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CRISPR/Cas9‐mediated targeting of the Rosa26 locus produces Cre reporter rat strains for monitoring Cre –loxP ‐mediated lineage tracing
Author(s) -
Ma Yuanwu,
Yu Lei,
Pan Shuo,
Gao Shan,
Chen Wei,
Zhang Xu,
Dong Wei,
Li Jing,
Zhou Rui,
Huang Lan,
Han Yunlin,
Bai Lin,
Zhang Li,
Zhang Lianfeng
Publication year - 2017
Publication title -
the febs journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.981
H-Index - 204
eISSN - 1742-4658
pISSN - 1742-464X
DOI - 10.1111/febs.14188
Subject(s) - cre recombinase , mcherry , crispr , cre lox recombination , biology , transgene , microbiology and biotechnology , green fluorescent protein , gene knockin , reporter gene , gene targeting , gene , genetically modified mouse , gene expression , genetics
The rat is an important laboratory animal for physiological, toxicological and pharmacological studies. Clustered regularly interspaced short palindromic repeats ( CRISPR )/CRISPR‐associated 9 (Cas9) is a simple and efficient tool to generate precise genetic modifications in rats, which will promote the accumulation of genetic resources and enable more precise studies of gene function. To monitor Cre– loxP ‐mediated excision in vivo , we generated a Cre reporter rat strain ( Rosa26‐imCherry ) by knockin of a Cre reporter cassette at the Rosa26 locus using CRISPR /Cas9. Rosa26‐imCherry rats exhibited inducible expression of the mC herry cassette ( imCherry ) using the Cre –loxP system, whereas normal rats exhibited ubiquitous expression of eGFP but not mC herry in the whole body. Injection of adeno‐associated virus serotype 9– Cre into the hippocampus and skeletal muscle resulted in mC herry expression in virus‐infected cells. Cre –loxP ‐mediated mC herry expression was then evaluated by crossing Rosa26‐imCherry rats with transgenic rats ubiquitously expressing CAG ‐ Cre , heart‐specific α‐ MHC‐Cre transgenic rats and liver‐specific Alb‐Cre knockin rats. Finally, using the established system the expression pattern of Cre driven by two endogenous gene promoters ( Wfs1‐Cre knockin rat, FabP2‐Cre knockin rat) was traced. In summary, we demonstrated excision of the loxP ‐flanked allele in Rosa26‐imCherry rats via activation of mC herry expression in the presence of Cre recombinase. This newly established Rosa26‐imCherry rat strain represents a useful tool to facilitate Cre ‐expression pattern determination and tracing experiments.