The level of expression of exogenous genes carried by transgenic mice typically varies from mouse to mouse and can be quite low. This behaviour is attributed to the influence of the mouse chromatin near the site of transgene integration. This 'position effect' has been seen in transgenic mice carrying the human beta-globin gene. It was however, abolished when DNase I hypersensitive sites (normally found 65 to 44 kilobases (kb) upstream) were linked to the human beta-globin transgene. Thus, the upstream DNA (previously named a dominant control or locus activation region, now denoted a locus control region) conferred the ability to express human beta-globin at high levels dependent on copy number on every mouse carrying the construct. We report here an investigation of chicken beta A-globin gene expression in transgenic mice. A 4.5-kb fragment carrying the beta A-globin gene and its downstream enhancer, without any far upstream elements, is sufficient to ensure that every transgenic mouse expresses chicken globin messenger RNA at levels proportional to the transgene copy number. Thus the chicken DNA elements that allow position-independent expression can function in mice. In marked contrast to the human beta cluster, these elements are no farther than 2 kb from the gene. The location of the elements within the cluster demonstrates that position independence can be mediated by DNA that does not define a gene cluster boundary.
"Transfection of drug resistance genes together with transgenes, each in separate expression cassette, to obtain stable transfectants has been a commonly used method. However, in this strategy, the drug resistance does not always appropriately reflect the expression level of the transgene because generally the stable expression levels of exogenous expression cassettes are highly sensitive to thier sites of integration, as a result of the local chromatin environment when the transgenes are randomly integrated into the host genome
, which affect the expression levels of the drug resistance gene cassette and the transgene cassette separately. "
[Show abstract][Hide abstract] ABSTRACT: Stable expression of transgenes is an important technique to analyze gene function. Various drug resistance genes, such as neo, pac, hph, zeo, bsd, and hisD, have been equally used as selection markers to isolate a transfectant without considering their dose-dependent characters.
We quantitatively measured the variation of transgene expression levels in mouse embryonic stem (mES) cells, using a series of bi-cistronic expression vectors that contain Egfp expression cassette linked to each drug resistant gene via IRES with titration of the selective drugs, and found that the transgene expression levels achieved in each system with this vector design are in order, in which pac and zeo show sharp selection of transfectants with homogenously high expression levels. We also showed the importance of the choice of the drug selection system in gene-trap or gene targeting according to this order.
The results of the present study clearly demonstrated that an appropriate choice of the drug resistance gene(s) is critical for a proper design of the experimental strategy.
"The CTCF-dependent enhancer-blocking element is also located at the downstream end of the domain in the area marked by the so-called 3′ constitutive HS
[10,11]. In addition to the LCR, the domain also possesses an internal enhancer (β/Ε-enhancer), which is located between the genes βA and Ε and is believed to contribute to the control of the activity of both genes
[12-14]. The partitioning of tasks between the LCR and the β/Ε-enhancer is not quite clear. "
[Show abstract][Hide abstract] ABSTRACT: Background
The β-globin gene domains of vertebrate animals constitute popular models for studying the regulation of eukaryotic gene transcription. It has previously been shown that in the mouse the developmental switching of globin gene expression correlates with the reconfiguration of an active chromatin hub (ACH), a complex of promoters of transcribed genes with distant regulatory elements. Although it is likely that observations made in the mouse β-globin gene domain are also relevant for this locus in other species, the validity of this supposition still lacks direct experimental evidence. Here, we have studied the spatial organization of the chicken β-globin gene domain. This domain is of particular interest because it represents the perfect example of the so-called ‘strong’ tissue-specific gene domain flanked by insulators, which delimit the area of preferential sensitivity to DNase I in erythroid cells.
Using chromosome conformation capture (3C), we have compared the spatial configuration of the β-globin gene domain in chicken red blood cells (RBCs) expressing embryonic (3-day-old RBCs) and adult (9-day-old RBCs) β-globin genes. In contrast to observations made in the mouse model, we found that in the chicken, the early embryonic β-globin gene, Ε, did not interact with the locus control region in RBCs of embryonic lineage (3-day RBCs), where this gene is actively transcribed. In contrast to the mouse model, a strong interaction of the promoter of another embryonic β-globin gene, ρ, with the promoter of the adult β-globin gene, βA, was observed in RBCs from both 3-day and 9-day chicken embryos. Finally, we have demonstrated that insulators flanking the chicken β-globin gene domain from the upstream and from the downstream interact with each other, which places the area characterized by lineage-specific sensitivity to DNase I in a separate chromatin loop.
Taken together, our results strongly support the ACH model but show that within a domain of tissue-specific genes, the active status of a promoter does not necessarily correlate with the recruitment of this promoter to the ACH.
"In addition the β A /ε enhancer and the upstream chicken β-globin LCR drive copy-numberdependent expression of a reporter gene in transgenic mice (Reitman et al., 1990). As mentioned before the β-globin LCR, containing wellcharacterized DHS, in collaboration with the β A /ε enhancer is able to drive position-independent gene expression in transgenic mice (Reitman et al., 1990; Abruzzo and Reitman, 1994; Mason et al., 1995). Individually, cHS2 and cHS3 showed significant enhancer activity while cHS1 did not. "
[Show abstract][Hide abstract] ABSTRACT: At the present time research situates differential regulation of gene expression in an increasingly complex scenario based on interplay between genetic and epigenetic information networks, which need to be highly coordinated. Here we describe in a comparative way relevant concepts and models derived from studies on the chicken alpha- and beta-globin group of genes. We discuss models for globin switching and mechanisms for coordinated transcriptional activation. A comparative overview of globin genes chromatin structure, based on their genomic domain organization and epigenetic components is presented. We argue that the results of those studies and their integrative interpretation may contribute to our understanding of epigenetic abnormalities, from beta-thalassemias to human cancer. Finally we discuss the interdependency of genetic-epigenetic components and the need of their mutual consideration in order to visualize the regulation of gene expression in a more natural context and consequently better understand cell differentiation, development and cancer.
Comparative Biochemistry and Physiology - Part A Molecular & Integrative Physiology 08/2007; 147(3):750-60. DOI:10.1016/j.cbpa.2006.10.037 · 1.97 Impact Factor
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