PurposeLentiviral vector that expresses an EGFP-Cre fusion protein with a nuclear localization signal from the CMV promoter
|Item||Catalog #||Description||Quantity||Price (USD)|
|Plasmid||86805||Standard format: Plasmid sent in bacteria as agar stab||1||$65|
This material is available to academics and nonprofits only.
Backbone manufacturerInder Verma
Vector typeMammalian Expression, Lentiviral, Cre/Lox
Growth in Bacteria
Growth Strain(s)NEB Stable
Gene/Insert nameCre recombinase
Insert Size (bp)1800
- Promoter CMV
/ Fusion Proteins
- EGFP (N terminal on insert)
- nuclear localization signal: PKKKRKV (N terminal on insert)
- Cloning method Restriction Enzyme
- 5′ cloning site AfeI (destroyed during cloning)
- 3′ cloning site SmaI (destroyed during cloning)
- 5′ sequencing primer CMV (Common Sequencing Primers)
Terms and Licenses
The plasmid pEGFP-cre (~5.75 Kb) was a gift from Brigitte Kieffer and Gregory Scherrer and was generated while they were at the University of Strasbourg and the Institut de Génétique et Biologie Moleculaire et Cellular (IGBMC), Illkirch, France.
A nuclear localization signal (NLS) PKKKRKV was added after the ATG start site of EGFP-cre with QuikChange II XL Site Directed Mutagenesis kit and protocol (Stratagene # 200521), and confirmed by sequencing. The ~1.8 Kb AfeI/SmaI blunt NLS-EGFP-cre fragment was then removed and ligated into blunted BamHI sites of the ~6.7 Kb backbone from the lentiviral plasmid vector LV-Lac (Addgene #12108) ie LV-Lac with the Lac removed. This ligation was transformed into SURE2 cells. DNA from potential subclones was screened for orientation with restriction digestions and checked with sequencing. Unfortunately the sequencing showed that this cloning strategy created a false ATG/start site 6 AAs downstream of the true ATG start of the EGFP-cre. To eliminate any potential problems this false ATG start site was mutated to ATC with QuikChange II XL Site Directed Mutagenesis and confirmed by sequencing. The corrected pLV-EGFP-cre subclone was subsequently transformed into TOP10F’ which gave better DNA yields than those from SURE2.
This plasmid was produced as part of the NIAAA-funded INIA Consortium.
Additional reference (PMID: 27986929):
Harris et al (2016) Genetic and pharmacologic manipulation of TLR4 has minimal impact on ethanol consumption in rodents. J. Neurosci., 37(5):1139-115.
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:pLV-EGFP-Cre was a gift from Amy Lasek (Addgene plasmid # 86805 ; http://n2t.net/addgene:86805 ; RRID:Addgene_86805)
For your References section:Inhibition of IKKbeta Reduces Ethanol Consumption in C57BL/6J Mice. Truitt JM, Blednov YA, Benavidez JM, Black M, Ponomareva O, Law J, Merriman M, Horani S, Jameson K, Lasek AW, Harris RA, Mayfield RD. eNeuro. 2016 Oct 31;3(5). pii: ENEURO.0256-16.2016. eCollection 2016 Sep-Oct. eN-NWR-0256-16 [pii] PubMed 27822501