Abstract
SummaryRat embryonic stem cells (ESCs) offer the potential for sophisticated genome engineering in this valuable biomedical model species. However, germline transmission has been rare following conventional homologous recombination and clonal selection. Here, we used the CRISPR/Cas9 system to target genomic mutations and insertions. We first evaluated utility for directed mutagenesis and recovered clones with biallelic deletions in Lef1. Mutant cells exhibited reduced sensitivity to glycogen synthase kinase 3 inhibition during self-renewal. We then generated a non-disruptive knockin of dsRed at the Sox10 locus. Two clones produced germline chimeras. Comparative expression of dsRed and SOX10 validated the fidelity of the reporter. To illustrate utility, live imaging of dsRed in neonatal brain slices was employed to visualize oligodendrocyte lineage cells for patch-clamp recording. Overall, these results show that CRISPR/Cas9 gene editing technology in germline-competent rat ESCs is enabling for in vitro studies and for generating genetically modified rats.
Highlights
The rat Rattus is a valuable and widely used model organism for studying cognition and behavior, physiology, toxicology, and various pathologies, such as metabolic and neurodegenerative diseases (Iannaccone and Jacob, 2009)
Genome engineering is mostly performed via embryonic stem cells (ESCs), and the ease of carrying out such work has been key to their widespread use as an animal model (Capecchi, 2005)
Rat ESCs are less robust than their mouse counterparts and demand expert handling to maintain robust growth and capacity for germline transmission (Blair et al, 2011), especially after clonal selection required for gene targeting (Hirabayashi et al, 2010b, 2013, 2014; Meek et al, 2010; Men et al, 2012; Men and Bryda, 2013; Tong et al, 2010)
Summary
The rat Rattus is a valuable and widely used model organism for studying cognition and behavior, physiology, toxicology, and various pathologies, such as metabolic and neurodegenerative diseases (Iannaccone and Jacob, 2009). Rat ESCs are less robust than their mouse counterparts and demand expert handling to maintain robust growth and capacity for germline transmission (Blair et al, 2011), especially after clonal selection required for gene targeting (Hirabayashi et al, 2010b, 2013, 2014; Meek et al, 2010; Men et al, 2012; Men and Bryda, 2013; Tong et al, 2010). These technical difficulties have hindered the widespread adoption of rat ESC transgenesis
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