title: Generation of transgenic pigs by cytoplasmic injection of piggyBac transposase-based pmGENIE-3 plasmids.
abstract: The process of transgenesis involves the introduction of a foreign gene, the transgene, into the genome of an animal. Gene transfer by pronuclear microinjection (PNI) is the predominant method used to produce transgenic animals. However, this technique does not always result in germline transgenic offspring and has a low success rate for livestock. Alternate approaches, such as somatic cell nuclear transfer using transgenic fibroblasts, do not show an increase in efficiency compared to PNI, while viral-based transgenesis is hampered by issues regarding transgene size and biosafety considerations. We have recently described highly successful transgenesis experiments with mice using a piggyBac transposase-based vector, pmhyGENIE-3. This construct, a single and self-inactivating plasmid, contains all the transpositional elements necessary for successful gene transfer. In this series of experiments, our laboratories have implemented cytoplasmic injection (CTI) of pmGENIE-3 for transgene delivery into in vivo-fertilized pig zygotes. More than 8.00% of the injected embryos developed into transgenic animals containing monogenic and often single transgenes in their genome. However, the CTI technique was unsuccessful during the injection of in vitro-fertilized pig zygotes. In summary, here we have described a method that is not only easy to implement, but also demonstrated the highest efficiency rate for nonviral livestock transgenesis.
authors.affiliation: Department of Animal Genetics, Breeding and Reproduction, College of Animal Science, South China Agricultural University, Guangzhou, Guangdong, People's Republic of China.
authors.forename: Zicong
authors.initials: Z
authors.lastname: Li
authors.forename: Fang
authors.initials: F
authors.lastname: Zeng
authors.forename: Fanming
authors.initials: F
authors.lastname: Meng
authors.forename: Zhiqian
authors.initials: Z
authors.lastname: Xu
authors.forename: Xianwei
authors.initials: X
authors.lastname: Zhang
authors.forename: Xiaoling
authors.initials: X
authors.lastname: Huang
authors.forename: Fei
authors.initials: F
authors.lastname: Tang
authors.forename: Wenchao
authors.initials: W
authors.lastname: Gao
authors.forename: Junsong
authors.initials: J
authors.lastname: Shi
authors.forename: Xiaoyan
authors.initials: X
authors.lastname: He
authors.forename: Dewu
authors.initials: D
authors.lastname: Liu
authors.forename: Chong
authors.initials: C
authors.lastname: Wang
authors.forename: Johann
authors.initials: J
authors.lastname: Urschitz
authors.forename: Stefan
authors.initials: S
authors.lastname: Moisyadi
authors.forename: Zhenfang
authors.initials: Z
authors.lastname: Wu
mesh_terms: D000818:Animals; D030801:Animals, Genetically Modified; D000831:Animals, Newborn; D015139:Blotting, Southern / Q000662:veterinary; D004247:DNA / Q000737:chemistry / Q000235:genetics; D004624:Embryo Transfer / Q000662:veterinary; D005260:Female; D018014:Gene Transfer Techniques / Q000662:veterinary*; D005822:Genetic Vectors / Q000008:administration & dosage / Q000235:genetics; D008297:Male; D008845:Microinjections / Q000662:veterinary; D010957:Plasmids / Q000008:administration & dosage* / Q000235:genetics; D016133:Polymerase Chain Reaction / Q000662:veterinary; D013552:Swine / Q000235:genetics* / Q000601:surgery*; D019076:Transgenes; D019895:Transposases / Q000008:administration & dosage / Q000235:genetics*; D015053:Zygote / Q000502:physiology
chemical_list: D004247:DNA; D019895:Transposases
keywords: pig; piggyBac; pmGENIE-3; transgenesis; transposase