The Experts below are selected from a list of 444 Experts worldwide ranked by ideXlab platform
Lei Wang - One of the best experts on this subject based on the ideXlab platform.
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Expanding the Genetic Code of Caenorhabditis elegans Using Bacterial Aminoacyl-tRNA Synthetase/tRNA Pairs
2016Co-Authors: Angela R. Parrish, Zhouxin Shen, Steven P Briggs, Andrew Dillin, Zheng Xiang, Irene Coin, Xingyu She, Lei WangAbstract:The genetic code specifies 20 common amino acids and is largely preserved in both single and multicellular organisms. Unnatural amino acids (Uaas) have been genetically incorporated into proteins by using engineered orthogonal tRNA/aminoacyl-tRNA synthetase (RS) pairs, enabling new research capabilities and precision inaccessible with common amino acids. We show here that Escherichia coli tyrosyl and leucyl amber suppressor tRNA/RS pairs can be evolved to incorporate different Uaas in response to the amber stop codon UAG into various proteins in Caenorhabditis elegans. To accurately report Uaa incorporation in worms, we found that it is crucial to integrate the UAG-containing reporter gene into the genome rather than to express it on an Extrachromosomal Array from which variable expression can lead to reporter activation independent of the amber-suppressing tRNA/RS. Synthesizing a Uaa in a dipeptide drives Uaa uptake and bioavailability. Uaa incorporation has dosage, temporal, tRNA copy, and temperature dependencies similar to those of endogenous amber suppression. Uaa incorporation efficiency was improved by impairing the nonsense-mediated mRNA decay pathway through knockdown of smg-1. We have generated stable transgenic worms capable of genetically encoding Uaas, enabling Uaa exploitation to address complex biological problems within a metazoan. We anticipate our strategies will be generally extendable to other multicellular organisms
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expanding the genetic code of caenorhabditis elegans using bacterial aminoacyl trna synthetase trna pairs
ACS Chemical Biology, 2012Co-Authors: Angela R Parrish, Zhouxin Shen, Steven P Briggs, Andrew Dillin, Zheng Xiang, Irene Coin, Lei WangAbstract:The genetic code specifies 20 common amino acids and is largely preserved in both single and multicellular organisms. Unnatural amino acids (Uaas) have been genetically incorporated into proteins by using engineered orthogonal tRNA/aminoacyl-tRNA synthetase (RS) pairs, enabling new research capabilities and precision inaccessible with common amino acids. We show here that Escherichia coli tyrosyl and leucyl amber suppressor tRNA/RS pairs can be evolved to incorporate different Uaas in response to the amber stop codon UAG into various proteins in Caenorhabditis elegans. To accurately report Uaa incorporation in worms, we found that it is crucial to integrate the UAG-containing reporter gene into the genome rather than to express it on an Extrachromosomal Array from which variable expression can lead to reporter activation independent of the amber-suppressing tRNA/RS. Synthesizing a Uaa in a dipeptide drives Uaa uptake and bioavailability. Uaa incorporation has dosage, temporal, tRNA copy, and temperature d...
Stuart K Kim - One of the best experts on this subject based on the ideXlab platform.
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mosaic analysis using a ncl 1 Extrachromosomal Array reveals that lin 31 acts in the pn p cells during caenorhabditis elegans vulval development
Genetics, 1996Co-Authors: L M Miller, D A Waring, Stuart K KimAbstract:We describe a genetic mosaic analysis procedure in which Caenorhabditis elegans mosaics are generated by spontaneous loss of an Extrachromosomal Array. This technique allows almost any C. elegans gene that can be used in germline transformation experiments to be used in mosaic analysis experiments. We identified a cosmid clone that rescues the mutant phenotype of ncl-1 , so that this cell-autonomous marker could be used to analyze mosaic animals. To determine the sites of action for unc-29 and lin-31 , an Extrachromosomal Array was constructed containing the ncl-1 (+) cosmid linked to lin-31 (+) and unc-29( +) cosmids. This Array is mitotically unstable and can be lost to produce a clone of mutant cells. The specific cell division at which the Extrachromosomal Array had been lost was deduced by scoring the Ncl phenotypes of individual cells in genetic mosaics. The Unc-29 and Lin-31 phenotypes were then scored in these animals to determine in which cells these genes are required. This analysis showed that unc-29 , which encodes a subunit of the acetylcholine receptor, acts in the body muscle cells. Furthermore, lin-31 , which specifies cell fates during vulval induction and encodes a putative transcription factor similar to HNF-3/fork head, acts in the Pn.p cells
Yuguang Zhao - One of the best experts on this subject based on the ideXlab platform.
