The beta-globin HS4 insulator confers copy-number dependent expression of IgH regulatory elements in stable B cell transfectants

Locus control regions (LCR) were first defined by their theoretical ability to enhance the expression of linked genes in a tissue-specific, position-independent and copy-number dependent manner. In fact, few of the so-called LCR identified completely fulfil this definition. For example, the regulatory elements located in 5' (Emu) and 3' (HS3a; HS1,2; HS3b; HS4) of the IgH locus display some properties of a LCR but lack a copy-number dependence and sometimes display position effects in transgenes. In order to study whether addition of insulators would allow to overcome such problems in transgenes, we studied constructs harboring a V(H) promoter-green fluorescent protein reporter gene linked to the 3' and/or 5' IgH elements, surrounded or not with the chicken beta-globin 5'HS4 insulator. When flanked with insulators it appeared that either 3' IgH and 5' IgH regulatory elements now behave as true LCR elements and noticeably display copy-number dependence in transfected pre-B or B cell lines.     

Locus control region elements HS2 and HS3 in combination with chromatin boundaries confer high-level expression of a human beta-globin transgene in a centromeric region

Expression constructs are subject to position-effects in transgenic assays unless they harbour elements that protect them from negative or positive influences exerted by chromatin at the site of integration. Locus control regions (LCRs) and boundary elements are able to protect from position effects by preventing heterochromatization of linked genes. The LCR in the human beta-globin gene locus is located far upstream of the genes and composed of several erythroid specific DNase I hypersensitive (HS) sites. Previous studies demonstrated that the LCR HS sites act synergistically to confer position-independent and high-level globin gene expression at different integration sites in transgenic mice. Here we show that LCR HS sites 2 and 3, in combination with boundary elements derived from the chicken beta-globin gene locus, confer high-level human beta-globin gene expression in different chromosomal integration sites in transgenic mice. Moreover, we found that the construct is accessible to nucleases and highly expressed when integrated in a centromeric region. These results demonstrate that the combination of enhancer, chromatin opening and boundary activities can establish independent expression units when integrated into chromatin.     

The beta-globin locus control region versus gene therapy vectors: a struggle for expression

Developmental control of gene expression has a major impact on the design of beta-globin retrovirus vectors for hematopoietic stem cell gene therapy of beta-thalassemia. It is obvious that the endogenous locus control region (LCR) elements that drive beta-globin gene expression in transgenic mice must be included in these vectors. However, the specific elements to use are not clear and require an understanding of LCR action. Moreover, retrovirus vectors contain silencer elements that function in stem cells and are dominant to LCR function. Recent studies on LCRbeta-globin transgenes and retrovirus silencing suggest ways to overcome this silencing effect after transfer into stem cells and carefully designed lentivirus vectors have exciting therapeutic benefit in animal models of beta-thalassemia. By building on 15 years of development, LCRbeta-globin vectors are now being tested in preclinical animal models and may ultimately lead to the long-sought cure for this genetic disease.     

Importance of globin gene order for correct developmental expression.

We have used transgenic mice to study the influence of position of the human globin genes relative to the locus control region (LCR) on their expression pattern during development. The LCR, which is located 5' of the globin gene cluster, is normally required for the activation of all the genes. When the human beta-globin gene is linked as a single gene to the LCR it is activated prematurely in the embryonic yolk sac. We show that the correct timing of beta gene activation is restored when it is placed farther from the LCR than a competing human gamma- or alpha-globin gene. Correct timing is not restored when beta is the globin gene closest to the LCR. Similarly, the human gamma-globin gene is silenced earlier when present farthest from the LCR. On the basis of this result, we propose a model of developmental gene control based on stage-specific elements immediately flanking the genes and on polarity in the locus. We suggest that the difference in relative distance to the LCR, which is a consequence of the ordered arrangement of the genes, results in nonreciprocal competition between the genes for activation by the LCR.     

Multiple interactions between regulatory regions are required to stabilize an active chromatin hub

The human beta-globin locus control region (LCR) is required for the maintenance of an open chromatin configuration of the locus. It interacts with the genes and the hypersensitive regions flanking the locus to form an active chromatin hub (ACH) transcribing the genes. Proper developmental control of globin genes is largely determined by gene proximal regulatory sequences. Here, we provide the first functional evidence of the role of the most active sites of the LCR and the promoter of the beta-globin gene in the maintenance of the ACH. When the human beta-globin gene promoter is deleted in the context of a full LCR, the ACH is maintained with the beta-globin gene remaining in proximity. Additional deletion of hypersensitive site HS3 or HS2 of the LCR shows that HS3, but not HS2, in combination with the beta-globin promoter is crucial for the maintenance of the ACH at the definitive stage. We conclude that multiple interactions between the LCR and the beta-globin gene are required to maintain the appropriate spatial configuration in vivo.     

慢病毒载体介导的转基因整合位点研究方法

慢病毒载体整合到宿主细胞的染色体上可以长期稳定表达,目前已成为基因治疗和转基因动物载体研究的热点.慢病毒载体整合的位置效应是影响外源基因表达的重要因素,慢病毒载体整合位点的研究是探索外源基因整合机制的手段之一.转基因整合位点研究的方法主要有5种:荧光原位杂交、个体基因组文库筛选法、反向PCR、接头PCR、锚定PCR.近来的研究后发现整合位点之间可能存在一些共同特征.     

Enhanced expression and stable transmission of transgenes flanked by inverted terminal repeats from adeno-associated virus in zebrafish.

Mosaic expression of transgenes in the F0 generation severely hinders the study of transient expression in transgenic fish. To avoid mosaicism, enhanced green fluorescent protein (EGFP) gene cassettes were constructed and introduced into one-celled zebrafish embryos. These EGFP gene cassettes were flanked by inverted terminal repeats (ITRs) from adeno-associated virus (AAV) and driven by zebrafish alpha-actin (palpha-actin-EGFP-ITR) or medaka beta-actin promoters (pbeta-actin-EGFP-ITR). EGFP was expressed specifically and uniformly in the skeletal muscle of 56% +/- 8% of the palpha-actin-EGFP-ITR-injected survivors and in the entire body of 1.3% +/- 0.8% of the pbeta-actin-EGFP-ITR-injected survivors. Uniform transient expression never occurred in zebrafish embryos injected with EGFP genes that were not flanked by AAV-ITRs. In the F0 generation, uniformly distributed EGFP could mimic the stable expression in transgenic lines early in development. We established five transgenic lines derived from palpha-actin-EGFP-ITR-injected embryos crossed with wild-type fish and 11 transgenic lines derived from pbeta-actin-EGFP-ITR-injected embryos crossed with wild-type fish. None of these transgenic lines failed to express the transgene, a result confirmed by polymerase chain reaction analysis. Stable mendelian transmission of the transgenes was achieved in both alpha-actin and beta-actin transgenic lines without changing the patterns of expression and integration. Progeny inheritance test and Southern blot analysis results strongly suggest that transgenes flanked by AAV-ITRs were integrated randomly into the genome at a single locus with a concatamerized multiplier. Thus, incorporating AAV-ITRs into transgenes results in uniform gene expression in the F0 generation and stable transmission of transgenes in zebrafish.