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Species: Mus musculus
Genetic Insert: Rag2
Vector Backbone Description: Vector Backbone:pLEXm; Vector Types:Mammalian Expression; Bacterial Resistance:Ampicillin
Defining Citation: PMID:21149691
Proper citation: RRID:Addgene_115650 Copy
Species: Mus musculus
Genetic Insert: JNK KTR-mCherry
Vector Backbone Description: Backbone Marker:Allan Bradley (Wellcome Sanger Institute); Backbone Size:6777; Vector Backbone:pPB; Vector Types:Mammalian Expression; Bacterial Resistance:Ampicillin
Defining Citation: PMID:30184500
Comments: See also: Regot et al Cell. 2014 Jun 19;157(7):1724-34. doi: 10.1016/j.cell.2014.04.039.
Proper citation: RRID:Addgene_115493 Copy
Species: Mus musculus
Genetic Insert: sgRNA M5, sgRNA M7, sgRNA M9
Vector Backbone Description: Vector Backbone:pT2K-CAGGS-IRES-CFP; Vector Types:Mammalian Expression, CRISPR; Bacterial Resistance:Ampicillin
Defining Citation: PMID:29880921
Proper citation: RRID:Addgene_114729 Copy
Species: Mus musculus
Genetic Insert: SENP3
Vector Backbone Description: Vector Backbone:pCDNA3; Vector Types:Mammalian Expression; Bacterial Resistance:Ampicillin
Defining Citation: PMID:18948745
Proper citation: RRID:Addgene_115521 Copy
Species: Mus musculus
Genetic Insert: SENP3-C526A
Vector Backbone Description: Vector Backbone:pCDNA3; Vector Types:Mammalian Expression; Bacterial Resistance:Ampicillin
Defining Citation: PMID:18948745
Proper citation: RRID:Addgene_115522 Copy
Species: Mus musculus
Genetic Insert: partial sequence of mannosidase II
Vector Backbone Description: Backbone Marker:Clontech; Vector Backbone:pIRESneo3; Vector Types:Mammalian Expression; Bacterial Resistance:Ampicillin
Defining Citation: PMID:30587510
Proper citation: RRID:Addgene_117274 Copy
Species: Mus musculus
Genetic Insert: Kif1A (1-489)
Vector Backbone Description: Vector Backbone:pcDNA3.1(+); Vector Types:; Bacterial Resistance:Ampicillin
Defining Citation: PMID:31061466
Comments: Addgene NGS results found S33N and E170G variants in Kif1A compared to the NCBI reference [XP_006529221.1]. These variants do not affect plasmid function.
Proper citation: RRID:Addgene_117833 Copy
Species: Mus musculus
Genetic Insert: Egr-1
Vector Backbone Description: Backbone Size:5400; Vector Backbone:pcDNA3; Vector Types:Mammalian Expression; Bacterial Resistance:Ampicillin
Defining Citation: PMID:15225550
Proper citation: RRID:Addgene_11729 Copy
Species: Mus musculus
Genetic Insert: CnA
Vector Backbone Description: Backbone Size:0; Vector Backbone:pBJ5-stop; Vector Types:Mammalian Expression; Bacterial Resistance:Ampicillin
Defining Citation: PMID:7523407
Proper citation: RRID:Addgene_11785 Copy
Species: Mus musculus
Genetic Insert: Epac2
Vector Backbone Description: Backbone Marker:clontech; Vector Backbone:peGFP-C1; Vector Types:Mammalian Expression; Bacterial Resistance:Kanamycin
Defining Citation: PMID:18824540
Proper citation: RRID:Addgene_118314 Copy
Species: Mus musculus
Genetic Insert: Src
Vector Backbone Description: Backbone Marker:invitrogen; Vector Backbone:pcDNA3; Vector Types:Mammalian Expression; Bacterial Resistance:Ampicillin
Defining Citation: PMID:11804588
Comments: Src constructs were subcloned into pcDNA3 containing Flag epitope with HindIII and BamHI.
