Transient transfection of human peripheral blood basophils.
The human basophil has resisted previous attempts at transient transfection. Basophils were transfected by nucleoporation and to test whether there was sufficient expression to modify cell function, the cells were transfected with a syk kinase tandem SH2 construct linked to GFP. This approach was taken because in RBL cells and murine mast cells syk kinase is known to play a very early role in signal transduction and previous studies in RBL cells demonstrated that expression of the tandem SH2 domains of syk would inhibit signaling, presumably by competition with endogenous syk for binding to ITAMs. Results from basophil transfections with SH2syk were compared to an empty construct. Basophils were stimulated with anti-IgE antibody and analyzed for single cell changes in cytosolic calcium levels. Basophils expressing the empty GFP construct showed a cytosolic calcium response similar to non-expressing cells. In contrast, basophils expressing the GFP-tandem SH2syk construct, on average, showed an anti-IgE-induced calcium response that was completely ablated. The transfection frequency was 8% (median), with an average viable recovery of 12% (n=18). While the procedure is not benign and is not always successful, these studies indicate that with gating techniques, the human basophil, a non-dividing primary leukocyte, can be transiently transfected to express high enough levels of an inhibitory protein to alter an IgE-mediated response.
Vilariño
Natalia
N
Johns Hopkins Asthma and Allergy Center, 5501 Hopkins Bayview Circle, Baltimore, MD 21224, USA.
MacGlashan
Donald
D
D000888:Antibodies, Anti-Idiotypic / Q000276:immunology / Q000494:pharmacology; D001491:Basophils / Q000187:drug effects / Q000276:immunology*; D002118:Calcium / Q000032:analysis / Q000378:metabolism; D002467:Cell Nucleus / Q000737:chemistry / Q000378:metabolism; D002478:Cells, Cultured; D003600:Cytosol / Q000737:chemistry / Q000378:metabolism; D004792:Enzyme Precursors / Q000032:analysis / Q000235:genetics / Q000502:physiology; D049452:Green Fluorescent Proteins / Q000032:analysis / Q000235:genetics; D006801:Humans; D007073:Immunoglobulin E / Q000276:immunology; D047908:Intracellular Signaling Peptides and Proteins; D011505:Protein-Tyrosine Kinases / Q000032:analysis / Q000235:genetics / Q000502:physiology; D000072377:Syk Kinase; D014162:Transfection / Q000379:methods*; D018909:src Homology Domains / Q000235:genetics / Q000502:physiology
D000888:Antibodies, Anti-Idiotypic; D004792:Enzyme Precursors; D047908:Intracellular Signaling Peptides and Proteins; C485184:enhanced green fluorescent protein; D049452:Green Fluorescent Proteins; D007073:Immunoglobulin E; D011505:Protein-Tyrosine Kinases; C000606896:SYK protein, human; D000072377:Syk Kinase; D002118:Calcium