PMID- 24199847
OWN - NLM
STAT- MEDLINE
DCOM- 20141112
LR  - 20220321
IS  - 1873-6823 (Electronic)
IS  - 0741-8329 (Print)
IS  - 0741-8329 (Linking)
VI  - 47
IP  - 8
DP  - 2013 Dec
TI  - Toll-like receptor 4 (Tlr4) knockout rats produced by transcriptional 
      activator-like effector nuclease (TALEN)-mediated gene inactivation.
PG  - 595-9
LID - S0741-8329(13)00160-2 [pii]
LID - 10.1016/j.alcohol.2013.09.043 [doi]
AB  - Genetically engineered mice are a valuable resource for studies of the behavioral 
      effects of ethanol. However, for some behavioral tests of ethanol action, the rat 
      is a superior model organism. Production of genetically engineered rats has been 
      severely hampered due to technical limitations. Here we utilized a promising new 
      technique for efficient site-specific gene modification to create a novel gene 
      knockout rat line. This approach is based on transcriptional activator-like 
      effector nucleases (TALENs). TALENs function in pairs and bind DNA in a 
      sequence-specific manner. Upon binding to the target sequence, a functional 
      nuclease is reconstituted that creates double-stranded breaks in the DNA that are 
      efficiently repaired by non-homologous end joining. This error-prone process 
      often results in deletions of varying lengths at the targeted locus. The 
      toll-like receptor 4 (Tlr4) gene was selected for TALEN-mediated gene 
      inactivation. Tlr4 has been implicated in ethanol-induced neuroinflammation and 
      neurodegeneration, as well as multiple ethanol-induced behavioral effects. To 
      generate Tlr4 knockout rats, a pair of TALEN constructs was created that 
      specifically target Exon 1 immediately downstream of the start of translation. 
      TALEN mRNAs were microinjected into the cytoplasm of one-cell Wistar rat embryos. 
      Of 13 live-born pups that resulted, one harbored a mutation in Exon 1 of Tlr4. 
      The mutated allele consisted of a 13 base-pair deletion that was predicted to 
      create a frameshift mutation after amino acid 25. This founder rat successfully 
      transmitted the mutation to F1 offspring. Heterozygous F1 offspring were 
      interbred to produce homozygous F2 animals. Homozygous mutants expressed the 
      13-bp deletion in Tlr4 mRNA. In contrast to control rats that produced a robust 
      increase in plasma tumor necrosis factor alpha in response to a 
      lipopolysaccharide challenge, homozygous rats had a markedly attenuated response. 
      Thus, the mutant Tlr4 allele generated by TALEN-mediated gene inactivation 
      represents a null allele. This knockout rat line will be valuable for studies of 
      ethanol action as well as more general inflammatory conditions including septic 
      shock. In conclusion, TALEN-mediated gene targeting in rat zygotes represents an 
      inexpensive, efficient, and rapid method for creating genetically engineered 
      rats.
CI  - Copyright (c) 2013 Elsevier Inc. All rights reserved.
FAU - Ferguson, Carolyn
AU  - Ferguson C
AD  - University of Pittsburgh, Department of Anesthesiology, 6060 Biomedical Science 
      Tower-3, 3501 Fifth Avenue, Pittsburgh, PA 15261, USA.
FAU - McKay, Matthew
AU  - McKay M
FAU - Harris, R Adron
AU  - Harris RA
FAU - Homanics, Gregg E
AU  - Homanics GE
LA  - eng
GR  - U01 AA020889/AA/NIAAA NIH HHS/United States
GR  - AA020889/AA/NIAAA NIH HHS/United States
PT  - Journal Article
PT  - Research Support, N.I.H., Extramural
DEP - 20131011
PL  - United States
TA  - Alcohol
JT  - Alcohol (Fayetteville, N.Y.)
JID - 8502311
RN  - 0 (Lipopolysaccharides)
RN  - 0 (RNA, Messenger)
RN  - 0 (Toll-Like Receptor 4)
RN  - 0 (Tumor Necrosis Factor-alpha)
RN  - EC 3.1.- (Deoxyribonucleases)
SB  - IM
MH  - Alleles
MH  - Animals
MH  - Deoxyribonucleases/*metabolism
MH  - Exons
MH  - Female
MH  - Gene Knockout Techniques/*methods
MH  - *Gene Silencing
MH  - Homozygote
MH  - Lipopolysaccharides/pharmacology
MH  - Male
MH  - Mutation/genetics
MH  - RNA, Messenger/genetics
MH  - Rats
MH  - Toll-Like Receptor 4/*deficiency/genetics
MH  - Tumor Necrosis Factor-alpha/blood
PMC - PMC3844088
MID - NIHMS531920
OTO - NOTNLM
OT  - Gene targeted rats
OT  - Genome editing
OT  - Knockout rats
OT  - Toll-like receptor 4 (Tlr4)
OT  - Transcriptional activator-like effector nuclease (TALEN)
EDAT- 2013/11/10 06:00
MHDA- 2014/11/13 06:00
PMCR- 2014/12/01
CRDT- 2013/11/09 06:00
PHST- 2013/08/22 00:00 [received]
PHST- 2013/09/27 00:00 [revised]
PHST- 2013/09/28 00:00 [accepted]
PHST- 2013/11/09 06:00 [entrez]
PHST- 2013/11/10 06:00 [pubmed]
PHST- 2014/11/13 06:00 [medline]
PHST- 2014/12/01 00:00 [pmc-release]
AID - S0741-8329(13)00160-2 [pii]
AID - 10.1016/j.alcohol.2013.09.043 [doi]
PST - ppublish
SO  - Alcohol. 2013 Dec;47(8):595-9. doi: 10.1016/j.alcohol.2013.09.043. Epub 2013 Oct 
      11.