Turborfp

Manufactured by Evrogen

TurboRFP is a fluorescent protein derived from the red fluorescent protein found in sea anemones. It exhibits high brightness and photostability, making it suitable for various fluorescence-based applications.

Automatically generated - may contain errors

Lab products found in correlation

Pcr topo 2, by Thermo Fisher Scientific (1 mentions) Phusion polymerase, by Thermo Fisher Scientific (1 mentions) Nucleobond bac 100, by Macherey-Nagel (1 mentions) Turbogfp, by Evrogen (1 mentions) Gibson assembly kit, by New England Biolabs (1 mentions)

5 protocols using turborfp

DNA Bait Amplification and Donor Plasmid Generation

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Genomic DNA from the wild type strain AB was used to amplify bait sequences by PCR (Phusion Polymerase, Thermo Fischer) using primers listed in the Supplementary Table S2. To generate donor plasmids, baits were cloned into a pCS2+ or Topo PCR II vectors (Invitrogen) containing the coding sequence for the Venus fluorescent protein or turboRFP (promoter-less tRFP plasmid, Evrogen). The CMV promoter was later removed from the pCS2+ vector. All constructs were verified by sequencing.

Kesavan G., Chekuru A., Machate A, & Brand M. (2017). CRISPR/Cas9-Mediated Zebrafish Knock-in as a Novel Strategy to Study Midbrain-Hindbrain Boundary Development. Frontiers in Neuroanatomy, 11, 52.

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Generation of Mx2 Knockout Reporter Mouse

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The BAC clone RP24-71I6 containing the murine Mx2 locus was obtained from BACPAC resource center. Homologs recombination was performed using the bacteriophage λ recombination system (23 (link)). Thereby, the open reading frame of the murine Mx2 gene was replaced by a linear fragment containing the amplified reporter TurboRFP (Evrogen) followed by an SV40 polyadenylation signal and an FRT (FLP recognition target) flanked cassette harboring a prokaryotic promoter, the PGK-promoter, a gene encoding for kanamycin/neomycin phosphotransferase and the bovine growth hormone polyadenylation signal. Primers used: Mx2Phom+Fluc2: 5′-TTA TAA TAT TCA TTT CCC ACA GAG TAC CCA ACT GAG AGA AGA AAT AAA AGA TGG AAG ATG CCA AAA ACA TTA AGA-3′ and Mx2Exon14hom+BamHI: 5′-AAA GAA AAG TGG TTT ATT AAG GAA TGC AAC AGG CAG CTC CCA TTT GTA CAC TCA AGG GCA TCG GTC GAC GGA TCC-3′. Modified BAC DNA was isolated using NucleoBond BAC100 (Macherey-Nagel).

Bhushal S., Wolfsmüller M., Selvakumar T.A., Kemper L., Wirth D., Hornef M.W., Hauser H, & Köster M. (2017). Cell Polarization and Epigenetic Status Shape the Heterogeneous Response to Type III Interferons in Intestinal Epithelial Cells. Frontiers in Immunology, 8, 671.

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Transgenic Mouse Models for Genetic Studies

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Genetically engineered mice used for this study were as follows: Pdx1^tTA/+ [42 (link)], tetO-HIST1H2BJ/GFP (tetO-H2B-GFP) [43 (link)], Hnf1bCreER [3 (link)], Gt(ROSA)26Sortm4(ACTB-tdTomato,-EGFP)Luo/J (mT/mG) [44 (link)], Neurog3-EYFP [29 (link)], and Neurog3-RFP (S3 Fig.). For embryonic stage, noon of the day when vaginal plug appeared was referred as E0.5.
The Neurog3-RFP transgenic construct (S3A Fig.) was generated by fusing 7.6 kb of the Neurog3-promoter [2 (link)] with a reporter construct composed of a chimeric intron; turbo RFP (Evrogen); a nuclear localization signal (NLS); a Myc-tagC; a bovine growth hormone polyadenylation signal (bGH-PolyA). Transgenic mouse lines were obtained by pronuclear injection of the construct (Transgenic Core Facility, EPFL, Switzerland). Two different lines were obtained initially, exhibiting similar levels of RFP signal detectable by a wide-field fluorescent microscope, and one of the lines was used for this study. All animals were handled humanely according to the authorized protocols of Switzerland and Denmark.

