Construction Of Goat Mammary Gland Specific Vectors And Their Transfection

Gene targeting leads foreign DNA sequence into target cell or tissue by means of DNA transfer, letting DNA Homologous recombination occur between foreign DNA sequence and target cell gene due to their homologous at early stage of splitting to modify target cell's genome on a precise base point, or gene seat. Mammary glands bioreactors are a class of transgenic animals collectively, which are based on embryonic operation and transgenic technology platform, by mediating the foreign genes into animal genomes, using milk protein genes or others'specific promoters to express foreign proteins in animal mammary glands and explore secretion capacity of mammary glands to harvest exogenous proteins including important medicinal proteins. The approach of integrating exogenous DNA random into the genome of target cell leads to " DNA position effect " which is easy to degrade foreign gene expression even silence, while gene targeting is to be designated to integrate exactly foreign gene into the genome of the pre-designed specific location. To enhance expression of foreign protein in transgenic animal, technologies such as gene targeting were used to build goat mammary gland specific vectors which were used to transfect goat fetal fibroblasts, and the targeting cells, collected by antibiotics, were used as nuclear donors in nuclear transfer to produce mammary glands bioreactors, which had resovled"position effect"in random integration and used milk protein genes activation components to improve expression of exogenic protein.This test treated goatβ-casein gene locus as gene targeting site, and goat DNA genome was extracted from Xi'Nong Saneng Dairy Goat's blood, then LA-PCR was used to amplifyβ-casein gene as two fragments, after TA cloning and identification, LA-PCR method was used again to harvest 5'region of CSN2 gene including regular control region and signal peptide DNA sequence totally 3 kb long of DNA sequence, and 2 kb long DNA sequence of 3'region of CSN2, both of them were treated as homologous arm. a Universal pA2T targeting vector was treated as the skeleton, with a neomycin phosphotransferase gene (neo) inserted between two LoxP sequences and two herpes virus thymidine kinase genes (HSV-tk) laid out of two LoxP sequences, to build a breast specific conditional gene targeting vectors.Human lysozyme gene whose product acts as a broad-spectrum antibiotic, anti-virus, immunity improving, tissue repairing, local blood circulation improving and pus decomposing effects and human tissue plasminogen activator gene defaulting finger domains whose product acts as blood clot dissolving, flow of blood vessels maintaining and the relatively long half-life effects were cloned from vectors using PCR. Both were digested and linked to gene targeting vectors. In addition, AcGFP gene containing nuclear location signal was used as marker gene, which was connected behind IRES sequence having an effect of initial translation of the subsequent gene.In the trial, plasmid pEGFP-C1 was treated as skeleton carrier, and bases were changed before GFP gene to improve its expression. Then, IVS which has the effect of enhancing RNA precursors splicing correctly and IRES was inserted before GFP gene, and another IVS taking with a multiple clone sites was inserted between CMV promoter and IVS. The vector was named p3I-GFP and expressed more GFP than pIRES2-AcGFP-Nuc bought from Clontech, so the p3I-GFPN whose MluI was removed from p3I-GFP. Human lysozyme gene was inserted into MCS of p3I-GFPN and CSN2 promoter was inserted between CMV promoter and lysozyme gene, the constructed vector pCL3I also showed green fluorescence in goat fetus fibroblasts and mammary gland cells. At last, the GFP gene was substituted by human interferon A gene, the vector named pC2-hL-hI was planed to study the effect of preventing and treating with mastitis.In order to obtain gene targeting goat fetus fibroblast cell line, liposome 2000 was used and transfected GFFs by pCLD or pCtD were cultured in 800μg / ml of Geneticin cell cultural solution. Seven days later, transfected GFFs were cultured in 400μg / ml of Geneticin and 200 nmol/ml Ganciclovir to get targeting cells. A single cell cultural system, goat fetal fibroblast feeder layer dealt with 5mg/ml mitomycin C and fresh nutrient solution, was established to culture single targeting cell to amplify cell quantity, at last, cells were cultured in selective solution containing 400μg / ml of Geneticin and 200 nmol/ml Ganciclovir and the survival cells were the purified targeted cell line.