Establishment Of Human HBV\HBx-Knockin-P53Rat Embryonic Stem Cell

Millions of people around the world are infected with HBV, particularly in China. Itis estimated about50%of people infected with HBV come from China, and they arehighly related with occurrence of cancer in liver. So some research on HBV are paidmuch attention, but we still have little knowledge of variation and molecular mechanismspartly owe to lack of appropriate animal models.It is difficult to establish ideal animal models because HBV only infect specificspecies and tissues. Some HBV transgenic mouse can not fully describe the process ofliver cancer. In contrast, rats are more proper, for they are close to human in many faces.For a long period, we cannot obtain rat embryonic stem cells which have much strains onthe development of animal models.Until2008, rat embryonic stem cells are established;and in2010, the first knockout rat is obtained. Through researches on knockout rats, moreresults are beneficial to understanding of occurrence.So establishments of rat embryonic stem cells and rat animal models on HBV\HBxknock-in P53locus will promote the illustration the process of HBV infection,thedevelopment and progression of liver cancer. Not only it will bring out new targets andmolecular pathways for therapy,but also as models to assess the drugs.In this article, it mainly include the construction of gene targeting vector, culture ofrat embryonic stem cells, electroporation and identifications of target embryonic stemcells. The gene targeting carrier take measures on dual selection of puromycin anddiphtheria toxin. During the process of construction, both homologous arms are added onthe basis of common carrier and finally introduced the HBV genome or X protein genesequences into the multiple cloning sites. Every step is identified by more than threegroups of enzymes, and also at last identified by sequencing. DAc8rat embryonic stemcell lines are cultured in2i and PBS-free medium, and are electroporated bylined-plasmid. Green fluorescence cell lines after several rounds of drug screening andfurther identified by PCR and Southern Blot. establishments of rat embryonic stem cellsand rat animal models on HBV\HBx knock-in P53locus will promote the illustration theprocess of HBV infection,the development and progression of liver cancer and will bringout new targets and molecular pathways for therapy.