Efficient generation of selection-gene-free rat knockout models by homologous recombination in ES cells.
Embryonic stem cell (ES cell)-based rat knockout technology, although successfully developed in 2010, has seen very limited usage to date due to low targeting efficiency and a lack of optimized procedures. In this study, we performed gene targeting in ES cells from the Sprague-Dawley (SD) and the Fischer 344 (F344) rat strains using an optimized procedure and the self-excising neomycin (neo)-positive selection cassette ACN to successfully generate Leptin and Trp53 knockout rats that did not carry the selection gene. These results demonstrate that our simplified targeting strategy using ACN provides an efficient approach to knock out many other rat genes.
State Key Laboratory of Agrobiotechnology, College of Biological Sciences, China Agricultural University, Beijing, China.
He
H
Lan
State Key Laboratory of Agrobiotechnology, College of Biological Sciences, China Agricultural University, Beijing, China.
Shuping
S
Li
State Key Laboratory of Agrobiotechnology, College of Biological Sciences, China Agricultural University, Beijing, China.
Zihang
Z
Guo
Veterinary Pathobiology, Rat Resource and Research Center, College of Veterinary Medicine, University of Missouri, Columbia, MO, USA.
Hongsheng
H
Men
Department of Human Genetics, University of Utah School of Medicine, Salt Lake City, UT, USA.
Yuanyuan
Y
Wu
State Key Laboratory of Agrobiotechnology, College of Biological Sciences, China Agricultural University, Beijing, China.
Ning
N
Li
Veterinary Pathobiology, Rat Resource and Research Center, College of Veterinary Medicine, University of Missouri, Columbia, MO, USA.
Elizabeth C
EC
Bryda
Department of Human Genetics, University of Utah School of Medicine, Salt Lake City, UT, USA.
Mario R
MR
Capecchi
State Key Laboratory of Agrobiotechnology, College of Biological Sciences, China Agricultural University, Beijing, China. swu@cau.edu.cn.
Sen
S
Wu
D000818:Animals; D002460:Cell Line; D053595:Embryonic Stem Cells / Q000166:cytology* / Q000378:metabolism; D055786:Gene Knockout Techniques / Q000379:methods*; D059765:Homologous Recombination; D020738:Leptin / Q000235:genetics / Q000378:metabolism; D008297:Male; D051381:Rats; D011916:Rats, Inbred F344; D017207:Rats, Sprague-Dawley; D016159:Tumor Suppressor Protein p53 / Q000235:genetics / Q000378:metabolism
D020738:Leptin; D016159:Tumor Suppressor Protein p53
ES cells; gene targeting; homologous recombination; rat; selection-gene-free