323 PRODUCTION OF Mx2-TRANSGENIC PIGS FOR INFLUENZA STUDY

Abstract

A worldwide flu pandemic occurred in 2009, resulting in many victims and high social damages. In the A (H1N1) virus spreading process, the pig is the intermediate host, and this virus is amplified and genetically changed through recombination in pigs. The objective of this study was to develop influenza-resistant pigs. In interferon-α and interferon-γ treated cells, the porcine Mx2 protein has been observed near the nuclear envelope, which consequently has been linked with inhibition of influenza virus proliferation. Therefore, we attempted to produce transgenic (TG) pigs overexpressing the Mx2 gene by somatic cell nuclear transfer (SCNT). Porcine fetal fibroblasts were transfected with the cytomegalovirus vector, which includes the porcine Mx2 gene. The established transgenic cell was injected into the enucleated ooplasm to produce Mx2-TG cloned embryos. In total, 511 female TG porcine embryos were transferred to 5 surrogates. Two recipients were diagnosed pregnant (pregnancy rate, 40%) on Day 25. On Day 114, 6 fetuses and 4 mummies were collected. The PCR analysis concluded that there was no integration of the Mx2 gene. Then, a male Mx2-TG cell line was established to use as donor cell of SCNT. In total, 547 male TG-SCNT embryos were produced. Of these, 38 embryos were cultured in vitro to confirm the developmental capacity of the embryos. Among these porcine SCNT-TG embryos, 26 embryos (68.4%) cleaved and 5 (13.2%) developed to the blastocyst stage. The PCR analysis confirmed that all male TG-SCNT blastocysts were for integration of the Mx2 gene. The remaining 509 male embryos were transferred to 5 surrogates. Two recipients (pregnancy rate, 40%) were diagnosed pregnant at Day 25. To date, 1 of the surrogate has maintained pregnancy and another recipient gave birth to 9 piglets. Two days after birth, 2 piglets died and the remaining piglets remain healthy. Verification analysis of gene targeting and resistance to influenza is in progress. This study has presented new possibilities of production of influenza virus resistant pig by SCNT for translational research. This work was supported by a grant from Next-Generation BioGreen 21 program (# PJ008121012011), Rural Development Administration, Republic of Korea.