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Retroviral Mediated Gene Transfer in Megakaryocytic Cell Lines
William R. Kiffmeyer, Peter J. Stambrook and Michael A. Lieberman
In Vitro Cellular & Developmental Biology. Animal
Vol. 30A, No. 11 (Nov., 1994), pp. 803-809
Published by: Society for In Vitro Biology
Stable URL: http://www.jstor.org/stable/4294332
Page Count: 7
You can always find the topics here!Topics: Cell lines, K562 cells, NIH 3T3 cells, 3T3 cells, DNA, Cell growth, PC12 cells, Megakaryocytes, Polymerase chain reaction, Bone marrow cells
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There have been no reports, to date, of successful introduction of foreign DNA into committed megakaryocyte precursor cells. We have successfully infected two megakaryocytic cell lines, one a committed cell line (CHRF-288-11) and the other a bipotential cell line (K562), with a retroviral vector containing the bacterial lacZ gene and a neomycin resistance marker. Modification of standard protocols was required for successful infection of the committed megakaryocyte cell line. Presence of the lacZ transgene was demonstrated at the molecular level by polymerase chain reaction (PCR), and its expression at the mRNA level by reverse transcriptase-PCR. Presence of the bacterial β-galactosidase was demonstrated by both immunofluorescence and enzyme activity. Treatment of the CHRF-288-11 infected cells with phorbol esters, which induces megakaryocytic differentiation, increases expression of the lacZ transgene. The staining pattern of the lacZ reaction product was perinuclear and punctate in the CHRF-288-11 cells, whereas it was uniform throughout the cytoplasm of the K562 cells, suggesting different sorting mechanisms for bacterial β-galactosidase in these two cell types. Overall, these results demonstrate the feasibility and provide a method for infecting cultured megakaryocytic cell lines with retroviral of vectors such that a molecular analysis of megakaryocyte differentiation can be accomplished.
In Vitro Cellular & Developmental Biology. Animal © 1994 Society for In Vitro Biology