Development of an interspecies somatic cell nuclear transfer system for establishing innovative medical treatment

Abstract

Recently, cell and tissue therapies with employing somatic cell nuclear transfer (SCNT) have been suggested to develop innovative medical technology, which greatly contributes to overcome degenerative diseases. After the successful birth of cloned sheep, ¡°Dolly¡±, SCNT is of significantly greater commercial and research importance. However, various concerns on human SCNT, which might cause human cloning, have been made. The objective of my master thesis was to develop an efficient SCNT, which could develop an alternative therapeutic cloning technology without sacrificing human oocytes. To achieve this purpose, I attempted to establish an interspecies nuclear transfer of human somatic cell into enucleated bovine oocytes. In the first series of experiment, I attempted to select the most efficient somatic cell type and preparation, fusion medium and culture strategy for interspecies clone embryos. As results, SCNT of fetal cord fibroblasts yielded more (P=0.0006) 2-cell embryos than SCNT of adult fibroblasts. A significant (P<0.05) batch effect of cord fibroblast on preimplantation development after SCNT was found and serum starvation greatly improved preimplantation development, regardless of the batches. No significant effect was observed after change of fusion medium (Ca2+- free mannitol solution) to Ca2+-containing mannitol or sorbitol solution, while embryo culture system greatly influenced the development. A continuous culture in modified synthetic oviduct fluid medium used in bovine embryo culture only supported blastocyst formation of interspecies clone embryos. In the second series of experiment, I further optimized the interspecies SCNT developed in my previous experiment by changing cord fibroblast cell batches and reconstruction method. Total 1,742 bovine oocytes were provided for SCNT. Reconstruction of a fibroblast by single DC pulse of 1.9 to 2.1 kV/cm for 20 ¥ìsecond yielded better rate of fusion (30 to 56%) and, development to the 2- cell (27 to 36%), 8-cell (7 to 14%), 16-cell (5 to 7%) and morula to blastocyst (3 to 5%) stages. Results from karyotyping and mitochondrial DNA analysis demonstrated that 56% of karyotyped embryos had presumptively normal human chromosome complements consisting of 23 pairs of autosomes with sex chromosome, while only bovine mtDNA was detected.