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
TLDR
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.Abstract:
Transgenesis is an important tool for assessing gene function. In zebrafish, transgenesis has suffered from three problems: the labor of building complex expression constructs using conventional subcloning; low transgenesis efficiency, leading to mosaicism in transient transgenics and infrequent germline incorporation; and difficulty in identifying germline integrations unless using a fluorescent marker transgene. The Tol2kit system uses site-specific recombination-based cloning (multisite Gateway technology) to allow quick, modular assembly of [promoter]-[coding sequence]-[3' tag] constructs in a Tol2 transposon backbone. It includes a destination vector with a cmlc2:EGFP (enhanced green fluorescent protein) transgenesis marker and a variety of widely useful entry clones, including hsp70 and beta-actin promoters; cytoplasmic, nuclear, and membrane-localized fluorescent proteins; and internal ribosome entry sequence-driven EGFP cassettes for bicistronic expression. 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.read more
Citations
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Evolution of vertebrate gill covers via shifts in an ancient Pou3f3 enhancer
Lindsey Barske,Lindsey Barske,Lindsey Barske,Peter Fabian,Christine Hirschberger,David Jandzik,David Jandzik,Tyler A. Square,Tyler A. Square,Pengfei Xu,Nellie Nelson,Haoze V Yu,Daniel Meulemans Medeiros,J. Andrew Gillis,J. Andrew Gillis,J. Gage Crump +15 more
TL;DR: The first essential gene for gill cover formation in modern vertebrates, Pou3f3, is identified and the genomic element that brought Pou2f3 expression into the pharynx more than 430 Mya is uncovered, and small changes in this deeply conserved sequence account for the single large gills cover in living bony fish versus the five separate covers of sharks and their brethren.
Journal ArticleDOI
Endocrine and local signaling interact to regulate spermatogenesis in zebrafish: follicle-stimulating hormone, retinoic acid and androgens
Diego Crespo,Luiz H.C. Assis,Henk J. G. van de Kant,Sjors de Waard,Diego Safian,Moline Severino Lemos,Jan Bogerd,Rüdiger W. Schulz +7 more
TL;DR: It is concluded that RA signaling is important for zebrafish spermatogenesis but is not of crucial relevance, and as Fsh stimulates androgen and RA production, germ cell-mediated, RA-dependent reduction of Leydig cell activity may form a hitherto unknown intratesticular negative-feedback loop.
Journal ArticleDOI
Transgenic zebrafish model of DUX4 misexpression reveals a developmental role in FSHD pathogenesis
Anna Pakula,Anna Pakula,Anna Pakula,Angela Lek,Jeffrey J. Widrick,Jeffrey J. Widrick,Hiroaki Mitsuhashi,Hiroaki Mitsuhashi,Hiroaki Mitsuhashi,Katlynn Bugda Gwilt,Katlynn Bugda Gwilt,Vandana Gupta,Vandana Gupta,Fedik Rahimov,Fedik Rahimov,June Criscione,June Criscione,Yuanfan Zhang,Yuanfan Zhang,Yuanfan Zhang,Devin E. Gibbs,Devin E. Gibbs,Quinn Murphy,Quinn Murphy,Anusha Manglik,Anusha Manglik,Lillian Mead,Lillian Mead,Louis M. Kunkel,Louis M. Kunkel,Louis M. Kunkel +30 more
TL;DR: It is shown that an induced burst of Dux4 expression during early development results in the onset of FSHD-like phenotypes in adulthood, even when DUX4 is no longer detectable.
Journal ArticleDOI
The Prx1 limb enhancers: Targeted gene expression in developing zebrafish pectoral fins
TL;DR: Zebrafish lines that express enhanced green fluorescent protein (EGFP) driven by the mouse Prx1 enhancer in developing pectoral fins are reported and it is shown that the mouse and zebrafish regulatory elements may be used to modify gene function in pECToral fins.
Journal ArticleDOI
Metformin rescues muscle function in BAG3 myofibrillar myopathy models.
Avnika A. Ruparelia,Emily A. McKaige,Caitlin Williams,Keith E. Schulze,Margit Fuchs,Viola Oorschot,Emmanuelle Lacène,Mirella Meregalli,Clara Yun Qi Lee,Rita J. Serrano,Emily C. Baxter,Keyne Monro,Yvan Torrente,Georg Ramm,Tanya Stojkovic,Josée N. Lavoie,Robert J. Bryson-Richardson +16 more
TL;DR: It is demonstrated metformin is not only able to bring about the removal of protein aggregates in zebrafish and human myoblasts but is also able to rescue the fiber disintegration and swimming deficit observed in the bag3 -/- fish, providing a promising therapy for BAG3 myopathy.
References
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TL;DR: A series of stages for development of the embryo of the zebrafish, Danio (Brachydanio) rerio is described, providing for flexibility and continued evolution of the staging series as the authors learn more about development in this species.
Journal ArticleDOI
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An Hsp70-like protein in the ER: identity with the 78 kd glucose-regulated protein and immunoglobulin heavy chain binding protein.
Sean Munro,Hugh R.B. Pelham +1 more
TL;DR: A cDNA clone is characterized that encodes a protein related to the 70 kd heat shock protein, but is expressed in normal rat liver, and it is identical with two previously described proteins: GRP78, whose synthesis is induced by glucose starvation, and BiP, which is found bound to immunoglobulin heavy chains in pre-B cells.
Journal ArticleDOI
GAL4-VP16 is an unusually potent transcriptional activator
TL;DR: It is shown that the hybrid protein (GAL4-VP16) activates transcription unusually efficiently in mammalian cells when bound close to, or at large distances from the gene, and suggested that the activating region of VP16 may be near-maximally potent.
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.