| Objective: Mitochondrial DNA disease(mt DNA)is a disorder of mitochondrial function caused by pathogenic mitochondrial DNA(mt DNA)mutations.Mitochondrial replacement therapy can prevent the transmission of pathogenic mt DNA to offspring.Most human blastocysts obtained during the experiment are of relatively poor quality and the rate of human blastocysts available for transplantation is extremely low,although there have been clinically babies born through nuclear plasmid replacement.Therefore,at present,improving the efficiency of nuclear plasmid replacement technology and reconstructing the quality of human blastocyst have become the focus and difficulty of our research.Do eggs spindle transplanted in mice,this study using different concentrations of Cytochalasin B(Cytochalasin B,CB)processing,first to compare the electrofusion and sendai virus(HVJ-E)integration efficiency of integration and test again to refactor mouse eggs cytoskeleton and statistics of mice embryonic development situation,select the optimal CB eggs spindle transplanted mice concentration,in order to improve human spindle transplantation techniques provide reliable experimental data.Background: Background: this study is in the state key development program "mitochondrial genetic disease treatment of assisted reproductive technology research" in the subject 3 "mitochondrial genetic disease screening and nuclear quality replacement technology transformation and application of clinical research",on the basis of the previous in Shanghai Jiaotong university and the Chinese academy of sciences has carried on the nuclear mass replacement technique training,mastered the technology of various operations,in the study of operation technology has been clear about the spindle transplantation and virus fusion method is applied to the highest efficiency,and the difficulties of this method is the choice of the CB concentration,therefore,against the problems in further research.Methods: Choose the same strain C57BL/6 mice oocyte spindle transplantation,to compare different concentration HVJ-E efficiency of fusion,and then respectively using 3.5μg/m L CB(group A),7.5μg/m L CB(group B),10μg/m L CB(group C),12.5μg/m L CB(group D)four different concentrations of CB treatment spindle migration process,including nuclear(every six to eight eggs in this study as A group,operation time control within 30 min.The prepared eggs were incubated in the working solution containing different concentrations of CB for 5min.With Holding needle fixed position of mouse eggs,put into spindle,the spindle in 12-1 o ’clock position,then use to remove spindle-chromosome compound needle),nuclear(to remove nuclear transfer to HVJ-E working liquid,immerse 10 to 15 s,then injected into another nuclear eggs,the note nucleus and cytoplasm of extrusion,conducive to integration)1.Compare the fusion efficiency of Sendai virus at different concentrations;2.Statistics of spindle transplantation efficiency in mice,including nuclear removal rate,nuclear injection rate and fusion rate;3.The fertilization rate,cleavage rate,blastocyst rate and hatching rate of normal and reconstructed mouse eggs were counted;4.Immunofluorescence staining was performed on mouse blastocysts in each group,and the total number of cells was counted under fluorescence microscope;5.The cytoskeleton of reconstructed mouse eggs in each group was detected by immunofluorescence staining,and the recovery of microfilaments and microtubules of reconstructed mouse eggs was observed under laser confocal scanning microscope.Results:1.HVJ-E fusion was performed on reconstructed mouse eggs in group A and B,and the efficiency of HVJ-E fusion was higher when diluted 1/20 times than 1/10 times;2.There was no difference in spindle transplantation efficiency between reconstructed mouse eggs in group A,B and D and those in group C(P > 0.05);3.Compared with normal mouse eggs in the control group,fertilization rate and cleavage rate of fused mouse eggs in group A,B,C and D showed no significant difference(P > 0.05),while blastocyst rate and hatching rate of fused mouse eggs in group D were significantly reduced(P < 0.05);4.Compared with normal mouse blastocysts in the control group,the total number of blastocysts in the reconstructed mice of group A,B and D was significantly reduced(P < 0.05).Compared with normal mice in the control group,the total number of blastocysts in the reconstructed mice in group C was not significantly different(P > 0.05);5.After the fusion of reconstructed mouse oocytes in group A,B,C and D,there was basically no difference in the ratio of microfilaments recovery at 0h,1h and 2h;6.The microfilaments of reconstructed mouse eggs in group A,B and D presented uneven distribution,while the microfilaments of reconstructed mouse eggs in group C presented uniform distribution.Conclusion:1.Mouse egg spindle transplantation,diluted 1/20 times of HVJ-E fusion efficiency is good;2.CB concentration selected in this study did not affect spindle transplantation efficiency,fertilization rate and cleavage rate of reconstructed mouse eggs,but affected blastocyst rate,hatchability rate and total number of blastocyst cells of reconstructed mouse eggs;3.10μg/mL CB was the optimal concentration for mouse egg spindle transplantation.4.The concentration of CB selected in this study did not affect the microfilaments recovery rate of reconstructed mouse eggs,but affected the microfilaments recovery state of reconstructed mouse eggs. |
PDF Full Text Request