title: Uptake of foreign nucleic acids in kidney tubular epithelial cells deficient in proapoptotic endonucleases.
abstract: 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.
authors: Division of Nephrology, Department of Internal Medicine, University of Arkansas for Medical Sciences, Little Rock, Arkansas, USA.
authors: Timea
authors: T
authors: Buzder
authors: Xiaoyan
authors: X
authors: Yin
authors: Xiaoying
authors: X
authors: Wang
authors: Gaspar
authors: G
authors: Banfalvi
authors: Alexei G
authors: AG
authors: Basnakian
mesh_terms: 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
chemical_list: 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