| Autor: |
K E, Matthews, G B, Mills, W, Horsfall, N, Hack, K, Skorecki, A, Keating |
| Rok vydání: |
1993 |
| Předmět: |
|
| Zdroj: |
Experimental hematology. 21(5) |
| ISSN: |
0301-472X |
| Popis: |
We report a simple, rapid, efficient and cost-effective method of gene transfer into bone marrow stromal and other adherent mammalian cells. Our approach involves brief incubation of cells with glass beads in a solution containing the DNA to be transferred. We optimized the technique using COS cells (SV40 transformed kidney cell line from African green monkey) and a transient expression assay for chloramphenicol acetyl transferase (CAT). Factors affecting gene transfer include size and condition of the beads and DNA concentration, but not DNA conformation. Gene transfer efficiency, assessed in a transient expression assay for beta-galactosidase activity, was 5 and 3% in nontransformed human bone marrow stromal cells and COS cells, respectively. Long-term stable expression with the selectable marker, neomycin phosphotransferase, was demonstrated in clonogenic COS cells at a frequency of 27%. Southern analysis of resistant clones revealed the transferred DNA to be integrated in low copy number at one or two sites in the host cell genome. Comparison with electroporation and DEAE-dextran indicates that bead transfection is more efficient than the latter and less costly than either of these methods. In view of its simplicity and because the use of retroviral sequences can be avoided, bead transfection may be an attractive means of gene insertion for gene therapy. |
| Databáze: |
OpenAIRE |
| Externí odkaz: |
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