A homozygous mutant embryonic stem cell bank applicable for phenotype-driven genetic screening

Journal name:
Nature Methods
Volume:
8,
Pages:
1071–1077
Year published:
DOI:
doi:10.1038/nmeth.1739
Received
Accepted
Published online

Abstract

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.

At a glance

Figures

  1. Experimental design.
    Figure 1: Experimental design.

    (a) Construction of the mutant ESC bank. (b) Modification of the Blm and Rosa26 loci in ESCs. Top, the tetracycline-controlled transactivator (tTA) and the tetracycline operator–minimal promoter (tetO-Pm) unit were knocked into both alleles of the second exon of the Blm gene. Bottom, the ERT2-iCre-ERT2 fusion gene was knocked into the Rosa26 locus. A single copy of the loxP or F3 site is left in the genome after removal of the selection cassettes used during the knock-in procedure. SA, splice acceptor; pA, polyadenylation signal; Dox, doxycycline. (c) Structure of the gene-trap vectors. Arrows indicate orientation of the neo (N) and puΔtk (P) genes. Pr, Pgk1 promoter. Note that two lox2272 sequences are inversely oriented and flipped upon Cre recombinase–mediated recombination, as depicted in d. For the retroviral vector, the structure of the provirus in which the 3′ FRT site is copied into the 5′ long terminal repeat (LTR) is presented. TIR, terminal inverted repeat. (d) Selection of homozygous mutants. Homozygous mutations are obtained by doxycycline-induced Blm suppression. The 4HT treatment activates Cre recombinase and inverts the orientation of neo and puΔtk genes. As a result, a fraction of homozygous mutants express both neo and puΔtk, allowing for double selection. The SNPs between 129 and C57BL/6 strains are rendered homozygous at the distal region of each vector insertion site, and this SNP homozygosity was used as a reference for the identification of homozygous mutation at the vector insertion site. One telomeric SNP was selected from each chromosome. Note that only the neo-puΔtk selection cassette is presented for the gene-trap vector. R, resistant; S, sensitive.

  2. Regulation of the cNP cassette.
    Figure 2: Regulation of the cNP cassette.

    (a) Experimental scheme. Regulation of the cNP cassette was analyzed in a mixed population of approximately 200 clones. (b) Inversion frequency was determined by the ratio of puromycin-resistant colonies to the total number of cells plated. The number of colonies in the absence of puromycin was used for normalization of plating efficiency.

  3. Isolation and characterization of homozygous mutants.
    Figure 3: Isolation and characterization of homozygous mutants.

    (a) Representative SNP analysis. PCR amplifications of C57BL/6 and 129 alleles were monitored for each double-resistant (G418 plus puromycin) clone with allele-specific SNP probes, which were labeled with different fluorescent reporter dyes (VIC or FAM). Both the homozygotes and non-homozygotes were derived from the same heterozygous clone. Genomic DNAs extracted from C57BL/6 and 129 mice were used as controls. (b) Summary of the screening for homozygous clones. (c) Representative results of locus-specific PCR analysis. Clones showing homozygosity by SNP analysis (as in a) were further analyzed at each gene-trap locus. Note that both homozygous (lane 2) and non-homozygous (lane 1) clones were observed at the Rap1b locus. p1–p3, primers; +/+, wild type/wild type; m/+, mutation/wild type. (d) Western blot analysis of homozygous mutant cells with insertions of the retroviral gene-trap vector. β-actin (Actb) was used as an internal control. m/m, mutation/mutation.

  4. Phenotypic analyses of Dgcr8 and Ptpn11 homozygous mutant cells.
    Figure 4: Phenotypic analyses of Dgcr8 and Ptpn11 homozygous mutant cells.

    (a) Scatter plots of the microRNA expression data from microarray analysis of wild-type and Dgcr8 homozygous mutant cells. Green lines indicate limits of the twofold change from the equivalent expression levels depicted by the red line. m/m, mutation/mutation. (b) Growth retardation of Dgcr8 homozygous mutant cells. Wild-type and Dgcr8 homozygous mutant cells were plated sparsely for clonal growth on feeder cells and cultured for 5 d. Doubling time of each cell type is shown with s.d. (n = 4). Scale bars, 500 μm. (c) Reduction of spontaneous differentiation in Ptpn11 homozygous mutant cells. ESCs were plated sparsely for clonal growth on gelatin-coated dishes in the absence of feeder cells and maintained for 7 d. Boxed areas in the top images are magnified in the bottom images. Large, flat-shaped differentiated cells were observed at the edge of the wild-type colonies, whereas no such cells were seen in the Ptpn11 homozygous mutant colonies. Scale bars, 500 μm (top) and 100 μm (bottom). (d) Top, sustained expression of Oct3/4 in embryoid bodies derived from Ptpn11 homozygous mutant cells. Embryoid bodies were immunostained after a 9-day suspension culture. Scale bars, 500 μm. Bottom, expression level of Oct3/4 mRNA was quantified by quantitative RT-PCR and normalized to β-actin mRNA expression level. (e) Continuous expression of SSEA-1 in Ptpn11 homozygous mutant cells. Embryoid bodies were dissociated into single cells and subjected to flow-cytometry analysis together with undifferentiated ESCs.

