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Species: Homo sapiens
Genetic Insert: SMURF1 WW domain #2
Vector Backbone Description: Vector Backbone:pHH0103; Vector Types:Bacterial Expression; Bacterial Resistance:Ampicillin
Comments: Amino acid sequence: Linker - ENLYFQGRRASV(gagctc) and WW domain - ELGPLPPGWEVRSTVSGRIYFVDHNNRTTQFTDPRLHHI
Proper citation: RRID:Addgene_104238 Copy
Species: Homo sapiens
Genetic Insert: TDP-43
Vector Backbone Description: Vector Backbone:pJ4M; Vector Types:Bacterial Expression; Bacterial Resistance:Kanamycin
Defining Citation: PMID:29438978
Proper citation: RRID:Addgene_104481 Copy
Species: Homo sapiens
Genetic Insert: CD9
Vector Backbone Description: Backbone Marker:Thermo Fisher; Backbone Size:9351; Vector Backbone:pLenti6.3/TO/V5-DEST; Vector Types:Lentiviral; Bacterial Resistance:Ampicillin
Defining Citation: PMID:29221804
Comments: Plasmid is used and named CD9-GFP in publication even though it contains mEmerald. Described in the methods of Boker et al 2018 (https://www.ncbi.nlm.nih.gov/pubmed/29221804). Please see publication for more information.
Also see the following article for additional information on using this plasmid: Schiller et al., 2018 https://www.sciencedirect.com/science/article/pii/S2329050118300378
Proper citation: RRID:Addgene_104402 Copy
Species: Homo sapiens
Genetic Insert: hnRNPA2
Vector Backbone Description: Vector Backbone:pJ411; Vector Types:Bacterial Expression; Bacterial Resistance:Kanamycin
Defining Citation: PMID:29358076
Proper citation: RRID:Addgene_104465 Copy
Species: Homo sapiens
Genetic Insert: hnRNPA2
Vector Backbone Description: Vector Backbone:pTHMT; Vector Types:Bacterial Expression; Bacterial Resistance:Kanamycin
Defining Citation: PMID:29358076
Proper citation: RRID:Addgene_104468 Copy
Species: Homo sapiens
Genetic Insert: hnRNPA2
Vector Backbone Description: Vector Backbone:pTHMT; Vector Types:Bacterial Expression; Bacterial Resistance:Kanamycin
Defining Citation: PMID:29358076
Proper citation: RRID:Addgene_104467 Copy
Species: Homo sapiens
Genetic Insert: CD95L leucine zipper
Vector Backbone Description: Backbone Marker:Invitrogen; Backbone Size:5428; Vector Backbone:pcDNA3.1(-); Vector Types:Mammalian Expression; Bacterial Resistance:Ampicillin
Defining Citation: PMID:32706785
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.
Proper citation: RRID:Addgene_104349 Copy
Species: Homo sapiens
Genetic Insert: Human IL-2 (C125S)
Vector Backbone Description: Backbone Marker:qIAGEN; Backbone Size:4751; Vector Backbone:pQE80L; Vector Types:Bacterial Expression; Bacterial Resistance:Ampicillin
Comments: Like the FDA approved Proleukin (Aldesleukin), the sequence contains a C125S mutation (TGT -> TCC) to prevent cysteine mispairing in E. coli, but this does not affect biological activity (Wang et al. 1984) Expression and inclusion body preparation was carried out essentially as described in Carmenate et al. (2013), except that the 6M guanidine solubilised IL-2 was applied directly to an NTA column at 1 column volume / minute. LPS was removed with 0.1% Triton X114 / TE washes following by on-column re-folding with TE buffer. NTA-purified His-IL-2 exhibited identical activity to commercially sourced (tag-free) IL-2 (Peprotech).
References:
Wang A, Lu SD, and Mark DF. Site-specific mutagenesis of the human interleukin-2 gene: structure-function analysis of the cysteine residues. Science 1984; 224:1431-1433.
Carmenate T, Pacios A, Enamorado M, Moreno E, Garcia-Martinez K, Fuente D, and Leon K. Human IL-2 mutein with higher antitumor efficacy than wild type IL-2. J. Immunol. 2013; 190:6230-6238.
