including doubly targeted ES cells ( 1a/2 ) showing the absence of protein expression, and failed targeting events ( 1a/+ ). ( D ) Reversion from null mutant ( 1a/2 ) to conditional mutant ( 1c/2 ) by Flp recombinase. ( E ) Western blots of mutant ( 1a/2 ) and reverted ( 1c/2 ) Cbx1 and Jarid2 ES cell lines showing re-expression of protein. Primers for LR-PCR genotyping are indicated by small arrows and α-tubulin was used for Western blot loading controls. ( F ) Rescue of Polycomb PRC1 recruitment to PRC2 target genes in Jarid2 revertant cell lines ( 1c/2 ), shown by reinstatement of Mel18 binding at known Jarid2-dependent gene promoter regions, assessed by chromatin immunoprecipitation (ChIP)-qRT-PCR. Hprt is a control locus known to be negative for PRC1 binding. Results show mean ± s.d. of three biological replicates (independent cell lines), where values are expressed as relative fold-enrichment over 10% input chromatin. Asterisks indicate statistically significant differences between Jarid2 revertant ( 1c/2 ) and null ( 1a/2 ) cell lines ( P < 0.05, one-tailed Student’s t -test).

Article Snippet: For each transformation, 50 μl of the electrocompetant cells were gently mixed with 2 μl of the Gateway reaction, transferred to a pre-cooled electroporation cuvette (0.1 cm gap, BioRad GenePulser) and electroporated using EC1 setting (BioRad MicroPulser).

Techniques: Biomarker Discovery, Plasmid Preparation, Knock-Out, Generated, Western Blot, Electroporation, Expressing, Mutagenesis, Long Range PCR, Binding Assay, Chromatin Immunoprecipitation, Quantitative RT-PCR, Control, One-tailed Test

Journal: Nucleic Acids Research

Article Title: An efficient method for generation of bi-allelic null mutant mouse embryonic stem cells and its application for investigating epigenetic modifiers

doi: 10.1093/nar/gkx811

Figure Lengend Snippet: Targeting frequencies following electroporation of insertion vectors pI_hygGFP into WT JM8 (parental or subcloned N4 or F6 lines as indicated) and heterozygous mutant tm1a/ + (knockout first) ES cells, selection in hygromycin and screening/genotyping by long range-PCR on genomic DNA

Article Snippet: For each transformation, 50 μl of the electrocompetant cells were gently mixed with 2 μl of the Gateway reaction, transferred to a pre-cooled electroporation cuvette (0.1 cm gap, BioRad GenePulser) and electroporated using EC1 setting (BioRad MicroPulser).

Techniques: Electroporation, Mutagenesis, Knock-Out, Selection, Long Range PCR, Clone Assay