53 EVALUATION OF NUCLEAR REPROGRAMMING IN CLONED MINIATURE PIG EMBRYOS USING GENES OF Oct-4 FAMILY

Abstract

Xenotransplantation as a source of organs is a rapidly expanding field which can save thousands of human lives each year. Cloned miniature pigs have been considered as a model system for xenotransplantation. However, the efficiency of somatic cell nuclear transfer (SCNT) is extremely low, with most clones resulting in early lethality and several aberrancies. Possible explanation of the developmental failure of SCNT embryos is related to insufficient reprogramming of the somatic cell nucleus. In order to test this, we analyzed the reprogramming capacity of differentiated fibroblast cell nuclei and undifferentiated germ cell nuclei with Oct-4 and four Oct-4-related genes (Ndp5211, Dppa2, Dppa3, and Dppa5) as molecular markers using quantitative reverse transcription-polymerase chain reaction (RT-PCR). Oct-4 expression patterns were similar among IVF-derived embryos and cloned embryos derived from fibroblasts or germ cells during pre-implantation embryo development. However, the expression level was significantly lower (P < 0.05) in hatched blastocysts of fibroblast clones compared to other hatched blastocysts. Also, 9 of 13 cloned morulae and 12 of 40 cloned blastocysts failed to reactivate at least one of the five tested genes, whereas all of the germ cell clones and control embryos correctly expressed these genes. Analysis with miniature pig fetuses collected at Day 30 of gestation revealed that normal and cloned fetuses successfully expressed these genes. In conclusion, our results suggest that analysis of expression of Oct-4 and related genes could be a reliable marker for evaluating the reprogramming status of transplanted donor nuclei in cloned embryos. The reprogramming of fibroblast cloned embryos is highly error-prone. This may contribute to their embryonic lethality because cloned embryos that fail to reactivate the marker genes may fail to be successfully implanted.

This study was supported by grants from the Ministry of Science and Technology (Top Scientist Fellowship), and the Biogreen 21-1000520030100000.