55 DIFFERENTIAL TRANSCRIPTIONAL GENE EXPRESSION BETWEEN MINIATURE AND LANDRACE PIG FETUSES: IMPLICATION FOR PRODUCTION OF CLONED MINIATURE PIGS USING LANDRACE PIGS AS OOCYTE DONORS AND SURROGATE MOTHERS

Abstract

Miniature pigs are regarded as a better organ donor breed for xenotransplantation because of their compatible organ size with human than any other pig breeds. The present study was performed to evaluate a somatic cell nuclear transfer (SCNT) system for producing cloned miniature pigs using Landrace pigs as oocyte donors and surrogate mothers. In Experiment 1, differential mRNA expression patterns of Day 30 gestation fetuses between miniature and Landrace breeds were compared using 13 610 cDNA microarray (based on Pig Genome Oligo sets; Qiagen, Valencia, CA, USA). In each breed, total mRNA from 3 fetuses was pooled before hybridization to minimize individual sample effect. With the fold-change test, 1551 cDNAs (11.40% of total) showed more than a 2-fold difference of intensity between the 2 breeds. In miniature fetuses, 252 genes were up-regulated and 1299 were down-regulated compared to Landrace ones. Among them some crucial genes related to implantation, including vascular endothelial growth factor (VEGF), vitronectin, and c-kit, were significantly down-regulated in miniature pig fetuses. In Experiment 2, in vitro developmental competence of SCNT embryos using fibroblasts from both breeds as nuclei donors were evaluated. In total, 352 miniature and 345 Landrace cloned embryos were cultured in vitro. There was no significant difference in fusion rate (78.78 vs. 77.48%), cleavage rate (69.8 vs. 65.3%), blastocyst rate (15.5 vs. 16.7%), and total cell number of blastocysts (48.0 ± 11.2 vs. 51.9 ± 17.5; all respectively). In Experiment 3, in vivo development was also monitored. In total, 1684 and 1354 SCNT embryos derived from miniature and Landrace pigs were transferred to 9 and 7 Landrace pig surrogate mothers, respectively. Overall, miniature embryos showed less in vivo developmental potency than Landrace ones; pregnancy rate at Day 30 of gestation (44 vs. 86%) and birth rate (11 vs. 43%) were low in miniature pig (based on the number of surrogates). Mean efficiency of SCNT embryo to term (0.24 vs. 1.55%) and mean litter size (4 vs. 7) were also low in miniature pigs. These results suggest that although in vitro development of SCNT embryos using recipient oocytes from Landrace pigs was similar between the 2 breeds, miniature pig embryos cannot interact with Landrace pig's reproductive tract properly and fail to implant, thus inhibiting fetal growth. In conclusion, cloned miniature pigs can be successfully produced using Landrace pigs as oocyte donors and surrogate mothers; however, the efficiency was very low due to transcriptional differences of fetuses between the 2 breeds.

The authors are grateful for a graduate fellowship provided by the Ministry of Education, through the BK21 program.