CGG Repeat Expansion, and Elevated Fmr1 Transcription and Mitochondrial Copy Number in a New Fragile X PM Mouse Embryonic Stem Cell Model

Frontiers in Cell and Developmental Biology
Inbal GazyK Usdin

Abstract

The Fragile-X related disorders (FXDs) are Repeat Expansion Diseases (REDs) that result from expansion of a CGG-repeat tract located at the 5' end of the FMR1 gene. While expansion affects transmission risk and can also affect disease risk and severity, the underlying molecular mechanism responsible is unknown. Despite the fact that expanded alleles can be seen both in humans and mouse models in vivo, existing patient-derived cells do not show significant repeat expansions even after extended periods in culture. In order to develop a good tissue culture model for studying expansions we tested whether mouse embryonic stem cells (mESCs) carrying an expanded CGG repeat tract in the endogenous Fmr1 gene are permissive for expansion. We show here that these mESCs have a very high frequency of expansion that allows changes in the repeat number to be seen within a matter of days. CRISPR-Cas9 gene editing of these cells suggests that this may be due in part to the fact that non-homologous end-joining (NHEJ), which is able to protect against expansions in some cell types, is not effective in mESCs. CRISPR-Cas9 gene editing also shows that these expansions are MSH2-dependent, consistent with those seen in vivo. While comparable human Gen...Continue Reading

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Datasets Mentioned

BETA
GSE81803
GSE114015
GSE48873

Methods Mentioned

BETA
transfections
transfection
PCR
genotyping
PCRs
electrophoresis
RNA-seq
Immunoprecipitation
ChIP
pull down

Key Resources (RRID) Mentioned

Addgene_62988

Software Mentioned

R script

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