Genetically modified pigs produced with a nonviral episomal vector

TLDR

Genetic modification of cells and animals is valuable, yet integrating vectors can cause insertional mutagenesis, variable expression, and silencing; scaffold/matrix attachment region-based nonviral vectors replicate autonomously, offering safer, reliable genetic modification. The study aimed to produce genetically modified pig fetuses using a scaffold/matrix attachment region-based vector. The vector pEPI was delivered to embryos via sperm‑mediated gene transfer. The pEPI vector was detected as an episome in 12 of 18 fetuses, the reporter gene expressed in 9 of those animals with an average of 79 % EGFP‑positive cells across tissues, indicating high expression, lack of mosaicism, and promising potential for biotechnological and germ‑line gene therapy applications.

Abstract

Genetic modification of cells and animals is an invaluable tool for biotechnology and biomedicine. Currently, integrating vectors are used for this purpose. These vectors, however, may lead to insertional mutagenesis and variable transgene expression and can undergo silencing. Scaffold/matrix attachment region-based vectors are nonviral expression systems that replicate autonomously in mammalian cells, thereby making possible safe and reliable genetic modification of higher eukaryotic cells and organisms. In this study, genetically modified pig fetuses were produced with the scaffold/matrix attachment region-based vector pEPI, delivered to embryos by the sperm-mediated gene transfer method. The pEPI vector was detected in 12 of 18 fetuses in the different tissues analyzed and was shown to be retained as an episome. The reporter gene encoded by the pEPI vector was expressed in 9 of 12 genetically modified fetuses. In positive animals, all tissues analyzed expressed the reporter gene; moreover in these tissues, the positive cells were on the average 79%. The high percentage of EGFP-expressing cells and the absence of mosaicism have important implications for biotechnological and biomedical applications. These results are an important step forward in animal transgenesis and can provide the basis for the future development of germ-line gene therapy.