Stable long-term germline transmission of GFP transgenic rat via PiggyBac transposon mediated gene transfer

• Published in: BMC veterinary research 2024-06, Vol.20 (1), p.275-10, Article 275
• Main Authors: Jeon, Beom-Jin, Kwon, Dong-Hyeok, Gim, Gyeong-Min, Kim, Hee-Kyoung, Lee, Jeong-Hwa, Jang, Goo
• Format: Article
• Language: English
• Subjects: Animal models; Animals; DNA methylation; DNA sequencing; DNA Transposable Elements - genetics; Embryos; Endometrium; Exons; Expression vectors; Female; Females; Gene amplification; Gene expression; Gene loci; Gene Silencing; Gene transfer; Gene Transfer Techniques - veterinary; Genes; Genetic aspects; Genetic engineering; Genetic research; Genetic transformation; Genetic vectors; Genetically modified mice; Genetically modified organisms; Genomes; Genomics; Germline Transmission; GFP-Rats; Green fluorescent protein; Green Fluorescent Proteins - genetics; Laboratory animals; Male; Methods; Nucleotide sequence; Nucleotide sequencing; Physiological aspects; Plasmids; Promoter Regions, Genetic; Quality control; Rats; Rats, Transgenic; Resources; Software; Sperm; Transgene Silencing; Transgenes; Transgenic animals; Transposon; Transposons; Trinucleotide repeats; Vectors (Biology); Whole genome sequencing
• ISSN: 1746-6148; 1746-6148
• DOI: 10.1186/s12917-024-04123-7

Transgene silencing provides a significant challenge in animal model production via gene engineering using viral vectors or transposons. Selecting an appropriate strategy, contingent upon the species is crucial to circumvent transgene silencing, necessitating long-term observation of in vivo gene expression. This study employed the PiggyBac transposon to create a GFP rat model to address transgene silencing in rats. Surprisingly, transgene silencing occurred while using the CAG promoter, contrary to conventional understanding, whereas the Ef1α promoter prevented silencing. GFP expression remained stable through over five generations, confirming efficacy of the Ef1α promoter for long-term protein expression in rats. Additionally, GFP expression was consistently maintained at the cellular level in various cellular sources produced from the GFP rats, thereby validating the in vitro GFP expression of GFP rats. Whole-genome sequencing identified a stable integration site in Akap1 between exons 1 and 2, mitigating sequence-independent mechanism-mediated transgene silencing. This study established an efficient method for producing transgenic rat models using PiggyBac transposon. Our GFP rats represent the first model to exhibit prolonged expression of foreign genes over five generations, with implications for future research in gene-engineered rat models.