title: Construction of a recombinant human insulin expression vector for mammary gland-specific expression in buffalo (Bubalus bubalis) mammary epithelial cell line.
abstract: The aim of the present study was construction of mammary gland specific expression vector for high level of human insulin (hINS) expression in transgenic buffalo for therapeutic use. We have constructed mammary gland specific vector containing human insulin gene and there expression efficiency was checked into in vitro cultured buffalo mammary epithelial cells (BuMECs). Human pro-insulin coding region was isolated from human genomic DNA by intron skipping PCR primer and furin cleavage site was inserted between B-C and C-A chain of human insulin by overlap extension PCR. A mammary gland-specific buffalo beta-lactoglobulin promoter was isolated from buffalo DNA and used for human insulin expression in BuMEC cells. The construct was transfected into BuMECs by lipofection method and positive transgene cell clones were obtained by G418 selection after 3 weeks. Expression of hINS in transfected cells were confirmed by RT-PCR, Immunocytochemistry, Western Blotting and ELISA. The pAcISUBC insulin-expressing clones secreted insulin at varying levels between 0.18 - 1.43 ng/ml/24 h/2.0 × 10(6) cells.
authors: Principal Scientist, Embryo Biotechnology Lab, Animal Biotechnology Centre, National Dairy Research Institute, Karnal, 132 001, India.
authors: Ramakant
authors: R
authors: Kaushik
authors: Karn Pratap
authors: KP
authors: Singh
authors: Archana
authors: A
authors: Kumari
authors: K
authors: K
authors: Rameshbabu
authors: Manoj Kumar
authors: MK
authors: Singh
authors: Radhey Shyam
authors: RS
authors: Manik
authors: Prabhat
authors: P
authors: Palta
authors: Suresh Kumar
authors: SK
authors: Singla
authors: Manmohan Singh
authors: MS
authors: Chauhan
mesh_terms: D000818:Animals; D030801:Animals, Genetically Modified; D001483:Base Sequence; D002020:Buffaloes / Q000235:genetics; D002478:Cells, Cultured; D003001:Cloning, Molecular; D004847:Epithelial Cells / Q000166:cytology; D005091:Exons; D005260:Female; D005786:Gene Expression Regulation; D005822:Genetic Vectors; D006801:Humans; D061385:Insulins / Q000096:biosynthesis*; D007782:Lactoglobulins / Q000235:genetics; D008321:Mammary Glands, Animal / Q000166:cytology*; D008969:Molecular Sequence Data; D011401:Promoter Regions, Genetic; D011994:Recombinant Proteins / Q000096:biosynthesis*; D014162:Transfection; D019076:Transgenes
chemical_list: D061385:Insulins; D007782:Lactoglobulins; D011994:Recombinant Proteins