Helper-independent piggyBac plasmids for gene delivery approaches: Strategies for avoiding potential genotoxic effects

Abstract

Efficient integration of functional genes is an essential prerequisite for successful gene delivery such as cell transfection, animal transgenesis, and gene therapy. Gene delivery strategies based on viral vectors are currently the most efficient. However, limited cargo capacity, host immune response, and the risk of insertional mutagenesis are limiting factors and of concern. Recently, several groups have used transposon-based approaches to deliver genes to a variety of cells. The piggyBac (pB) transposase in particular has been shown to be well suited for cell transfection and gene therapy approaches because of its flexibility for molecular modification, large cargo capacity, and high transposition activity. However, safety considerations regarding transposase gene insertions into host genomes have rarely been addressed. Here we report our results on engineering helper-independent pB plasmids. The single-plasmid gene delivery system carries both the piggyBac transposase (pBt) expression cassette as well as the transposon cargo flanked by terminal repeat element sequences. Improvements to the helper-independent structure were achieved by developing new plasmids in which the pBt gene is rendered inactive after excision of the transposon from the plasmid. As a consequence, potentially negative effects that may develop by the persistence of an active pBt gene posttransposition are eliminated. The results presented herein demonstrate that our helper-independent plasmids represent an important step in the development of safe and efficient gene delivery methods that should prove valuable in gene therapy and transgenic approaches.

Structure–function analysis of mouse Sry reveals dual essential roles of the C-terminal polyglutamine tract in sex determination

“…This conclusion is supported by recent evidence that truncated polyQ domains containing three glutamine blocks are sufficient to enable Sry to induce Sox9 expression in a rat pre-Sertoli cell line (9). (22,23) in which the expression of the transgene is controlled by a constitutively active human ubiquitin C (UBC) promoter (Fig. 5A).…”

Hyperactive self-inactivating piggyBac for transposase-enhanced pronuclear microinjection transgenesis

“…Previously, we used a polyclonal antibody to assess piggyBac localization expressed from newly developed constructs synthesized with chimeric piggyBac transposases (19). To gain an understanding of the functional competence of the newly developed constructs synthesized with chimeric piggyBac transposases, we performed immunolocalization with a newly produced monoclonal antibody against the piggyBac protein.…”

“…These features were shown to reduce the construct's genotoxic potential while simultaneously improving transposon integration efficiency. We have demonstrated that this design allowed us to reduce the number of transgene integrations markedly, often to one per genome (19). Here, we report the development of a series of unique hyperactive plasmid vectors (pmhyGENIE-3) based on a previously described hyperactive mammalian codon-optimized piggyBac transposase variant (20).…”

“…We have recently developed an improved ICSI-Tr method that uses the piggyBac transposase for efficient gene transfer into the mouse genome (18,19). We have engineered unique piggyBac plasmid vectors that contain the transposon cargo and the piggyBac transposase gene on a single-helper independent plasmid (pmGENIE-3).…”

Insertional Mutagenesis by a Hybrid PiggyBac and Sleeping Beauty Transposon in the Rat

“…Previous transposon mutagenesis studies have generated initial transgenic mice or rats by standard pronuclear injection to create single integrations with transposon concatemers (Dupuy et al 2001;Fischer et al 2001;Horie et al 2001;Dupuy et al 2002;Ding et al 2005;Kitada et al 2007;Lu et al 2007;Urschitz et al 2010). These have been crossed with transgenic animals that express the corresponding transposase in the germline, using male germ cell-specific regulatory sequences.…”