Gateway compatible vectors for analysis of gene function in the zebrafish.
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TLDR
This work describes the construction and validation of Gateway compatible vectors based on commonly used zebrafish plasmids and demonstrates the use of this system to rapidly identify tissue‐specific cis elements to aid the establishment of blood vessel‐specific transgenic constructs.Abstract:
The recent establishment of recombination-based cloning systems has greatly facilitated the analysis of gene function by allowing rapid and high-efficiency generation of plasmid constructs. However, the use of such an approach in zebrafish requires the availability of recombination-compatible plasmids that are appropriate for functional studies in zebrafish embryos. In this work, we describe the construction and validation of Gateway compatible vectors based on commonly used zebrafish plasmids. We have generated pCS-based plasmids that allow rapid generation of both N-terminal and C-terminal fusion proteins, and we demonstrate that mRNA synthesized from these plasmids encodes functional native or fusion proteins in injected zebrafish embryos. In parallel, we have established similar Gateway plasmids containing Tol2 cis elements that promote efficient integration into the zebrafish genome and allow expression of native or fusion proteins in a tissue-specific manner in the zebrafish embryo. Finally, we demonstrate the use of this system to rapidly identify tissue-specific cis elements to aid the establishment of blood vessel-specific transgenic constructs. Taken together, this work provides an important platform for the rapid functional analyses of open reading frames in zebrafish embryos.read more
Citations
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The Tol2kit: a multisite gateway-based construction kit for Tol2 transposon transgenesis constructs.
Kristen M. Kwan,Esther Fujimoto,Clemens Grabher,Benjamin D. Mangum,Melissa Hardy,Douglas Simon Campbell,John M. Parant,H. Joseph Yost,John P. Kanki,Chi Bin Chien +9 more
TL;DR: The Tol2kit greatly facilitates zebrafish transgenesis, simplifies the sharing of clones, and enables large‐scale projects testing the functions of libraries of regulatory or coding sequences.
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Role of PFKFB3-Driven Glycolysis in Vessel Sprouting
Katrien De Bock,Maria Georgiadou,Sandra Schoors,Anna Kuchnio,Brian W. Wong,Anna Rita Cantelmo,Annelies Quaegebeur,Bart Ghesquière,Sandra Cauwenberghs,Guy Eelen,Li-Kun Phng,Inge Betz,Bieke Tembuyser,Katleen Brepoels,Jonathan Welti,Ilse Geudens,Inmaculada Segura,Bert Cruys,Franscesco Bifari,Ilaria Decimo,Raquel Blanco,Sabine Wyns,Jeroen Vangindertael,Susana Rocha,Russel T Collins,Sebastian Munck,Dirk Daelemans,Hiromi Imamura,Roland Devlieger,Mark H. Rider,Paul P. Van Veldhoven,Frans Schuit,Ramon Bartrons,Johan Hofkens,Peter Fraisl,Sucheta Telang,Ralph J. DeBerardinis,Luc Schoonjans,Stefan Vinckier,Jason Chesney,Holger Gerhardt,Holger Gerhardt,Mieke Dewerchin,Peter Carmeliet +43 more
TL;DR: It is shown that ECs relied on glycolysis rather than on oxidative phosphorylation for ATP production and that loss of the gly colytic activator PFKFB3 in ECs impaired vessel formation.
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Guided self-organization and cortical plate formation in human brain organoids
Madeline A. Lancaster,Madeline A. Lancaster,Nina S. Corsini,Simone Wolfinger,E. Hilary Gustafson,Alex W Phillips,Thomas R Burkard,Tomoki Otani,Frederick J. Livesey,Juergen A. Knoblich +9 more
TL;DR: Microfilament-engineered cerebral organoids (enCORs) model the distinctive radial organization of the cerebral cortex and allow for the study of neuronal migration and demonstrate that combining 3D cell culture with bioengineering can increase reproducibility and improve tissue architecture.
