title: The piggyBac-Based Gene Delivery System Can Confer Successful Production of Cloned Porcine Blastocysts with Multigene Constructs.
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.
authors: Section of Gene Expression Regulation, Frontier Science Research Center, Kagoshima University, Kagoshima 890-8544, Japan. masasato@ms.kagoshima-u.ac.jp.
authors: Masahiro
authors: M
authors: Sato
authors: Laboratory of Animal Reproduction, Faculty of Agriculture, Kagoshima University, Kagoshima 890-0065, Japan. k7504084@kadai.jp.
authors: Kosuke
authors: K
authors: Maeda
authors: Laboratory of Animal Reproduction, Faculty of Agriculture, Kagoshima University, Kagoshima 890-0065, Japan. k3326891@kadai.jp.
authors: Miyu
authors: M
authors: Koriyama
authors: Department of Pediatric Dentistry, Graduate School of Medical and Dental Sciences, Kagoshima University, Kagoshima 890-8544, Japan. inada@dent.kagoshima-u.ac.jp.
authors: Emi
authors: E
authors: Inada
authors: Division of Pediatric Dentistry, Department of Oral Health Sciences, Course for Oral Life Science, Graduate School of Medical and Dental Sciences, Niigata University, Niigata 951-8514, Japan. isaito@dent.niigata-u.ac.jp.
authors: Issei
authors: I
authors: Saitoh
authors: Department of Regenerative Medicine, Basic Medical Science, School of Medicine, Tokai University, Kanagawa 259-1193, Japan. rascal511520531@yahoo.co.jp.
authors: Hiromi
authors: H
authors: Miura
authors: Division of Basic Molecular Science and Molecular Medicine, School of Medicine, Tokai University, Kanagawa 259-1193, Japan. masato@is.icc.u-tokai.ac.jp.|The Institute of Medical Sciences, Tokai University, Kanagawa 259-1193, Japan. masato@is.icc.u-tokai.ac.jp.
authors: Masato
authors: M
authors: Ohtsuka
authors: Division of Biomedical Engineering, National Defense Medical College Research Institute, Saitama 359-8513, Japan. snaka@ndmc.ac.jp.
authors: Shingo
authors: S
authors: Nakamura
authors: Department of Cardiovascular Research, Graduate school of Medicine, Shinshu University, Nagano 390-8621, Japan. tsakurai@shinshu-u.ac.jp.
authors: Takayuki
authors: T
authors: Sakurai
authors: Animal Genome Research Unit, Division of Animal Science, National Institute of Agrobiological Sciences, Ibaraki 305-8602, Japan. kettle@affrc.go.jp.
authors: Satoshi
authors: S
authors: Watanabe
authors: Laboratory of Animal Reproduction, Faculty of Agriculture, Kagoshima University, Kagoshima 890-0065, Japan. kmiyoshi@agri.kagoshima-u.ac.jp.
authors: Kazuchika
authors: K
authors: Miyoshi
mesh_terms: D000818:Animals; D001755:Blastocyst / Q000378:metabolism; D004251:DNA Transposable Elements / Q000235:genetics*; D004351:Drug Resistance / Q000235:genetics; D005260:Female; D018014:Gene Transfer Techniques; D005822:Genetic Vectors / Q000235:genetics; D049452:Green Fluorescent Proteins / Q000235:genetics; D053652:Nuclear Transfer Techniques; D011247:Pregnancy; D013552:Swine; D013556:Swine, Miniature / Q000235:genetics*; D019076:Transgenes
chemical_list: D004251:DNA Transposable Elements; D049452:Green Fluorescent Proteins
keywords: drug selection; multiple transgenes; piggyBac; porcine embryonic fibroblasts; somatic cell nuclear transfer; transposon