DNA uptake and repair enzyme access to transfected DNA is under reported by gene expression.
Gene expression is the typical biological end point of interest following transfection. However, transcription may not accurately assess DNA uptake, or the ability of transfected DNA to be acted on by other enzymatic pathways. We have compared DNA uptake to gene expression and the unrelated enzymatic process of DNA double strand break (DSB) repair. Transfection efficiency (at limiting DNA concentration) was assessed as a function of DNA uptake and gene expression in the DSB repair proficient WI38VA13 and MO59K cell lines and the DSB repair defective cell line MO59J, by comparing eGFP expression from the pHygEGFP expression vector with uptake of rhodamine labeled linear pSP189 plasmid (3:1). Repair proficient cells expressed eGFP most efficiently, but never approached DNA uptake levels (>or=90%). Although transfected DNAs were stable in repair proficient cells and degraded in MO59J cells, most cells did not express eGFP, but in the repair proficient cells linear DNA did undergo DSB repair.
Pastwa
Elzbieta
E
Nuclear Medicine Department, Warren Grant Magnuson Clinical Center, National Institutes of Health, Bethesda, MD 20892, USA.
Lubner
Erin M
EM
Mezhevaya
Katherina
K
Neumann
Ronald D
RD
Winters
Thomas A
TA
D015139:Blotting, Southern; D002460:Cell Line; D002461:Cell Line, Transformed; D004247:DNA / Q000378:metabolism / Q000493:pharmacokinetics*; D004249:DNA Damage; D004260:DNA Repair; D004587:Electrophoresis, Agar Gel; D005347:Fibroblasts / Q000378:metabolism; D015870:Gene Expression; D005786:Gene Expression Regulation; D005821:Genetic Techniques; D049452:Green Fluorescent Proteins; D006801:Humans; D008164:Luminescent Proteins / Q000378:metabolism; D010957:Plasmids / Q000378:metabolism; D013997:Time Factors; D014162:Transfection
D008164:Luminescent Proteins; D049452:Green Fluorescent Proteins; D004247:DNA