Isolation and culture of bovine mammary epithelial cells and establishment of gene transfection conditions in the cells.
Bovine mammary epithelial cells (BMEC) were isolated as acinous fragments from a mammary gland of a lactating cow. They grew well on plastic substratum, showed the characteristic cobblestone morphology of epithelial cells, and secreted alpha s1-, beta-, and kappa-caseins even when grown on plastic substratum. A plasmid containing the bacterial chloramphenicol acetyl transferase (CAT) gene was transfected to the isolated BMEC by calcium phosphate precipitation and electroporation methods. The transfection efficiency of BMEC by the calcium phosphate method was greatly improved by post-transfection osmotic shock with glycerol or polyethylene glycol. An about 700 bp DNA fragment containing 5'-flanking sequence of bovine alpha s1-casein gene showed promoter activity in the transfected BMEC. The primary culture of BMEC might be useful for studies on regulation of bovine milk-protein gene expression.
Ahn
J Y
JY
Department of Applied Biological Sciences, School of Agricultural Sciences, Nagoya University, Japan.
Aoki
N
N
Adachi
T
T
Mizuno
Y
Y
Nakamura
R
R
Matsuda
T
T
D000818:Animals; D001483:Base Sequence; D002130:Calcium Phosphates / Q000737:chemistry; D002364:Caseins / Q000235:genetics / Q000378:metabolism; D002417:Cattle; D002455:Cell Division / Q000235:genetics / Q000502:physiology; D002478:Cells, Cultured; D015500:Chloramphenicol O-Acetyltransferase / Q000235:genetics*; D004247:DNA / Q000235:genetics / Q000378:metabolism; D017931:DNA Primers / Q000737:chemistry; D004591:Electrophoresis, Polyacrylamide Gel; D018274:Electroporation; D004847:Epithelial Cells; D004848:Epithelium / Q000378:metabolism; D015971:Gene Expression Regulation, Enzymologic / Q000235:genetics*; D005990:Glycerol / Q000737:chemistry; D015151:Immunoblotting; D007774:Lactation; D008321:Mammary Glands, Animal / Q000166:cytology* / Q000378:metabolism; D008858:Microscopy, Phase-Contrast; D008892:Milk / Q000378:metabolism; D008969:Molecular Sequence Data; D009997:Osmotic Pressure; D010957:Plasmids; D011092:Polyethylene Glycols / Q000737:chemistry; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D014162:Transfection
D002130:Calcium Phosphates; D002364:Caseins; D017931:DNA Primers; D011993:Recombinant Fusion Proteins; C485828:alpha-tricalcium phosphate; C485830:tetracalcium phosphate; D011092:Polyethylene Glycols; C485838:calcium phosphate, monobasic, anhydrous; D004247:DNA; C020243:calcium phosphate; D015500:Chloramphenicol O-Acetyltransferase; C485829:calcium phosphate, dibasic, anhydrous; D005990:Glycerol