TY - JOUR
T1 - Transfer of MHC Genes Into Hematopoietic Stem Cells by Electroporation
T2 - A Model for Monitoring Gene Expression
AU - Eustis-Turf, E. P.
AU - Wang, X. M.
AU - Schook, L. B.
N1 - Funding Information:
ACKNOWLEDGEMENTS We wish to acknowledge S. Grossberg (IFN-7), E. Choi (A/""12 gene), I. D. Goldman (methotrexate) andL. Hood (cosmid 17.2)fortheir generous contributions ofreagents used inthese studies. We also thank Genentech for their supplying oftheplasmid pFR400. Ourgratitude isalso extendedto H. Lewin, K. Kelley, D. Sehy, B. White, andJ.Auger for their assistance, discussions andencouragement. These studies were supported inpart bytheNIH (ES-04348 andES-04347), the University ofIllinois BiotechnologyCenter and an IBM Research Award from the University ofIllinois Research Board.
PY - 1990/1
Y1 - 1990/1
N2 - Previous attempts in using recombinant viral vectors to transfer genes into bone marrow cells have resulted in expression frequencies of 2-15% (8, 9, 12, 13, 14, 24). These vectors, however, require complex gene vector constructions and culture methods and have been difficult to utilize. We therefore have used electroporation to establish a less complicated and equally efficient technique for gene transfer. Conditions yielding high gene transfer into bone marrow precursor cells by electroporation were determined using a mutant dihydrofolate reductase gene and/or murine MHC class II genes. Successful transfer and expression of these genes were assessed by (1) fluorescence staining using monoclonal antibodies, (2) acquired resistance to the anti-folate drug methotrexate and (3) Southern blot analysis. Stable integration of the newly acquired mutant dihydrofolate reductase gene was observed in > 25% of murine hematopoietic progenitor cells (CFU-M). Electroporation of class II genes in conjunction with the mutant dihydrofolate reductase and subsequent selection with methotrexate resulted in expression of transfected Ia molecules in 12-15% of bone marrow derived macrophages. Integration of the transferred major histocompatibility genes was verified by Southern blot analysis of high molecular weight cellular DNA following electroporation and methotrexate selection. The frequencies of gene transfer and expression achieved in this study suggest that electroporation is a viable option for monitoring gene expression in bone marrow derived cells.
AB - Previous attempts in using recombinant viral vectors to transfer genes into bone marrow cells have resulted in expression frequencies of 2-15% (8, 9, 12, 13, 14, 24). These vectors, however, require complex gene vector constructions and culture methods and have been difficult to utilize. We therefore have used electroporation to establish a less complicated and equally efficient technique for gene transfer. Conditions yielding high gene transfer into bone marrow precursor cells by electroporation were determined using a mutant dihydrofolate reductase gene and/or murine MHC class II genes. Successful transfer and expression of these genes were assessed by (1) fluorescence staining using monoclonal antibodies, (2) acquired resistance to the anti-folate drug methotrexate and (3) Southern blot analysis. Stable integration of the newly acquired mutant dihydrofolate reductase gene was observed in > 25% of murine hematopoietic progenitor cells (CFU-M). Electroporation of class II genes in conjunction with the mutant dihydrofolate reductase and subsequent selection with methotrexate resulted in expression of transfected Ia molecules in 12-15% of bone marrow derived macrophages. Integration of the transferred major histocompatibility genes was verified by Southern blot analysis of high molecular weight cellular DNA following electroporation and methotrexate selection. The frequencies of gene transfer and expression achieved in this study suggest that electroporation is a viable option for monitoring gene expression in bone marrow derived cells.
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U2 - 10.1080/10495399009525728
DO - 10.1080/10495399009525728
M3 - Article
AN - SCOPUS:84959997350
SN - 1049-5398
VL - 1
SP - 47
EP - 60
JO - Animal Biotechnology
JF - Animal Biotechnology
IS - 1
ER -