The Functionality of Minimal PiggyBac Transposons in Mammalian Cells

Troyanovsky, Boris; Bitko, Vira; Pastukh, Viktor M.; Fouty, Brian; Solodushko, Viktoriya

doi:10.1038/mtna.2016.76

Cited by 18 publications

(14 citation statements)

References 42 publications

(57 reference statements)

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“…Notably, de novo ICR methylation occurred in accordance with the silencing of reprogramming factors. A previous study demonstrated that de novo DNA methylation plays a role in PB silencing (Troyanovsky et al., 2016). It is thus possible that the same machinery is involved in de novo ICR methylation and PB silencing.…”

Section: Discussionmentioning

confidence: 99%

De Novo DNA Methylation at Imprinted Loci during Reprogramming into Naive and Primed Pluripotency

et al. 2019

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Summary CpG islands (CGIs) including those at imprinting control regions (ICRs) are protected from de novo methylation in somatic cells. However, many cancers often exhibit CGI hypermethylation, implying that the machinery is impaired in cancer cells. Here, we conducted a comprehensive analysis of CGI methylation during somatic cell reprogramming. Although most CGIs remain hypomethylated, a small subset of CGIs, particularly at several ICRs, was often de novo methylated in reprogrammed pluripotent stem cells (PSCs). Such de novo ICR methylation was linked with the silencing of reprogramming factors, which occurs at a late stage of reprogramming. The ICR-preferred CGI hypermethylation was similarly observed in human PSCs. Mechanistically, ablation of Dnmt3a prevented PSCs from de novo ICR methylation. Notably, the ICR-preferred CGI hypermethylation was observed in pediatric cancers, while adult cancers exhibit genome-wide CGI hypermethylation. These results may have important implications in the pathogenesis of pediatric cancers and the application of PSCs.

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Section: Discussionmentioning

confidence: 99%

De Novo DNA Methylation at Imprinted Loci during Reprogramming into Naive and Primed Pluripotency

et al. 2019

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“…A potential improvement to the system is a re-excision deficient piggyBac vector. By moving the majority of the TRE sequence outside of the transposon to the non-inserted backbone of the donor plasmid, the size of the inserted TRE can be reduced with minimal loss of transposition efficiency (52, 53). Once inserted into the genome, the transposon lacks full-length TREs and cannot be efficiently excised.…”

Section: Discussionmentioning

confidence: 99%

RNA-guided piggyBac transposition in human cells

et al. 2019

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Safer and more efficient methods for directing therapeutic genes to specific sequences could increase the repertoire of treatable conditions. Many current approaches act passively, first initiating a double-stranded break, then relying on host repair to uptake donor DNA. Alternatively, we delivered an actively integrating transposase to the target sequence to initiate gene insertion. We fused the hyperactive piggyBac transposase to the highly specific, catalytically dead SpCas9-HF1 (dCas9) and designed guide RNAs (gRNAs) to the CCR5 safe harbor sequence. We introduced mutations to the native DNA-binding domain of piggyBac to reduce non-specific binding of the transposase and cause the fusion protein to favor binding by dCas9. This strategy enabled us, for the first time, to direct transposition to the genome using RNA. We showed that increasing the number of gRNAs improved targeting efficiency. Interestingly, over half of the recovered insertions were found at a single TTAA hotspot. We also found that the fusion increased the error rate at the genome-transposon junction. We isolated clonal cell lines containing a single insertion at CCR5 and demonstrated long-term expression from this locus. These vectors expand the utility of the piggyBac system for applications in targeted gene addition for biomedical research and gene therapy.

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“…An all-in-one-type plasmid, called mPB-GLuc-mCherry, which confers simultaneous expression of both mCherry (red fluorescent protein) and GLuc (secretory Gaussia luciferase) together with PB transposase, was injected subcutaneously into the tails of mice followed by in vivo EP locally across the injection site [64]. They observed a GLuc signal six months after gene delivery.…”

Section: Gene Delivery To Tailmentioning

confidence: 99%

piggyBac-Based Non-Viral In Vivo Gene Delivery Useful for Production of Genetically Modified Animals and Organs

et al. 2020

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In vivo gene delivery involves direct injection of nucleic acids (NAs) into tissues, organs, or tail-veins. It has been recognized as a useful tool for evaluating the function of a gene of interest (GOI), creating models for human disease and basic research targeting gene therapy. Cargo frequently used for gene delivery are largely divided into viral and non-viral vectors. Viral vectors have strong infectious activity and do not require the use of instruments or reagents helpful for gene delivery but bear immunological and tumorigenic problems. In contrast, non-viral vectors strictly require instruments (i.e., electroporator) or reagents (i.e., liposomes) for enhanced uptake of NAs by cells and are often accompanied by weak transfection activity, with less immunological and tumorigenic problems. Chromosomal integration of GOI-bearing transgenes would be ideal for achieving long-term expression of GOI. piggyBac (PB), one of three transposons (PB, Sleeping Beauty (SB), and Tol2) found thus far, has been used for efficient transfection of GOI in various mammalian cells in vitro and in vivo. In this review, we outline recent achievements of PB-based production of genetically modified animals and organs and will provide some experimental concepts using this system.

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The Functionality of Minimal PiggyBac Transposons in Mammalian Cells

Cited by 18 publications

References 42 publications

De Novo DNA Methylation at Imprinted Loci during Reprogramming into Naive and Primed Pluripotency

De Novo DNA Methylation at Imprinted Loci during Reprogramming into Naive and Primed Pluripotency

RNA-guided piggyBac transposition in human cells

piggyBac-Based Non-Viral In Vivo Gene Delivery Useful for Production of Genetically Modified Animals and Organs

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