The Observation In White Blood Cell Nucleolus Of Rat Transfer Into Enucleared Mouse Oocytes To Generate Blastocysts

Interspecies somatic cell nuclear transfer(ISCNT) is using donor cells and recipient oocytes which are from different speciethe to construct cloning somatic cell nuclear transfer embryos. through nuclear transplantation micromanipulation,somatic cell from one species is transplanted to another species enucleation oocytes to construct nucleus-entosarc complex,and culture it to normal blastula or embryotic sphere,then transfer them to acceptor of female elder or donator of female elder to keep on developing,extremely eventually to bring about new individuality.The performance of their descendants for the main body of the genetic traits of nuclear material. Inter-species nuclear transfer technology in the protection of endangered animals,to cultivate new varieties of human embryonic stem cells and biological basis of academic research has demonstrated a bright future.In recent years,with the successful cloning the sheep of Dolly,it is attempted between cattle and buffalo,mouse and rabbit,pig and mice,mice and bovine,rat and mouse,rat and cow and sheep cattle and pigs,cattle and antelope,cow and man,people - goats,people - the rabbit,human - pig and other animal species somatic cell nuclear transfer research,in addition to ibex a wild goat,wild argali sheep access to the individual a wild giant panda occurrence of a species of rabbit early pregnancy,other animals have been,respectively,at different stages of in vitro development of early embryos,in particular,is a cow,people - goats,people - the rabbit and human - pig embryos and others have been reports of nuclear development to the blastocyst stage.It is clear that heterologous recombinant have a certain degree of heterogeneity to receptor,for example embryo,namely the reorganization of the nrclei and cytoplasm of embryo genetic material can not be the source of the dams at the same time with exactly the same receptor,immune rejection of xenogeneic pregnancy rely solely on the issue at this stage,embryos with recombinant xenogeneic receptor affinity natural mother and maintain their pregnancy,to produce the results of future generations is difficult to achieve,and genetic relationship among animals farther away from this natural affinity the more difficult Some people will try to clone a giant panda research individual criticized as contrary to natural law of "pseudo-science." However,embryonic stem cells are known to mainly come from the blastocyst,using a variety of animals to nuclear oocyte nuclear blastocyst Adult Health for human embryonic stem cell lines for the study of somatic cell nuclear transfer technology dissimilar to open significant new research.The use of such heterologous nuclear transfer technology pluripotent human stem cell lines,and then combined with tissue engineering technology,theoretically capable of forming the treatment and rehabilitation of human disease with the need for a variety of tissues and organs,is expected to resolve the current organizations and source of the shortage of organs,many patients can not be a good treatment of problems.The use of cloning in human ways of treating tissue engineering and cell replacement therapy study,leukemia patients will be the most pressing one of the study.A large number of studies have shown that cord blood stem cells than bone marrow and peripheral blood transplantation better advantage,but because each of cord blood hematopoietic cells contained in a relatively small number,mainly for the treatment of patients with body weight less than 45kg,they are using umbilical cord blood transplantation in adults has been more restricted.In order to make umbilical cord blood transplantation for adults,to explore ex vivo expansion of cord blood stem cells is particularly urgent,heterologous nuclear transfer may be the next best way to explore,and is expected to be able to solve this fundamental problem of an important research topic.However,the choice has yet to see the blood cells of heterologous nuclear transfer is reported,select the white blood cells as a heterologous nuclear transfer for the nuclear medicine in the human body not only on the clinical significance of relatively large,and easy to collect white blood ceils obtained relatively large nucleus,is expected to replace the terminal differentiation of other cells to become more efficient to generate blastocysts for heterogeneous nuclear nuclear transfer donor cells.Instead of rats and mice belong to the same family,close relationship,but under natural conditions,interspecific mating will be no public dams.Have shown in rats - mice embryos to the blastocyst stage of development can be,this blastocyst can not be in each other and the mucous membrane of the uterus to establish normal contacts with the difficulties caused by the pregnancy. Our laboratory in the design of therapeutic cloning for embryonic stem cells will be ways to generate blastocysts xenograft nuclear transfer as a new research direction,the use of somatic cell nuclear transfer technique in the