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MicroRNA-mediated integration of haemodynamics and Vegf signalling during angiogenesis
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Consensus guidelines for the use and interpretation of angiogenesis assays
Patrycja Nowak-Sliwinska,Kari Alitalo,Elizabeth Allen,Andrey Anisimov,Alfred C. Aplin,Robert Auerbach,Hellmut G. Augustin,Hellmut G. Augustin,David O. Bates,Judy R. van Beijnum,R. Hugh F. Bender,Gabriele Bergers,Gabriele Bergers,Andreas Bikfalvi,Joyce Bischoff,Barbara C. Böck,Barbara C. Böck,Peter C. Brooks,Federico Bussolino,Bertan Cakir,Peter Carmeliet,Daniel Castranova,Anca Maria Cimpean,Ondine Cleaver,George Coukos,George E. Davis,Michele De Palma,Anna Dimberg,Ruud P.M. Dings,Valentin Djonov,Andrew C. Dudley,Neil Dufton,Sarah-Maria Fendt,Napoleone Ferrara,Marcus Fruttiger,Dai Fukumura,Bart Ghesquière,Bart Ghesquière,Yan Gong,Robert J. Griffin,Adrian L. Harris,Christopher C.W. Hughes,Nan W. Hultgren,M. Luisa Iruela-Arispe,Melita Irving,Rakesh K. Jain,Raghu Kalluri,Joanna Kalucka,Robert S. Kerbel,Jan Kitajewski,Ingeborg Klaassen,Hynda K. Kleinmann,Pieter Koolwijk,Elisabeth Kuczynski,Brenda R. Kwak,Koen Marien,Juan M. Melero-Martin,Lance L. Munn,Roberto F. Nicosia,Agnès Noël,Jussi Nurro,Anna-Karin Olsson,Tatiana V. Petrova,Kristian Pietras,Roberto Pili,Jeffrey W. Pollard,Mark J. Post,Paul H.A. Quax,Gabriel A. Rabinovich,Marius Raica,Anna M. Randi,Domenico Ribatti,Curzio Rüegg,Reinier O. Schlingemann,Reinier O. Schlingemann,Stefan Schulte-Merker,Lois E.H. Smith,Jonathan W. Song,Steven A. Stacker,Jimmy Stalin,Amber N. Stratman,Maureen Van de Velde,Victor W.M. van Hinsbergh,Peter B. Vermeulen,Johannes Waltenberger,Brant M. Weinstein,Hong Xin,Bahar Yetkin-Arik,Seppo Ylä-Herttuala,Mervin C. Yoder,Arjan W. Griffioen +90 more
TL;DR: In vivo, ex vivo, and in vitro bioassays that are available for the evaluation of angiogenesis are described and critical aspects that are relevant for their execution and proper interpretation are highlighted.
References
More filters
Journal ArticleDOI
Improved monomeric red, orange and yellow fluorescent proteins derived from Discosoma sp. red fluorescent protein.
Nathan C. Shaner,Robert E. Campbell,Robert E. Campbell,Paul Steinbach,Ben N G Giepmans,Amy E. Palmer,Roger Y. Tsien +6 more
TL;DR: The latest red version matures more completely, is more tolerant of N-terminal fusions and is over tenfold more photostable than mRFP1, and three monomers with distinguishable hues from yellow-orange to red-orange have higher quantum efficiencies.
Journal ArticleDOI
Effective targeted gene ‘knockdown’ in zebrafish
TL;DR: It is shown here that antisense, morpholino-modified oligonucleotides (morpholinos) are effective and specific translational inhibitors in zebrafish, and conserved vertebrate processes and diseases are now amenable to a systematic, in vivo, reverse-genetic paradigm using zebra fish embryos.
Journal ArticleDOI
In vivo imaging of embryonic vascular development using transgenic zebrafish.
TL;DR: It is found that the zebrafish fli1 promoter is able to drive expression of enhanced green fluorescent protein (EGFP) in all blood vessels throughout embryogenesis, and these transgenic lines allow detailed analysis of both wild type and mutant embryonic vasculature.
Journal ArticleDOI
The Tol2kit: a multisite gateway-based construction kit for Tol2 transposon transgenesis constructs.
Kristen M. Kwan,Esther Fujimoto,Clemens Grabher,Benjamin D. Mangum,Melissa Hardy,Douglas Simon Campbell,John M. Parant,H. Joseph Yost,John P. Kanki,Chi Bin Chien +9 more
TL;DR: The Tol2kit greatly facilitates zebrafish transgenesis, simplifies the sharing of clones, and enables large‐scale projects testing the functions of libraries of regulatory or coding sequences.
Journal ArticleDOI
DNA Cloning Using In Vitro Site-Specific Recombination
TL;DR: A method called recombinational cloning is described that uses in vitro site-specific recombination to accomplish the directional cloning of PCR products and the subsequent automatic subcloning of the DNA segment into new vector backbones at high efficiency.