MAC-T cells as a tool to evaluate lentiviral vector construction targeting recombinant protein expression in milk.
Prior to generating transgenic animals for bioreactors, it is important to evaluate the vector constructed to avoid poor protein expression. Mammary epithelial cells cultured in vitro have been proposed as a model to reproduce the biology of the mammary gland. In the present work, three lentiviral vectors were constructed for the human growth hormone (GH), interleukin 2 (IL2), and granulocyte colony-stimulating factor 3 (CSF3) genes driven by the bovine β-casein promoter. The lentiviruses were used to transduce mammary epithelial cells (MAC-T), and the transformed cells were cultured on polystyrene in culture medium with and without prolactin. The gene expression of transgenes was evaluated by PCR using cDNA, and recombinant protein expression was evaluated by Western-blotting using concentrated medium and cellular extracts. The gene expression, of the three introduced genes, was detected in both induced and non induced MAC-T cells. The human GH protein was detected in the concentrated medium, whereas CSF3 was detected in the cellular extract. Apparently, the cellular extract is more appropriate than the concentrated medium to detect recombinant protein, principally because concentrated medium has a high concentration of bovine serum albumin. The results suggest that MAC-T cells may be a good system to evaluate vector construction targeting recombinant protein expression in milk.
a Centro de Ciencias Biológicas e da Saúde , Universidade Norte do Paraná , Londrina , Paraná , Brazil.
Paulo S
PS
Monzani
Samuel
S
Guemra
Paulo R
PR
Adona
Otavio M
OM
Ohashi
Flávio V
FV
Meirelles
Matthew B
MB
Wheeler
D000818:Animals; D002417:Cattle; D002460:Cell Line; D004847:Epithelial Cells; D005260:Female; D005822:Genetic Vectors / Q000235:genetics*; D016086:Lentivirus / Q000235:genetics*; D008321:Mammary Glands, Animal / Q000166:cytology; D008892:Milk / Q000737:chemistry* / Q000378:metabolism; D011994:Recombinant Proteins / Q000032:analysis / Q000235:genetics / Q000378:metabolism*; D014162:Transfection
D011994:Recombinant Proteins
CSF-3; Growth hormone; Interleukin 2; Lentivirus; MAC-T cells; Protein expression