A homozygous mutant embryonic stem cell bank applicable for phenotype-driven genetic screening.
Genome-wide mutagenesis in mouse embryonic stem cells (ESCs) is a powerful tool, but the diploid nature of the mammalian genome hampers its application for recessive genetic screening. We have previously reported a method to induce homozygous mutant ESCs from heterozygous mutants by tetracycline-dependent transient disruption of the Bloom's syndrome gene. However, we could not purify homozygous mutants from a large population of heterozygous mutant cells, limiting the applications. Here we developed a strategy for rapid enrichment of homozygous mutant mouse ESCs and demonstrated its feasibility for cell-based phenotypic analysis. The method uses G418-plus-puromycin double selection to enrich for homozygotes and single-nucleotide polymorphism analysis for identification of homozygosity. We combined this simple approach with gene-trap mutagenesis to construct a homozygous mutant ESC bank with 138 mutant lines and demonstrate its use in phenotype-driven genetic screening.
Department of Social and Environmental Medicine, Graduate School of Medicine, Osaka University, Suita, Osaka, Japan. horie@mr-envi.med.osaka-u.ac.jp
Kyoji
K
Horie
Chikara
C
Kokubu
Junko
J
Yoshida
Keiko
K
Akagi
Ayako
A
Isotani
Akiko
A
Oshitani
Kosuke
K
Yusa
Ryuji
R
Ikeda
Yue
Y
Huang
Allan
A
Bradley
Junji
J
Takeda
D000818:Animals; D018070:Biological Specimen Banks; D053595:Embryonic Stem Cells / Q000166:cytology / Q000378:metabolism*; D023281:Genomics; D005839:Gentamicins / Q000494:pharmacology; D006720:Homozygote; D051379:Mice; D009154:Mutation / Q000235:genetics*; D010641:Phenotype; D020641:Polymorphism, Single Nucleotide / Q000235:genetics; D054592:Protein Tyrosine Phosphatase, Non-Receptor Type 11 / Q000235:genetics; D011506:Proteins / Q000235:genetics; D011691:Puromycin / Q000494:pharmacology; D016601:RNA-Binding Proteins; D014015:Tissue Banks
C474044:Dgcr8 protein, mouse; D005839:Gentamicins; D011506:Proteins; D016601:RNA-Binding Proteins; D011691:Puromycin; C010680:antibiotic G 418; D054592:Protein Tyrosine Phosphatase, Non-Receptor Type 11; C516725:Ptpn11 protein, mouse