Production of p53 gene knockout rats by homologous recombination in embryonic stem cells

Abstract

The use of homologous recombination to modify genes in embryonic stem (ES) cells provides a powerful means to elucidate gene function and create disease models1. Application of this technology to engineer genes in rats has not previously been possible because of the absence of germline-competent ES cells in this species. We have recently established authentic rat ES cells2,3. Here we report the generation of gene knockout rats using the ES-cell-based gene targeting technology. We designed a targeting vector to disrupt the tumour suppressor gene p53 (also known as Tp53) in rat ES cells by means of homologous recombination. p53 gene-targeted rat ES cells can be routinely generated. Furthermore, the p53 gene-targeted mutation in the rat ES-cell genome can transmit through the germ line via ES-cell rat chimaeras to create p53 gene knockout rats. The rat is the most widely used animal model in biological research4,5,6,7. The establishment of gene targeting technology in rat ES cells, in combination with advances in genomics and the vast amount of research data on physiology and pharmacology in this species, now provide a powerful new platform for the study of human disease.

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Figure 1: Schematic diagram showing the strategy to disrupt the rat p53 gene via homologous recombination.
Figure 2: Confirmation of p53 gene targeting in rat ES cells.
Figure 3: Germline transmission of the p53 gene-targeted mutation in the rat.
Figure 4: Generation of p53 gene knockout rats.

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Acknowledgements

This work was supported by a NIH/NCRR grant to Q.-L.Y. (1R01 RR025881). We thank members of the Ying laboratory and the USC Stem Cell Core for technical assistance; G. Chester for ordering rats; R. Montano and colleagues for rat husbandry; T. Saunders, E. Bryda and M. Pera for critical reading of the manuscript; E. Kho for financial support. We are also grateful to M. Pera and R. Maxson for their support and encouragement.

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Contributions

C.T. designed and performed most of the experiments described in the paper. P.L. and Q.-L.Y. derived and cultured rat ES cells. N.L.W. and Y.Y. performed blastocyst injections. Q.-L.Y. conceived the study and wrote 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 Inc.

Additional information

The p53 gene-targeted rats generated in this study will be deposited in the Rat Resource and Research Center at University of Missouri.

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Supplementary Information

This file contains Supplementary Table 1 and Supplementary Figures 1-5. (PDF 371 kb)

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Tong, C., Li, P., Wu, N. et al. Production of p53 gene knockout rats by homologous recombination in embryonic stem cells. Nature 467, 211–213 (2010). https://doi.org/10.1038/nature09368

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