Study Of Knocking-out Or Silencing The Integrin αv Gene In Dairy Cattle Fetal Fibroblast Cells

Foot and mouth disease(FMD)is one of the most acute, febrile, highly contagious disease caused by foot and mouth disease virus(FMDV). The virus affects many domestic livestock such as pigs,sheeps,goats,and cattle, all of them are cloven-hoofed animals and Camelidae. FMD outbreaks can lead to devastating economic consequence. FMDV is the prototype member of the Aphthovirus genus of the Picornaviridae family, which has a single-stranded, positive-sense RNA genome. There are seven serotypes:A,O,C,Asia-1,and South African Territories (SAT1),SAT2,and SAT3, no cross-protection between the serotypes, so inoculation one serotype vaccine can't prevent and control other field serotypes FMD.The virus receptor molecules is a prerequisits for the virus infecting cells. It has been demonstrated that at least four members of theαv integrin family of cellular receptors,αvβ1,αvβ3,αvβ6,αvβ8, can serve as receptors for FMDV in vitro.αv is common subunit, knockout or silencing ofαv gene will make the cell loss or lower expressing of all four integrins, and possibly lead to be not sensitive to FMDV. Thus, knockout or silence ofαv gene will become a new way to control FMD.In gene targeting research, we amplified 1.8kb and 2.4kb homologous arms by polymerase chain reaction (PCR). The two homologous arms were cloned into the gene targeting plasimd pLn, in which the neo was inserted between the two homologous sequences and HSV-tk was placed outside the homologous arms. The dairy cattle integrinαv gene targeting vector of pln-R-P1 was constructed. This vector was linearized and transfected into dairy cattle fetal fibroblast cells through electroporation. After transfection, G418 and Ganciclovir drugs were used for screening positive cells based on positive and negative selection. Four different dairy cattle fetal fibroblast cell lines were used, 268 double-resistant clones obtained and 20 clones were theαv gene knock out clones by PCR analysis.We also used RNA interference technology to silenceαv gene. The cDNA ofαv gene was obtained from the dairy cattle fetal fibroblast cell through RT-PCR, and cloned into pEASY-T3 to construct pEASY-T3-360. After DNA sequence analysis, three small hairpin RNA(shRNA) aganistαv were designed and synthesised, then recombinant lentiviral shRNA expression vectors were constracted. After the shRNA recombinant vectors was transfected into dairy cattle fetal fibroblast cell, semi-quantitative reverse transcription and polymerase Chain reaction(SqRT-PCR) is used to detect the expression of theαv gene, Our result showed thatαv gene was knockdown.