Proper citation: RRID:Addgene_104348 Copy
Species: Homo sapiens
Genetic Insert: TDP-43
Vector Backbone Description: Vector Backbone:pJ4M; Vector Types:Bacterial Expression; Bacterial Resistance:Kanamycin
Defining Citation: PMID:29438978
Proper citation: RRID:Addgene_104480 Copy
Species: Homo sapiens
Genetic Insert: FRBx2
Vector Backbone Description: Backbone Marker:Stratagene; Backbone Size:2958; Vector Backbone:pBlueScript KS (-); Vector Types:phagemid; Bacterial Resistance:Ampicillin
Defining Citation: PMID:26709839
Proper citation: RRID:Addgene_104476 Copy
Species: Homo sapiens
Genetic Insert: hnRNPA2
Vector Backbone Description: Vector Backbone:pJ411; Vector Types:Bacterial Expression; Bacterial Resistance:Kanamycin
Defining Citation: PMID:29358076
Proper citation: RRID:Addgene_104470 Copy
Species: Homo sapiens
Genetic Insert: TDP-43
Vector Backbone Description: Vector Backbone:pJ411; Vector Types:Bacterial Expression; Bacterial Resistance:Kanamycin
Defining Citation: PMID:29438978
Proper citation: RRID:Addgene_104479 Copy
Species: Homo sapiens
Genetic Insert: MYCN proto-oncogene, bHLH transcription factor
Vector Backbone Description: Vector Backbone:PiggyBac; Vector Types:Mammalian Expression, Bacterial Expression; Bacterial Resistance:Ampicillin
Defining Citation: PMID:29320761
Proper citation: RRID:Addgene_104542 Copy
Species: Homo sapiens
Genetic Insert: Human IgG1 CH1/CH2/CH3
Vector Backbone Description: Backbone Marker:Invitrogen (Thermo Fisher); Backbone Size:10143; Vector Backbone:pCEP4; Vector Types:Mammalian Expression; Bacterial Resistance:Ampicillin
Defining Citation: PMID:30196504
Comments: Addgene NGS results found A169G, A261G, A292G, and A295G within the EBNA1 translation on the pCEP4 backbone compared to YP_401677.1
Proper citation: RRID:Addgene_104583 Copy
Species: Homo sapiens
Genetic Insert: Caspase-3
Vector Backbone Description: Backbone Marker:Scott Gradia (Addgene plasmid # 29653); Vector Backbone:pET His6 TEV LIC cloning vector; Vector Types:Bacterial Expression; Bacterial Resistance:Kanamycin
Defining Citation: PMID:28893998
Comments: LOV2 domain was inserted into the intersubunit linker of the caspase, with linker variation N7C-7
Proper citation: RRID:Addgene_104629 Copy
Species: Homo sapiens
Genetic Insert: Caspase-3
Vector Backbone Description: Backbone Marker:Scott Gradia (Addgene plasmid # 29653); Vector Backbone:pET His6 TEV LIC cloning vector; Vector Types:Bacterial Expression; Bacterial Resistance:Kanamycin
Defining Citation: PMID:28893998
Comments: Please note that during Addgene's quality control process 2 mismatches were found in lacI which result in mutations S219F and Q311P.
LOV2 domain was inserted into the intersubunit linker of the caspase, with linker variation N7C-7 and mutation C450A, which is a dark-locked and light-insensitive mutation
Proper citation: RRID:Addgene_104628 Copy
Species: Homo sapiens
Genetic Insert: major histocompatibility complex, class I, B
Vector Backbone Description: Backbone Size:5000; Vector Backbone:pMP71; Vector Types:Mammalian Expression, Retroviral; Bacterial Resistance:Ampicillin
Proper citation: RRID:Addgene_104848 Copy
Species: Homo sapiens
Genetic Insert: major histocompatibility complex, class I, B
Vector Backbone Description: Backbone Size:5000; Vector Backbone:pMP71; Vector Types:Mammalian Expression, Retroviral; Bacterial Resistance:Ampicillin
Defining Citation: PMID:31022685
Proper citation: RRID:Addgene_104850 Copy
Species: Homo sapiens
Genetic Insert: major histocompatibility complex, class I, B
Vector Backbone Description: Backbone Marker:Invitrogen; Backbone Size:2580; Vector Backbone:pENTR; Vector Types:entry vector for Gateway system; Bacterial Resistance:Kanamycin
Proper citation: RRID:Addgene_104837 Copy
Species: Homo sapiens
Genetic Insert: Tripartite Motif containing 21
Vector Backbone Description: Backbone Marker:Mark Allen (MRC LMB); Backbone Size:3113; Vector Backbone:HLTV; Vector Types:Bacterial Expression; Bacterial Resistance:Ampicillin
Defining Citation: PMID:29153837
Proper citation: RRID:Addgene_104973 Copy
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