Insulators: many functions, many mechanisms

West, Adam G.; Gaszner, Miklos; Felsenfeld, Gary

doi:10.1101/gad.954702

Cited by 578 publications

(482 citation statements)

References 132 publications

(133 reference statements)

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“…37 However, a recent study by Recillas-Targa et al 18 indicated that different domains of cHS4 are responsible for position effect and long-term silencing protections. In our study, ArsI in a plus orientation prevented variability of expression, while it failed to maintain expression in the long term.…”

Section: Discussionmentioning

confidence: 98%

“…Various insulators have been found from Drosophila and vertebrates, including chicken, mouse and human. [10][11][12][13] These insulators have two conserved properties, 14 one is an enhancer-blocking activity in which the insulator blocks enhancer/promoter interaction when placed in between them and the other is the protection from the position effect. Specifically, by flanking the promoter/reporter cassette with two insulators, the cassette will be isolated from the local chromosomal environment and therefore be protected from the position effect.…”

Section: Introductionmentioning

confidence: 99%

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Sea urchin insulator protects lentiviral vector from silencing by maintaining active chromatin structure

et al. 2004

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Suppressed expression of transgenes in vivo is the major obstacle in the gene therapy. For the long-term expression, we utilized a chromatin insulator from sea urchin arylsulfatase (Ars) gene locus (Ars insulator, ArsI), which has been shown to epigenetically regulate gene expression across species. ArsI was able to prevent silencing of the transgene in a myeloid cell line, HL-60, and a murine embryonic stem cell line, CCE, in an orientation-dependent manner, but not in Huh-7, K562 and MCF-7 cells, indicating that the effect of ArsI on gene silencing was cell type dependent. Although anti-silencing effect of ArsI was almost equivalent to that of chicken b-globin insulator, incorporation of ArsI into lentiviral vector had little effect on the virus titer compared with chicken b-globin insulator. Clonal analysis of transduced HL-60 cells revealed that ArsI protects the lentiviral vector from position effects regardless of its orientation. Furthermore, chromatin immunoprecipitation assays revealed that a high acetylation level was observed in the promoter of the insulated vector, whereas that of ArsI was independent of its anti-silencing capacity. In addition to it having little deteriorative effect on the virus titer, the identified anti-silencing effect of ArsI suggested its possibility for application in gene therapy.

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Section: Discussionmentioning

confidence: 98%

Section: Introductionmentioning

confidence: 99%

Sea urchin insulator protects lentiviral vector from silencing by maintaining active chromatin structure

et al. 2004

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“…As cells differentiate, multicellular eukaryotes render regions of chromosomes transcriptionally inactive by a process called silencing 16,17 . Chromatin silencing can spread to neighboring loci and the insertion of a transgene into a We inserted duplicated copies of the chicken β-globin 5′HS4 250 base-pair insulator flanking the CMV driven GFP into our reporter plasmid.…”

Section: Introductionmentioning

confidence: 99%

“…The transcriptional silencing (or the inappropriate activation by insertion next to an enhancer) of a transgene based on where it inserts into the genome is referred to as chromosome position effect 18 . Insulators are DNA sequences that protect genes from chromosome position effects 16,17 . One of the best characterized insulators is the chicken β-globin 5′ HS4 insulator 2,19 .…”

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confidence: 99%

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Transgenic Xenopus laevis embryos can be generated using φC31 integrase

2005

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Phage ϕC31 encodes an integrase that can mediate the insertion of extrachromosomal DNA into genomic DNA 1 . Here we show ϕC31 integrase can be used to generate transgenic Xenopus laevis embryos. mRNA encoding integrase was co-injected with a reporter plasmid containing a CMV promoter driven GFP into one cell embryos. The reporter plasmid was integrated into the genome. GFP expression, though robust, was in a limited number of tissues and varied among the embryos analyzed. We attributed this restriction to transcriptional silencing by chromosome position effects. Modification of the reporter plasmid by bracketing the CMV-GFP region with tandem copies of the chicken β-globin 5′ HS4 insulator 2 relieved position effects. Embryos transgenic with insulated CMV-GFP expressed GFP uniformly. Tissue specific expression was achieved when the CMV promoter was replaced with a 551 base-pair minimal gamma crystallin lens promoter from Xenopus. Embryos transgenic with this plasmid had GFP expression limited to the lens of the eye. We observed that about a third of embryos assayed one week after fertilization were transgenic. These experiments demonstrate that the integration of insulated gene sequences using ϕC31 integrase can be used to efficiently create transgenic embryos in Xenopus laevis and may increase the practical use of ϕC31 integrase in other systems as well.The ability to generate transgenic animals has revolutionized studies on gene function. Transgenic frogs of the genus Xenopus have been made using methods based on a restriction-enzyme mediated sperm integration approach described by Kroll and Amaya 3 . This method has been remarkably successful, but can be technically demanding. In addition, many embryos contain multiple copies of the transgene inserted at random insertion sites 3, 4 .We have tried an alternative approach, creating transgenic Xenopus embryos by using ϕC31 integrase mediated recombination. ϕC31 is a bacteriophage that encodes an integrase that mediates sequence directed recombination between a 34 nucleotide long bacterial attachment site (attB) and a 39 base-pair long phage attachment site (attP). ϕC31 integrase has a high efficiency of recombination, requires no accessory factors 5,6 and has been effectively used to integrate genes into plant cells 7 , mammalian cells 1,[8][9][10][11][12][13] , and Drosophila 14 . The ϕC31 integrase does not require an attP site to have perfect sequence fidelity for it to be recognized and cleaved 9 . These imperfect attP sites, or psuedo-attP sites, may have a similarity as low as 24% to an attP site and still allow recombination 9 . It has been estimated that a mammalian genome may contain 100-1000 psuedo attP sites 9 . Since the Xenopus laevis genome is approximately the same size as a mammalian genome (3×10 9 base pairs), we hypothesized that there would also be psuedo-attP sites in its genome that could be used to create transgenic Xenopus embryos. In the experiments that follow all the figure 1e. The half life of the GFP protein has been estimate...

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Animal Transgenesis and Cloning

Houdebine¹

2003

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Insulators: many functions, many mechanisms

Cited by 578 publications

References 132 publications

Sea urchin insulator protects lentiviral vector from silencing by maintaining active chromatin structure

Sea urchin insulator protects lentiviral vector from silencing by maintaining active chromatin structure

Transgenic Xenopus laevis embryos can be generated using φC31 integrase

Animal Transgenesis and Cloning

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