A comparison of calcium phosphate coprecipitation and electroporation. Implications for studies on the genetic effects of DNA damage.
Plasmid-based transfection assays provide a rapid means to measure homologous and nonhomologous recombination in mammalian cells. Often it is of interest to examine the stimulation of recombination by DNA damage induced by radiation, genotoxic chemicals, or nucleases. Transfection is frequently performed by using calcium phosphate coprecipitation (CPP), because this method is well suited for handling large sample sets, and it does not require expensive reagents or equipment. Alternative transfection methods include lipofection, microinjection, and electroporation. Since DNA strand breaks are known to stimulate both homologous and nonhomologous recombination, the induction of nonspecific damage during transfection would increase background recombination levels and thereby reduce the sensitivity of assays designed to detect the stimulation of recombination by experimentally induced DNA damage. In this article, we compare the stimulatory effects of nuclease-induced double-strand breaks (DSBs) on homologous and nonhomologous recombination for molecules transfected by CPP and by electroporation. Although electroporation yielded fewer transfectants, both nonhomologous and homologous recombination were stimulated by nuclease-induced DSBs to a greater degree than with CPP. Ionizing radiation is an effective agent for inducing DNA strand breaks, but previous studies using CPP generally showed little or no stimulation of homologous recombination among plasmids damaged with ionizing radiation. By contrast, we found clear dose-dependent enhancement of recombination with irradiated plasmids transfected using electroporation. Thus, electroporation provides a higher signal-to-noise ratio for transfection-based studies of damage-induced recombination, possibly reflecting less nonspecific damage to plasmid DNA during transfection of mammalian cells.
Nickoloff
J A
JA
Department of Molecular Genetics and Microbiology, University of New Mexico School of Medicine, Albuquerque 87131, USA. jnickoloff@salud.unm.edu
Spirio
L N
LN
Reynolds
R J
RJ
D000818:Animals; D016466:CHO Cells / Q000166:cytology / Q000187:drug effects / Q000378:metabolism; D002130:Calcium Phosphates / Q000494:pharmacology*; D006224:Cricetinae; D004247:DNA / Q000187:drug effects / Q000235:genetics; D004249:DNA Damage; D004270:DNA, Circular / Q000187:drug effects / Q000235:genetics; D018274:Electroporation; D005822:Genetic Vectors / Q000187:drug effects / Q000235:genetics / Q000528:radiation effects; D009154:Mutation; D010957:Plasmids / Q000187:drug effects / Q000235:genetics / Q000528:radiation effects; D011995:Recombination, Genetic / Q000187:drug effects / Q000235:genetics / Q000528:radiation effects; D014162:Transfection / Q000187:drug effects / Q000235:genetics / Q000379:methods*
D002130:Calcium Phosphates; D004270:DNA, Circular; C485828:alpha-tricalcium phosphate; C485830:tetracalcium phosphate; C485838:calcium phosphate, monobasic, anhydrous; D004247:DNA; C020243:calcium phosphate; C485829:calcium phosphate, dibasic, anhydrous