Lineage-specific transgene expression in hematopoietic cells using a versatile 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. Constitutive expression of retroviral gene constructs in hematopoietic cells can be achieved using the endogenous 5? LTR promoter, but this is often undesirable due to potential perturbations of hematopoiesis and unrestricted expression in the various hematopoietic lineages. We describe here a single tri-cistronic retroviral vector (MGfI4) that facilitates Cre-dependent, lineage-specific gene expression within hematopoietic cells. Bone marrow stem cells transduced with MGfI4 co-express a loxP-flanked (floxed) eGFP cDNA together with truncated human CD4 (hCD4?). 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 Lck-cre, Cd19-cre or Cr2¬-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 with specific patterns of expression within the hematopoietic system is a versatile tool for exploring gene function within the immune system.
|ISBN||1872-7905 (Electronic) 0022-1759 (Linking)|
|Authors||Turner, V.M; Gardam, S.; Brink, R.|
|Publisher Name||J IMMUNOL METHODS|
|Published Date||2010-08-16 00:00:00|
|OpenAccess Link||https://publications.gimr.garvan.org.au/download.php?10590_10988/10 Turner J Imm Meth.pdf|