High cleavage efficiency of a 2A peptide derived from porcine teschovirus-1 in human cell lines, zebrafish and mice.
Jin Hee Kim,Sang Rok Lee,Li Hua Li,Li Hua Li,Hye Jeong Park,Hye Jeong Park,Jeong Hoh Park,Kwang Youl Lee,Myeong Kyu Kim,Boo Ahn Shin,Seok-Yong Choi +10 more
TLDR
Western blotting and confocal microscopic analyses revealed that among the four 2As, the one derived from porcine teschovirus-1 (P2A) has the highest cleavage efficiency in all the contexts examined.Abstract:
When expression of more than one gene is required in cells, bicistronic or multicistronic expression vectors have been used. Among various strategies employed to construct bicistronic or multicistronic vectors, an internal ribosomal entry site (IRES) has been widely used. Due to the large size and difference in expression levels between genes before and after IRES, however, a new strategy was required to replace IRES. A self-cleaving 2A peptide could be a good candidate to replace IRES because of its small size and high cleavage efficiency between genes upstream and downstream of the 2A peptide. Despite the advantages of the 2A peptides, its use is not widespread because (i) there are no publicly available cloning vectors harboring a 2A peptide gene and (ii) comprehensive comparison of cleavage efficiency among various 2A peptides reported to date has not been performed in different contexts. Here, we generated four expression plasmids each harboring different 2A peptides derived from the foot-and-mouth disease virus, equine rhinitis A virus, Thosea asigna virus and porcine teschovirus-1, respectively, and evaluated their cleavage efficiency in three commonly used human cell lines, zebrafish embryos and adult mice. Western blotting and confocal microscopic analyses revealed that among the four 2As, the one derived from porcine teschovirus-1 (P2A) has the highest cleavage efficiency in all the contexts examined. We anticipate that the 2A-harboring cloning vectors we generated and the highest efficiency of the P2A peptide we demonstrated would help biomedical researchers easily adopt the 2A technology when bicistronic or multicistronic expression is required.read more
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mScarlet: a bright monomeric red fluorescent protein for cellular imaging
Daphne S. Bindels,Lindsay Haarbosch,Laura van Weeren,Marten Postma,Katrin E. Wiese,Marieke Mastop,S. Aumonier,S. Aumonier,Guillaume Gotthard,Guillaume Gotthard,Antoine Royant,Antoine Royant,Mark A. Hink,Theodorus W. J. Gadella +13 more
TL;DR: The engineering of mScarlet is reported, a truly monomeric red fluorescent protein with record brightness, quantum yield, and fluorescence lifetime and it is especially useful as a Förster resonance energy transfer (FRET) acceptor in ratiometric imaging.
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High-throughput screening of a CRISPR/Cas9 library for functional genomics in human cells
TL;DR: The development of a focused CRISPR/Cas-based (clustered regularly interspaced short palindromic repeats/CRISPR-associated) lentiviral library in human cells and a method of gene identification based on functional screening and high-throughput sequencing analysis are reported.
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ATR Prohibits Replication Catastrophe by Preventing Global Exhaustion of RPA
Luis I. Toledo,Matthias Altmeyer,Maj-Britt Rask,Claudia Lukas,Dorthe Helena Larsen,Lou Klitgaard Povlsen,Simon Bekker-Jensen,Niels Mailand,Jiri Bartek,Jiri Lukas +9 more
TL;DR: ATR-mediated suppression of dormant origins shields active forks against irreversible breakage via preventing exhaustion of nuclear RPA, elucidates how replicating genomes avoid destabilizing DNA damage and provides a molecular rationale for their hypersensitivity to ATR inhibitors.
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Improving cytidine and adenine base editors by expression optimization and ancestral reconstruction.
Luke W. Koblan,Jordan L. Doman,Jordan L. Doman,Jordan L. Doman,Christine D. Wilson,Christine D. Wilson,Christine D. Wilson,Jonathan M. Levy,Jonathan M. Levy,Jonathan M. Levy,Tristan Tay,Tristan Tay,Tristan Tay,Gregory A. Newby,Gregory A. Newby,Gregory A. Newby,Juan Pablo Maianti,Juan Pablo Maianti,Juan Pablo Maianti,Aditya Raguram,Aditya Raguram,Aditya Raguram,David R. Liu,David R. Liu,David R. Liu +24 more
TL;DR: It is shown that expression levels are a bottleneck in base-editing efficiency, and cytidine and adenine base editors are optimized by modification of nuclear localization signals and codon usage, and ancestral reconstruction of the deaminase component.
Journal ArticleDOI
Long-term expanding human airway organoids for disease modeling
Norman Sachs,Angelos Papaspyropoulos,Domenique D. Zomer-van Ommen,Inha Heo,Lena Böttinger,Dymph Klay,Fleur Weeber,Guizela Huelsz-Prince,Nino Iakobachvili,Gimano D. Amatngalim,Joep de Ligt,Arne Van Hoeck,Natalie Proost,Marco C. Viveen,Anna Lyubimova,Luc Teeven,Sepideh Derakhshan,Jeroen Korving,Harry Begthel,Johanna F. Dekkers,Kuldeep Kumawat,Emilio Ramos,Matthijs F.M. van Oosterhout,G. Johan A. Offerhaus,Dominique J Wiener,Eduardo P. Olimpio,Krijn K. Dijkstra,Egbert F. Smit,Maarten van der Linden,Sridevi Jaksani,Marieke van de Ven,Jos Jonkers,Anne C. Rios,Emile E. Voest,Coline H.M. van Moorsel,Cornelis K. van der Ent,Edwin Cuppen,Alexander van Oudenaarden,Frank E. J. Coenjaerts,Linde Meyaard,Louis Bont,Peter J. Peters,Sander J. Tans,Jeroen S. van Zon,Sylvia F. Boj,Robert G.J. Vries,Jeffrey M. Beekman,Hans Clevers +47 more
TL;DR: It is concluded that human airway organoids represent versatile models for the in vitro study of hereditary, malignant, and infectious pulmonary disease.
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