41 PRODUCTION OF CLONED MINIATURE PIGS USING BONE MARROW MESENCHYMAL STEM CELLS

The cloning of miniature pigs (Mini-pig) are considered to advance in genetic engineering technology and xenotransplantation. A few researches have recently been reported successfully produce cloned Mini-pigs using somatic cells, however its efficiency is still low. The present study was aimed to successfully produce cloned Mini-pigs derived from bone marrow mesenchymal stem cells (MSCs) by NT, and compared the developmental ability of cloned Mini-pigs between fetal fibroblast (FF) and differentiated MSCs. For the production of the cloned Mini-pig derived from MSCs, MSCs were isolated from a 1 month old of female Mini-pigs (T-type, PWG Micro-pig®, PWG Genetics Korea, Ltd.). MSCs were differentiated into osteocytes, adipocytes, and chondrocytes under controlled conditions and characterized by cell surface antigen profile using specific markers. These differentiated or undifferentiated MSCs, FFs of Mini-pig were transferred into enucleated oocytes of domestic pigs, and 2-cell stage of 100 NT embryos were surgical transferred to the synchronized recipients. Statistical analysis was performed using one-way ANOVA, t-test, Duncan’s and Tukey’s multiple comparisons test by SPSS (SPSS, Inc., Chicago, IL, USA). The developmental potential of NT embryos derived from MSCs (differentiated and undifferentiated), the rates of blastocyst formation was significantly (P < 0.05) higher than NT embryos derived from FFs, however the NT embryos derived from three different types of differentiated MSCs were significantly (P < 0.05) lower than undifferentiated MSCs. In addition, total cell numbers in NT blastocysts derived from MSCs were significantly (P < 0.05) higher than NT blastocysts derived from FFs, but it did not significantly (P < 0.05) differ between differentiated or undifferentiated MSCs. NT embryos derived from MSCs were transferred to 5 domestic pig recipients, and 5 cloned Mini-pigs were obtained from 2 recipients (one stillbirth and 4 viable offspring). All of them were confirmed by the microsatellite analysis (8 markers) of the genomes of cloned offspring, donor MSCs and recipients. Physical and histological studies are in the process for the characterization of a cloned Mini-pig derived from MSCs as animal model. The results demonstrated that, in vitro developmental ability of NT embryos derived from undifferentiated MSCs were a higher than those from differentiated MSCs or FFs. Moreover, multipotent MSC might have a potential for the production of viable cloned Mini-pigs. Therefore, MSCs as a nuclear donor might a key to improving the production of cloned Mini-pig as animal model for xenotransplantation. This work was supported by Grant No. 20070301034040 from Bio-organ, Republic of Korea.