Proper citation: RRID:Addgene_118308 Copy
Species: Mus musculus
Genetic Insert: Alpha Catulin
Vector Backbone Description: Backbone Size:4700; Vector Backbone:pEGFP-N1; Vector Types:Mammalian Expression; Bacterial Resistance:Kanamycin
Defining Citation: PMID:26769899
Proper citation: RRID:Addgene_118979 Copy
Species: Mus musculus
Genetic Insert: OptoTGFBRs
Vector Backbone Description: Backbone Marker:Clontech; Backbone Size:3925; Vector Backbone:ptdToamto-N1; Vector Types:Mammalian Expression; Bacterial Resistance:Kanamycin
Defining Citation: PMID:29241005
Proper citation: RRID:Addgene_118942 Copy
Species: Mus musculus
Genetic Insert: Kif5a cDNA
Vector Backbone Description: Backbone Marker:Clontech; Backbone Size:4710; Vector Backbone:Clontech C3; Vector Types:Mammalian Expression; Bacterial Resistance:Kanamycin
Proper citation: RRID:Addgene_118744 Copy
Species: Mus musculus
Genetic Insert: Snapin cDNA
Vector Backbone Description: Backbone Marker:Clontech; Backbone Size:5978; Vector Backbone:Clontech C2; Vector Types:Mammalian Expression; Bacterial Resistance:Kanamycin
Proper citation: RRID:Addgene_118743 Copy
Species: Mus musculus
Genetic Insert: Borcs7 cDNA
Vector Backbone Description: Backbone Marker:Clontech; Backbone Size:4062; Vector Backbone:Clontech C5; Vector Types:Mammalian Expression; Bacterial Resistance:Kanamycin
Proper citation: RRID:Addgene_118746 Copy
Species: Mus musculus
Genetic Insert: Vegfr3
Vector Backbone Description: Vector Backbone:pCDNA3.1; Vector Types:Mammalian Expression; Bacterial Resistance:Ampicillin
Proper citation: RRID:Addgene_119232 Copy
Species: Mus musculus
Genetic Insert: Id1
Vector Backbone Description: Backbone Marker:Clontech; Backbone Size:7800; Vector Backbone:pLVX-tight-Puro; Vector Types:Mammalian Expression; Bacterial Resistance:Ampicillin
Defining Citation: PMID:31582374
Proper citation: RRID:Addgene_119280 Copy
Species: Mus musculus
Genetic Insert: Type I procollagen α2 chain
Vector Backbone Description: Backbone Size:5400; Vector Backbone:pcDNA3.1; Vector Types:Mammalian Expression; Bacterial Resistance:Ampicillin
Defining Citation: PMID:30287488
Comments: These plasmids were constructed after the G610C mouse model of OI in which the Gly610 residue (starting from the N-terminal end of the triple helix) was substituted with Cys.
These mouse type I procollagen plasmids were designed for immortalized and primary murine osteoblasts and fibroblasts. Their expression in other cells might cause significant disruption of assembly, folding and trafficking of type I procollagen, resulting in artifacts. Even in osteoblasts and fibroblasts a fraction of transfected chains, particularly proα2, might be trafficked/degraded as monomers instead of being integrated into procollagen heterotrimers. In our experience, the best evidence of normal behavior of the transfected chains is the appearance of fluorescent extracellular collagen fibers ~ 12 h after transfection. Fluorescent molecules should be incorporated into fibers even when the transfected chains contain the N-propeptide cleavage site, because the cleavage is highly conformation sensitive and always incomplete.
We recommend following these guidelines when using the plasmids
1) Only mouse cells that have high expression of endogenous type I procollagen are suitable for studies of physiologically-relevant processes with these constructs.
2) At least 100 μM ascorbic acid at and after transfection is required to ensure proper procollagen folding (for some cells, preincubation with ascorbic acid before transfection might be needed).
3) Optimization of transfection efficiency is needed for all cell types.
4) Experiments beyond 24h after transfection are not recommended, to avoid excessive accumulation of aggregates and cell malfunction caused by increased procollagen synthesis.
5) Lack of extracellular fluorescent fibers 12-24 h after transfection indicates improper procollagen synthesis/trafficking or cellular malfunction.
6) Excessive expression of transfected chains might cause rapid accumulation of large procollagen aggregates in the ER, resulting in cellular malfunction and death.
Contact information: Shakib Omari ([email protected]), Sergey Leikin ([email protected]).
Proper citation: RRID:Addgene_119827 Copy
Species: Mus musculus
Genetic Insert: Type I procollagen α1 chain
Vector Backbone Description: Backbone Size:5300; Vector Backbone:pcDNA3.1; Vector Types:Mammalian Expression; Bacterial Resistance:Ampicillin
Defining Citation: PMID:30287488
Comments: These mouse type I procollagen plasmids were designed for immortalized and primary murine osteoblasts and fibroblasts. Their expression in other cells might cause significant disruption of assembly, folding and trafficking of type I procollagen, resulting in artifacts. Even in osteoblasts and fibroblasts a fraction of transfected chains, particularly proα2, might be trafficked/degraded as monomers instead of being integrated into procollagen heterotrimers. In our experience, the best evidence of normal behavior of the transfected chains is the appearance of fluorescent extracellular collagen fibers ~ 12 h after transfection. Fluorescent molecules should be incorporated into fibers even when the transfected chains contain the N-propeptide cleavage site, because the cleavage is highly conformation sensitive and always incomplete.
We recommend following these guidelines when using the plasmids
1) Only mouse cells that have high expression of endogenous type I procollagen are suitable for studies of physiologically-relevant processes with these constructs.
2) At least 100 μM ascorbic acid at and after transfection is required to ensure proper procollagen folding (for some cells, preincubation with ascorbic acid before transfection might be needed).
3) Optimization of transfection efficiency is needed for all cell types.
4) Experiments beyond 24h after transfection are not recommended, to avoid excessive accumulation of aggregates and cell malfunction caused by increased procollagen synthesis.
5) Lack of extracellular fluorescent fibers 12-24 h after transfection indicates improper procollagen synthesis/trafficking or cellular malfunction.
6) Excessive expression of transfected chains might cause rapid accumulation of large procollagen aggregates in the ER, resulting in cellular malfunction and death.
Contact information: Shakib Omari ([email protected]), Sergey Leikin ([email protected]).
Proper citation: RRID:Addgene_119843 Copy
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