Uptake of foreign nucleic acids in kidney tubular epithelial cells deficient in proapoptotic endonucleases.
Degradation of DNA during gene delivery is an obstacle for gene transfer and for gene therapy. DNases play a major role in degrading foreign DNA. However, which of the DNases are involved and whether their inactivation can improve gene delivery have not been studied. We have recently identified deoxyribonuclease I (DNase I) and endonuclease G (EndoG) as the major degradative enzymes in the mouse kidney proximal tubule epithelial (TKPTS) cells. In this study, we used immortalized mouse TKPTS cells and primary tubular epithelial cells isolated from DNase I or EndoG knockout (KO) mice and examined the degradation of plasmid DNA during its uptake. DNase I and EndoG KO cells showed a higher rate of transfection by pECFP-N1 plasmid than wild-type cells. In addition, EndoG KO cells prevented the uptake of fluorescent-labeled RNA. Complete inhibition of secreted DNase I by G-actin did not improve plasmid transfection, indicating that only intracellular DNase I affects DNA stability. Data demonstrate the importance of DNase I and EndoG in host cell defense against gene and RNA delivery to renal tubular epithelial cells in vitro.
Buzder
Timea
T
Division of Nephrology, Department of Internal Medicine, University of Arkansas for Medical Sciences, Little Rock, Arkansas, USA.
Yin
Xiaoyan
X
Wang
Xiaoying
X
Banfalvi
Gaspar
G
Basnakian
Alexei G
AG
D000818:Animals; D002461:Cell Line, Transformed; D002478:Cells, Cultured; D004247:DNA / Q000235:genetics / Q000378:metabolism; D003850:Deoxyribonuclease I / Q000235:genetics / Q000378:metabolism*; D004706:Endodeoxyribonucleases / Q000235:genetics / Q000378:metabolism*; D004847:Epithelial Cells / Q000166:cytology / Q000378:metabolism*; D049452:Green Fluorescent Proteins / Q000235:genetics / Q000378:metabolism; D007684:Kidney Tubules / Q000166:cytology / Q000378:metabolism; D008055:Lipids / Q000737:chemistry; D051379:Mice; D008810:Mice, Inbred C57BL; D008815:Mice, Inbred Strains; D018345:Mice, Knockout; D008856:Microscopy, Fluorescence; D009696:Nucleic Acids / Q000235:genetics* / Q000378:metabolism / Q000493:pharmacokinetics; D010957:Plasmids / Q000235:genetics / Q000378:metabolism; D012313:RNA / Q000235:genetics / Q000378:metabolism; D034741:RNA, Small Interfering / Q000235:genetics; D020133:Reverse Transcriptase Polymerase Chain Reaction; D014162:Transfection
C485182:Cyan Fluorescent Protein; D008055:Lipids; C086724:Lipofectamine; D009696:Nucleic Acids; D034741:RNA, Small Interfering; D049452:Green Fluorescent Proteins; D012313:RNA; D004247:DNA; D004706:Endodeoxyribonucleases; C052391:endonuclease G; D003850:Deoxyribonuclease I