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

Authors :
Masahiro Sato
Kosuke Maeda
Miyu Koriyama
Emi Inada
Issei Saitoh
Hiromi Miura
Masato Ohtsuka
Shingo Nakamura
Takayuki Sakurai
Satoshi Watanabe
Kazuchika Miyoshi
Source :
International Journal of Molecular Sciences, Vol 17, Iss 9, p 1424 (2016)
Publication Year :
2016
Publisher :
MDPI AG, 2016.

Abstract

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.

Details

Language :
English
ISSN :
14220067
Volume :
17
Issue :
9
Database :
Directory of Open Access Journals
Journal :
International Journal of Molecular Sciences
Publication Type :
Academic Journal
Accession number :
edsdoj.fdb88fc136894b67ae55cac96f0766c9
Document Type :
article
Full Text :
https://doi.org/10.3390/ijms17091424