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The piggyBac-Based Gene Delivery System Can Confer Successful Production of Cloned Porcine Blastocysts with Multigene Constructs

The introduction of multigene constructs into single cells is important for improving the performance of domestic animals, as well as understanding basic biological processes. In particular, multigene constructs allow the engineering and integration of multiple genes related to xenotransplantation i...

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Autores principales: Sato, Masahiro, Maeda, Kosuke, Koriyama, Miyu, Inada, Emi, Saitoh, Issei, Miura, Hiromi, Ohtsuka, Masato, Nakamura, Shingo, Sakurai, Takayuki, Watanabe, Satoshi, Miyoshi, Kazuchika
Formato: Online Artículo Texto
Lenguaje:English
Publicado: MDPI 2016
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5037703/
https://www.ncbi.nlm.nih.gov/pubmed/27589724
http://dx.doi.org/10.3390/ijms17091424
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author Sato, Masahiro
Maeda, Kosuke
Koriyama, Miyu
Inada, Emi
Saitoh, Issei
Miura, Hiromi
Ohtsuka, Masato
Nakamura, Shingo
Sakurai, Takayuki
Watanabe, Satoshi
Miyoshi, Kazuchika
author_facet Sato, Masahiro
Maeda, Kosuke
Koriyama, Miyu
Inada, Emi
Saitoh, Issei
Miura, Hiromi
Ohtsuka, Masato
Nakamura, Shingo
Sakurai, Takayuki
Watanabe, Satoshi
Miyoshi, Kazuchika
author_sort Sato, Masahiro
collection PubMed
description The introduction of multigene constructs into single cells is important for improving the performance of domestic animals, as well as understanding basic biological processes. In particular, multigene constructs allow the engineering and integration of multiple genes related to xenotransplantation into the porcine genome. The piggyBac (PB) transposon system allows multiple genes to be stably integrated into target genomes through a single transfection event. However, to our knowledge, no attempt to introduce multiple genes into a porcine genome has been made using this system. In this study, we simultaneously introduced seven transposons into a single porcine embryonic fibroblast (PEF). PEFs were transfected with seven transposons containing genes for five drug resistance proteins and two (red and green) fluorescent proteins, together with a PB transposase expression vector, pTrans (experimental group). The above seven transposons (without pTrans) were transfected concomitantly (control group). Selection of these transfected cells in the presence of multiple selection drugs resulted in the survival of several clones derived from the experimental group, but not from the control. PCR analysis demonstrated that approximately 90% (12/13 tested) of the surviving clones possessed all of the introduced transposons. Splinkerette PCR demonstrated that the transposons were inserted through the TTAA target sites of PB. Somatic cell nuclear transfer (SCNT) using a PEF clone with multigene constructs demonstrated successful production of cloned blastocysts expressing both red and green fluorescence. These results indicate the feasibility of this PB-mediated method for simultaneous transfer of multigene constructs into the porcine cell genome, which is useful for production of cloned transgenic pigs expressing multiple transgenes.
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spelling pubmed-50377032016-09-29 The piggyBac-Based Gene Delivery System Can Confer Successful Production of Cloned Porcine Blastocysts with Multigene Constructs Sato, Masahiro Maeda, Kosuke Koriyama, Miyu Inada, Emi Saitoh, Issei Miura, Hiromi Ohtsuka, Masato Nakamura, Shingo Sakurai, Takayuki Watanabe, Satoshi Miyoshi, Kazuchika Int J Mol Sci Article The introduction of multigene constructs into single cells is important for improving the performance of domestic animals, as well as understanding basic biological processes. In particular, multigene constructs allow the engineering and integration of multiple genes related to xenotransplantation into the porcine genome. The piggyBac (PB) transposon system allows multiple genes to be stably integrated into target genomes through a single transfection event. However, to our knowledge, no attempt to introduce multiple genes into a porcine genome has been made using this system. In this study, we simultaneously introduced seven transposons into a single porcine embryonic fibroblast (PEF). PEFs were transfected with seven transposons containing genes for five drug resistance proteins and two (red and green) fluorescent proteins, together with a PB transposase expression vector, pTrans (experimental group). The above seven transposons (without pTrans) were transfected concomitantly (control group). Selection of these transfected cells in the presence of multiple selection drugs resulted in the survival of several clones derived from the experimental group, but not from the control. PCR analysis demonstrated that approximately 90% (12/13 tested) of the surviving clones possessed all of the introduced transposons. Splinkerette PCR demonstrated that the transposons were inserted through the TTAA target sites of PB. Somatic cell nuclear transfer (SCNT) using a PEF clone with multigene constructs demonstrated successful production of cloned blastocysts expressing both red and green fluorescence. These results indicate the feasibility of this PB-mediated method for simultaneous transfer of multigene constructs into the porcine cell genome, which is useful for production of cloned transgenic pigs expressing multiple transgenes. MDPI 2016-08-30 /pmc/articles/PMC5037703/ /pubmed/27589724 http://dx.doi.org/10.3390/ijms17091424 Text en © 2016 by the authors; licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC-BY) license (http://creativecommons.org/licenses/by/4.0/).
spellingShingle Article
Sato, Masahiro
Maeda, Kosuke
Koriyama, Miyu
Inada, Emi
Saitoh, Issei
Miura, Hiromi
Ohtsuka, Masato
Nakamura, Shingo
Sakurai, Takayuki
Watanabe, Satoshi
Miyoshi, Kazuchika
The piggyBac-Based Gene Delivery System Can Confer Successful Production of Cloned Porcine Blastocysts with Multigene Constructs
title The piggyBac-Based Gene Delivery System Can Confer Successful Production of Cloned Porcine Blastocysts with Multigene Constructs
title_full The piggyBac-Based Gene Delivery System Can Confer Successful Production of Cloned Porcine Blastocysts with Multigene Constructs
title_fullStr The piggyBac-Based Gene Delivery System Can Confer Successful Production of Cloned Porcine Blastocysts with Multigene Constructs
title_full_unstemmed The piggyBac-Based Gene Delivery System Can Confer Successful Production of Cloned Porcine Blastocysts with Multigene Constructs
title_short The piggyBac-Based Gene Delivery System Can Confer Successful Production of Cloned Porcine Blastocysts with Multigene Constructs
title_sort piggybac-based gene delivery system can confer successful production of cloned porcine blastocysts with multigene constructs
topic Article
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5037703/
https://www.ncbi.nlm.nih.gov/pubmed/27589724
http://dx.doi.org/10.3390/ijms17091424
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