Intravenous Delivery of piggyBac Transposons as a Useful Tool for Liver-Specific Gene-Switching.

Shingo Nakamura,Masahiro Sato,Masato Ohtsuka,Naoko Ando,Masayuki Ishihara,Satoshi Watanabe

doi:10.3390/ijms19113452

Abstract

Hydrodynamics-based gene delivery (HGD) is an efficient method for transfecting plasmid DNA into hepatocytes in vivo. However, the resulting gene expression is transient, and occurs in a non-tissue specific manner. The piggyBac (PB) transposon system allows chromosomal integration of a transgene in vitro. This study aimed to achieve long-term in vivo expression of a transgene by performing hepatocyte-specific chromosomal integration of the transgene using PB and HGD. Using this approach, we generated a novel mouse model for a hepatic disorder. A distinct signal from the reporter plasmid DNA was discernible in the murine liver approximately two months after the administration of PB transposons carrying a reporter gene. Then, to induce the hepatic disorder, we first administered mice with a PB transposon carrying a CETD unit (loxP-flanked stop cassette, diphtheria toxin-A chain gene, and poly(A) sites), and then with a plasmid expressing the Cre recombinase under the control of a liver-specific promoter. We showed that this system can be used for in situ manipulation and analysis of hepatocyte function in vivo in non-transgenic (Tg) animals.

Highlights

The production of transgenic (Tg) animals has been one of the most powerful tools for exploring the function of genes of interest (GOIs) for analyzing pathogenic mechanisms and for developing therapeutic approaches [1]
To examine whether PB transposons introduced into murine liver through hydrodynamics-based gene delivery (HGD) can guarantee the long-term expression of a GOI (EGFP cDNA in this case), adult Institute of Cancer Research (ICR) male mice were intravenously injected with a solution containing two PB-related vectors and a non-PB vector ptdTomato (Figure 1A) using HGD
Polymerase chain reaction (PCR) analysis using genomic DNA isolated from the right median lobes demonstrated the presence of Enhanced green fluorescent protein (EGFP) cDNA on the samples isolated from 2 to 56 days after HGD (Figure 1D)

Summary

Introduction

The production of transgenic (Tg) animals has been one of the most powerful tools for exploring the function of genes of interest (GOIs) for analyzing pathogenic mechanisms and for developing therapeutic approaches [1]. To bypass this step, intravenous injection of naked DNA (usually plasmids) (or DNA usually complexed with delivery vehicles (e.g., cationic lipids)) or the direct administration of DNA using surgical techniques into tissues/organs has often been employed [2]. Hydrodynamics-based gene delivery uses the rapid injection of a relatively large volume of solution containing naked plasmid

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