Establishment Of Vill-Transgenic Mice

VILL full named VILLIN-like is a member of VILLIN superfamily. VILLIN is an extraordinarily versatile actin regulatory protein that can nucleate, cap, bind and sever actin in epithelial cells depending on Ca2+ concentration. Phosphorylated villin regulates cell morphology and migration by interacting with PIP2, PLC-γ1 and Jak3. It plays a major role in cancer and anti-apoptosis. The structure of VILL likes VILLIN containing 6 repeated gelsolin-like domains and a headpiece (HP). It suggests that VILL and VILLIN have the same functions. But, there was few research on VILL reported. Meanwhile, our laboratory obtained a kind of recessive scant hair mouse (snth-1Bao) by chemomorphosis and the mutation has been identified as a deletion of Vill and Plcdl on chromosome 9. To validate (or to preclude) the relation between Vill and the phenotype of scant hair, the exogenous Vill gene was planned to be imported into the mutant mice to rescue the phenotype of scant hair. The key is that Vill-transgenic mice must be made for redeeming the missing function of Vill in snthr-1Bao mice. This article is principal to construct the Vill expression vector and create Vill-transgenic mice by microinjection.Part1 Construction of Expression Vector pEF6V5His-VillTo construct expression vector containing Vill was the most important first step for making Vill-transgenic mice. The whole length of Vill gene was amplifyed by RT-PCR using total RNA from the kidney of C57BL/6 mouse. After the DNA fragment of Vill cloned into the pMD19-T Vector, the plasmid was transformed into DH5α. The positive plasmid was selected by blue-white selection, PCR and sequencing. Two restriction enzyme cutting sites of SpeI and BstBI and the modified termination codon were introduced into Vill Then the DNA fragment of Vill was amplifyed by PCR using the well-designed primers and pMD19-T-Vill as the template. PCR product containing Vill and pEF6V5His were digested by SpeI and BstBI in the same time. The products of restriction enzyme digestionthey were connected and then translated into the DH5a competent cells. Recombinant expression vector of pEF6V5His-Vill was identificated by restriction endonuclease digestion of SpeI and BstBI, PCR and sequencing. Then pEF6V5His-Vill was transfected into L929 cell using electroporation method. Following the common steps of indirect immunofluorescence (IFT) using Mouse Anti-His primary antibody and Cy3-labeled Goat Anti-Mouse IgG, the expression of pEF6V5His-Vill in L929 can be detected under fluorescence microscope. The results were as follows. The Vill gene about 2605bp was successfully amplifyed by RT-PCR. The pMD19-T-Vill and further the pEF6V5His-Vill had been constructed one after another. In vitro, the electroporation condition about 80V 35ms was choosed and adopted, the red fluorescence could be detected on plasmalemma and in cytoplasm stimulated by green fluorescence. It suggested that VILL had been has be successfully expressed in L929 cells. Obtaining pEF6V5His-Vill lays a solid foundation for Vill-transgenic mice.Part2 Obtaining Vill-Transgenic Mice by MicroinjectionThe designed DNA fragment of Vill gene was obtained by restriction endonuclease digestion of AseI and NruI from the expression vector of pEF6V5His-Vill constructed in part 1, and depurated using the Whatman Kit. Then DNA solution was microinjected into the 0.5-day-old male pronucleus of fertilized eggs from 4-week-old B6CF1 female mice treated by superovulation. The survived embryos were transplanted into oviducts of 8-week-old ICR mice with vaginal plug checked in the same day.19 days later, the potential Vill-transgene mice were preliminary detected by PCR. The integration efficiency of microinjection were calculated and their exophenotype was observed. 3ng/μl DNA solution was microinjected into 390 fertilized eggs, and 155 fertilized eggs survived culturing in vitro for a couple of hours, with a survival ratio of 39.74%. At last, 11 ICR surrogate mothers transplanted into 155 treated eggs gave birth to 77 little mice who were detected by PCR using two kinds of primers respectively,19 mice were primarily determined as the Vill-transgenic positive. The integration efficiency was high as 24.68%. General appearance was not found difference between Vill-transgenic mice and the widetype. Although confirmed detection of transgenic mice, breeding and in-depth phenotypic analysis should be done in the future, Vill transgenic mice provide a good foundation material for the functional studies of Vill gene and the identification of mutation gene responsible for the scant hair mouse (snthr-1Bao).