  5. Phenotypic analyses of homozygous mutant ESC lines.
    Figure 5: Phenotypic analyses of homozygous mutant ESC lines.

    (af) Defective neural differentiation of the Axin1 homozygous mutant cells. m/+, mutation/wild type; m/m, mutation/mutation. (a) Impaired neurosphere formation from Axin1 homozygous mutant ESCs. Scale bars, 500 μm. (b) Removal of mutagenic vector sequences by FLPo recombinase. Axin1r, revertant allele. Asterisk indicates nonspecific PCR bands. (c,d) Differential expression of lineage-specific markers in Axin1 mutants. The monolayer culture was stained with antibodies to Nestin (c) and TuJ-1 (d) 7 d and 14 d after induction of neural differentiation, respectively. Scale bars, 100 μm. (e,f) Expression of Nestin and TuJ-1 mRNAs was examined by quantitative RT-PCR and normalized by β-actin expression level. (g) Impaired self-renewal of Csnk2b and Ilf2 homozygous mutant ESCs. These clones were identified from the screen in Supplementary Figure 5. ESCs were cultured in 2i medium (MEK inhibitor and GSK3 inhibitor) for 5 d in the absence of feeder cells. Revertant clones (Csnk2br/r and Ilf2r/r) were obtained by the same procedure shown in b. Doubling time of each cell type is shown with s.d. (n = 3). Scale bars, 500 μm.

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Author information

Affiliations

  1. Department of Social and Environmental Medicine, Graduate School of Medicine, Osaka University, Suita, Osaka, Japan.

    • Kyoji Horie,
    • Chikara Kokubu,
    • Junko Yoshida,
    • Kosuke Yusa,
    • Ryuji Ikeda &
    • Junji Takeda
  2. Precursory Research for Embryonic Science and Technology, Japan Science and Technology Agency, Kawaguchi, Saitama, Japan.

    • Kyoji Horie
  3. Center for Advanced Science and Innovation, Osaka University, Suita, Osaka, Japan.

    • Chikara Kokubu,
    • Akiko Oshitani &
    • Junji Takeda
  4. Mouse Cancer Genetics Program, Center for Cancer Research, National Cancer Institute, Frederick, Maryland, USA.

    • Keiko Akagi
  5. Comprehensive Cancer Center, Ohio State University, Columbus, Ohio, USA.

    • Keiko Akagi
  6. Research Institute for Microbial Diseases, Osaka University, Suita, Osaka, Japan.

    • Ayako Isotani
  7. The Wellcome Trust Sanger Institute, Hinxton, Cambridge, UK.

    • Yue Huang &
    • Allan Bradley
  8. National Laboratory of Medical Molecular Biology, Institute of Basic Medical Sciences, Department of Medical Genetics, Peking Union Medical College & Chinese Academy of Medical Sciences, Beijing, China.

    • Yue Huang
  9. Present addresses: The Wellcome Trust Sanger Institute, Hinxton, Cambridge, UK (K.Y.) and Osaka Isen College of Medical Care & Welfare, Osaka, Osaka, Japan (R.I.).

    • Kosuke Yusa &
    • Ryuji Ikeda

Contributions

K.H. designed experiments, constructed vectors, performed ESC culture and phenotypic analyses of mutant ESCs, and wrote the manuscript. C.K. performed bioinformatics analyses and contributed to writing the manuscript. J.Y. conducted vector construction and ESC culture. K.A. performed bioinformatics analyses and constructed the database. A.I. generated chimeric mice. A.O. conducted ESC culture. K.Y. performed gene targeting of ESCs. R.I. conducted ESC culture and PCR genotyping of mutant ESCs. Y.H. and A.B. contributed to the vector design for the selection of homozygous mutants. J.T. conducted vector construction and gene targeting of ESCs, and contributed to writing the manuscript.

Competing financial interests

The authors declare no competing financial interests.

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Supplementary information

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    Supplementary Figs. 1-5, Supplementary Tables 1-6

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  1. Supplementary Data (504K)

    Vector insertion sites.

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