pMCP-MU-Luc (MCP-1 mut promoter in pGL3-basic)
|Item||Catalog #||Description||Quantity||Price (USD)|
|Plasmid||40325||Standard format: Plasmid sent in bacteria as agar stab||1||$75|
This material is available to academics and nonprofits only.
- Backbone size w/o insert (bp) 4818
- Total vector size (bp) 6018
Vector typeMammalian Expression, Luciferase
Growth in Bacteria
Gene/Insert nameMCP-1 promoter (mutated)
Alt namemacrophage chemoattractant protein-1 promoter (mutated)
Alt nameCCL2 promoter (mutated)
SpeciesM. musculus (mouse)
Insert Size (bp)1200
Mutationcontains MCP-1 promoter region through the nucleotides 28-bp downstream of the MCP-1 TATA site. Sequence has two base changes from the wild-type MCP-1 promoter around the -150 bp position (see note below).
- Promoter MCP-1
/ Fusion Protein
- Luciferase (C terminal on backbone)
- Cloning method Restriction Enzyme
- 5′ cloning site XmaI (unknown if destroyed)
- 3′ cloning site XhoI (not destroyed)
- 5′ sequencing primer RVprimer3
- 3′ sequencing primer LucNrev (Common Sequencing Primers)
A plasmid containing the mouse MCP-1 promoter driving luciferase (pMCP-Luc) was constructed from pGL3-basic (Promega, Madison, WI) and a PCR-generated MCP-1 promoter fragment. A proofreading competent Taq reagent (AccuTaq, Sigma) was used to ensure high fidelity PCR. 1.2 kb of the MCP-1 promoter (GenBank Accession no. U12470) was obtained by PCR of C57BL/6 mouse DNA with the following primers:
5′–TCATGTATATGGCTTTCCAGGTCT–3′ (sense strand, distal to transcription start)
5′–TGCACTTCTGGCTGCTCTGAG GCA–3′ (antisense strand, proximal to transcription start)
The PCR product terminates 28-bp downstream of the MCP-1 TATA site. XmaI and XhoI sites were added to the MCP-1 promoter by a second round of PCR with the primers:
and XmaI and XhoI were used to clone the MCP-1 promoter fragment into pGL3-basic.
For the construction of the MCP-1 mutant promoter, the BCL-6 site was mutated by a two step PCR–based strategy using the above flanking primers and the following complementary primers to mutate the BCL-6 site:
5′–TGTTT[AA]AGGAAGTGGAAGAGAGA–3′ (mutated bases are in brackets)
The mutant promoter was cloned into pGL3 via XhoI and XmaI. The wild-type and mutant MCP-1 promoter sequences were verified by sequencing.
These plasmids were created by your colleagues. Please acknowledge the Principal Investigator, cite the article in which the plasmids were described, and include Addgene in the Materials and Methods of your future publications.
For your Materials & Methods section:pMCP-MU-Luc (MCP-1 mut promoter in pGL3-basic) was a gift from Alexander Dent (Addgene plasmid # 40325 ; http://n2t.net/addgene:40325 ; RRID:Addgene_40325)
For your References section:BCL-6 regulates chemokine gene transcription in macrophages. Toney LM, Cattoretti G, Graf JA, Merghoub T, Pandolfi PP, Dalla-Favera R, Ye BH, Dent AL. Nat Immunol. 2000 Sep;1(3):214-20. 10.1038/79749 PubMed 10973278