Parameters influencing high-efficiency transfection of bacterial artificial chromosomes into cultured mammalian cells.
Although bacterial artificial chromosomes (BACs) provide a well-characterized resource for the analysis of large chromosomal domains, low transfection rates have proven a significant limitation for their use in cell culture models. Using TP53 BAC clones that contain expression cassettes for enhanced green fluorescent protein or red fluorescent protein, we have examined conditions that promote BAC transfection in hamster, human, and mouse cell lines. Atomic force microscopy shows that BAC transfection efficiency correlates with the generation of small, highly condensed but dispersed lipid: BAC DNA transfection complexes. BAC DNA purity and concentration are critical for good transfection; debris from purification columns induces the formation of large aggregates that do not gain entry into the cell, and DNA concentrations must be optimized to promote intramolecular condensation rather than intermolecular linking, which also causes aggregation and diminished transfection efficiency. The expression of both markers and genes within BACs initially occurs at lower levels than observed with plasmids, requiring 3-5 days to evaluate the transfection results. We also show that BACs can be co-transfected with other BACs, which provides for increased experimental flexibility.
University of Vermont, College of Medicine, Burlington, VT, USA.
William J
WJ
Montigny
Stephanie F
SF
Phelps
Sharon
S
Illenye
Nicholas H
NH
Heintz
D000818:Animals; D002460:Cell Line; D022202:Chromosomes, Artificial, Bacterial / Q000235:genetics* / Q000648:ultrastructure*; D006224:Cricetinae; D003412:Cricetulus; D020869:Gene Expression Profiling / Q000379:methods*; D006801:Humans; D008322:Mammals; D051379:Mice; D012516:Osteosarcoma / Q000235:genetics / Q000648:ultrastructure; D011786:Quality Control; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity; D014162:Transfection / Q000379:methods*