Pgl4.21 luc2p puro vector

Manufactured by Promega

Sourced in United States

The PGL4.21[luc2P/puro] vector is a plasmid designed for gene expression studies. It contains the luc2P luciferase reporter gene and a puromycin resistance marker. This vector provides a tool for monitoring and selecting cells expressing the gene of interest.

Automatically generated - may contain errors

Lab products found in correlation

Primestar gxl dna polymerase, by Takara Bio (2 mentions) One glo luciferase assay system, by Promega (1 mentions) Dual glo luciferase assay system, by Promega (1 mentions) Puromycin, by Merck Group (1 mentions) Jetprime reagent, by Polyplus Transfection (1 mentions) Luciferase assay system, by Promega (1 mentions)

Spelling variants of this product

PGL4 vector (65 citations) PGL4.21[luc2P/Puro] (3 citations) PGL4.21 vector (3 citations)

5 protocols using pgl4.21 luc2p puro vector

Wnt-Regulated Transcription Assay

Check if the same lab product or an alternative is used in the 5 most similar protocols

The pGL4.21 [luc2P/Puro] vector was purchased from Promega (Madison, WI, USA). The M50 Super 8× TOPflash and M51 Super 8× FOPFlash vectors were obtained from the Randall T. Moon laboratory. The sequences of the TCF/LEF binding sites in the M50 Super 8× TOPflash and M51 Super 8× FOPFlash vectors were cloned into the pGL4.21 vector. The pGL4.21-TOPflash and pGL4.21-FOPflash vectors were generated and used in this study.
To establish the Huh6/M50 stable cell lines, Huh6 cells were transfected with the pGL4.21-TOPflash vector and incubated for 2 days. The transfected cells were selected with 0.5 mg/mL puromycin (Sigma-Aldrich) until colonies formed. A ONE-Glo Luciferase Assay System (Promega, Madison, WI, USA) was used to select the cell colonies with high luciferase activity.
To assess the effect of shRNA and DsiRNA on Wnt-regulated transcription, cells were cotransfected with the pGL4.21 vector containing the TOPflash or FOPflash sequence and the indicated shRNA or DsiRNA. The experiments required transfection of the pRL-TK Renilla luciferase vector as the control for the transfection efficiency. The firefly and Renilla luciferase activities were measured by a Dual-Glo Luciferase Assay System (Promega). The relative activity of TOPflash or FOPflash was normalized to the Renilla luciferase activity and indicated the fold change in Wnt/β-catenin pathway activation.

Wu J.Y., Shih Y.L., Lin S.P., Hsieh T.Y, & Lin Y.W. (2019). YC-1 Antagonizes Wnt/β-Catenin Signaling Through the EBP1 p42 Isoform in Hepatocellular Carcinoma. Cancers, 11(5), 661.

+ Open protocol

+ Expand

Hypoxia-Responsive Luc Reporter Cell Line

Cited 1 time

Check if the same lab product or an alternative is used in the 5 most similar protocols

Detailed methods and validation of the system were previously reported [25] (link). Briefly, to construct the plasmid encoding the luc gene fused to ODD under the regulation of a five–tandem repeat of HRE sequence (5xHRE) with a puromycin resistance cassette, the 5xHRE-ODD-luc (firefly luciferase fused with ODD) sequence from a 5HREp-ODD-luc plasmid (kindly provided by Dr. H. Harada) was digested with KpnI and XbaI and subcloned between KpnI and EcoRI of a pGL4.21[luc2P/Puro] vector (Promega, Madison, WI). Human PCa PC-3 cells expressing EGFP variant pd2EGFP under the promotion of a 5xHRE sequence (PC3-HRE-EGFP) were transfected with the 5xHRE-ODD-luc construct (PC3-HRE-ODD-luc and PC3-HRE-EGFP/HRE-ODD-luc) using jetPRIME reagent (Polyplus transfection) following the manufacturer's protocol. Transfected cells were selected using 1 μg/ml of puromycin. PC3-HRE-EGFP/HRE-ODD-luc cells constitutively expressing the bright red fluorescent protein tdTomato (PC3-HRE-EGFP/HRE-ODD-luc/tdTomato) were generated by lentiviral infection with the pRRL-Δluc-tdtomato plasmid [25] (link).

Bharti S.K., Kakkad S., Danhier P., Wildes F., Penet M.F., Krishnamachary B, & Bhujwalla Z.M. (2019). Hypoxia Patterns in Primary and Metastatic Prostate Cancer Environments. Neoplasia (New York, N.Y.), 21(2), 239-246.

