Liposome mediated in vitro transfection of pancreatic islet cells.
The aim of this study was to evaluate the suitability of the liposome technique for transfection of pancreatic islet cells in vitro. For this purpose, fetal islets were isolated and cultured free floating for two days after which they were further cultured, either intact or dispersed into islet cell suspensions, with different DNA-liposome preparations. The DNA-construct used were the control plasmid (pSP65) and the viral oncogene v-src contained in the plasmid pSPRIsrc. A previous study showed that islet cells transfected by means of electroporation with pSPRIsrc displayed an increased thymidine incorporation rate, making this plasmid suitable for further transfection studies. The DNA was associated with the liposomes by means of surface adhesion. The liposomes used were either conventional phosphatidylcholine-containing liposomes, phosphatidylethanolamine/oleic acid containing liposomes (pH-sensitive liposomes) or Lipofectin. After the exposure of islet cells to the DNA-liposome preparations, the transfection efficiency was assessed by determination of the uptake of the DNA-constructs (Southern blot analysis) and expression of the gene construct into an mRNA (Northern blot analysis). In addition, the impact of the different DNA-liposome preparations on islet DNA replication (thymidine incorporation rates) was determined. It was found that two days after exposure to the DNA-liposomes, the v-src construct was located in islet cell nuclei and that v-src derived transcripts were transiently expressed in the islet cells. The Lipofectin liposomes were more efficient in transfecting islet cells than the pH-sensitive liposomes as assessed by the blotting techniques.(ABSTRACT TRUNCATED AT 250 WORDS)
Welsh
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N
Department of Medical Cell Biology, Uppsala University, Sweden.
Oberg
C
C
Hellerström
C
C
Welsh
M
M
D000818:Animals; D015152:Blotting, Northern; D015139:Blotting, Southern; D004247:DNA / Q000235:genetics; D066298:In Vitro Techniques; D007515:Islets of Langerhans / Q000378:metabolism*; D008081:Liposomes; D010957:Plasmids; D011233:Precipitin Tests; D012333:RNA, Messenger / Q000032:analysis; D051381:Rats; D014162:Transfection
D008081:Liposomes; D012333:RNA, Messenger; D004247:DNA