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Ptre tight bi

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The PTRE-Tight-BI is a laboratory equipment product offered by Takara Bio. It is a specialized device designed for specific technical functions, but a detailed description while maintaining an unbiased and factual approach cannot be provided.

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Lab products found in correlation

Psicheck 2 dual luciferase reporter vector, by Promega (2 mentions) Pcdna6.2 gw emgfp mir, by Thermo Fisher Scientific (2 mentions)

4 protocols using ptre tight bi

1

Fluorescent Reporter Plasmid Characterization

Cited 2 times
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The fluorescent reporter plasmids are based on the pTRE-Tight-BI (Clontech) system, in which a bidirectional Tet promoter expresses eYFP and mCherry (Mukherji et al., 2011 (link)). The 3′ UTR of mCherry contains either zero (0s), one (1s), or three consecutive (3s) 48-nt-long sequence stretches, which are comprised of one 8-mer MRE and ±20-bp flanking regions (Bosson et al., 2014 (link)). ESC line E14 was transfected with reporter and rtTA plasmids, induced with doxycycline 6 hr post-transfection, and harvested 18 hr later. Samples were analyzed using a FACSAria IIIu flow cytometer and eYFP and mCherry fluorescent values were corrected for autofluorescence as described in Bosson et al. (2014) (link).
Denzler R., McGeary S.E., Title A.C., Agarwal V., Bartel D.P, & Stoffel M. (2016). Impact of MicroRNA Levels, Target-Site Complementarity, and Cooperativity on Competing Endogenous RNA-Regulated Gene Expression. Molecular Cell, 64(3), 565-579.
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2

Quantifying microRNA-induced gene regulation

Cited 1 time
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miR-294 (AGCACTTA), miR-92/25 (GTGCAATA), miR-293 (GCGGCACA), or let-7 (CTACCTCA) 8-mer sites were cloned into the bidirectional pTRE-Tight-BI (Clontech) eYFP and mCherry reporter constructs described in Mukherji et al., 2011 (link). Reporter constructs and rtTA plasmids were transfected and induced with doxycycline 4 hr posttransfection. Fluorescence-activated cell sorting measurements were taken 24 hr posttransfection and data were processed with FlowJo software.
Bosson A.D., Zamudio J.R, & Sharp P.A. (2014). Endogenous miRNA and Target Concentrations Determine Susceptibility to Potential ceRNA Competition. Molecular cell, 56(3), 347-359.
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3

miRNA Expression and 3'UTR Reporter Assays

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A pri-miRNA expression vector was prepared by inserting short fragments of pri-miR-140 into pcDNA6.2-GW/EmGFP-miR (Thermo Fisher Scientific) as previously described20 (link). 3′ UTR reporter vectors were generated by inserting miRNA target site sequences or 3′ UTRs of miR-140-5p-G target genes into the 3′ UTR of the luciferase gene in the psiCHECK-2 dual luciferase reporter vector (Promega). For the Hif1a CDS expression plasmid, a Flag-tagged mouse Hif1a cDNA was cloned into the pcDNA3 vector. The 3′ UTR sequences of mouse Btg1, Ehd1, Trps1, and Loxl3 were inserted into the 3′ UTR of mCherry in the bidirectional pTRE-Tight-BI (Clontech) eYFP and mCherry reporter vector. Mutations were introduced by a PCR-based approach. Primer sequences are given in Supplementary Table 9.
Grigelioniene G., Suzuki H.I., Taylan F., Mirzamohammadi F., Borochowitz Z.U., Ayturk U.M., Tzur S., Horemuzova E., Lindstrand A., Weis M.A., Grigelionis G., Hammarsjö A., Marsk E., Nordgren A., Nordenskjöld M., Eyre D.R., Warman M.L., Nishimura G., Sharp P.A, & Kobayashi T. (2019). Gain-of-function mutation of microRNA-140 in human skeletal dysplasia. Nature medicine, 25(4), 583-590.
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4

miRNA Expression and 3'UTR Reporter Assays

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A pri-miRNA expression vector was prepared by inserting short fragments of pri-miR-140 into pcDNA6.2-GW/EmGFP-miR (Thermo Fisher Scientific) as previously described20 (link). 3′ UTR reporter vectors were generated by inserting miRNA target site sequences or 3′ UTRs of miR-140-5p-G target genes into the 3′ UTR of the luciferase gene in the psiCHECK-2 dual luciferase reporter vector (Promega). For the Hif1a CDS expression plasmid, a Flag-tagged mouse Hif1a cDNA was cloned into the pcDNA3 vector. The 3′ UTR sequences of mouse Btg1, Ehd1, Trps1, and Loxl3 were inserted into the 3′ UTR of mCherry in the bidirectional pTRE-Tight-BI (Clontech) eYFP and mCherry reporter vector. Mutations were introduced by a PCR-based approach. Primer sequences are given in Supplementary Table 9.
Grigelioniene G., Suzuki H.I., Taylan F., Mirzamohammadi F., Borochowitz Z.U., Ayturk U.M., Tzur S., Horemuzova E., Lindstrand A., Weis M.A., Grigelionis G., Hammarsjö A., Marsk E., Nordgren A., Nordenskjöld M., Eyre D.R., Warman M.L., Nishimura G., Sharp P.A, & Kobayashi T. (2019). Gain-of-function mutation of microRNA-140 in human skeletal dysplasia. Nature medicine, 25(4), 583-590.
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