Establishment a knock-in mouse model with conditional overexpression of XBP1s gene

Abstract

Objective To establish a heterozygous knock-in mouse model with conditional overexpression of spiced X-box-binding protein 1 (XBP1s) gene at the Rosa 26 site. Methods By use of the in-fusion cloning technique, an embryonic stem (ES) cell-targeting vector was constructed, linearized, and transfected into the JM8A3 ES cells through electroporation. The positive clones of JM8A3 ES cells with antibiotic resistance were screened, and identified with the long-fragment PCR method, and were then injected into blastocysts of C57BL/6J mice to get the chimeric mice. The high-percentage-chimeric mice hybridized with wild type C57BL/6J mice to get positive mice of F1 generation. The positive F1 generation mice were identified by PCR and sequencing. Results The ES cell-targeting vector constructed was identified with enzyme digestion mothed. Total 144 positive ES cell clones with antibiotic resistance were obtained, among which 21 positive clones were identified with long-fragment PCR mothed and sequencing. The positive E3 clone and C6 clone were amplified, and injected into 96 blastocysts of C57BL/6J mice. After embryonic implantation, 2 high-percentage-chimeric mice were obtained, which then hybridized with wild type C57BL/6J mice to get 3 mice of F1 generation recognized with PCR and sequencing. Conclusion A heterozygous knock-in mouse model with conditional overexpression of XBP1s gene at the Rosa 26 site was successfully established, and F1 generation mice were obtained, which built up a good basis for getting conditional XBP1s gene knock-in mice of immunological cells, renal intrinsic cells, and peritoneal mesothelial cells in the future. Key words: Spliced X-box-binding protein 1 (XBP1s); Unfolded protein response/ER stress; Homologous recombination; Gene targeting; Conditional gene knock-in