ObjectiveTo establish human HBV/HBx-knockin-p53 rat embryonic stem (ES) cell line, so as to lay a foundation for establishment of animal models. MethodsFirst we constructed the targeting vector pKO-gHBV or pKO-X with HBV whole genome or X and p53 homologue arms by PCR and connection with common gene targeted vector pKO; then after linearized with SalⅠ the vector was transferred into rat ES cells through electroporation. And 2i ES culture medium containing puromycin was used for a three-cycle selection of puromycin resistant clones. Then PCR was used to screen the obtained positive clones; mycoplasma examination and karyotypic analysis were used to confirm targeted ES cell clones. ResultsWe successfully obtained two targeted vectors: pKO-gHBV and pKO-X; then after three-cycle drug-selection we obtained 2 pKO-X clones and 1 pKO-gHBV clone, which were confirmed by PCR, mycoplasma examination and karyotypic analysis. ConclusionWe have successfully established human HBV/HBx-knockin-p53 rat ES cells, paving a way for future establishment of HBV/HBx-knockin-p53 rat model.