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Generating gene knockout rats by homologous recombination in embryonic stem cells


We describe here a detailed protocol for generating gene knockout rats by homologous recombination in embryonic stem (ES) cells. This protocol comprises the following procedures: derivation and expansion of rat ES cells, construction of gene-targeting vectors, generation of gene-targeted rat ES cells and, finally, production of gene-targeted rats. The major differences between this protocol and the classical mouse gene-targeting protocol include ES cell culture methods, drug selection scheme, colony picking and screening strategies. This ES cell–based gene-targeting technique allows sophisticated genetic modifications to be performed in the rat, as many laboratories have been doing in the mouse for the past two decades. Recently we used this protocol to generate Tp53 (also known as p53) gene knockout rats. The entire process requires 1 year to complete, from derivation of ES cells to generation of knockout rats.

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Figure 1
Figure 2: Schematic diagram showing the strategy for constructing a rat gene-targeting vector.
Figure 3
Figure 4: Diagram of the strategy for constructing a control vector used for optimizing PCR screening conditions.
Figure 5: ES cell–rat chimera and its germline pups.
Figure 6: Confirmation of p53 gene targeting in rat ES cells and rats by Southern blot and genotyping.
Figure 7: Rat ES cells derived and maintained in the 2i condition.
Figure 8: DA rat ES colonies formed after electroporation (ac).


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We thank N. Wu and Y. Yan for blastocyst injection; G. Chester for ordering rats; R. Montano and colleagues for rat husbandry; and T. Saunders for scientific input. This work was funded by a US National Institutes of Health National Center for Research Resouces grant (R01 RR025881).

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Authors and Affiliations



C.T. and Q.-L.Y. designed the study. C.T., G.H. and P.L. conducted the experiments. G.H. and C.T. wrote a draft of the paper. C.A. and Q.-L.Y. proofread and finalized the paper.

Corresponding author

Correspondence to Qi-Long Ying.

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Competing interests

Q.-L.Y. is an inventor on a patent relating to this study filed by the University of Edinburgh and licensed to StemCells.

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Tong, C., Huang, G., Ashton, C. et al. Generating gene knockout rats by homologous recombination in embryonic stem cells. Nat Protoc 6, 827–844 (2011).

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