Reconstruction Of Embryos From Lymphocytes And Pilot Study Of Its CD Molecules Expression

Two types of nuclear transfer are mentioned in the mammalian cloning study depending on the origin of nuclear donor cells, which are nuclear transfer with embryonic cleavage cells and nuclear transfer with somatic cells. As model animals, mice play an important role in nuclear transfer. There are kinds of methods to complete nuclear transfer with mice's oocytes. While most of them either need special apparatuses (for example, Piezoimpact equipment, and Spindle-View system), or have side effects on the following development of reconstructed embryos. In this study, an improved method was used to reconstructed embryos. Following the puncture of zona pellucida with two holes by injection pipette that contained donor nuclei or cells, the injection pipette was pulled back to the perivitelline space while the negative pressure was increased in the holding pipette until the polar body and karyoplasm were wiped off completely. Then a reconstructed embryo was completed by direct injection of the donor nucleus or cell without pulling out the injection pipette. The enucleation rate and survival rate were analyzed, and it demonstrated that the nucleus was completely eliminated and another was injected only using the inverted microscope and micromanipulator. Moreover, the efficiency of nuclear transplantation and the survival rate of reconstructed embryos were greatly improved. Furthermore, it is very easy to manipulate and popularize in practice .In this study, reconstructed embryos were also obtained from oocytic cytoplasm with lymphocyte nuclei. The states of donor nuclei were observed during the embryo' development, and the expressions of CD molecules were inspected in reconstructed embryos and donor cell. The results demonstrated that the genome reprogramming of reconstructed lymphocyte genome took place within 24 hours after the reconstructed embryos were activated, and that reconstructed cells maybe dying if the genome reprogramming did not happene during this time. Lymphocyte nucleus still expressed its differentiation antigen during the early times of reconstructed embryoic development. while this expression was closed along with those embryos' development. It indicated that during the reconstructed lymphocyte genome reprogramming there were certain programs which control the conversion of gene expression. As nuclear genome was adjusted to a certain level in early embryo development, it would express towards the way of normal embryo development. This study has provided necessary techniche and theory for the further study of molecular mechenisms of nuclear genome reprogremming. Furthermore it will greatly help to illustrate the rules of reconstructed nuclear genome program and the molecular mechanism of cell differentiation. Moreover, it has great practical meaning in the field of biomedicine as well.