The Nucleus Isolation And DNA Methylation Study Of Neuron In Special Neural Circuit Traced With Adeno-associated Virus

Brain,the most complex and precision organ human body,is constituted with billions of neurons.Those neuron connects with each others by synaptic junctions,and it is those connections that form the functional neural network which allow us to own individual consciousness and behaviour.The original library construction method for NGS which used tissue sample as input will cover up the difference between the neurons.Besides,the different forms,functions and gene expressions are determined by the epigenome.So in this study,a method for single neuron tracing and isolation of its nucleus was introduced,and DNA methylation,a important form of epigenome was selected as study subject.During the construction of recombinational adeno-associated virus,the vectors pAAV-CMV/hSyn-SUN1-GFP-antibody tag which express the recombinational nuclear membrane protein were constructed firstly.The general promoter CMV was used in cell transfection experiment in vitro and the neuron-specificity promoter hSyn was used in mouse brain injection experiment in vivo for recombinational nuclear membrane protein transcription.The recombinational vector was transfected into HEK-293 T cells as a test,and the green fluorescence signal could be observed under fluorescence microscope.The signal was circularly distributed and the surrounding was lighter than the middle,which meant the recombinational nuclear membrane protein was targeted on the nuclear membrane as expectation.However,after recombinational virus injection,no signal could be observed on the brain slices.Based on the consideration that the changing of promoter or the operation during the experiment may caused for the negative result,an other recombinational adeno-associated virus rAAV-hSyn-eGFP-NLS was constructed and injected.Enough signals could be observed on brain slices,so the speculation above couldn't be ruled out.When isolate the tagged nucleus,the transfected cells were used as materials to simulate the neurons infected by the recombinational virus.The transfected cells were homogenized and centrifuged and the isolated nucleuses were intact and with little impurity.The co-immunoprecipitation assay was been done to capture the recombinational nuclear membrane protein tagged nucleuses.The positive rates of purified nucleuses were nearly 70%,and the capture efficiencies were between 65% to85%.On the other hand,the residual rate of untagged nucleuses in negative control group is only 0.18%.So it could be said that it method could be applied in further study.For single-cell DNA methylation sequencing library construction,the single-cellbisulfite sequencing method had been used.A small amount of cells and single neuron were introduced as input sample one after another,and the libraries were constructed successfully.The size distribution of fragment in final library was as expected,checked with agarose gel electrophoresis assay and the bands were bright that meant the library were qualified for sequencing.Besides,the bio-informatics analysis of sequencing result of testing sample had also been done and it was found that the sequencing qualities were good and the difference caused by the operation between each sample were little,which meant the repeatability of this method is good.According to the problem met in this study that the package of AAV is limited,the existing methods for AAV package extension were discussed and a new idea was also introduced that the capsid protein coding gene cap could be modified by artificial evolution method.Besides,the applications of single-cell DNA methylation analysis in medical test were also discussed.