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J Genet Genomics. 2012 Jun 20;39(6):275-80. doi: 10.1016/j.jgg.2012.04.004. Epub 2012 May 9.

Rapid and cost-effective gene targeting in rat embryonic stem cells by TALENs.

Author information

1
Eli and Edythe Broad Center for Regenerative Medicine and Stem Cell Research at USC, Department of Cell and Neurobiology, Keck School of Medicine, University of Southern California, Los Angeles, CA 90033, USA.

Abstract

The rat is the preferred animal model in many areas of biomedical research and drug development. Genetic manipulation in rats has lagged behind that in mice due to the lack of efficient gene targeting tools. Previously, we generated a knockout rat via conventional homologous recombination in rat embryonic stem (ES) cells. Here, we show that efficient gene targeting in rat ES cells can be achieved quickly through transcription activator-like effector nuclease (TALEN)-mediated DNA double-strand breaks. Using the Golden Gate cloning technique, we constructed a pair of TALEN targeting vectors for the gene of interest in 5 days. After gene transfection, the targeted rat ES cell colonies were isolated, screened, and confirmed by PCR without the need of drug selection. Our results suggest that TALEN-mediated gene targeting is a superior means of establishing genetically modified rat ES cell lines with high efficiency and short turnaround time.

PMID:
22749015
PMCID:
PMC3856761
DOI:
10.1016/j.jgg.2012.04.004
[Indexed for MEDLINE]
Free PMC Article
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