Gene Specificity of Suppression of Transgene-Mediated Insertional Transcriptional Activation by the Chicken HS4 Insulator
| Autor: | Romain Desprat, Eric E. Bouhassira |
|---|---|
| Jazyk: | angličtina |
| Rok vydání: | 2009 |
| Předmět: |
Transcriptional Activation
Transgene Genetics and Genomics/Nuclear Structure and Function lcsh:Medicine Cell Separation beta-Globins Biology Viral vector Insertional mutagenesis 03 medical and health sciences 0302 clinical medicine Genetics and Genomics/Epigenetics Gene expression Animals Globin Gene Silencing Transgenes Enhancer lcsh:Science Molecular Biology Locus control region Cell Biology/Gene Expression Genetics and Genomics/Genetics of Disease 030304 developmental biology Genetics Regulation of gene expression Molecular Biology/Recombination 0303 health sciences Multidisciplinary Reverse Transcriptase Polymerase Chain Reaction Genetics and Genomics/Functional Genomics Genetics and Genomics/Gene Therapy lcsh:R Hematology Genetic Therapy Molecular Biology/Transcription Initiation and Activation Flow Cytometry Genetics and Genomics/Gene Function Hemoglobinopathies Mutagenesis Insertional Gene Expression Regulation Genetic Techniques Mutagenesis 030220 oncology & carcinogenesis lcsh:Q Hematology/Hemoglobinopathies Chickens Research Article Biotechnology |
| Zdroj: | PLoS ONE PLoS ONE, Vol 4, Iss 6, p e5956 (2009) |
| ISSN: | 1932-6203 |
| Popis: | Insertional mutagenesis has emerged as a major obstacle for gene therapy based on vectors that integrate randomly in the genome. Reducing the genotoxicity of genomic viral integration can, in first approximation, be equated with reducing the risk of oncogene activation, at least in the case of therapeutic payloads that have no known oncogenic potential, such as the globin genes. An attractive solution to the problem of oncogene activation is the inclusion of insulators/enhancer-blockers in the viral vectors. In this study we have used Recombinase-Mediated Cassette Exchange to characterize the effect of integration of globin therapeutic cassettes in the presence or absence of the chicken HS4 and three other putative insulators inserted near Stil, Tal1 and MAP17, three well-known cellular proto-oncogenes in the SCL/Tal1 locus. We show that insertion of a Locus Control Region-driven globin therapeutic globin transgene had a dramatic activating effect on Tal1 and Map17, the two closest genes, a minor effect on Stil, and no effect on Cyp4x1, a non-expressed gene. Of the four element tested, cHS4 was the only one that was able to suppress this transgene-mediated insertional transcriptional activation. cHS4 had a strong suppressive effect on the activation expression of Map17 but has little or no effect on expression of Tal1. The suppressive activity of cHS4 is therefore promoter specific. Importantly, the observed suppressive effect of cHS4 on Map17 activation did not depend on its intercalation between the LCR and the Map 17 promoter. Rather, presence of one or two copies of cHS4 anywhere within the transgene was sufficient to almost completely block the activation of Map17. Therefore, at this complex locus, suppression of transgene-mediated insertional transcriptional activation by cHS4 could not be adequately explained by models that predict that cHS4 can only suppress expression through an enhancer-blocking activity that requires intercalation between an enhancer and a promoter. This has important implications for our theoretical understanding of the possible effects of the insertion of cHS4 on gene therapy vectors. We also show that cHS4 decreased the level of expression of the globin transgene. Therefore, the benefits of partially preventing insertional gene activation are in part negated by the lower expression level of the transgene. A cost/benefit analysis of the utility of incorporation of insulators in gene therapy vectors will require further studies in which the effects of insulators on both the therapeutic gene and the flanking genes are determined at a large number of integration sites. Identification of insulators with minimal promoter specificity would also be of great value. |
| Databáze: | OpenAIRE |
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