Technology development in mouse genetics and epigenetics

Abstract

The importance and significance of a model organism in biological research cannot be overstated. The mouse in particular has been very useful in understanding questions in many areas of research such as developmental biology, cancer biology, neuroscience and genetics. However, even though the methods to make transgenic mice and gene knockin and knockouts have been successful, they are very inefficient, labor intensive and costly. Therefore, in this thesis we developed a novel methodology to rapidly and efficiently modify the mouse genome. Using CRISPR/Cas9, a novel genome-engineering technology developed from bacteria, we were able to genetically modify mouse embryonic stem cells and make mice that carried genetic modification by zygotic injections. Using CRISPR/Cas9 we were able to make mice in as little as three weeks that contained multiple gene knockouts, single nucleotide modifications, GFP and mCherry reporter alleles, epitope-tagged alleles, and conditional alleles. Another interesting area of research in mouse genetics is epigenetic regulation, specifically how DNA methylation regulates development, gene expression, and cell state. Multiple studies have shown that this epigenetic modification plays an important regulatory role in these processes; however, the technology that has existed so far to investigate DNA methylation has only been able to look at snapshots of methylation patterns in fixed cell populations. In this thesis we have developed a novel technology named Reporter of Genomic Methylation (RGM), which allows for the investigation of methylation dynamics at single cell-resolution in vivo. The RGM technology was developed using a minimal synthetic secondary DMR promoter that drives the expression of a florescent protein. Using CRISPR/Cas9 the RGM reporter can be integrated into any genomic locus where it can report on the methylation state of its surroundings. We further show that the RGM reporter activity reflects the methylation state of non-coding regulatory elements such as promoters and enhancers. Furthermore, we show that the RGM technology allows for the dynamics of methylation and demethylation to be observed at these non-coding loci as cells transition between a pluripotent and differentiated state.