Chromosomal sequences flanking an efficiently expressed transgene dramatically enhance its expression

Cranston, Anona Jane; Chen, Dong; Howcroft, Jill; Clark, Anthony

doi:10.1016/s0378-1119(01)00459-0

Cited by 13 publications

(7 citation statements)

References 26 publications

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“…In this study, the transgene integration sites in K25-2 and K25-3 were checked and single different integration sites were detected in the two transgenic pigs. It is consistent with previous reports that when the integration is within a permissive region as in K25-3, the transgene can be actively transcribed (Cranston et al, 2001). While, interestingly, in K25-2, the integration is in an L1M LINE element where the transgene should be repressed (Mehta et al, 2009); however, we observed transgene expression in all tissues detected.…”

Section: Discussionsupporting

confidence: 93%

“…The phenomena which govern this effect seem rather complex. Subtle interactions between transgene and genomic DNA sequences seem to influence expression (Cranston et al, 2001). Thus, PEV is often an obstacle to obtaining transgene stable expression cell lines, and the same problem is common in the application of transgenic animals (Whitelaw et al, 2001).…”

Section: Introductionmentioning

confidence: 99%

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Position effect variegation and epigenetic modification of a transgene in a pig model

Yin

Kong²,

Zhao³

et al. 2012

Genet. Mol. Res.

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ABSTRACT. Sequences proximal to transgene integration sites are able to regulate transgene expression, resulting in complex position effect variegation. Position effect variegation can cause differences in epigenetic modifications, such as DNA methylation and histone acetylation. However, it is not known which factor, position effect or epigenetic modification, plays a more important role in the regulation of transgene expression. We analyzed transgene expression patterns and epigenetic modifications of transgenic pigs expressing green fluorescent protein, driven by the cytomegalovirus (CMV) promoter. DNA hypermethylation and loss of acetylation of specific histone H3 and H4 lysines, except H4K16 acetylation in the CMV promoter, were consistent with a low level of transgene expression. Moreover, the degree of DNA methylation and histone H3/H4 acetylation in the promoter region depended on the integration site; consequently, position effect variegation caused variations in epigenetic modifications. The transgenic pig fibroblast cell lines were treated with DNA methyltransferase inhibitor 5-Aza-2'-deoxycytidine and/or histone deacetylase inhibitor trichostatin A. Transgene expression was promoted by reversing the DNA hypermethylation and histone hypoacetylation status. The differences in DNA methylation and histone acetylation in the CMV promoter region in these cell lines were not significant; however, significant differences in transgene expression were detected, demonstrating that variegation of transgene expression is affected by the integration site. We conclude that in this pig model, position effect variegation affects transgene expression.

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Section: Discussionsupporting

confidence: 93%

Section: Introductionmentioning

confidence: 99%

Position effect variegation and epigenetic modification of a transgene in a pig model

Yin

Kong²,

Zhao³

et al. 2012

Genet. Mol. Res.

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“…HIV-based lentiviral vectors may therefore, provide a more stable platform to deliver genes controlled by various cis-acting elements, such as LCRs. When used in transgenic animals, the globin LCR is able to confer an open and accessible chromatin structure at ectopic sites and is able to prevent the strong position-ofintegration effects (position effect variegation) observed in the absence of the LCR [Cranston et al, 2001].…”

Section: Locus Control Regions (Lcrs)mentioning

confidence: 99%

Promoters and Control Elements: Designing Expression Cassettes for Gene Therapy

Papadakis

Nicklin²,

Baker³

et al. 2004

CGT

146

121

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It has become apparent that the clinical success anticipated in the field of gene therapy has been limited by progress in several of the fundamental areas of genetics, molecular and cellular biology relevant to its application. Whilst a great deal of effort has been made in the evaluation of transgenes, it is only more recently with the advance of vector systems that attention has begun to be focused upon the means and control of transgene expression. Until recently, the majority of constructs have employed ubiquitous viral promoters to drive expression from simple gene expression cassettes using viral promoters and lacking introns, 3' untranslated regions (UTRs), locus control regions (LCR's), matrix attachment regions (MAR's) and other such genetic components. It has consequently emerged that these elements may have a key role in determining the levels and longevity of gene expression attainable in vivo, irrespective of the vector system utilised. The majority of gene therapy applications would also benefit from the specific optimisation of 'tailormade' expression cassettes to optimise their therapeutic efficacy. In conjunction with modification of vector tropism and strategies to limit their immunogenicity, this should create vectors suitable for the clinical application of gene therapy. This review aims to highlight some of the principle considerations of gene expression in vivo, and the means by which it may most effectively be achieved, whether this is via the minimal modification of an existing eukaryotic promoter or by the more extensive design of a novel promoter and associated elements.

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“…20,21 To minimise variations in transfection efficiencies due to plasmid size and vector sequence, DNA-containing promoter sequences were individually cloned into the pd2EGFP-1 upstream of the coding sequence for destabilised GFP. The rapid turnover of destabilised GFP enables reporter activity to be tracked at finer intervals, providing a closer examination of promoter strength in a transient expression assay.…”

Section: Discussionmentioning

confidence: 99%

A transient expression vector for recombinant protein production in Chinese hamster ovary cells

Liao

Sunstrom

2005

J of Chemical Tech & Biotech

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An expression vector was specifically designed for use in Chinese hamster ovary (CHO) cells to enhance the level of protein production in a transient expression system. Two key components that can increase protein production transiently are the promoter used to drive recombinant gene expression and the template copy number. In this study the modified and metal-inducible metallothionein (M2.6) promoter was shown to be superior to the human cytomegalovirus (CMV) and to the simian virus SV40 promoters. Plasmid replication was achieved using the Polyoma (

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Chromosomal sequences flanking an efficiently expressed transgene dramatically enhance its expression

Cited by 13 publications

References 26 publications

Position effect variegation and epigenetic modification of a transgene in a pig model

Position effect variegation and epigenetic modification of a transgene in a pig model

Promoters and Control Elements: Designing Expression Cassettes for Gene Therapy

A transient expression vector for recombinant protein production in Chinese hamster ovary cells

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