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. 2011 Apr;53(4):331-8.
doi: 10.1016/j.ymeth.2010.12.025. Epub 2011 Feb 1.

Bi-allelic gene targeting in mouse embryonic stem cells

Peri H Tate  1 , William C Skarnes
Affiliations

Bi-allelic gene targeting in mouse embryonic stem cells

Peri H Tate et al. Methods. 2011 Apr.

Abstract

The EUCOMM and KOMP programs have generated targeted conditional alleles in mouse embryonic stem cells for nearly 10,000 genes. The availability of these stem cell resources will greatly accelerate the functional analysis of genes in mice and in cultured cells. We present a method for conditional ablation of genes in ES cells using vectors and targeted clones from the EUCOMM and KOMP conditional resources. Inducible homozygous cells described here provide a precisely controlled experimental system to study gene function in a model cell.

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Figures

Fig. 1
Fig. 1
Bi-allelic targeting strategy. Step 1: Heterozygous KO-first alleles (tm1a) are converted to conditional alleles (tm1c) by transient expression of Flp recombinase. Step 2: The second allele is targeted by re-cycling the knockout-first targeting vector, generating bi-allelic ES cells that carry a conditional allele (tm1c) and a knockout-first allele (tm1a). Step 3: Inducible Cre-recombinase is introduced into cells by high efficiency targeted insertion into the Rosa26 locus. Step 4: Gene activity is eliminated in undifferentiated or differentiated ES cell cultures through activation of Cre-recombinase with tamoxifen (4-OHT), generating deletion alleles (tm1d and tm1b).
Fig. 2
Fig. 2
Summary information for Chd4 knockout-first targeted clones. The IKMC webportal (www.knockoutmouse.org/martsearch) displays the current status of genes assigned to individual targeting pipelines with order links for vectors, targeted ES cell clones and mutant mice available from affiliated repositories. Links to detailed molecular information are provided, including annotated sequences (genbank files) and maps (allele images) of the targeted alleles. Gene-specific primers used for long-range PCR genotyping can be found by following the ‘Design ID’ link.
Fig. 3
Fig. 3
LacZ staining of Flp-treated ES cell clones. X-gal staining of expanded clones after transient transfection of Chd4 knockout-first cells with Flp recombinase. A complete loss of β-galactosidase expression is observed in most clones (top and bottom left panels). Contamination of LacZ-positive cells can be detected in some of the wells (bottom right panel).
Fig. 4
Fig. 4
Identification of bi-allelic targeted clones by long-range PCR. The knockout-first allele is detected with a gene-specific primers outside of the 5′ homology (GF) and 3′ homology (GR) arms in combination with vector-specific primers to the En2 splice acceptor (En2R) and neo gene (NF), respectively. The conditional allele, but not the knockout-first allele is detected with GF and a primer to the 3× loxP site (LR). The knockout-first allele is >5 kb larger than the conditional allele, too large to be amplified with the GF/LR primer pair. In this example, clone 3 is positive for the knockout-first and conditional allele, indicating bi-allelic targeting. Clones 1 and 4 have re-targeted the conditional allele. The size of the amplified product in clone 5 is smaller than expected, suggesting a deletion has occurred in the locus.
Fig. 5
Fig. 5
Tamoxifen-induced ablation of Chd4 protein expression. Western blot of two Chd4 bi-allelic targeted clones (tm1c/tm1a) and a heterozygous targeted clone (tm1a/+) after two passages following treatment with ethanol (−4OHT) and tamoxifen (+4OHT). Activation of Cre recombinase in homozygous targeted clones removes the critical exon from the conditional targeted allele (tm1c) to generate the deletion allele (tm1d), eliminating expression of wild-type Chd4 protein (w.t.). The Chd4 antibody (Bethyl Laboratories A301-081A) also detects truncated Chd4 protein expressed from the tm1a and tm1b alleles.
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References

    1. Evans MJ, Kaufman MH. Nature. 1981;292:154–156. - PubMed
    1. Thomson JA, Itskovitz-Eldor J, Shapiro SS, Waknitz MA, Swiergiel JJ, Marshall VS, Jones JM. Science. 1998;282:1145–1147. - PubMed
    1. Bradley A, Evans M, Kaufman MH, Robertson E. Nature. 1984;309:255–256. - PubMed
    1. Guan K, Rohwedel J, Wobus AM. Cytotechnology. 1999;30:211–226. - PMC - PubMed
    1. Murry CE, Keller G. Cell. 2008;132:661–680. - PubMed

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