DNA methylation is a critical mechanism for the epigenetic control of tissue differentiation in organisms, particularly when it comes to the development of sperm and testes. There is little information, however, concerning specific methylation patterns and their effects on these regions. One gene of interest is SLC9B1, which codes for NHA1, a sodium-hydrogen exchanger that has been connected to sperm motility and male fertility. This gene is unmethylated in mice with normal fertility and it has been shown that abnormal methylation in the area surrounding this gene can lead to reduced or complete loss of fertility. This study investigates the effects of targeted methylation using dCas9-DNMT3A-3L in MK4 cells in combination with 4 different guide RNAs that target different parts of the gene, including the promoter and nearby CpG islands. Though much of this study is still in progress, it has been concluded that the guide RNAs being used are able to target their assigned region of DNA effectively and that the isolated use of dCas9-DNMT3A-3L does not have any effect on the methylation status of the MK4 cells. As this research continues, the next steps include assessing the MK4 dCas9-DNMT3A-3L cells that have been transfected with a guide RNA for changes in methylation status using bisulfite sequencing. If these results show that there has been targeted methylation, an analysis of the mRNA and protein products found in cells would follow. This research helps to bring about a better understanding of the epigenetic function of methylation, which could be applied to both new fertility treatments and new forms of contraception.
Author(s): Caroline Reckers, Biology Major
Advisor(s): Paul James, Department of Biology
Cameron Gardner, Department of Biology
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