Efficient and sustained transgene expression in mature rat oligodendrocytes in primary culture.
In order to evaluate the characteristics and efficiency of gene transfer in primary cultures of oligodendrocytes, four different techniques including particle bombardment (Accell gene gun), cationic liposome-mediated transfection (lipofection), calcium phosphate co-precipitation and retroviral infection were compared using the LacZ and luciferase reporter genes. Highly purified postnatal adult rat oligodendrocytes were obtained by sequential immunopanning, plated in culture, and transfected using various reporter and promoter genes. The most efficient expression of LacZ and luciferase genes was found with particle mediated gene delivery. The transgene expression level obtained with gene gun delivery was at least two- to 100-fold greater than three other tested gene transfer methods. Comparison of the relative strength of four viral and two cellular promoters in these primary oligodendrocytes cultures demonstrated that the CMV promoter was the strongest. Using a human growth hormone (hGH) reporter gene, a long-term transgene expression pattern in primary oligodendrocytes was demonstrated to be sustained in culture for the entire experimental period (4 weeks) after particle-mediated gene transfer. These results demonstrate that expression of a foreign gene can be effectively achieved in primary cultures of adult oligodendrocytes, especially by using the particle bombardment method. The results also suggest that the current ex vivo gene transfer system may be used to manipulate oligodendrocytes for future application in gene therapy studies.
Guo
Z
Z
Department of Medical Sciences, University of Wisconsin-Madison 53706, USA.
Yang
N S
NS
Jiao
S
S
Sun
J
J
Cheng
L
L
Wolff
J A
JA
Duncan
I D
ID
D000256:Adenoviridae / Q000235:genetics; D000818:Animals; D001358:Avian Sarcoma Viruses / Q000235:genetics; D019470:Biolistics; D002130:Calcium Phosphates; D002412:Cations; D002478:Cells, Cultured; D003587:Cytomegalovirus / Q000235:genetics; D015870:Gene Expression; D018014:Gene Transfer Techniques / Q000295:instrumentation; D017930:Genes, Reporter; D005813:Genes, Synthetic; D005822:Genetic Vectors; D013006:Growth Hormone / Q000096:biosynthesis / Q000235:genetics; D008081:Liposomes; D051379:Mice; D009836:Oligodendroglia / Q000378:metabolism*; D010729:Phosphoenolpyruvate Carboxykinase (GTP) / Q000235:genetics; D010735:Phosphoglycerate Kinase / Q000235:genetics; D011401:Promoter Regions, Genetic; D051381:Rats; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis*; D012091:Repetitive Sequences, Nucleic Acid; D012190:Retroviridae; D013539:Simian virus 40 / Q000235:genetics; D013997:Time Factors; D014162:Transfection / Q000379:methods; D001616:beta-Galactosidase / Q000096:biosynthesis / Q000235:genetics
D002130:Calcium Phosphates; D002412:Cations; D008081:Liposomes; D011993:Recombinant Fusion Proteins; C485828:alpha-tricalcium phosphate; C485830:tetracalcium phosphate; C485838:calcium phosphate, monobasic, anhydrous; D013006:Growth Hormone; C020243:calcium phosphate; D010735:Phosphoglycerate Kinase; D001616:beta-Galactosidase; D010729:Phosphoenolpyruvate Carboxykinase (GTP); C485829:calcium phosphate, dibasic, anhydrous