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URL: http://www.addgene.org/104349
Proper Citation: RRID:Addgene_104349
Insert Name: CD95L leucine zipper
Organism: Homo sapiens
Bacterial Resistance: Ampicillin
Defining Citation: PMID:32706785
Vector Backbone Description: Backbone Marker:Invitrogen; Backbone Size:5428; Vector Backbone:pcDNA3.1(-); Vector Types:Mammalian Expression; Bacterial Resistance:Ampicillin
Comments: Synthetic DNA encoding a fusion of an improved IL2 signal sequence (Zhang et al. 2005 & Naito et al. 2013), an octahistidine-tagged-leucine zipper motif, a flexible 17aa linker (Naito et al. 2013) and the death inducing, c-terminal extracellular domain (aa137-208) of CD95L. The cloning strategy omits the proteolytically-susceptible extracellular stalk region (aa102-136), but includes the extracellular self-assembly domain (aa137-183; Orlinick et al. 1997). NTA column purified LZ-CD95L produced in expi393 cells is highly active (IC50 Jurkat E6-1 cells < 50ng/ml). Human CD95L is also active in inducing cell death in murine thymocytes (McLellan et al. 2000). Note, during following transient transfection, rising levels of LZ-CD95L in the culture supernatant induce cell death in HEK293FT and expi293. Users could explore co-transfection of this vector with a c-FLIP-encoding vector, select CD95L resistant HEK293 variants, or use other CD95L-resistant mammalian cell lines to maximise LZ-CD95L yields. References: McLellan AD, Terbeck G, Mengling T, Starling GC, Kiener PA, Gold R, Bröcker EB, Leverkus M, Kämpgen E. Differential susceptibility to CD95 (Apo-1/Fas) and MHC class II-induced apoptosis during murine dendritic cell development. Cell Death Differ. 2000 Oct;7(10):933-8. Naito M, Hainz U, Burkhardt UE, Fu B, Ahove D, Stevenson KE, Rajasagi M, Zhu B, Alonso A, Witten E, Matsuoka K, Neuberg D, Duke-Cohan JS, Wu CJ, and Freeman GJ. CD40L-Tri, a novel formulation of recombinant human CD40L that effectively activates B cells. Cancer Immunol Immunother 2013; 62:347-357. Orlinick JR, Elkon KB, and Chao MV. Separate domains of the human fas ligand dictate self-association and receptor binding. J Biol Chem 1997; 272:32221-32229. Zhang L, Leng Q, and Mixson AJ. Alteration in the IL-2 signal peptide affects secretion of proteins in vitro and in vivo. The Journal of Gene Medicine 2005; 7:354-365.
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Source: Addgene (RRID:SCR_002037)
Description: Non-profit plasmid repository dedicated to helping scientists around the world share high-quality plasmids. Facilitates archiving and distributing DNA-based research reagents and associated data to scientists worldwide. Repository contains over 65,000 plasmids, including special collections on CRISPR, fluorescent proteins, and ready-to-use viral preparations. There is no cost for scientists to deposit plasmids, which saves time and money associated with shipping plasmids themselves. All plasmids are fully sequenced for validation and sequencing data is openly available. We handle the appropriate Material Transfer Agreements (MTA) with institutions, facilitating open exchange and offering intellectual property and liability protection for depositing scientists. Furthermore, we curate free educational resources for the scientific community including a blog, eBooks, video protocols, and detailed molecular biology resources.
URL: http://www.addgene.org
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