Transient promoter activity in primary rat mammary epithelial cells evaluated using particle bombardment gene transfer |
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Authors: | T Anthony Thompson Michael N Gould Joseph K Burkholder Ning-Sun Yang |
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Institution: | (1) Environmental Toxicology Center, University of Wisconsin-Madison, 53792 Madison, Wisconsin;(2) Department of Human Oncology, University of Wisconsin-Madison, 53792 Madison, Wisconsin;(3) Department of Mammalian Genetics, Agracetus, 8520 University Green, 53562 Middleton, Wisconsin |
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Abstract: | Summary 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 ≈ CMV ≈ SV40 > MLV > MMTV. Tissue-specific activity of the MMTV-LTR was demonstrated, although this promoter conferred
the lowest expression level among the promoters tested. |
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Keywords: | gene transfection mammary gland particle bombardment gene transfer tissue-specific expression viral promoters |
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