文章基本信息

标题：Efficient targeted integration into the bovine Rosa26 locus using TALENs
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作者：Ming Wang ; Zhaolin Sun ; Zhiyuan Zou 等
期刊名称：Scientific Reports
电子版ISSN：2045-2322
出版年度：2018
卷号：8
期号：1
页码：10385
DOI：10.1038/s41598-018-28502-x
语种：English
出版社：Springer Nature
摘要：The genetic modification of cattle has many agricultural and biomedical applications. However, random integration often results in the unstable expression of transgenes and unpredictable phenotypes. Targeting genes to the "safe locus" and stably expressing foreign genes at a high level are desirable methods for overcoming these hurdles. The Rosa26 locus has been widely used to produce genetically modified animals in some species expressing transgenes at high and consistent levels. For the first time, we identified a bovine orthologue of the mouse Rosa26 locus through a genomic sequence homology analysis. According to 5' rapid-amplification of cDNA ends (5'RACE), 3' rapid-amplification of cDNA ends (3'RACE), reverse transcription PCR (RT-PCR) and quantitative PCR (Q-PCR) experiments, this locus encodes a long noncoding RNA (lncRNA) comprising two exons that is expressed ubiquitously and stably in different tissues. The bovine Rosa26 (bRosa26) locus appears to be highly amenable to transcription activator-like effector nucleases (TALENs)-mediated knock-in, and ubiquitous expression of enhanced green fluorescent protein (EGFP) inserted in the bRosa26 locus was observed in various stages, including cells, embryos, fetus and cattle. Finally, we created a valuable master bRosa26-EGFP fetal fibroblast cell line in which any gene of interest can be efficiently introduced and stably expressed using recombinase-mediated cassette exchange (RMCE). The new tools described here will be useful for a variety of studies using cattle.