Cell-specific and efficient expression in mouse and human B cells by a novel hybrid immunoglobulin promoter in a lentiviral vector.
The expression of genes specifically in B cells is of great interest in both experimental immunology as well as in future clinical gene therapy. We have constructed a novel enhanced B cell-specific promoter (Igk-E) consisting of an immunoglobulin kappa (Igk) minimal promoter combined with an intronic enhancer sequence and a 3' enhancer sequence from Ig genes. The Igk-E promoter was cloned into a lentiviral vector and used to control expression of enhanced green fluorescent protein (eGFP). Transduction of murine B-cell lymphoma cell lines and activated primary splenic B cells, with IgK-E-eGFP lentivirus, resulted in expression of eGFP, as analysed by flow cytometry, whereas expression in non-B cells was absent. The specificity of the promoter was further examined by transducing Lin(-) bone marrow with Igk-E-eGFP lentivirus and reconstituting lethally irradiated mice. After 16 weeks flow cytometry of lymphoid tissues revealed eGFP expression by CD19+ cells, but not by CD3+, CD11b+, CD11c+ or Gr-1+ cells. CD19+ cells were comprised of both marginal zone B cells and recirculating follicular B cells. Activated human peripheral mononuclear cells were also transduced with Igk-E-eGFP lentivirus under conditions of selective B-cell activation. The Igk-E promoter was able to drive expression of eGFP only in CD19+ cells, while eGFP was expressed by both spleen focus-forming virus and cytomegalovirus constitutive promoters in CD19+ and CD3+ lymphocytes. These data demonstrate that in these conditions the Igk-E promoter is cell specific and controls efficient expression of a reporter protein in mouse and human B cells in the context of a lentiviral vector.
Laurie
K L
KL
Molecular Immunology Unit, Wolfson Centre for Gene Therapy of Childhood Disease, UCL Institute of Child Health, London, UK.
Blundell
M P
MP
Baxendale
H E
HE
Howe
S J
SJ
Sinclair
J
J
Qasim
W
W
Brunsberg
U
U
Thrasher
A J
AJ
Holmdahl
R
R
Gustafsson
K
K
D000818:Animals; D001402:B-Lymphocytes / Q000821:virology*; D002460:Cell Line; D005260:Female; D005434:Flow Cytometry; D015870:Gene Expression; D005818:Genetic Engineering; D015316:Genetic Therapy / Q000379:methods*; D005822:Genetic Vectors / Q000008:administration & dosage* / Q000235:genetics; D049452:Green Fluorescent Proteins / Q000235:genetics; D015497:HIV-1 / Q000235:genetics*; D006801:Humans; D007136:Immunoglobulins / Q000235:genetics*; D008297:Male; D051379:Mice; D008810:Mice, Inbred C57BL; D011401:Promoter Regions, Genetic; D020133:Reverse Transcriptase Polymerase Chain Reaction; D014161:Transduction, Genetic / Q000379:methods; D019076:Transgenes
C014609:IgK; D007136:Immunoglobulins; C485184:enhanced green fluorescent protein; D049452:Green Fluorescent Proteins