Knock-in of Enhanced Green Fluorescent Protein or/and Human Fibroblast Growth Factor 2 Gene into β-Casein Gene Locus in the Porcine Fibroblasts to Produce Therapeutic Protein
نویسندگان
چکیده
Transgenic animals have become important tools for the production of therapeutic proteins in the domestic animal. Production efficiencies of transgenic animals by conventional methods as microinjection and retrovirus vector methods are low, and the foreign gene expression levels are also low because of their random integration in the host genome. In this study, we investigated the homologous recombination on the porcine β-casein gene locus using a knock-in vector for the β-casein gene locus. We developed the knock-in vector on the porcine β-casein gene locus and isolated knock-in fibroblast for nuclear transfer. The knock-in vector consisted of the neomycin resistance gene (neo) as a positive selectable marker gene, diphtheria toxin-A gene as negative selection marker, and 5' arm and 3' arm from the porcine β-casein gene. The secretion of enhanced green fluorescent protein (EGFP) was more easily detected in the cell culture media than it was by western blot analysis of cell extract of the HC11 mouse mammary epithelial cells transfected with EGFP knock-in vector. These results indicated that a knock-in system using β-casein gene induced high expression of transgene by the gene regulatory sequence of endogenous β-casein gene. These fibroblasts may be used to produce transgenic pigs for the production of therapeutic proteins via the mammary glands.
منابع مشابه
Investigation of an Optimized Context for the Expression of GFP as a Reporter Gene in Chlamydomonas Reinhardtii
Background: Chlamydomonas reinhardtii is a novel recombinant eukaryotic expression system with many advantages including fast growth rate, rapid scalability, absence of human pathogens and the ability to fold and assemble complex proteins accurately, however, obstacle relatively low expression level necessitates optimizing foreign gene expression in this system. The Green Fluorescent Protein (G...
متن کاملComparison of Effects of High-Fat, High-Protein and Low-Calorie Diets on Klotho Gene Expression and TGF-β Level in Serum and Liver of Old Male Wistar Rats
Background and Objectives: Different diets have important roles in human health and aging. In the current study, we aimed to characterize the Klotho, an anti-aging protein, gene expression and protein level in the serum and assess the protein levels of Transforming growth factor-β and Platelet-derived growth factor in old rats which treated with different diets (high-fat, high-protein, low calo...
متن کاملA New Reporter Gene Technology: Opportunities and Perspectives
The paper summarizes the current status of the reporter gene technology and their basics. Reporter gene technology is widely used to monitor cellular events associated with gene expression and signal transduction. Based upon the splicing of transcriptional control elements to a variety of reporter genes, it “reports” the effects of a cascade of signaling events on gene expression inside cells. ...
متن کاملGenetically Engineered Mesenchymal Stem Cells Stably Expressing Green Fluorescent Protein
Objective(s) Mesenchymal stem cells (MSCs) are nonhematopoietic stromal cells that are capable of differentiating into and contribute to the regeneration of mesenchymal tissues. Human mesenchymal stem cells (hMSCs) are ideal targets in cell transplantation and tissue engineering. Enhanced green fluorescent protein (EGFP) has been an important reporter gene for gene therapy. The aim of this stu...
متن کاملEffect of Simulated Microgravity Conditions on Differentiation of Adipose Derived Stem Cells towards Fibroblasts Using Connective Tissue Growth Factor
Background: Mesenchymal stem cells (MSCs) are multipotent cells able to differentiating into a variety of mesenchymal tissues including osteoblasts, adipocytes and several other tissues. Objectives: Differentiation of MSCs into fibroblast cells in vitro is an attractive strategy to achieve fibroblast cell and use them for purposes such as regeneration medicine. The goal of this s...
متن کامل