Pcpgfree promoter lucia vector

Manufactured by InvivoGen

Sourced in France

The PCpGfree-promoter-Lucia vector is a plasmid that contains a synthetic promoter sequence designed to drive the expression of the secreted Lucia luciferase reporter gene. The vector is free of CpG motifs, which are known to activate the Toll-like receptor 9 (TLR9) pathway and potentially interfere with gene expression studies.

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Lab products found in correlation

E coli gt115 cells, by InvivoGen (2 mentions) Hpych4iv digestion, by New England Biolabs (2 mentions) Cpg methyltransferase m sssi, by New England Biolabs (1 mentions) Mspji, by New England Biolabs (1 mentions) Lipofectamine, by Thermo Fisher Scientific (1 mentions) Sssi methylase, by New England Biolabs (1 mentions) Renilla luciferase, by Promega (1 mentions) Quanti luc, by InvivoGen (1 mentions) Dpni digestion, by New England Biolabs (1 mentions) Methyltransferase m sss 1, by New England Biolabs (1 mentions) Lipofectamine 3000 transfection reagent, by Thermo Fisher Scientific (1 mentions) Dual luciferase reporter assay system, by Promega (1 mentions) Qiaquick gel extraction kit, by Qiagen (1 mentions) M sssi methyltransferase, by New England Biolabs (1 mentions)

Close spelling variants of this product

PCpGfree-promoter-lucia PCpGfree-Lucia PCpGfree-promoter

5 protocols using pcpgfree promoter lucia vector

ANKRD26 Promoter Methylation Assay

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ANKRD26 promoter (− 716/− 597 bp) was amplified by PCR. The purified PCR fragment was cloned into the firefly luciferase reporter pCpG-free-promoter-Lucia vector (Invivogen, Toulouse, France). This vector is completely void of CpG dinucleotides in all the elements required for replication and selection of the plasmid in E. coli and gene expression in mammalian cells. Also, it is devoid of all Dam methylation sites (GATC) to prevent prokaryotic methylation [51 ]. The pCpG-Ankrd26 vector was amplified in E. coli GT115 cells (Invivogen). In vitro methylation was performed using the M.SssI CpG methyltransferase following manufacturer’s protocol (New England BioLabs, Ipswich, MA). Un-methylated DNA was obtained in the absence of M.SssI. Methylation was confirmed by digestion with MspJI (New England BioLabs). HEK-293 cells were transfected with the CpG methylated or un-methylated pCpG-ANKRD26 vector and Renilla control vector (Promega, Madison WI) by lipofectamine (Life Technologies), following manufacturer’s instructions. Firefly luciferase activity of each transfection was normalized for transfection efficiency against Renilla luciferase activity.

Desiderio A., Longo M., Parrillo L., Campitelli M., Cacace G., de Simone S., Spinelli R., Zatterale F., Cabaro S., Dolce P., Formisano P., Milone M., Miele C., Beguinot F, & Raciti G.A. (2019). Epigenetic silencing of the ANKRD26 gene correlates to the pro-inflammatory profile and increased cardio-metabolic risk factors in human obesity. Clinical Epigenetics, 11, 181.

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Functional Role of CpG Sequences in Promoter Activity

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To assess any functional role for CpG sequences of interest, the target sequence (613 bp) was amplified from 69,991,078 to 69,991,690 of human chromosome 10 (hg18) using the primer set described in Table S1 and inserted into the AvrII/SpeI sites of the pCpGfree-promoter-Lucia vector (InvivoGen, Toulouse, France), which is a reporter plasmid completely devoid of CpG dinucleotides. For in vitro methylation, 10 μg of the reporter constructs was incubated with 32 U SssI methylase (NEB) and 32 mM S-adenosylmethionine (NEB) at 37°C overnight. After confirmation of the methylation status of methylated or unmethylated constructs with pyrosequencing, luciferase activity was measured using QUANTI-Luc (InvivoGen) and normalized to that of Renilla luciferase (Promega). The experiment was performed in triplicate.

Park J.L., Kim H.J., Seo E.H., Kwon O.H., Lim B., Kim M., Kim S.Y., Song K.S., Kang G.H., Kim H.J., Choi B.Y, & Kim Y.S. (2015). Decrease of 5hmC in gastric cancers is associated with TET1 silencing due to with DNA methylation and bivalent histone marks at TET1 CpG island 3′-shore. Oncotarget, 6(35), 37647-37662.

