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Tdtomato n1

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TdTomato-N1 is a plasmid vector that expresses the tandem dimer Tomato (tdTomato) fluorescent protein. The tdTomato protein is a variant of the Discosoma sp. red fluorescent protein that has been engineered for improved brightness and photostability. The TdTomato-N1 plasmid can be used for fluorescent labeling and visualization of proteins in living cells.

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Lab products found in correlation

Tdtomato c1, by Addgene (1 mentions) Nebuilder hifi dna assembly kit, by New England Biolabs (1 mentions) Gblock, by Integrated DNA Technologies (1 mentions) Phenol red, by Thermo Fisher Scientific (1 mentions) Fetal calf serum (fcs), by Thermo Fisher Scientific (1 mentions) Neurobasal a, by Thermo Fisher Scientific (1 mentions) Primocin, by InvivoGen (1 mentions)

3 protocols using tdtomato n1

1

Stable Tau Overexpression in HEK293T and SH-SY5Y Cells

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Plasmids used for tdTomato-Tau overexpression in HEK293T cells and the tdTomato-C1 control plasmid were a gift from Michael Davidson: tdTomato-MAPTau-C-10 (RRID: Addgene_58112), tdTomato-MAPTau-N-10 (RRID:Addgene_58113) and tdTomato-C1 (RRID:Addgene_54653). Control tdTomato-N1 plasmid was a gift from Michael Davidson, Nathan Shaner and Roger Tsien (RRID:Addgene_54642) [129 (link)]. For Tau overexpression in SH-SY5Y cells, the cDNA sequence of either the 441 amino acids-long human 2N4R Tau isoform or that of the 758 amino acids-long human Big Tau isoform, respectively, was cloned into the pSMPUW-IRES-Bsd vector backbone (Cell Biolabs, cat. no. VPK-219). The empty vector was used as a control. Lentiviral particles generated using these plasmids were used to create SH-SY5Y cell lines that stably overexpress Tau, as well as corresponding control cell lines.
Ellis M.J., Lekka C., Holden K.L., Tulmin H., Seedat F., O’Brien D.P., Dhayal S., Zeissler M.L., Knudsen J.G., Kessler B.M., Morgan N.G., Todd J.A., Richardson S.J, & Stefana M.I. (2024). Identification of high-performing antibodies for the reliable detection of Tau proteoforms by Western blotting and immunohistochemistry. Acta Neuropathologica, 147(1), 87.
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2

Fluorescent Protein Expression Constructs

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Protein sequences for each of the following fluorescent proteins were obtained from their respective publications, and DNA sequences were synthesized with optimized codon usage for murine and human cells (gBLOCKs; Integrated DNA Technologies, Coralville, Iowa): Venus, mIFP, LssmOrange, TagRFP657, mOrange2, mApple, Sapphire, mTagBFP2 and mCerulean3. Notably, while the encoded proteins are the same as the published protein sequence, the DNA sequence encoding them is different than the published sequence. Overhangs were designed to flank the fluorescent proteins, 43 bp on the 5’ end and 37 bp on the 3’ end, in order to insert the sequences into the MSCV Puro using the NEBuilder HiFi DNA Assembly Kit (New England Biolabs Ipswich, MA). All MSCVpuro constructs have been submitted to AddGene (AG; Cambridge, MA) and are available for distribution [Venus (AG #96940), mIFP (AG #96951), LssmOrange (AG #96937), TagRFP657 (AG #96939), mOrange2 (AG #96938), mApple (AG #96934), Sapphire (AG #96950), mTagBFP2 (AG #96935), tdTomato (AG #97079), and mCerulean3 (AG #96936)]. NEB 5-alpha Competent E. coli were transformed, and DNA was prepped (Qiagen Hilden, Germany) and sequence verified. EYFP (pMSCV-IRES-YFP II) was a generous gift from Mohammed Azam (CCHMC). EGFP (MIGR1; AddGene #27490), tdTomato-N1 (Addgene #54642) and pMSCV-mCherry (AddGene #52114) were obtained from AddGene.
Kleeman B., Olsson A., Newkold T., Kofron M., DeLay M., Hildeman D, & Grimes H.L. (2018). A Guide to Choosing Fluorescent Protein Combinations for Flow Cytometric Analysis Based on Spectral Overlap. Cytometry. Part A : the journal of the International Society for Analytical Cytology, 93(5), 556-562.
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3

Neuron-Astrocyte Co-Culture Transfection

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Neurons and astrocytes were derived from cortices of wild-type Sprague Dawley rats at 0–1 days postnatal. Neurons were grown on a confluent monolayer of astrocytes at 5% CO2. Astrocytes were plated on 18 mm coverslips made of German glass (Carolina Scientific) that were acid cleaned then coated with a mixture of collagen and poly-D-lysine and grown in MEM without phenol red (Gibco), supplemented with N2 (Gibco), 10% fetal calf serum (Gibco or HyClone), 20% glucose, glutamax (Gibco), and Primocin (Invivogen). When astrocytes formed a confluent monolayer, neurons were plated on top of the astrocytes. Co-cultures were maintained in neuronal medium: Neurobasal-A (Gibco) with B27 (Gibco) and without antibiotics or phenol red, as previously described (Bury and Sabo, 2011 (link), 2014 (link); Sceniak et al., 2012 (link)). Half of the medium was replaced with fresh, equilibrated neuronal medium every 3–5 days.
Neurons were co-transfected with pEGFP-GluN2B constructs (2.6 μg DNA/coverslip) along with tdTomato (0.4 μg DNA/coverslip) at 2 DIV using the calcium phosphate method (Berry et al., 2012 (link); Sceniak et al., 2019 (link)). pEGFP-GluN2B constructs were previously described (Sceniak et al., 2019 (link)). EGFP is inserted in the amino-terminal extracellular domain after the signal peptide. tdTomato-N1 was Addgene plasmid 54642.
Bahry J.A., Fedder-Semmes K.N., Sceniak M.P, & Sabo S.L. (2021). An Autism-Associated de novo Mutation in GluN2B Destabilizes Growing Dendrites by Promoting Retraction and Pruning. Frontiers in Cellular Neuroscience, 15, 692232.
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