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 models. 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 cells. 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 research. 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.

Figure 1. Schematic diagram showing the strategy to disrupt the rat p53 gene via homologous recombination

a, Structures of the wild-type (WT) rat p53 gene allele and the rat p53 gene targeting vector. b, The predicted structure of the gene-targeted rat p53^{tm1(EGFP-pac)} allele.

Figure 2. Confirmation of p53 gene targeting in rat ES cells

a, Phase-contrast and fluorescence images of DAc8-p53-#1 rat ES cells. Scale bar, 50μm. b, Southern-blot analysis of p53 gene-targeted rat ES cells using 5′, 3′ or internal probes. For Southern blot analysis with 5′ or internal probes, genomic DNA from rat ES cells was digested with SpeI. For Southern blot analysis using 3′ probe, rat ES cell genomic DNA was digested with HindIII. The expected sizes of wild-type and p53 gene-targeted bands with different probes are shown in Fig. 1. c, The diagram showing the positions of two PCR primer pairs. d-g, Sequence alignment of PCR products amplified from DAc8-p53-#1 rat ES cells. The sequence of the PCR product (Query) amplified using the first pair of primers (F1 and R1) was aligned with the sequences of the rat genome (d) or the p53 gene targeting vector (e). The sequence of the PCR product amplified using the second pair of primers (F2 and R2) was aligned with the sequences of the p53 gene targeting vector (f) or the rat genome (g). The highlighted sequences represent the junctions between the homology arms and the rat genome (d and g) or the CAG-EGFP-IRES-Pac cassette (e and f).

Figure 3. Germline transmission of the p53 gene-targeted mutation in the rat

a, The male chimera generated from the DAc8-p53-#1 subclone. The agouti coat color and the appearance of GFP-positive tissues indicate that p53 gene-targeted DAc8 rat cells were present in the ES cell-rat chimera. b, Offspring produced by breeding the male chimera shown in (a) with SD female rats. c, Diagrams showing the positions of three PCR primers designed for genotyping p53 gene-targeted offspring. F1: gcg ttg ctc tga tgg tga c; F2: tgc ggt ggg ctc tat ggc ttc t; R: cag cgt gat gat ggt aag gat. The expected sizes of PCR products for wild-type and p53 gene-targeted alleles are 309bp and 498bp, respectively. d, PCR genotyping analysis of the p53 gene-targeted allele. M, 100bp DNA marker; 1, DAc8 rat ES cells; 2, DAc8-p53-#1 rat ES cells; 3, 6, and 8, the three GFP-negative germline offspring; 4, 5, and 7, the three GFP-positive offspring; 9 and 10, the two albino littermates. e, Southern blot analysis for the p53 gene-targeted allele using the 5′ probe. 1, 4, and 6 are the three GFP-negative germline offspring; 2, 3, and 5 are the three GFP-positive offspring.

Figure 4. Generation of p53 gene knockout rats

a, Offspring generated by intercrossing p53^{tm1(EGFP-pac)} heterozygote rats. b, Genotyping analysis on tail biopsies using PCR primers shown in Fig. 3c. M, 100bp DNA marker; 1–9, the nine GFP-positive offspring; 3 and 7, the two p53^{tm1(EGFP-pac)} homozygote pups; 10–12, the three GFP-negative offspring. c, Detection of p53 mRNA by Northern blot. Northern analysis was performed by sequential hybridization with probes for p53 and β-actin. d. Detection of p53 protein by Western blot.