The pTETRIS-Cargo vector was created from components of a cumate-inducible piggyBAC transposon vector (System Biosciences), pGl4.10-Luciferase (Promega), and pTRE-Tight (Clontech). Briefly, a 567bp fragment containing a minimal mouse PGK promoter was cloned into a SacI site in pGl4.10-Luciferase to generate pGI4-PGK-Luc-pA. The reverse complement of PGK-Luc-pA was cloned into a vector containing the bovine growth hormone polyA site. The entire bGHpa-[reversePGK-Luc-pA] was cloned into NotI and SalI sites of the piggyBAC vector (System Biosciences). The cumate-inducible promoter in the piggyBAC vector was then replaced with the Tetracycline Responsive Element (TRE) from pTRE-Tight (Clontech) via Gibson assembly to generate pTETRIS-Cargo in Fig. 4A , in which the lncRNA, the luciferase gene, and a gene encoding puromycin resistance are all flanked by chicken HS4 insulator elements, and inverted terminal repeats (ITRs) recognized by the piggyBAC transposase. The rtTA-cargo vector from Fig. 4A was generated by cloning the hUbiC-rtTA3-IRES-Neo cassette from pSLIK-Neo (Addgene Plasmid #25735) into SfiI and SalI sites in a piggyBAC transposon vector (System Biosciences). The piggyBAC transposase from System Biosciences was cloned into SmaI and HindIII sites into pUC19 (NEB) to allow propagation of the transposase on ampicillin plates.
Pgl4.10 luciferase
Manufactured by Promega
Sourced in United States
PGl4.10-Luciferase is a reporter gene that produces the luciferase enzyme from the firefly. This enzyme catalyzes a bioluminescent reaction, emitting light that can be detected and quantified. The PGl4.10 promoter drives the expression of the luciferase gene.
Automatically generated - may contain errors
Lab products found in correlation
Piggybac vector, by System Biosciences (4 mentions)
Ptre tight, by Takara Bio (2 mentions)
Piggybac transposase, by System Biosciences (2 mentions)
Piggybac transposon vector, by System Biosciences (2 mentions)
Puc19, by New England Biolabs (2 mentions)
Pgl4.74 hrluc, by Promega (1 mentions)
Dual luciferase reporter assay system, by Promega (1 mentions)
Fugene 6, by Promega (1 mentions)
E1910, by Promega (1 mentions)
4 protocols using pgl4.10 luciferase
1
Modular Inducible Genetic Cargo System
2
Luciferase Reporter Assay in 293 Cells
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A total of 50 ng of each (CMV, CMV random (CGG)4, or CMV (CGG)4) pGL4.10 luciferase (Promega, Madison, WI, USA, Cat. No. E6651) was transfected along with 50 ng of pGL4.74 hRluc (Promega, Madison, WI, USA, Cat. No. E6921) using FuGENE 6 (Promega, Madison, WI, USA, Cat. No. E2691) in 293 cells using the manufacturer’s instructions. Firefly and renilla luciferase activity were taken 48 h later by using the Dual-Luciferase® Reporter Assay System (Promega, Madison, WI, USA, Cat. No. E1910) and a SpectraMax iD5 plate reader.
3
Modular Inducible Genetic Cargo System
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The pTETRIS-Cargo vector was created from components of a cumate-inducible piggyBAC transposon vector (System Biosciences), pGl4.10-Luciferase (Promega), and pTRE-Tight (Clontech). Briefly, a 567bp fragment containing a minimal mouse PGK promoter was cloned into a SacI site in pGl4.10-Luciferase to generate pGI4-PGK-Luc-pA. The reverse complement of PGK-Luc-pA was cloned into a vector containing the bovine growth hormone polyA site. The entire bGHpa-[reversePGK-Luc-pA] was cloned into NotI and SalI sites of the piggyBAC vector (System Biosciences). The cumate-inducible promoter in the piggyBAC vector was then replaced with the Tetracycline Responsive Element (TRE) from pTRE-Tight (Clontech) via Gibson assembly to generate pTETRIS-Cargo in Fig. 4A , in which the lncRNA, the luciferase gene, and a gene encoding puromycin resistance are all flanked by chicken HS4 insulator elements, and inverted terminal repeats (ITRs) recognized by the piggyBAC transposase. The rtTA-cargo vector from Fig. 4A was generated by cloning the hUbiC-rtTA3-IRES-Neo cassette from pSLIK-Neo (Addgene Plasmid #25735) into SfiI and SalI sites in a piggyBAC transposon vector (System Biosciences). The piggyBAC transposase from System Biosciences was cloned into SmaI and HindIII sites into pUC19 (NEB) to allow propagation of the transposase on ampicillin plates.
4
Luciferase Assay for NSD2 and DGCR8 Promoters
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The promoter sequences of NSD2 and DGCR8 were inserted in the pGL4.10 luciferase vector (E6651, Promega Corporation, Madison, WI, USA) to construct corresponding luciferase reporter vectors. The constructed vectors were transfected into SCC-9 and CAL27 cells along with sh-NRIP1, sh-NSD2, or sh-NC. After 48 h, the luciferase activity in cells was analyzed according to the protocol of the dual luciferase reporter system (E1910, Promega).
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