Retroviral mediated gene transfer in megakaryocytic cell lines.
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 beta-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 beta-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.
Kiffmeyer
W R
WR
Department of Anatomy and Cell Biology, University of Cincinnati College of Medicine, OH 45267-0524.
Stambrook
P J
PJ
Lieberman
M A
MA
D001483:Base Sequence; D002454:Cell Differentiation / Q000502:physiology; D002455:Cell Division / Q000502:physiology; D002460:Cell Line; D003124:Colorimetry; D017931:DNA Primers / Q000032:analysis / Q000737:chemistry / Q000235:genetics; D004273:DNA, Neoplasm / Q000032:analysis / Q000737:chemistry / Q000235:genetics; D015672:Erythroid Precursor Cells / Q000166:cytology; D005455:Fluorescent Antibody Technique; D015971:Gene Expression Regulation, Enzymologic; D018014:Gene Transfer Techniques; D006801:Humans; D007947:Leukemia, Megakaryoblastic, Acute / Q000235:genetics* / Q000473:pathology; D008533:Megakaryocytes / Q000473:pathology* / Q000502:physiology*; D008969:Molecular Sequence Data; D016133:Polymerase Chain Reaction; D012333:RNA, Messenger / Q000032:analysis / Q000235:genetics; D012190:Retroviridae / Q000502:physiology*; D014407:Tumor Cells, Cultured; D001616:beta-Galactosidase / Q000235:genetics
D017931:DNA Primers; D004273:DNA, Neoplasm; D012333:RNA, Messenger; D001616:beta-Galactosidase