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

Nakamura, Shingo; Watanabe, Satoshi; Ando, Naoto; Ohtsuka, Masato

doi:10.3390/ijms19113452

Cited by 12 publications

(14 citation statements)

References 71 publications

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“…Furthermore, only three of the six fetuses carrying pCGSap1‐EGFP exhibited the targeted mutation in their hearts (see Table ). To improve this low efficiency TPGD‐GEF, subjecting the females to TPGD‐GEF at a different stage of pregnancy, employment of reagents suitable for in vivo gene delivery, administration of increased amounts of DNA, and employment of hydrodynamics‐based gene delivery (HGD) are methods that may be examined. In our previous experiments, females at days 4.5–10.5 of gestation were refractory to transfection with the exogenous DNA .…”

Section: Discussionmentioning

confidence: 99%

Transplacental delivery of genome editing components causes mutations in embryonic cardiomyocytes of mid‐gestational murine fetuses

et al. 2019

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Genome editing, as exemplified by CRISPR/Cas9, is now recognized as a powerful tool for the engineering of endogenous target genes. It employs only two components, namely, Cas9 in the form of DNA, mRNA, or protein; and guide RNA (gRNA), which is specific to a target gene. When these components are transferred to cells, they create insertion/deletion mutations (indels) within a target gene. Therefore, when fetuses within the uteri of pregnant murine females are exposed to these reagents, fetal cells incorporating them should show mutations in the target gene. To examine a possible genome editing of fetal cells in vivo, we intravenously administered a solution containing plasmid DNA–FuGENE complex to pregnant wild‐type female mice [which had been successfully mated with enhanced green fluorescent protein (EGFP)‐expressing male transgenic mice] on day 12.5 of gestation. The plasmid DNA induces the expression of gRNA, which was targeted at the EGFP cDNA, and that of the Cas9 gene. All fetuses in the pregnant females should express EGFP systemically, since they are heterozygous (Tg/+) for the transgene. Thus, the delivery of CRISPR system targeted at EGFP in the fetuses will cause a reduced expression of EGFP as a result of the genome editing of EGFP genomic sequence. Of the 24 fetuses isolated from three pregnant females 2 days after gene delivery, 3 were found to have reduced fluorescence in their hearts. Genotyping of the dissected hearts revealed the presence of the transgene construct (Cas9 gene) in all the samples. Furthermore, all the three samples exhibited mutations at the target loci, although normal cells were also present. Thus, transplacental delivery of gene editing components may be a useful tool for developing animal models with heart disorder for heart‐related disease research, and gene therapy in congenital heart defects such as hypertrophic cardiomyopathy (HCM). © 2019 IUBMB Life, 9999(9999):1–10, 2019

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Section: Discussionmentioning

confidence: 99%

Transplacental delivery of genome editing components causes mutations in embryonic cardiomyocytes of mid‐gestational murine fetuses

et al. 2019

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“…Nakamura et al [43] generated a novel mouse model for a hepatic disorder. To induce chromosomal integration of the PB transposon, pT-CETD (carrying a CETD unit (loxP-flanked stop cassette, diphtheria toxin-A chain (DT-A) gene, and poly(A) sites) ( Figure 2B) in murine hepatocytes, HGD-based tail-vein injection of a TransIT-EE Hydrodynamic Delivery Solution (Takara Bio Inc., Shiga, Japan; hereafter referred to as TransIT-EE) containing pT-CETD + pTrans (PB transposase expresion plasmid) was performed using ICR mice ( Figure 2A).…”

Section: Useful For Regulated Gene Expression In Vivomentioning

confidence: 99%

“…Hydrodynamics-based gene delivery (HGD) and piggyBac (PB) transposon system enable long-term gene expression in murine liver, according to the article by Nakamura et al[43]. (A) Schematic representation of experimental outline.…”

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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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“…Prior to injection, mice were subjected to sufficient anesthesia by intraperitoneal (IP) injection of three combined anesthetics [medetomidine (0.75 mg/kg; Nippon Zenyaku Kogyo Co. Ltd., Fukushima, Japan), midazolam (4 mg/kg; Sandoz K.K., Tokyo, Japan), and butorphanol (5 mg/kg; Meiji Seika Pharma Co., Ltd., Tokyo, Japan)]. HGD was performed according to methods described previously [30][31][32]. Briefly, anesthetized mice were intravenously injected with PBS(-) containing 10 µL of stock solution using a syringe (3 mL Luer lock type; Nipro, Inc., Osaka, Japan) fitted with a 30-gauge needle (Dentronics Co., Ltd., Tokyo, Japan).…”

Section: Protein Delivery Into Murine Hepatocytes Via Hgdmentioning

confidence: 99%

Development of Novel Heparin/Protamine Nanoparticles Useful for Delivery of Exogenous Proteins In Vitro and In Vivo

Nakamura

Ando

2020

Nanomaterials

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We previously reported that heparin/protamine particles (LHPPs) produced as nanoparticles through simple mixing of raw materials exhibit sustained protein release and can be retained in cells. In the present study, we modified LHPPs without employing any organic synthetic approach. The resulting LHPPs were re-named as improved LHPPs (i-LHPPs) and have the ability to retain cell-penetrating peptides (GRKKRRQRRRPPQ) based on electrostatic interactions. We examined whether i-LHPPs can introduce exogenous proteins (i.e., lacZ protein encoding bacterial β-galactosidase) into cultured cells in vitro, or into murine hepatocytes in vivo through intravenous injection to anesthetized mice. We found an accumulation of the transferred protein in both in vitro cultured cells and in vivo hepatocytes. To the best of our knowledge, reports of successful in vivo delivery to hepatocytes are rare. The i-LHPP-based protein delivery technique will be useful for in vivo functional genetic modification of mouse hepatocytes using Cas9 protein-mediated genome editing targeting specific genes, leading to the creation of hepatic disease animal models for research that aims to treat liver diseases.

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

Cited by 12 publications

References 71 publications

Transplacental delivery of genome editing components causes mutations in embryonic cardiomyocytes of mid‐gestational murine fetuses

Transplacental delivery of genome editing components causes mutations in embryonic cardiomyocytes of mid‐gestational murine fetuses

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

Development of Novel Heparin/Protamine Nanoparticles Useful for Delivery of Exogenous Proteins In Vitro and In Vivo

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