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Effect of the Em IgH enhancer on expression of a GFP reporter gene in transfected B cells and transgenic mice

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Effect of the Em IgH enhancer on expression of a GFP reporter gene in transfected B cells and transgenic mice Laurence Guglielmi a, Marc Le Bert b, Michel Cogne a, Yves Denizot a,* a UMR CNRS 6101, Faculte de Medecine, 2 rue Dr. Marcland, 87025 Limoges, France b CDTA, UPS CNRS 44, Orleans, France Received 19 September 2002; accepted 31 October 2002 Abstract Transgenic mice were generated to identify the first B cell maturation stage showing expression of an immunoglobulin transcriptional enhancer element (Em)-green fluorescent protein (GFP) transgene, and to check the ability of the Em element to behave as a locus control region. Flow cytometry experiments indicated that stably transfected 18/81 cells (a murine pre-B cell line) and A20 cells (a murine IgM B cell line) maintained a constant GFP expression for several months in culture. Contrasting with in vitro results, flow cytometry experiments did not highlight GFP B cells in spleen and bone marrow of Em-GFP transgenic mice and no GFP transcripts were detected by Northern blot and reverse transcriptase polymerase chain reaction analysis. In transgenic mice, the lack of GFP expression seemed related to transgene DNA methylation occurring within all organs. Our results show dramatic differences for expression of the Em-GFP transgene in vitro and in vivo. Despite that Em was reported to efficiently control the in vivo expression of other associated transgenes, it is not sufficient to sustain GFP expression in transgenic mice and to counteract developmental silencing programs that occur in the embryo.

  • genomic dna

  • kb afiii genomic

  • transfected

  • em-gfp transgenic

  • kb pcr

  • a20 cells

  • stably transfected


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Immunology Letters 86 (2003) 7783 /
www.elsevier.com/locate/
Effect of the EmIgH enhancer on expression of a GFP reporter gene in transfected B cells and transgenic mice
Abstract
a b a a, LaurenceGuglielmi,MarcLeBert,MichelCogn´e,Yves Denizot* a UMRCNRS6101,Facult´edeM´edecine,2rueDr.Marcland,87025Limoges,France b CDTA, UPS CNRS 44, Orleans, France
Receiv31 October 2002September 2002; accepted ed 19
Transgenic mice were generated to identify the first B cell maturation stage showing expression of an immunoglobulin transcriptional enhancer element (Em)green fluorescent protein (GFP) transgene, and to check the ability of the Emelement to behave as a locus control region. Flow cytometry experiments indicated that stably transfected 1881 cells (a murine preB cell line) / and A20 cells (a murine IgM B cell line) maintained a constant GFP expression for several months in culture. Contrasting with in vB cells in spleen and bone marrow of Eitro results, flow cytometry experiments did not highlight GFP mGFP transgenic mice and no GFP transcripts were detected by Northern blot and reverse transcriptase polymerase chain reaction analysis. In transgenic mice, the lack of GFP expression seemed related to transgene DNA methylation occurring within all organs. Our results show dramatic differences for expression of the EmGFP transgene invitro and invivo. Despite that Emwas reported to efficiently control the in vivo expression of other associated transgenes, it is not sufficient to sustain GFP expression in transgenic mice and to counteract developmental silencing programs that occur in the embryo. #2002 Elsevier Science B.V. All rights reserved.
Keywords:B lymphocytes; Transgenic mouse; Gene regulation; Emenhancer; Methylation
1. Introduction
A complex interplay of multiple regulatory elements is responsible for tissuespecific and stage specific regula tion of both transcription and rearrangements of the IgH chain locus. Several events such as the germline transcription of the Cmregion and the initiation of VDJ rearrangements are regulated by upstream elements including the VHand DQ52 promoters and the Em intronic enhancer[1]. Emis located between the JHgene segment cluster and the constant Cmcoding sequence. The key role of Emin early B cell development has been highlighted in knockout mice[2]. The core region of the mouse Emenhancer, which contains multiple transcrip tion factor binding sites, is flanked by two matrix attachment region (MARs)[3]. Our knowledge concern
* Corresponding author. Tel.:33555435896; fax:33555 / / 435897 Email address:yves.denizot@unilim.fr(Y. Denizot).
ing the role of the Emintronic enhancer derives from in vitro and invivo studies and this elements is usually reported as a strong enhancer, supposedly able to behave as a locus control region when it is flanked by its MARs. Thus, the Emelement has been widely used in transgenes in order to target expression in cells of the B and T lineages[46]. The green fluorescent protein / (GFP) is a 238 amino acid protein that requires no host cofactor and emits a green fluorescence (lmax507 nm) / in living cells transfected with GFP cDNA after stimulation with UV light (lmax488 nm)[7]. Taking / advantage of the spontaneous GFP fluorescence, we therefore undertook the generation of transgenic mice harboring a VHpromoterGFP reporter gene linked to the Emelement in order to identify precisely the first B cell maturation stage showing expression of the EmGFP transgene. It was found that the EmGFP transgene was not expressed in transgenic mice, a result that markedly differs from data obtained with stably EmGFPtrans fected B cell lines.
01652478/02/$  see front matter#2002 Elsevier Science B.V. All rights reserved. PII: S 0 1 6 5  2 4 7 8 ( 0 2 ) 0 0 2 6 4  X