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Gene targeting in mosquito cells: a demonstration of 'knockout' technology in Extrachromosomal gene Arrays
BMC Genetics, 2001Co-Authors: Paul Eggleston, Yuguang ZhaoAbstract:Background Gene targeting would offer a number of advantages over current transposon-based strategies for insect transformation. These include freedom from both position effects associated with quasi-random integration and concerns over transgene instability mediated by endogenous transposases, independence from phylogenetic restrictions on transposon mobility and the ability to generate gene knockouts. Results We describe here our initial investigations of gene targeting in the mosquito. The target site was a hygromycin resistance gene, stably maintained as part of an Extrachromosomal Array. Using a promoter-trap strategy to enrich for targeted events, a neomycin resistance gene was integrated into the target site. This resulted in knockout of hygromycin resistance concurrent with the expression of high levels of neomycin resistance from the resident promoter. PCR amplification of the targeted site generated a product that was specific to the targeted cell line and consistent with precise integration of the neomycin resistance gene into the 5' end of the hygromycin resistance gene. Sequencing of the PCR product and Southern analysis of cellular DNA subsequently confirmed this molecular structure. Conclusions These experiments provide the first demonstration of gene targeting in mosquito tissue and show that mosquito cells possess the necessary machinery to bring about precise integration of exogenous sequences through homologous recombination. Further development of these procedures and their extension to chromosomally located targets hold much promise for the exploitation of gene targeting in a wide range of medically and economically important insect species.
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Gene targeting in mosquito cells: a demonstration of 'knockout' technology in Extrachromosomal gene Arrays
2001Co-Authors: Paul Eggleston, Yuguang ZhaoAbstract:© 2001 Eggleston and Zhao; licensee BioMed Central Ltd. Verbatim copying and redistribution of this article are permitted in any medium for any noncommercial purpose, provided this notice is preserved along with the article's original URL. For commercial use, contact info@biomedcentral.com Background: Gene targeting would offer a number of advantages over current transposon-based strategies for insect transformation. These include freedom from both position effects associated with quasi-random integration and concerns over transgene instability mediated by endogenous transposases, independence from phylogenetic restrictions on transposon mobility and the ability to generate gene knockouts. Results: We describe here our initial investigations of gene targeting in the mosquito. The target site was a hygromycin resistance gene, stably maintained as part of an Extrachromosomal Array. Using a promoter-trap strategy to enrich for targeted events, a neomycin resistance gene was integrated into the target site. This resulted in knockout of hygromycin resistance concurrent with the expression of high levels of neomycin resistance from the resident promoter. PCR amplification of the targeted site generated a product that was specific to the targeted cell line and consistent with precise integration of the neomycin resistance gene into the 5 ' end of the hygromyci
L M Miller - One of the best experts on this subject based on the ideXlab platform.
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mosaic analysis using a ncl 1 Extrachromosomal Array reveals that lin 31 acts in the pn p cells during caenorhabditis elegans vulval development
Genetics, 1996Co-Authors: L M Miller, D A Waring, Stuart K KimAbstract:We describe a genetic mosaic analysis procedure in which Caenorhabditis elegans mosaics are generated by spontaneous loss of an Extrachromosomal Array. This technique allows almost any C. elegans gene that can be used in germline transformation experiments to be used in mosaic analysis experiments. We identified a cosmid clone that rescues the mutant phenotype of ncl-1 , so that this cell-autonomous marker could be used to analyze mosaic animals. To determine the sites of action for unc-29 and lin-31 , an Extrachromosomal Array was constructed containing the ncl-1 (+) cosmid linked to lin-31 (+) and unc-29( +) cosmids. This Array is mitotically unstable and can be lost to produce a clone of mutant cells. The specific cell division at which the Extrachromosomal Array had been lost was deduced by scoring the Ncl phenotypes of individual cells in genetic mosaics. The Unc-29 and Lin-31 phenotypes were then scored in these animals to determine in which cells these genes are required. This analysis showed that unc-29 , which encodes a subunit of the acetylcholine receptor, acts in the body muscle cells. Furthermore, lin-31 , which specifies cell fates during vulval induction and encodes a putative transcription factor similar to HNF-3/fork head, acts in the Pn.p cells
Angela R Parrish - One of the best experts on this subject based on the ideXlab platform.
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expanding the genetic code of caenorhabditis elegans using bacterial aminoacyl trna synthetase trna pairs
ACS Chemical Biology, 2012Co-Authors: Angela R Parrish, Zhouxin Shen, Steven P Briggs, Andrew Dillin, Zheng Xiang, Irene Coin, Lei WangAbstract:The genetic code specifies 20 common amino acids and is largely preserved in both single and multicellular organisms. Unnatural amino acids (Uaas) have been genetically incorporated into proteins by using engineered orthogonal tRNA/aminoacyl-tRNA synthetase (RS) pairs, enabling new research capabilities and precision inaccessible with common amino acids. We show here that Escherichia coli tyrosyl and leucyl amber suppressor tRNA/RS pairs can be evolved to incorporate different Uaas in response to the amber stop codon UAG into various proteins in Caenorhabditis elegans. To accurately report Uaa incorporation in worms, we found that it is crucial to integrate the UAG-containing reporter gene into the genome rather than to express it on an Extrachromosomal Array from which variable expression can lead to reporter activation independent of the amber-suppressing tRNA/RS. Synthesizing a Uaa in a dipeptide drives Uaa uptake and bioavailability. Uaa incorporation has dosage, temporal, tRNA copy, and temperature d...
