Evaluation of expression of transferred genes in differentiating myeloid cells: expression of human glucocerebrosidase in murine macrophages.
The retroviral vector LGSN, in which the human glucocerebrosidase (GC) cDNA is driven by the Moloney murine leukemia virus (MoMLV) long terminal repeat (LTR), was tested for expression in the murine myelomonocytic leukemia cell line M1 before and after induction of differentiation with interleukin-6 (IL-6). Southern analysis of the seven transduced clones selected for neomycin resistance in Geneticin (G-418 sulfate) demonstrated one to eight copies of intact provirus with rearrangements in only two clones. Absolute levels of human GC RNA and protein increased with increased copy numbers of provirus in the clones. Upon induction with IL-6 of the seven transduced clones to the macrophage phenotype, there was no significant change, overall, in RNA levels but some increase in human GC protein levels could be detected. Although this was the average trend, considerable clonal variation in RNA and protein levels was observed upon induction. Transduction of the M1 cells did not interfere with the ability of the cells to differentiate from blasts to macrophages as seen by the appearance of membrane receptors for the constant region of immunoglobulins (Fc gamma RI) and lysozyme production in the differentiated M1 cells. Thus, the M1 cell line can be used for testing retroviral vector expression in myeloid lineages at early and late stages of differentiation. This rapid in vitro testing of potential retroviral vectors will be beneficial for gene therapy of disorders that affect differentiated macrophages such as Gaucher's disease.
Freas
D L
DL
Molecular and Medical Genetics Section, National Institutes of Neurological Disorders and Stroke, Bethesda, MD 20892.
Correll
P H
PH
Dougherty
S F
SF
Karlsson
S
S
Pluznik
D H
DH
D000818:Animals; D002454:Cell Differentiation / Q000187:drug effects / Q000235:genetics; D004247:DNA; D005822:Genetic Vectors; D005962:Glucosylceramidase / Q000096:biosynthesis / Q000235:genetics*; D006801:Humans; D015850:Interleukin-6 / Q000494:pharmacology; D015479:Leukemia, Myelomonocytic, Acute; D008264:Macrophages / Q000166:cytology / Q000378:metabolism*; D051379:Mice; D008979:Moloney murine leukemia virus / Q000235:genetics; D012313:RNA / Q000378:metabolism; D012091:Repetitive Sequences, Nucleic Acid; D014162:Transfection; D014407:Tumor Cells, Cultured
D015850:Interleukin-6; D012313:RNA; D004247:DNA; D005962:Glucosylceramidase