title: Expression of naked plasmids by cultured myotubes and entry of plasmids into T tubules and caveolae of mammalian skeletal muscle.
abstract: Plasmid DNA or artificial mRNA injected intramuscularly into skeletal muscle via a 27 g needle expressed transgenes at relatively efficient levels in skeletal myofibers and cardiac cells. In the present study, several approaches were used to determine the mechanism of cellular uptake. After exposure of naked plasmid DNA, primary rat muscle cells in vitro expressed transgenes to a much greater extent than other types of immortalized or primary cells. In vivo light microscope studies showed that intramuscularly injected plasmid DNA was distributed throughout the muscle and was able to diffuse through the extracellular matrix, cross the external lamina, and enter myofibers. Electron microscope studies showed that colloidal gold conjugated to plasmid DNA traversed the external lamina and entered T tubules and caveolae, while gold complexed with polylysine, polyethylene glycol or polyglutamate primarily remained outside of the myofibers. The results indicate that it is highly unlikely that the plasmid DNA enters the myofiber simply by the needle grossly disrupting the sarcolemma. In addition, transient membrane disruptions do not appear to be responsible for the uptake of DNA. Furthermore, no evidence for endocytosis could be found. The possible uptake of plasmid DNA by some type of cell membrane transporter, in particular via potocytosis, is discussed.
authors: Department of Pediatrics, Waisman Center, University of Wisconsin, Madison 53705.
authors: J A
authors: JA
authors: Wolff
authors: M E
authors: ME
authors: Dowty
authors: S
authors: S
authors: Jiao
authors: G
authors: G
authors: Repetto
authors: R K
authors: RK
authors: Berg
authors: J J
authors: JJ
authors: Ludtke
authors: P
authors: P
authors: Williams
authors: D B
authors: DB
authors: Slautterback
mesh_terms: D016475:3T3 Cells; D000818:Animals; D002478:Cells, Cultured; D004274:DNA, Recombinant / Q000008:administration & dosage / Q000378:metabolism* / Q000494:pharmacology; D004285:Dogs; D015870:Gene Expression; D006046:Gold / Q000008:administration & dosage; D007150:Immunohistochemistry; D007273:Injections, Intramuscular; D008081:Liposomes; D008253:Macaca mulatta; D051379:Mice; D008807:Mice, Inbred BALB C; D009132:Muscles / Q000166:cytology / Q000378:metabolism*; D010957:Plasmids; D012333:RNA, Messenger / Q000378:metabolism* / Q000494:pharmacology; D051381:Rats; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000235:genetics; D014162:Transfection; D014170:Transformation, Genetic
chemical_list: D004274:DNA, Recombinant; D008081:Liposomes; D012333:RNA, Messenger; D011993:Recombinant Fusion Proteins; D006046:Gold