laboratory rat cumulus granulosa cells for nuclear the establishment of a donor rat - mouse species nuclear transfer technology research platform,and rat white blood cell nucleus into enucleated mouse oocytes of species means of nuclear transfer blastocysts to generate a preliminary exploration.The purpose of this study to explore the adoption of experimental animal models of white blood cells as nuclear donor cells for xenotransplantation and the possibility of nuclear transfer blastocysts generated by the efficiency,in order to further the application of human white blood cells to carry out the production of nuclear transfer blastocysts xenotransplantation experiments to explore for the treatment of leukemia patients in vitro cord blood stem cells to generate new ways to lay the foundation.Test are summarized as follows:1 The study of mouse oocytes to enucleate quickly.Enucleation of sucrose in this experiment were pre-treatment to nuclear law,micro-fluorescence staining,sucrose pretreatment + micro-fluorescence staining to the nuclear method of comparison, the aim is to determine an effective approach to nuclear.The results showed that:sucrose pretreatment - fluorescence microscopy staining method to the nuclear core to the highest,reaching 41.86%for the other significant difference between the two groups(P＜0.05).It can be seen that in the oocytes to the use of sucrose pre-treatment nuclear - nuclear micro-fluorescence staining method to reach better results to nuclear2 Observation on activation effect of different activator to reconstructed embryo of rat granulosa cell-mouse enucleation oocyteThis experiment used four kinds of chemical activation method,of rat granulosa cells - in mouse oocytes enucleated embryos to activate the deal,aimed at finding a more reasonable way to activate.Treatment group 1:with 7%ethanol raM16 activation of oocytes deal 5min;treatment group 2:containing 10mmol / L of cycloheximide mM16 activation of oocytes deal 4h;treatment group 3:containing 10mmol / L SrCl₂ the mM16 activation of oocytes after 4h to deal with to deal with,and then 2.5mmol / L 6-DMAP deal 4h;deal with Group 4:ethanol containing 7%of the oocytes after the activation processing 5rain,and then 2.5 mmol / L 6-DMAP to deal with 4h.The results showed that:in a few of the activation program to deal with the activation of group 4 the best,reaching 40.42 percent,with other differences between the groups were significantly(P＜0.05).Therefore,in activated embryos to ethanol(7%,5rain) + CB(5μg / ml) +6- DMAP(2.5 mmol / L,4h) would be better to deal with the effect of the activation.3 Effect of reconstructed embryo culture in different culture solution to in vitroThis experiment with KSOM,M16,mM16,mCZB culture medium of activated embryos cultured in vitro to explore the reconfiguration of the in vitro embryo culture conditions and how to optimize.The results showed that:In a few embryos to cultivate the program,the reconstruction of embryos cultured in medium mCZB blastocyst development rate of the highest,4.34%,with other significant difference between the groups(P＜0.05),M16 and KSOM 4-8cells differences in growth rates was not significant(P＞0.05).It can be seen in the heterologous nuclear transfer embryos cultured in vitro in order to adopt more appropriate mCZB. 4 The observation in white blood cell nucleolus of rat transfer into enucleared mouse oocytes to generate blastocystsPretreatment of the use of sugar - micro-fluorescence staining to nuclear law for mouse oocytes to nuclear processing,and then use the micro-injection method to inject white blood cells to carry out a nuclear deal with the formation of embryos on the use of ethanol(7%,5 min) + CB(5μg / ml) +6- DMAP(2.5 mmol / L,4h activate and then activate the embryos to be used in vitro mCZB.In this experiment aims to rat cumulus granulosa cells for nuclear body established in rats - mice species nuclear transfer technology research platform,based on rat white blood cell nucleus into enucleated mouse oocytes of species means of nuclear transfer blastocysts generated exploratory research.The results show that:rat white blood cell nucleus into enucleated mouse oocytes species can be formed between the nuclear transfer blastocysts,blastocyst rate was 2.04%.Conclusion:(1) In this experiment,whether the nucleolus of rat Cumulus cells or white blood cells,the nuclear receptor xenograft mouse oocytes to nuclear approach to the use of sucrose pre-treatment - to micro-fluorescence staining to be better(2) Carried out on embryos activated by ethanol(7%,5min) + CB(5μg / ml) +6- DMAP(2.5 mmol / L,4h)be a better co-activation method.(3) In the heterogeneous nuclear transfer embryos cultured in vitro with the use of embryos in vitro mCZB as a more appropriate medium.(4)Rat cumulus granulosa cells for the nuclear donor test results compared to rat white blood cells for the nuclear donor somatic cell nuclear transplantation to xenotransplantation the effect of relatively poor,but access to blastocysts,blastocyst rate was 2.04%,4.34%with the control group rate of the blastocysts to generate significant differences(P＜0.05).