Transduction of human hematopoietic cells and cell lines using a retroviral vector containing a modified murine CD4 reporter gene.
To investigate conditions for improving transduction efficiencies of human hematopoietic stem or progenitor cells using retroviral vectors, we constructed a retroviral vector containing a modified murine CD4 cDNA reporter gene with a truncated cytoplasmic domain to prevent signaling. The advantages of using this truncated murine CD4 reporter gene include: (i) CD4 is well characterized with well-known cell signaling pathways, (ii) truncation of the cytoplasmic domain of CD4 has been demonstrated to abrogate signaling, (iii) the truncated murine CD4 is easily detectable on the cell surface with no cross-reaction to human CD4, (iv) a variety of monoclonal antibodies directed against the murine CD4 molecule are available commercially, and (v) expression of a truncated CD4 molecule in a transgenic mouse in vivo does not interfere with hematopoiesis. We cloned the truncated murine CD4 reporter gene into the retroviral vector LXSN, packaged this vector using PG13 retrovirus packaging cells, and transduced hematopoietic cell lines representing erythroid, myeloid, megakaryocyte, and lymphoid lineages using vector-containing medium harvested from the murine CD4 producer line. After seven daily exposures to vector-containing medium, all cell lines expressed murine CD4 on the cell surface, and 5-7% of human CD34+ cells expressed murine CD4 on the cell surface after 3 days of exposure to murine CD4 vector-containing medium. Colony-forming cell assays assessing progenitor cells demonstrated the presence of transduced cells in the CD34+ population. These results demonstrate the utility of using a modified murine CD4 gene in a retroviral vector to allow optimization of in vitro transduction conditions of human hematopoietic cells and to facilitate identification of the lineages that have been transduced using different growth factors, prior to clinical trials using retroviral vectors.
Bauer
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Medical Research Service, VA Puget Sound Health Care System, Seattle, WA 98108, USA.
Hickstein
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D000595:Amino Acid Sequence; D000818:Animals; D018952:Antigens, CD34 / Q000276:immunology; D015704:CD4 Antigens / Q000235:genetics* / Q000276:immunology; D003114:Colony-Forming Units Assay; D005434:Flow Cytometry; D018014:Gene Transfer Techniques; D017930:Genes, Reporter / Q000235:genetics* / Q000276:immunology; D005822:Genetic Vectors / Q000737:chemistry*; D018922:HL-60 Cells; D006412:Hematopoietic Stem Cells / Q000276:immunology / Q000378:metabolism*; D006801:Humans; D004915:Leukemia, Erythroblastic, Acute; D015464:Leukemia, Myelogenous, Chronic, BCR-ABL Positive; D016403:Lymphoma, Large B-Cell, Diffuse; D051379:Mice; D008969:Molecular Sequence Data; D016133:Polymerase Chain Reaction; D012190:Retroviridae / Q000235:genetics*; D014407:Tumor Cells, Cultured
D018952:Antigens, CD34; D015704:CD4 Antigens