Construction Of Porcine CASQ2 Gene Knockout Vector And Fetal Fibroblast Line

CASQ2 is the major Ca²⁺ storage protein in heart , located in the junctional sarcoplasmic reticulum of mammalian myocardium . It serves as the principal site of Ca²⁺ storage and Ca²⁺ release. The mutations or deletions of conservative part will directly affect the excitement-contraction coupling process of heart and lead to severe CPVT. And now CASQ2-null mice has been generated, the CASQ2-null mice had premature spontaneous Ca²⁺ release and phenocopy the human CPVT. However, due to the greater differences of physiological structure of mouse and human,the experimental results in mice can not be a complete reflection of the pathogenesis of human diseases, in addition, there are many studies can not be carried out on mice, but the physiological structure of porcine is very similar to that of human ,In particular the cardiovascular system. So, it is feasible for us to make CASQ2 gene knockout pig. Then to elucidate the activity of CASQ2 in CPVT; This work provides a basis for the construction of CASQ2 gene knockout pigs.This will provide a scientific basis for further study of human CPVT ,exploring the measures of prevention, diagnosis and treatment of human CPVT.Gene knockout is a useful way to modify the genome based on the homologous recombination. It can modify the genome of target cell on a precise base point or gene seat by homologous recombination between foreign DNA sequence and target cell genome due to their homologous at the early stage of splitting . The animal model of human disease are a class of transgenic animals collectively,which are based on embryonic operation and transgenic,by mediating the foreign genes into animal genomes to change the genetic information of the animals,and it is possible to study the molecular mechanism,pathological conditions and treatment of human disease. The successful isolation,culture of embryonic stem cells and the phenomenon of homologous recombination provide theory basic for gene targeting. Transgenic cloning technology developed rapidly,kinds of animals were cloned,including mice,sheep,cow and pig. Somatic cell cloning is a technique in which the nucleus of a somatic cell is transferred into an enucleated oocyte for the generation of a new individual,genetically identical to the somatic cell donor.This technique gives us a new way to generate cloning big mammalians. The post-genome era came since the genome of human beings had been sequenced . Gene knockout is becoming one of the most direct and effective method to study the function of genes. This study has successfully established CASQ2 gene targeting vector and Fetal Fibroblast to lay the foundation for construction CASQ2 knockout of porcine.Two pairs of primers were designed by Primer Premier 5.0 and Oligo 6.0 and synthesized according to pig CASQ2 gene sequence from GenBank. Firstly, homologous arms were amplified from the pig genome by the long fragment PCR technique,the length are 1.7kb and 4.7kb,respectively. This generated a 615-bp deletion that removes the entire exon 9 as well as 514 bp of upstream sequences. The homologous arms were cloned into the pMD-18T vector for the sequencing analysis. Homologous sequence alignment indicated 99% homology with the corresponding sequence of GenBank. These data proved that the amplified fragment was the pig CASQ2 gene which could be used for the gene targeting research. Sarm fragment with cohesive ends was then obtained by SalI and XhoI double digestion; Larm fragment with cohesive ends was then obtained by HindIII and ClaI double digestion. The two arms were cloned into a universal gene knockout vector pSSC-9 on either side of the positive selective marker-Neo. The pig CASQ2 gene knockout vector (pSSC-CASQ2) was identified successfully by PCR and restriction endonuclease.Porcine fetal fibroblast were transfected with FuGENEHD Transfection Reagent,and selected with G418 for about 7～10 days, G418 and GANC for about 7～10 days. The survival cells were resistance cells. Then cells were cultured in selective solution containing Geneticin and Ganciclovir and the survival cells were the purified resistance cell line. PCR method was used to identify positive cells from resistance cells.One of neomycin resist clones transfected with pSSC-9-CASQ2 were identified to be positive by PCR examination. These results indicate that site-specific integration homologous recombination occurred in these cells. The positive cells were the purified targeted cell line.