Optimization of piggyBac transposon-mediated gene transfer method in common marmoset embryos

• Published in: PloS one Vol. 18; no. 6; p. e0287065
• Main Authors: Kohri, Nanami, Ota, Mitsuo, Kousaku, Hikaru, Minakawa, Eiko N, Seki, Kazuhiko, Tomioka, Ikuo
• Format: Journal Article
• Language: English
• Published: United States Public Library of Science 09.06.2023; Public Library of Science (PLoS)
• Subjects: Abdomen; Alzheimer's disease; Analysis; Animal genetic engineering; Animal models; Biology and Life Sciences; Disease; Electroporation; Embryo cells; Embryonic development; Embryonic stem cells; Embryos; Engineering and Technology; Ethics; Experiments; Females; Gene transfer; Genes; Genetic aspects; Genetic modification; Genetic research; Genetic vectors; Genetically modified organisms; Genomes; Genomics; Growth; Health aspects; Laboratory animals; Marmosets; Methods; Monkeys & apes; mRNA; Mutation; Nervous system diseases; Neurodegenerative diseases; Optimization; Physiological aspects; Primates; Proteins; Research and Analysis Methods; RNA; Scientific equipment and supplies industry; Sperm; Stem cells; Tetracycline; Tetracyclines; Transgenes; Transgenic animals; Transplantation; Transposase; Transposons; Japan; United States; Denmark; United States > US
• ISSN: 1932-6203; 1932-6203
• DOI: 10.1371/journal.pone.0287065

Generating non-human primate models of human diseases is important for the development of therapeutic strategies especially for neurodegenerative diseases. The common marmoset has attracted attention as a new experimental animal model, and many transgenic marmosets have been produced using lentiviral vector-mediated transgenesis. However, lentiviral vectors have a size limitation of up to 8 kb in length for transgene applications. Therefore, the present study aimed to optimize a piggyBac transposon-mediated gene transfer method in which transgenes longer than 8 kb were injected into the perivitelline space of marmoset embryos, followed by electroporation. We constructed a long piggyBac vector carrying the gene responsible for Alzheimer's disease. The optimal weight ratio of the piggyBac transgene vector to the piggyBac transposase mRNA was examined using mouse embryos. Transgene integration into the genome was confirmed in 70.7% of embryonic stem cells established from embryos injected with 1000 ng of transgene and transposase mRNA. Under these conditions, long transgenes were introduced into marmoset embryos. All embryos survived after transgene introduction treatment, and transgenes were detected in 70% of marmoset embryos. The transposon-mediated gene transfer method developed in this study can be applied to the genetic modification of non-human primates, as well as large animals.