Lineage-specific transgene expression in hematopoietic cells using a Cre-regulated retroviral vector.
Transduction of bone marrow stem cells with retroviral expression vectors represents a cheaper and more rapid alternative to conventional transgenesis for studies of in vivo gene function. However, achieving tissue-specific expression of genes inserted into retroviral vectors is notoriously difficult. We have developed a single tri-cistronic retroviral vector (MG(f)I4) that facilitates Cre-dependent, lineage-specific gene expression within hematopoietic cells. Bone marrow stem cells transduced with MG(f)I4 co-express a loxP-flanked (floxed) eGFP cDNA together with truncated human CD4 (hCD4Delta). Open reading frames (ORFs) cloned between these two cDNAs are not constitutively translated but are activated upon Cre-mediated removal of the eGFP cDNA. Mice reconstituted with transduced bone marrow stem cells obtained from Cd19-Cre, Cr2-Cre or Lck-Cre, donors were shown to specifically express an ORF insert in the appropriate lymphocyte subsets. Cells that had activated ORF expression were identifiable by transition from a GFP+, hCD4+ to a GFP(-), hCD4+ phenotype. The use of this novel vector in conjunction with the wide range of well-characterized Cre-transgenic lines will be a versatile tool for exploring gene function within the immune system. In particular, this approach will provide a convenient way to test the functional significance of naturally occurring genetic mutations linked to human disease.
Turner
Vivian M
VM
Garvan Institute of Medical Research, 384 Victoria St, Darlinghurst NSW 2010, Australia.
Gardam
Sandra
S
Brink
Robert
R
D048868:Adaptor Proteins, Signal Transducing / Q000235:genetics / Q000378:metabolism; D000818:Animals; D018941:Antigens, CD19 / Q000235:genetics / Q000378:metabolism; D001854:Bone Marrow Cells / Q000473:pathology; D016026:Bone Marrow Transplantation; D015704:CD4 Antigens / Q000235:genetics / Q000378:metabolism; D019070:Cell Lineage / Q000235:genetics; D005822:Genetic Vectors; D049452:Green Fluorescent Proteins / Q000235:genetics; D006412:Hematopoietic Stem Cells / Q000276:immunology / Q000378:metabolism* / Q000473:pathology; D006801:Humans; D019426:Integrases / Q000235:genetics / Q000378:metabolism*; D016131:Lymphocyte Subsets / Q000276:immunology / Q000378:metabolism* / Q000473:pathology; D051379:Mice; D008810:Mice, Inbred C57BL; D008822:Mice, Transgenic; D009928:Organ Specificity / Q000235:genetics / Q000276:immunology; D017464:Receptors, Complement 3d / Q000235:genetics / Q000378:metabolism; D012190:Retroviridae / Q000235:genetics*; D017384:Sequence Deletion
D048868:Adaptor Proteins, Signal Transducing; D018941:Antigens, CD19; D015704:CD4 Antigens; D017464:Receptors, Complement 3d; C402726:Sh2d2a protein, mouse; D049452:Green Fluorescent Proteins; C045073:Cre recombinase; D019426:Integrases