A DNA transposon-based approach to functional screening in neural stem cells

Albieri, Ilaria; Onorati, Marco; Calabrese, Giovanna; Moiana, Alessia; Biasci, Daniele; Badaloni, Aurora; Camnasio, Stefano; Spiliotopoulos, Dimitrios; Ivics, Zoltán; Cattaneo, Elena; Consalez, G. Giacomo

doi:10.1016/j.jbiotec.2010.07.027

Cited by 8 publications

(5 citation statements)

References 47 publications

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“…DNA transposon is well established to generate target gene overexpression in rodents, particularly gene function via mutagenesis 14 or cancer study 8 . Additionally, in human cells, SB or PB delivery have been used for gene therapy 15 16 17 .…”

Section: Discussionmentioning

confidence: 99%

Efficient generation of transgenic cattle using the DNA transposon and their analysis by next-generation sequencing

Yum

Lee²,

Kim

et al. 2016

Sci Rep

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Here, we efficiently generated transgenic cattle using two transposon systems (Sleeping Beauty and Piggybac) and their genomes were analyzed by next-generation sequencing (NGS). Blastocysts derived from microinjection of DNA transposons were selected and transferred into recipient cows. Nine transgenic cattle have been generated and grown-up to date without any health issues except two. Some of them expressed strong fluorescence and the transgene in the oocytes from a superovulating one were detected by PCR and sequencing. To investigate genomic variants by the transgene transposition, whole genomic DNA were analyzed by NGS. We found that preferred transposable integration (TA or TTAA) was identified in their genome. Even though multi-copies (i.e. fifteen) were confirmed, there was no significant difference in genome instabilities. In conclusion, we demonstrated that transgenic cattle using the DNA transposon system could be efficiently generated, and all those animals could be a valuable resource for agriculture and veterinary science.

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Section: Discussionmentioning

confidence: 99%

Efficient generation of transgenic cattle using the DNA transposon and their analysis by next-generation sequencing

Yum

Lee²,

Kim

et al. 2016

Sci Rep

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“…The DNA-binding capacity of E2C in the context of a transposase fusion was examined in competitive luciferase assays that measure reporter gene expression in the presence of ZF/transposase fusions (Figure 1c) that compete with ZF/AD fusions for binding to the ZF binding sites (Figure 2a). As fusion partner, a hyperactive version of the SB transposase, M3a, 15 was used. Cotransfection of E2C/M3a reduced reporter gene expression to as low as ~10% (Figure 2b).…”

Section: Dna Binding and Transpositional Activities Of E2c/sb Fusionsmentioning

confidence: 99%

Retargeting Sleeping Beauty Transposon Insertions by Engineered Zinc Finger DNA-binding Domains

et al. 2012

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The Sleeping Beauty (SB) transposon is a nonviral, integrating vector system with proven efficacy in preclinical animal models, and thus holds promise for future clinical applications. However, SB has a close-to-random insertion profile that could lead to genotoxic effects, thereby presenting a potential safety issue. We evaluated zinc finger (ZF) DNA-binding domains (DBDs) for their abilities to introduce a bias into SB's insertion profile. E2C, that binds a unique site in the erbB-2 gene, mediated locus-specific transposon insertions at low frequencies. A novel ZF targeting LINE1 repeats, ZF-B, showed specific binding to an 18-bp site represented by ~12,000 copies in the human genome. We mapped SB insertions using linear-amplification (LAM)-PCR and Illumina sequencing. Targeted insertions with ZF-B peaked at approximately fourfold enrichment of transposition around ZF-B binding sites yielding ~45% overall frequency of insertion into LINE1. A decrease in the ZF-B dataset with respect to transposon insertions in genes was found, suggesting that LINE1 repeats act as a sponge that "soak up" a fraction of SB insertions and thereby redirect them away from genes. Improvements in ZF technology and a careful choice of targeted genomic regions may improve the safety profile of SB for future clinical applications.

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“…For example, a modified Sleeping Beauty transposon was generated to randomly trap genes in the neural stem cell genome and modify their expression or tag them with fluorescent markers and selectable genes, allowing recombinant cells to be isolated and expanded clonally. This approach may facilitate the identification of novel determinants of stem cell biology and neural cell fate specification in NSCs [273]. A modified Tol2 transposon was also designed to allow for conditional disruption of a broad spectrum of genes.…”

Section: Dna Transposons As Tools For Genetic Engineering Of Stem Cellsmentioning

confidence: 99%

The impact of transposable element activity on therapeutically relevant human stem cells

et al. 2019

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Human stem cells harbor significant potential for basic and clinical translational research as well as regenerative medicine. Currently ~ 3000 adult and ~ 30 pluripotent stem cell-based, interventional clinical trials are ongoing worldwide, and numbers are increasing continuously. Although stem cells are promising cell sources to treat a wide range of human diseases, there are also concerns regarding potential risks associated with their clinical use, including genomic instability and tumorigenesis concerns. Thus, a deeper understanding of the factors and molecular mechanisms contributing to stem cell genome stability are a prerequisite to harnessing their therapeutic potential for degenerative diseases. Chemical and physical factors are known to influence the stability of stem cell genomes, together with random mutations and Copy Number Variants (CNVs) that accumulated in cultured human stem cells. Here we review the activity of endogenous transposable elements (TEs) in human multipotent and pluripotent stem cells, and the consequences of their mobility for genomic integrity and host gene expression. We describe transcriptional and post-transcriptional mechanisms antagonizing the spread of TEs in the human genome, and highlight those that are more prevalent in multipotent and pluripotent stem cells. Notably, TEs do not only represent a source of mutations/CNVs in genomes, but are also often harnessed as tools to engineer the stem cell genome; thus, we also describe and discuss the most widely applied transposon-based tools and highlight the most relevant areas of their biomedical applications in stem cells. Taken together, this review will contribute to the assessment of the risk that endogenous TE activity and the application of genetically engineered TEs constitute for the biosafety of stem cells to be used for substitutive and regenerative cell therapies.

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A DNA transposon-based approach to functional screening in neural stem cells

Cited by 8 publications

References 47 publications

Efficient generation of transgenic cattle using the DNA transposon and their analysis by next-generation sequencing

Efficient generation of transgenic cattle using the DNA transposon and their analysis by next-generation sequencing

Retargeting Sleeping Beauty Transposon Insertions by Engineered Zinc Finger DNA-binding Domains

The impact of transposable element activity on therapeutically relevant human stem cells

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