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Le Bert M Comte I Dessain ML Drouet M Ayer Le Lievre C Cogné M Denizot Y Combination of and IgH regulatory elements mimics the B specific endogenous expression pattern of IgH genes from pro B cells to mature B cells in a transgenic mouse model

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Niveau: Supérieur, Doctorat, Bac+8
lato gH gen Co Mic , 2 ru S 44, VH promoter-GFP reporter gene linked to the 3VLCR region and the EA ta 164 A complex interplay of multiple regulatory elements is responsible for tissue-specific and stage-specific regulation of both transcription and rearrangements of the IgH chain locus. Several events such as the germline transcription of the CA region and the initiation of VDJ rearrangements are regulated by upstream elements including the VH promoter and the EA intronic enhancer [1]. In addition, four lym- phoid-specific transcriptional enhancers (namely hs3a, hs1,2, hs3b and hs4) have been located in a 3V regulatory region downstream the locus. It has been suggested that the latter altogether act as a locus control region (LCR). The [3] reported that transgenic mice bearing a h-globin report- er gene linked to the 3VLCR displayed a high level, position-independent and B cell-specific expression. Our knowledge of the role of these 3Venhancers mostly derives from in vitro studies [4]. Thus, transient transfection experi- ments and reporter gene assays revealed that the four 3V enhancers are rather weak by themselves but that their combination display a strong transcriptional synergy espe- cially when the normal palindromic arrangement is respected [4–6]. All these studies provided important information about synergies between enhancers and sug- gested a B cell-restricted activity of the 3V IgH elements.

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  • vlcr transgenic

  • ea- gfp

  • yielded both

  • gene

  • kb pcr

  • gfp


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Biochimica et Biophysica Acta 1642 (2003) 181 – 190
Combination of 3Vand 5VIgH regulatory elements mimics the endogenous expression pattern of IgH genes from proB cells B cells in a transgenic mouse model
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Bspecific to mature
a, b a b a * Laurence Guglielmi , Marc Le Bert , Isabelle Comte , Marie Laure Dessain , Mireille Drouet , a a a Christiane AyerLe Lievre , Michel Cogne´ , Yves Denizot a UMR CNRS 6101, Faculte´ de Me´decine, 2 rue Dr. Marcland, 87025 Limoges, France b CDTA,UPSCNRS44,Orl´eans,France Received 14 April 2003; received in revised form 31 July 2003; accepted 12 August 2003
Abstract
To ensure the B cell differentiation stage specificity of the intronic EAelement and of the locus control region (LCR) that lies downstream of the IgH chain locus, we generated transgenic mice harboring a VHpromoterGFP reporter gene linked to the 3VLCR region and the EA + + + + element. By flow cytometry, GFP lymphocytes were observed amongst proB cells (B220 CD43 CD117 ) and at all stages of + + + differentiation up to mature B cells (B220 IgM IgD ). Expression was strictly confined to cells committed to the B lymphocyte lineage as + + ++ + judged by the lack of GFP Thy1,2 cells (T lymphocytes) and GFP B220 CD117 CD43 cells (uncommitted lymphohematopoietic progenitors). Therefore, the EAGFP3VLCR transgene is not expressed by hematopoietic stem cells, begins its expression in proB cells and is specifically active at all stages of B cell maturation. The combination of 3Vand 5VIgH regulatory elements thus appears as a potentially useful cassette in transgenes that require a stringent and early B lineagespecific expression. D2003 Elsevier B.V. All rights reserved.
Keywords:B lymphocyte; Transgenic; Gene regulation; EA; 3VLCR
1. Introduction
A complex interplay of multiple regulatory elements is responsible for tissuespecific and stagespecific regulation of both transcription and rearrangements of the IgH chain locus. Several events such as the germline transcription of the CAregion and the initiation of VDJ rearrangements are regulated by upstream elements including the VHpromoter and the EAintronic enhancer[1]. In addition, four lym phoidspecific transcriptional enhancers (namely hs3a, hs1,2, hs3b and hs4) have been located in a 3Vregulatory region downstream the locus. It has been suggested that the latter altogether act as a locus control region (LCR). The presence of three of these elements (hs1,2, hs3b and hs4) confers a positionindependent and a copydependent ex pression to a cmyc transgene transfected in a plasmacy
* Corresponding author. Tel.: +33555435896; fax: +3355543 5897. Email address:laurence.guglielmi@unilim.fr (L. Guglielmi).
01674889/$  see front matterD2003 Elsevier B.V. All rights reserved. doi:10.1016/j.bbamcr.2003.08.005
toma cell line[2]. Confirming these results, Chauveau et al. [3]reported that transgenic mice bearing ahglobin report er gene linked to the 3VLCR displayed a high level, positionindependent and B cellspecific expression. Our knowledge of the role of these 3Venhancers mostly derives from in vitro studies[4]. Thus, transient transfection experi ments and reporter gene assays revealed that the four 3V enhancers are rather weak by themselves but that their combination display a strong transcriptional synergy espe cially when the normal palindromic arrangement is respected[4 – 6]. All these studies provided important information about synergies between enhancers and sug gested a B cellrestricted activity of the 3VIgH elements. However, they analyzed only bulk of transfected cells or whole organs and did not allow to definitely assess at what stage of B cell development and in which percentage of cells of the B or T lineage transgenes were expressed. The EAintronic enhancer has been already used in DNA constructs in order to target expression to the B cell lineage. However, EAdriven transgenes are often transcribed at high