title: Rapid and cost-effective gene targeting in rat embryonic stem cells by TALENs.
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.
authors: 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.
authors: Chang
authors: C
authors: Tong
authors: Guanyi
authors: G
authors: Huang
authors: Charles
authors: C
authors: Ashton
authors: Hongping
authors: H
authors: Wu
authors: Hexin
authors: H
authors: Yan
authors: Qi-Long
authors: QL
authors: Ying
mesh_terms: D000818:Animals; D001483:Base Sequence; D002460:Cell Line; D053595:Embryonic Stem Cells / Q000378:metabolism*; D023061:Gene Order; D018390:Gene Targeting; D005822:Genetic Vectors; D051379:Mice; D008969:Molecular Sequence Data; D051381:Rats; D011995:Recombination, Genetic