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CpG Methylation of Ankrd26 Promoter

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Ankrd26 promoter (−733 bp/−344 bp) was amplified by PCR. The purified PCR fragment was cloned into the firefly luciferase reporter pCpGfree-promoter-Lucia vector (Invivogen, Toulouse, France). The following site-specific mutated constructs were generated by PCR-based mutagenesis: pCpG-Ankrd26-436, pCpG-Ankrd26-431, pCpG-Ankrd26-391. The wilde type (Wt) pCpG-Ankrd26 vector, used as template, was removed from the PCR reaction by DpnI digestion (New England BioLabs, Ipswich, MA). Wt and mutated (mut) vectors were amplified into E. coli GT115 cells (Invivogen). Site-specific mutagenesis of each construct was validated by sequencing. In vitro methylation was performed using the M.SsI CpG methyltransferase following manufacturer’s protocol (New England BioLabs). Un-methylated DNA was obtained in the absence of M.SsI. Methylation was confirmed by resistance to HpyCH4IV digestion (New England BioLabs).

Raciti G.A., Spinelli R., Desiderio A., Longo M., Parrillo L., Nigro C., D’Esposito V., Mirra P., Fiory F., Pilone V., Forestieri P., Formisano P., Pastan I., Miele C, & Beguinot F. (2017). Specific CpG hyper-methylation leads to Ankrd26 gene down-regulation in white adipose tissue of a mouse model of diet-induced obesity. Scientific Reports, 7, 43526.

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Methylation of miR-9-3 Promoter

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Genomic DNA was isolated from lung cancer A549 cells, and the segment of the miR-9-3 promoter (−129 ~ +232 bp) was amplified using the following primers: 5′-AATGCATGTGTGCGTGTGTCTGTCCATCCC-3′ (forward) and 5′-CCACTAGTGGCACTGCAAGTGTCCCCAGAGA-3′ (reverse). The PCR products were digested with Nsi I and Spe I and then inserted into the pCpGfree-promoter-Lucia vector (InvivoGen, San Diego, CA, USA). After sequencing confirmation, the recombinant plasmid was treated with methyltransferase M.Sss I at 37°C for 2 h (New England Biolabs, Beverly, MA, USA). The methylated plasmid was purified using the QIAquick gel extraction kit (Qiagen, Valencia, CA, USA). The efficiency of the methylation process was verified using the methylation-insensitive Hpa II and Hha I restriction endonucleases.
A549, HEK293 and HT29 cells were seeded in 12-well plates for 24 h, and 500 ng of the recombinant plasmids was transfected using Lipofectamine 3000 transfection reagent (Invitrogen, Carlsbad, CA, USA) according to manufacturer’s instructions. A total of 50 ng of pGL 4.13 was co-transfected as the internal control. After 24 h, the luciferase activity was measured using the Dual-Luciferase Reporter Assay System (Promega, Madison, WI, USA).

Gao L., Cheng D., Yang J., Wu R., Li W, & Kong A.N. (2018). Sulforaphane epigenetically demethylates the CpG sites of the miR-9-3 promoter and reactivates miR-9-3 expression in human lung cancer A549 cells. The Journal of nutritional biochemistry, 56, 109-115.

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Zfp423 Promoter Mutagenesis and Methylation

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Zfp423 promoter (−1037/−1002) was amplified by PCR and cloned into the firefly luciferase reporter pCpGfree-promoter-Lucia vector (InvivoGen). A one-step PCR-based mutagenesis technique was used to generate site-specific mutation [28 (link)] and produce a mutated construct. One complementary pair of primers was designed that contained the desired mutation, replacing the cytosine at position −1016 with adenine. The wild-type and mutated constructs were transformed into E. coli GT115 cells. These cells were purchased from InvivoGen and are mycoplasma-free. In vitro methylation was performed using M.SssI methyltransferase following the manufacturer’s protocol (New England BioLabs). Unmethylated wild-type and mutated constructs were obtained in the absence of M.SssI. Methylation was confirmed by resistance to HpyCH4IV digestion (New England Biolabs). After 48 h, firefly and Renilla luciferase activity were assayed using a luciferase reporter assay kit, as reported in the previous paragraph.

Longo M., Raciti G.A., Zatterale F., Parrillo L., Desiderio A., Spinelli R., Hammarstedt A., Hedjazifar S., Hoffmann J.M., Nigro C., Mirra P., Fiory F., Formisano P., Miele C., Smith U, & Beguinot F. (2017). Epigenetic modifications of the Zfp/ZNF423 gene control murine adipogenic commitment and are dysregulated in human hypertrophic obesity. Diabetologia, 61(2), 369-380.

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