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Pnlcoi1 luc2 p2a nlucp hygro vector

Manufactured by Promega
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The PNLCoI1 [luc2-P2A-NlucP/Hygro] vector is a laboratory tool that contains the luc2 and NlucP reporter genes separated by a P2A sequence. It also includes a hygromycin resistance marker. This vector is designed for use in cell-based assays and other research applications.

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Lab products found in correlation

Lipofectamine, by Thermo Fisher Scientific (1 mentions) Hygromycin, by InvivoGen (1 mentions) In fusion kit, by Takara Bio (1 mentions) Clonamp hifi, by Takara Bio (1 mentions)

2 protocols using pnlcoi1 luc2 p2a nlucp hygro vector

1

Human LMNA Promoter Luciferase Assay

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The human promoter region of LMNA was recognized from the earlier published rat LMNA promoter sequence (Tiwari et al., 1998 (link)). The promoter sequence was chosen to include the TATA box, GC box, and activator protein 1 (AP1) binding site. The upstream -1.3 kB promoter region (sequence -1334 to-16) was synthesized and subcloned into a pNLCoI1 [luc2-P2A-NlucP/Hygro] vector (Promega, Madison, Wisconsin, United States) at GenScript, Netherlands. The vector (50 ng/ml) was transfected into fibroblasts using Lipofectamine (Thermo Fischer Scientific) and, after 72 h, Dual Glo luciferase assay reagent was added. After an incubation time of 15 min at RT, the firefly luciferase was measured on a luminometer. Then Dual Glo Stop and Go reagent was added, incubated for 15 min, and renilla fluorescence was measured on a luminometer. The background was subtracted and the values were normalized to renilla (firefly/renilla).
West G., Turunen M., Aalto A., Virtanen L., Li S.P., Heliö T., Meinander A, & Taimen P. (2022). A heterozygous p.S143P mutation in LMNA associates with proteasome dysfunction and enhanced autophagy-mediated degradation of mutant lamins A and C. Frontiers in Cell and Developmental Biology, 10, 932983.
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2

Cloning SV40 Enhancer and KL Promoters

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To clone the SV40 enhancer into pNLCoI1[luc2-P2A-NlucP/Hygro] Vector (Promega), the SV40 enhancer was amplified from pGL3 using the following primers using Clontech HiFi according to the manufacturer’s protocol:
The PCR product was isolated and ligated with pNL vector by digestion with BamHI to generate the pNLCoI1-SV40 vector. The KL 1.8 kb promoter was digested from pGL3-KL1800 (King et al., 2012 (link)) with HindIII and XhoI, and subcloned into pNLCoI1-SV40 vector using the same restriction sites to generate pNLCoI1-SV40-KL1800.
To clone the KL 4 kb promoter, the additional 2.2Kb (−1800 to −4000) and the vector were amplified from HEK293 genomic DNA and pNLCoI1-SV40-KL1800, respectively, with the following primers and ClonAmp HiFi polymerase according to the manufacturer’s protocol:
The insert and vector bands (100 ng each) were ligated together using In Fusion kit (Clontech) to generate pNLCoI1-SV40-KL4000. Stable HEK-293 cells expressing 4kb of the KL promoter or the PGK promoter with the coincidence reporter were generated from single clones and selected using Hygromycin (Invivogen, USA) at a concentration of 75μg/ml for two weeks following Hygromycin 25μg/ml for maintenance.
Chen C.D., Zeldich E., Li Y., Yuste A, & Abraham C.R. (2018). Activation of the anti-aging and cognition-enhancing gene Klotho by CRISPR-dCas9 transcriptional effector complex. Journal of molecular neuroscience : MN, 64(2), 175-184.
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