Efficient gene transfer into primary murine lymphocytes obviating the need for drug selection.
The efficient introduction of exogenous genes into primary lymphocytes is potentially important both for somatic cell gene therapy and for studying lymphocyte biology. We describe the use of retroviral vectors to efficiently introduce exogenous genes into primary, mature murine lymph node T and B cells, and primary, immature murine CD4- CD8- double-negative (DN) thymocytes. Efficient infection of primary cells was achieved by cocultivation of target cells with lethally irradiated helper cells that produce high titers of retroviral vectors containing either the neomycin phosphotransferase II (neo) gene, or both the neo and the human adenosine deaminase (ADA) genes, in the presence of lymphokines and/or mitogens. Two days postinfection, without neomycin selection, one to five copies of the exogenous genes per cell were detected by Southern blot analysis. Expression of the exogenous human ADA protein was detected at levels comparable to the endogenous murine ADA protein in the mature T and B lymphocytes, and was somewhat lower for the immature DN thymocytes.
Department of Molecular Genetics and Microbiology, Robert Wood Johnson Medical School, University of Medicine and Dentistry, Piscataway, NJ 08854-5635.
M L
ML
Kuo
N
N
Sutkowski
Y
Y
Ron
J P
JP
Dougherty
D000243:Adenosine Deaminase / Q000235:genetics; D000818:Animals; D005260:Female; D015870:Gene Expression; D005796:Genes; D005822:Genetic Vectors; D019868:Kanamycin Kinase; D008198:Lymph Nodes / Q000166:cytology; D016131:Lymphocyte Subsets / Q000502:physiology*; D008297:Male; D051379:Mice; D008807:Mice, Inbred BALB C; D010770:Phosphotransferases / Q000235:genetics; D012333:RNA, Messenger / Q000235:genetics; D012190:Retroviridae / Q000235:genetics; D013950:Thymus Gland / Q000166:cytology; D014162:Transfection / Q000379:methods*
D012333:RNA, Messenger; D010770:Phosphotransferases; D019868:Kanamycin Kinase; D000243:Adenosine Deaminase