Transient promoter activity in primary rat mammary epithelial cells evaluated using particle bombardment gene transfer.
The relative strengths of several commonly used viral promoters in primary cultures of rat mammary epithelial cells were studied using a particle bombardment gene transfer method. NIH 3T3 cells were also examined as a representative cell line. Initially, the conditions necessary for efficient gene transfer using particle bombardment were determined. Discharge voltage for particle bombardment was evaluated to maximize the levels of gene expression and cell viability. After transfection, transgene expression decreased over a 5-day period in both mammary cells and NIH 3T3 cells. Particle bombardment gene transfer was at least fivefold more efficient than lipofection, calcium phosphate co-precipitation, or electroporation. The activity of five viral enhancer/promoters was compared using a luciferase gene assay system. The relative promoter strengths in mammary cells were determined to be: RSV approximately CMV approximately SV40 > MLV > MMTV. Tissue-specific activity of the MMTV-LTR was demonstrated, although this promoter conferred the lowest expression level among the promoters tested.
Environmental Toxicology Center, University of Wisconsin-Madison 53792.
T A
TA
Thompson
M N
MN
Gould
J K
JK
Burkholder
N S
NS
Yang
D016475:3T3 Cells; D000818:Animals; D001358:Avian Sarcoma Viruses / Q000235:genetics; D002470:Cell Survival; D002478:Cells, Cultured; D015500:Chloramphenicol O-Acetyltransferase / Q000235:genetics / Q000378:metabolism; D003587:Cytomegalovirus / Q000235:genetics; D004558:Electric Stimulation / Q000379:methods; D004847:Epithelial Cells; D004848:Epithelium / Q000502:physiology; D005260:Female; D007700:Kinetics; D008156:Luciferases / Q000235:genetics / Q000378:metabolism; D008321:Mammary Glands, Animal / Q000166:cytology / Q000502:physiology*; D008324:Mammary Tumor Virus, Mouse / Q000235:genetics; D051379:Mice; D008979:Moloney murine leukemia virus / Q000235:genetics; D010957:Plasmids; D011401:Promoter Regions, Genetic; D051381:Rats; D012091:Repetitive Sequences, Nucleic Acid; D013539:Simian virus 40 / Q000235:genetics; D014162:Transfection / Q000379:methods*
D008156:Luciferases; D015500:Chloramphenicol O-Acetyltransferase