Protocol For Chromatin Accessibility Of Testicular Cells In Mouse Model Of Klinefelter Syndrome

Klinefelter syndrome(KS),a disease with high incidence but low diagnostic rate,is one the most common chromosomal abnormalities.Patients suffer with typical bisexual characteristics,accompanied by substantial variation in metabolic syndromes.Azoospermia-related male infertility is a substantial issue of KS.However,the mechanism of germ cell loss in KS is still unknown.In this study,we aim to contrive a protocol for investigating the mechanism of germ cell loss in KS based on the KS mouse model and Assay for Transposase Accessible Chromatin with high-throughput sequencing(ATAC-seq)analysis.Establishment of model mice using four-generation cross-breeding method is the basis of this experiment.PCR,the technique we used to identify target mice with different karyotypes with the copy number or expression profiles of specific genes.Then crosses specific offspring to produce 41,XXY mice in F4 generation.After breeding enough mice,Leydig cells and Sertoli cells were selected as candidates for research according for the background information of azoospermia in KS and the changes of different testicular cells.Then separation,purification,and identification of different testicular cells are illustrated for each kind of cell.According to the library construction results of the original kit and the sequencing requirements,the construction process of the accessibility library was optimized.Subsequently,the optimized library construction protocol was used to testicular cells from model mice and control mice to establish an accessibility region library.The fourth chapter interprets the results of ATAC-seq library sequencing analysis and the analysis results of plasma metabolomics.At the same time,relevant literature was reviewed to further confirm the analysis results.We found that the testicular immune microenvironment changes in KS model mice.Then,through the review of existing research,we found that the changes in the testis of KS mice were similarly to those of autoimmune orchitis mice,suggesting that changes in the immune environment were the potential cause of the germ cell loss in KS.In conclusion,in this thesis,a chromatin accessibility library of Sertoli Cell and Leydig Cell of KS model mice was constructed,and the library was sequenced and analyzed.