Construction Of Goat GH Mammary Gland Specific Gene Targeting Vector And Modified Fetal Fibroblast Cell Line

Prior studies demonstrated that growth hormone (GH) could interact with the GH receptor (GHR) on the breast stromal cell and induce the formation of insulin-like growth factor-I (IGF-I), promote the proliferation of mammary epithelial cells. GH could act as the central regulator of blood flow and metabolism of mammary gland, and GH was related to the length of lactation period and the milk production. The Gene targeting technique could help mediating the goat GH site-specific integrated into the beta-casein locus in this study. We firstly constructed a mammary glands specific Gene targeting vector pLG, verificating its function in cells and mammary glands respectively. The pLG vector was transfected into goat fibroblast and screened. The gene-targeted goat fibroblasts will be used as donor cells for nuclear transfer to produce GH transgenic goats.The main experiments in this study were as follows:1. Cloning of goat beta-casein gene5’and3’regulatory element and its function verificationThe5’and3’regulatory element sequences of goat beta-casein (CSN2) was cloned by PCR to construct the gene targeting vector pGFP, verifying the function of regulatory elements cloned by the expression of green fluorescent protein (GFP). The5’and3’flanking sequences were isolated from goat genomic DNA by PCR as homologous arms and inserted into the Sal I/Cla I and Not I/Xho I multiple clone site (MCS) and we named the vector as pL5. Then we inserted the GFP cDNA into the Xho I MCS of pL5and got the gene targeting vector pGFP. The pGFP plasmid was transfected into Bcap-37cells with lipofectamine2000and we observated the GFP expression under Inverted fluorescence microscope. The observation results demonstrated that the GFP expressed in Bcap-37cells transfected with pGFP plasmid was conspicuous than cells not transfected with pGFP plasmid, which indicate that the5’and3’regulatory elements sequence had a biological function of mammary gland specific expressing exogenous gene. 2. Construction of goat GH gene mammary specific Gene targeting vector pLG and its biological function verificationThe GFP cDNA was inserted into the Xho I MCS of pL5and got the mammary gland specific gene targeting vector pLG We transfected the pLG plasmid into Bcap-37cells and injected pLG plasmid into goat mammary glands respectively. The transcription and expression of GH in cell lysate and mammary glands tissues were detected by RT-PCR and ELISA. Results showed that Bcap-37cells and mammary glands transfected or injected with pLG plasmid had a hiGHer transcription and expression efficiency than cells and mammarys tissues not transfected or injected with pLG plasmid, which demonstrated that pLG gene tarteting vector had the ability of regulating the GH expression in vivo and vitro respectively. We could use the pLG gene targeting vector could be used in further research about producing GH transgenic goat with hiGH milk production.3.Transfecting the goat fibroblast with pLG Gene targeting vector and cell screeningThe pLG plasmid was transfected into goat fetal fibroblast and the G418was added into cell culture fluid36h after the transfection. When the monoclonal cell formed10d after the transfection, the GANC was also added and we passaged the monoclonal cell into48-wells cell culture plates7days later. The monoclonal cell genomic DNA was isolated and the monoclonal cells were freezed about15days after the cell passaging. Then the monoclonal cells genomic DNA was detected by PCR to verify the monoclonal cell. Our results showed that there were5monoclonal cells correctly gene targeted by pLG gene targeting vector.