+ Open protocol

+ Expand

Construction of Cytokine Promoter Reporter Plasmids

Check if the same lab product or an alternative is used in the 5 most similar protocols

The reporter plasmids carrying the firefly luciferase cDNA driven by a human TNFα, IL6, and IL33 gene promoters were constructed as follows. The 5′-flanking region of human TNFα, IL6, and IL33 genes were the amplified forms of genomic DNAs derived from human HEK293 cells with polymerase chain reaction (PCR) using PrimeSTAR GXL DNA polymerase (TaKaRa BIO, Shiga, Japan) and specific primers as described in Table 1. The amplified DNA fragments were digested with KpnI and XhoI and ligated into the complementary sites of pGL4.21[luc2P/puro] vector (Promega, Madison, WI, USA).Table 1

Primer sequences for the reporter plasmids of cytokine genes.

Table 1

Gene	Position	Orientation	Sequence
TNFα	nt −1335 to +79	Sense	5′-CGCGGTACCGCTGTCTGCTTGTGTGTGTG-3′
		Antisense	5′-CGCCTCGAGTGTCCTTTCCAGGGGAGAGAGA-3′
IL6	nt −800 to +105	Sense	5′-CGCGGTACCCAGTGAAACAGTGGTGAAGA-3′
		Antisense	5′-CGCCTCGAGTGGAGGGGAGATAGAGCTTC-3′
IL33 V1	nt −2524 to +37	Sense	5′-CGCGGTACCCCATGCTTTCATCTTCATTC-3′
		Antisense	5′-CGCCTCGAGAGAGCTGCAGCTCTGTGTTC-3′
IL33 V5	nt −1956 to +48	Sense	5′-CGCGGTACCTAAACTTCTGGGCTCAGGTG-3′
		Antisense	5′-CGCCTCGAGGCTGGTCTCAGATGATGAGG-3′

Yamagishi N., Yamaguchi T., Kuga T., Taniguchi M., Khan M.S., Matsumoto T., Deguchi Y., Nagaoka H., Wakabayashi K, & Watanabe T. (2020). Development of a system for the detection of the inflammatory response induced by airborne fine particulate matter in rat tracheal epithelial cells. Toxicology Reports, 7, 900-908.

+ Open protocol

+ Expand

Cloning and Characterization of EPB41L5 Promoter

Check if the same lab product or an alternative is used in the 5 most similar protocols

The human EPB41L5 gene promoter was prepared and purified by PCR amplification of human genomic DNA from HEK293T cells for a specific region (−900 to 100 nucleotides) of the EPB41L5 gene. It was cloned into the pGL4.21 (luc2P/Puro) vector (Promega, E6761) using KpnI and XhoI restriction enzymes. Luciferase analysis of the EPB41L5 reporter gene and β-galactosidase gene was transiently transfected with 100 ng and 400 ng per well, respectively. After incubation for 24 h, the luciferase assay system (Promega, E1501) was used according to the manufacturer’s instructions. The colorimetric substrate ortho-nitrophenyl-galactoside was used to measure β-galactosidase activity at 405 nm absorbance. Normalized activity was expressed as luciferase activity/β-galactosidase activity (fold change).

Jeong J.H., Park S.H., Kim H., Nam H.Y., Kim S.H., Jeong M., Kong M.J., Son J., Jeong J.E., Song J.H., Kim S.W, & Choi K.C. (2023). ZBTB7A suppresses glioblastoma tumorigenesis through the transcriptional repression of EPB41L5. Experimental & Molecular Medicine, 55(1), 43-54.

+ Open protocol

+ Expand

Construction of Cytokine Reporter Plasmids

Check if the same lab product or an alternative is used in the 5 most similar protocols

Primer sequences for the reporter plasmids of cytokine genes.

Table 1

Gene	Position	Orientation	Sequence
TNFα	nt −1335 to +79	Sense	5′-CGCGGTACCGCTGTCTGCTTGTGTGTGTG-3′
		Antisense	5′-CGCCTCGAGTGTCCTTTCCAGGGGAGAGAGA-3′
IL6	nt −800 to +105	Sense	5′-CGCGGTACCCAGTGAAACAGTGGTGAAGA-3′
		Antisense	5′-CGCCTCGAGTGGAGGGGAGATAGAGCTTC-3′
IL33 V1	nt −2524 to +37	Sense	5′-CGCGGTACCCCATGCTTTCATCTTCATTC-3′
		Antisense	5′-CGCCTCGAGAGAGCTGCAGCTCTGTGTTC-3′
IL33 V5	nt −1956 to +48	Sense	5′-CGCGGTACCTAAACTTCTGGGCTCAGGTG-3′
		Antisense	5′-CGCCTCGAGGCTGGTCTCAGATGATGAGG-3′

+ Open protocol

+ Expand

About PubCompare

Our mission is to provide scientists with the largest repository of trustworthy protocols and intelligent analytical tools, thereby offering them extensive information to design robust protocols aimed at minimizing the risk of failures.

We believe that the most crucial aspect is to grant scientists access to a wide range of reliable sources and new useful tools that surpass human capabilities.

However, we trust in allowing scientists to determine how to construct their own protocols based on this information, as they are the experts in their field.

Ready to get started?

Revolutionizing how scientists
search and build protocols!