Genetically modified pigs produced with a nonviral episomal vector

doi:10.1073/pnas.0604938103

Genetically modified pigs produced with a nonviral episomal vector

*Department of Surgical Sciences, University of Milano-Bicocca, 20052 Milan,Italy;
^†Institute of Cell Biology, Witten/Herdecke University, 58448 Witten, Germany;
^‡Department of Veterinary Morphophysiology and Animal Production, University of Bologna, 40064 Bologna, Italy;
^§Centro Regionale di Competenza Applicazioni Tecnologico-Industriali di Biomolecole e Biosistemi-BioTekNet-Seconda Università di Napoli, 80138 Naples, Italy; and
^¶Department of Cellular and Developmental Biology, La Sapienza University, 00185 Rome, Italy

Edited by Mark T. Groudine, Fred Hutchinson Cancer Research Center, Seattle, WA, and approved October 2, 2006 (received for review June 13, 2006)

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.

Footnotes

^‖To whom correspondence should be addressed. E-mail: marialuisa.lavitrano{at}unimib.it
Author contributions: S.M. and A.V. contributed equally to the work; M.L.B., M.F., P.D.,M.P., H.J.L., and M.L. designed research; S.M., A.V., I.M.S., M.L.B., M.G.C., R.G., A.Z., M.R.B.,M.F., M.P., and M.L. performed research; S.M., A.V., I.M.S., M.L.B., M.G.C., R.G., A.Z., M.R.B.,M.F., P.D., M.P., H.J.L., and M.L. analyzed data; and P.D., H.J.L., and M.L. wrote the paper.
The authors declare no conflict of interest.
This article is a PNAS direct submission.
Abbreviations:

SFM,

swine fertilization medium;

S/MAR,

scaffold/matrix attachment region;

SMGT,

sperm-mediated gene transfer.
Freely available online through the PNAS open access option.