Repressible antisense inhibition in B lymphocytes.
The tetracycline-responsive promoter (TRP) system has been adopted in an attempt to obtain repressible antisense inhibition in a B lymphocyte model in vitro. Levels of secreted IgM protein and mRNA were assessed following the stable transfection of B cell line, HO-2.2, with a series of plasmid constructs containing antisense or sense target sequence DNA (the 3'-untranslated region adjacent to the secreted exon of IgM gene) under the control of the TRP. Significant reduction (approximately 90%) in IgM secretion was observed for clones transfected with antisense plasmids driven by the TRP and containing the IgH enhancer element and the polyadenylation signal sequence from membrane IgM, when compared with untransfected and sense controls. Tetracycline (1 microgram/ml) addition to the culture medium restored the level of IgM secretion in these clones to control values, demonstrating repressibility of antisense inhibition. Transfection of HO-2.2 cells with antisense (or sense) constructs had no detectable effect on membrane IgM protein levels. Hybridisation studies demonstrated that decreased protein production observed in the antisense-transfected clones was most likely attributable to reduced RNA levels. These data show that the TRP can be used for repressible and specific antisense inhibition of gene product expression in B lymphocytes.
McCall
M N
MN
Centenary Institute of Cancer Medicine and Cell Biology, New South Wales, Australia. m.mccall@centenary.usyd.edu.au
D000818:Animals; D016375:Antisense Elements (Genetics) / Q000494:pharmacology*; D001402:B-Lymphocytes / Q000187:drug effects / Q000502:physiology*; D002462:Cell Membrane / Q000378:metabolism; D003587:Cytomegalovirus / Q000235:genetics; D004742:Enhancer Elements, Genetic; D007075:Immunoglobulin M / Q000187:drug effects / Q000235:genetics* / Q000378:metabolism; D017403:In Situ Hybridization; D051379:Mice; D016376:Oligonucleotides, Antisense / Q000235:genetics / Q000494:pharmacology; D010957:Plasmids / Q000235:genetics; D011061:Poly A; D011401:Promoter Regions, Genetic / Q000187:drug effects; D016372:RNA, Antisense / Q000187:drug effects / Q000235:genetics; D012045:Regulatory Sequences, Nucleic Acid; D013752:Tetracycline / Q000494:pharmacology; D014158:Transcription, Genetic; D014162:Transfection
D016375:Antisense Elements (Genetics); D007075:Immunoglobulin M; D016376:Oligonucleotides, Antisense; D016372:RNA, Antisense; D011061:Poly A; D013752:Tetracycline