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Establishment of Vill-Transgenic Mice

Author: YinLiJing
Tutor: XueZhengFeng；WuBaoJin
School: Yangzhou University
Course: Zoology
Keywords: Vill gene pEF6V5His-Vill Microinjection Transgenic mice
CLC: Q78
Type: Master's thesis
Year: 2010
Downloads: 24
Quote: 0
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Abstract

The VILL called the VILLIN-like, fluff-like proteins, one of the members of the of VILLIN protein superfamily. VILLIN a pluripotent actin regulatory proteins able to rely on Ca2 nucleation, combined with capping, cutting actin in epithelial cells. After phosphorylation, by interacting with PIP2, PLC-γ1, Jak3 to influence cell morphology and migration plays an important role in tumor cancer and anti-apoptotic. Very similar to the protein structure VILL, and VILLIN have the same repeated six gelsolin-like domain and a headpiece (HP) structure, which indicates that the two have similar functionality, but very few reports of relevant VILL. At the same time, the laboratory obtained by chemical mutagenesis scant hair mice was recessive genetic Positional identification on the the Vill and Plcdl two gene deletion in chromosome 9 to verify (or exclude) Vill gene with dilute The hair phenotype relationship, plans to import exogenous the Vill gene to rescue the mutant mice \The first part pEF6V5His-Vill expression vector construct the expression vector construct expression Vill transgenic mice produced Vill work. First mouse total RNA extracted from the kidney of C57BL / 6 mice Vill complete coding sequence was amplified by RT-PCR method. PCR products pMD19-T Vector transformed into DH5a E. coli by the blue-white screening, PCR detection and sequencing positive monoclonal plasmid. SpeI and BstBI restriction sites introduced by the primer and the termination codon to be modified, and then as a template for PCR amplification to pMD19-T-Vill. Expression vector containing the gene of PCR products and pEF6V5His SpeI and BstBI digested connection transformed into DH5a E. coli after the of ampicillin, digestion, PCR detection and sequencing reorganization of pEF6V5His-Vill. The expression plasmid pEF6V5His-Vill by electrically transfection methods import L929 mouse fibroblast cells, using anti-His mouse monoclonal antibody as an antibody, Cy3 red fluorescence-labeled goat anti-mouse IgGH L monoclonal antibody as the second antibody, indirect immunofluorescence assay pEF6/V5-H expression is-Vill recombinant plasmid in cells. A result, we successfully amplified by RT-PCR method 2605bp Vill objective gene fragment constructed cloning vector pMD19-T-Vill, by further subcloning, successfully obtained pEF6V5His-Vill recombinant expression vector. The expression was detected by in vitro transfection of cell level, electric transfection conditions for 80V35ms obvious red fluorescence excitation of the green fluorescence detected the cytoplasmic membrane of the L929 cells. pEF6V5His-Vill recombinant expression vector was successfully expressed in L929 cells, lay the foundation for the next step microscopy the injection preparation Vill transgenic mice. The second portion of the microinjection method obtained Vill transgenic mice successful pEF6V5His-Vill recombinant expression vector based on the extracted expression plasmid pEF6V5His-Vill double digestion with restriction enzymes Asel and Nrul purified by Whatman method, DNA fragment for microinjection. 0.5 days for microinjection of fertilized eggs collected from superovulated female weeks old B6CF1, tubal. Microinjection of exogenous DNA, after a brief in vitro cultivation, and select viable embryos cultured overnight. Transplanted into 8-week-old ICR pseudopregnant recipient mice tubal see the day tied, conventional breeding. PCR analysis of mice born, the preliminary screening positive mice, calculated the efficiency of integration, and observe the appearance of the phenotype of mice. A result, the digested purified DNA fragment was obtained for microinjection, the fragment containing the containing the HEF-1α promoter, Vill target gene fragments, V5, His-tag, and BGH 3ng/μl transgenic member microinjected into 390 gold fertilized eggs male pronucleus, after a brief in vitro cultivation, 155 survived, the survival rate was 39.74%, the final 11 recipient mice a total of 77 mice born by PCR on the genomic DNA of these mice, using two primers, The preliminary confirmed 17 mice were strongly positive, two weak positive integration rate was 24.68%. And appearance-positive mice and wild-type mice there was no significant difference in the detection, breeding transgenic positive mice and in-depth analysis of the phenotype to be further expanded. The Vill transgenic mice provide a good foundation material obtained for the Vill gene functional studies and the laboratory scant hair mice appraisal.

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