Gene transfer by electroporation: a model for gene therapy.
We examined electroporation for the introduction of DNA into hematopoietic cells in order to develop an in vitro model for human gene therapy. We demonstrated that electroporation is a relatively efficient and reproducible method of gene transfer in permanent hematopoietic lines. Characterization of DNA transfer revealed that genes are integrated in single or low copy number. We showed that human normal granulopoietic progenitors can express the transferred gene at a frequency of more than two percent. Although the progeny of erythroid progenitors failed to express transferred genes, the frequency of transfer to erythroid clones, as detected by the polymerase chain reaction, was moderately high (16%). In order to improve efficiency of expression, we examined the effect of different viral and cellular promoters in the hematopoietic lines K562 and KG1a as well as in marrow stromal cells. We found that Rous Sarcoma virus LTR was the most active promoter in K562 while the murine cytomegalovirus immediate early promoter was highly active in KG1a and marrow stromal cells. Further improvements in the efficiency of DNA transfer and expression with electroporation are likely to result in a successful hematopoietic model for gene therapy.
Keating
A
A
University of Toronto Autologous Bone Marrow Transplant Program, Toronto General Hospital, Ontario, Canada.
Toneguzzo
F
F
D001853:Bone Marrow / Q000378:metabolism; D002460:Cell Line; D002463:Cell Membrane Permeability; D004247:DNA / Q000378:metabolism*; D004560:Electricity; D015870:Gene Expression; D015316:Genetic Therapy / Q000379:methods*; D006412:Hematopoietic Stem Cells / Q000378:metabolism; D006801:Humans; D008957:Models, Genetic; D011401:Promoter Regions, Genetic / Q000502:physiology; D014162:Transfection / Q000235:genetics*
D004247:DNA