Stable knockdown of MAFA expression in RBL-2H3 cells by siRNA retrovirus-delivery system.
The mast cell function-associated antigen (MAFA) is a type II membranal glycoprotein first discovered on the surface of the rat mucosal-type mast cells of the RBL-2H3 line. A C-type lectin domain and an immunoreceptor tyrosine-based inhibitory motif (ITIM) are located in the extracellular and intracellular domains of MAFA, respectively. MAFA clustering by its specific monoclonal antibody G63, has been previously shown to cause a dose-dependent inhibition of the secretory response of these cells to the FcRI stimulus. More recently, human and mouse homologues of MAFA have also been discovered. However, they are expressed also or only by NK- and T cells, suggesting that MAFA may play additional functional role(s). To further pursue MAFA's functional significance in mast cells, we have employed the recently developed siRNA (small inhibitory RNA)-dependent gene-specific silencing protocol. We have first incorporated the key elements of the well-defined and commercially available siRNA vector (pSilencer1.0-U6) together with anti-MAFA hairpin into a retroviral vector (pLXSNneo). This enabled generating retroviruses that induced a stable and efficient downregulation (knockdown) of MAFA's expression. RBL-2H3 cells with either normal or knocked-down MAFA expression levels are currently examined for their biological responses e.g. FcRI mediated secretion, cell proliferation and cell adhesion. So far however, no significant changes were resolved in the above biological functions.
Abramson
Jakub
J
Department of Immunology, The Weizmann Institute of Science, 761 00 Rehovot, Israel.
Rozenblum
Guido
G
Pecht
Israel
I
D000818:Animals; D001483:Base Sequence; D002448:Cell Adhesion / Q000276:immunology / Q000502:physiology; D002455:Cell Division / Q000276:immunology / Q000502:physiology; D005822:Genetic Vectors; D006801:Humans; D037181:Lectins, C-Type / Q000235:genetics* / Q000276:immunology / Q000378:metabolism; D008407:Mast Cells / Q000276:immunology / Q000378:metabolism*; D008562:Membrane Glycoproteins / Q000235:genetics* / Q000276:immunology / Q000378:metabolism; D051379:Mice; D008969:Molecular Sequence Data; D034741:RNA, Small Interfering; D051381:Rats; D017455:Receptors, IgE / Q000276:immunology; D011971:Receptors, Immunologic; D012190:Retroviridae; D015534:Trans-Activators
C113413:KLRG1 protein, human; C451496:Klrg1 protein, mouse; C094696:Klrg1 protein, rat; D037181:Lectins, C-Type; D008562:Membrane Glycoproteins; D034741:RNA, Small Interfering; D017455:Receptors, IgE; D011971:Receptors, Immunologic; D015534:Trans-Activators