Kim Y.H., Larsen H.L., Rué P., Lemaire L.A., Ferrer J, & Grapin-Botton A. (2015). Cell Cycle–Dependent Differentiation Dynamics Balances Growth and Endocrine Differentiation in the Pancreas. PLoS Biology, 13(3), e1002111.

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Specific Knockdown of H3F3A Mutant

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Initial attempts to knockdown K27M mutant H3F3A without also targeting the wild type allele were unsuccessful. Therefore, we designed shRNAs highly specific for either H3F3A or H3F3B, the latter serving as a control for the knockdown of wild type H3.3. Sequences for specific shRNAs were cloned into the pGIPZ vector, which utilizes the backbone of miR30 for improved knockdown efficiency. We also adapted the pGIPZ vector to replace the miR30 backbone a miR-E backbone [14 (link)], and exchanged turboGFP with turboRFP (Evrogen). Specific shRNA sequences were either custom designed or obtained from the pGIPZ library of constructs, and adapted for cloning into the vector using the shRNA Retriever online tool. shRNA sequences are provided in [Supp. Table 1 (Online Resource 1)]. H3F3A specific shRNAs are referred to as sh^K27M when used in K27M mutant xenografts and sh^H3F3A when used in the H3 wild type xenograft. The notation sh^K27M refers to both H3F3A hairpins sh^K27M#1 and sh^K27M#2.

Silveira A.B., Kasper L.H., Fan Y., Jin H., Wu G., Shaw T.I., Zhu X., Larson J.D., Easton J., Shao Y., Yergeau D.A., Rosencrance C., Boggs K., Rusch M.C., Ding L., Zhang J., Finkelstein D., Noyes R.M., Russell B.L., Xu B., Broniscer A., Wetmore C., Pounds S.B., Ellison D.W., Zhang J, & Baker S.J. (2019). H3.3 K27M Depletion Increases Differentiation and Extends Latency of Diffuse Intrinsic Pontine Glioma Growth In Vivo. Acta neuropathologica, 137(4), 637-655.

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Engineering Transcriptional Reporters in Pristionchus pacificus

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We selected the genes Ppa-stdh-1 and Ppa-acs-19.1 to generate transcriptional reporters and established transgenic lines necessary for their use as dietary sensors. For Ppa-stdh-1, a 2.3 kb interval encompassing the upstream region and the first two exons was amplified. For Ppa-acs-19.1, a 1.4 kb region upstream of the first predicted exon was amplified. These promoters were fused to TurboRFP (Evrogen), together with the 3′ UTR sequence of the gene Ppa-rpl-23 using the primers listed in Supplementary Table 1.
PCR fragments were assembled using Gibson assembly kit (NEB) and verified by Sanger sequencing. The Ppa-stdh-1::RFP and Ppa-acs-19.1::RFP constructs were amplified with the addition of restriction sites (XmaI and PstI) for subsequent digestion. To form stable lines via the formation of complex arrays, the expression construct Ppa-stdh-1::RFP was digested with PstI and 5 ng/μl of this, co-injected into the germlines of young adult P. pacificus worms with the marker Ppa-egl-20::Venus (10 ng/μl), and genomic carrier DNA (60 ng/μl), also digested with PstI [32 (link)]. For the Ppa-acs-19.1::RFP construct, 10 ng/μl of the construct cut with PstI, was injected with the marker Ppa-egl-20::RFP (10 ng/μl), and genomic carrier DNA (60 ng/μl) also cut with PstI. At least two independent lines were obtained from microinjections for both transgenes.

Akduman N., Lightfoot J.W., Röseler W., Witte H., Lo W.S., Rödelsperger C, & Sommer R.J. (2020). Bacterial vitamin B12 production enhances nematode predatory behavior. The ISME Journal, 14(6), 1494-1507.

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