A reporter system for template bias of meiotic repair in mammals.

Emily Bramel

Document Type

Article

Publication Date

Summer 2017

JAX Location

In: Student Reports, Summer 2017, Jackson Laboratory

Abstract

The purpose of this study is to develop a reporter system to investigate the phenomenon of preferential double-strand break (DSB) repair form the homolog, rather than sister chromatid, during meiosis in mammals. While previous studies have examined the regulation of recombinational repair in yeast, a novel method is required to fully understand repair template bias in mammals. An effective reporter system will provide insight into the mechanism of template choice in inter-sister (IS) and inter-homolog (IH) repair during meiosis. We designed a two-part reporter system that employs CRISPR-Cas9 to induce DSBs and fluorescent proteins with distinct color and localization of green (GFP) and blue (BFP) fluorescence to distinguish DSB repair events during mouse meiosis. In this study we tested the components of this system in cell culture to validate the design and correct expression of fluorescent proteins. HeLa cells were transfected with mutant BFP (no fluorescence), BFP, and GFP and demonstrated strong color and localization of fluorescence. We established stable cell lines with integration of fluorescent gene cassettes into the HeLa cell genome. We successfully used Cas9 and guiding RNA to induce DSBs in mutant BFP expressing cells and observed the result of DSB repair using a provided GFP DNA template resulting in vibrant green fluorescence. After further validation in mammalian cell lines, this system will be introduced to a mouse model to identify genes involved in repair template bias.

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