Effect Of Reprogramming On Donor Nucleus Of Tumour Cell Nuclear Transfer

Tumour is the heavest threat to human being. Although a great deal of research work has been done, there isn't a theory that can completely explain the occurrence mechanism of tumor. There are five characteristics of tumour cells: limitless replicative potential, evading apoptosis, dedifferenciation, sustained angiogenesis, tissue invasion and metastasis. Researchers tried to find out ways to restrain or reverse the five characteristics for treatment purpose, this kind of work showed great application potential, but we haven't yet find out one that can completely fulfill the reversion. In 2004, Rudolf Jaenisch got blastocysts by use of nuclear transfer technique with embryonal carcinoma cells as nuclei donor cells, he picked up the inner cell masses from the blastocysts , injected them into normal blastocysts, at last he got chimera mice. This research work showed the possibility and feasibility of reversing tumor cells by use of nuclear transfer.Nuclear transfer(or nuclear transplantation) means injecting donor nucleus into recipient enucleated oocyte or zygote through micromanipulation, after activation, donor nucleus was reprogrammed by recipient cell cytoplasm, reconstructed embryo then come into being. In 1997, Wilmut cloned out the sheep Dolly, which is the first time a mammal animal was cloned from somatic cell. The cloned one shares same genetic characters with the animal which donor the nucleus.Reprogramming donor nucleus is involved in nuclear transfer. Whether the donor nucleus is fully reprogrammed is a key to succedent development of the reconstructed egg. Gurdon injected nuclei of tadpole intestine epithelium cells into enucleated zygotes, the reconstructed eggs stopped development soon, and many embryonic cells showed chromosomal aberrancy, Gurdon then picked up cells with normal chromosomes as nuclear donor cells, cloned out frogs with a success rate of 7%. This work showed that nuclei have been modified as cloned embryos are developing, only nuclei reprogrammed to appropriate state can support to fully development.We improved success rate of nuclear transfer by exploring appropriate ways tocomplete every step of nuclear transfer: 1) Mouse oocytes were pretreated with 3% sucrose to visualize the metaphase spindles and chromosomes under common light microscopy and led to a 100% enucleation rate, and reduced cytoplasm loss for higher development rate. 2) Five ways of activating reconstructed eggs were compared: electrical pulse, electrical pulse added with cycloheximide(CHX), alcohol added with CHX, Sr2+added with CCB, Sr2* added with CHX and CCB. There are evident differences in activation rates and development rates to blastocysts among the five groups. Reconstructed eggs activated by Sr2* added with CHX and CCB got highest development rate to blastocysts. 3). Effect of length of serum starvation on nuclei donor cells were observed, and nuclear transfer success rates among the groups were compared. Results show that highest development rate can be achieved by serum starvation for 3-5 days. 4). Electrical fuse rates of different electrical pulse composed on donor cell-recipient cell compounds were compared. There is no evident difference when pulse voltage and pulse time vary among a proper range. But adverse effect can be induced by inappropriate pulse voltage or too long or too short of pulse time.For exploring effect of reprogramming on donor nuclei, chromosomal states of five cell groups (nuclear transfer embryos of tumor cells, nuclear transfer embryos of somatic cells, parthenogenetic embryos, B16 cells, granulosa cells) were detected by comet assay, resultant images were analysed with software of COMET V2.3.5. Nuclear transfer embryos of tumor cells show difference from other cell groups as follows: 1). Image intensity of nuclear transfer embryos derived from B16 cells varied extensively, means the chromosomal aberrancy rate is high. 2). Comet assay images of 2-cell stage embryos to 8-cell stage embryos show tails, the percentage of tail DNA reach the uppermost in 2-cell embryos, then descend as embryos reaching latter development stage, disappearing in morula stage embryos. This may be due to chromosomal damage or exposure of DNA strain vulnerable points to alkaline condition caused by reprogramming.