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Species: Mus musculus
Genetic Insert: Mecom
Vector Backbone Description: Vector Backbone:FUW; Vector Types:Mammalian Expression, Lentiviral; Bacterial Resistance:Ampicillin
Defining Citation: PMID:30318302
Proper citation: RRID:Addgene_118590 Copy
Species: Mus musculus
Genetic Insert: eSOX2NRR
Vector Backbone Description: Vector Backbone:pMX; Vector Types:Mammalian Expression, Retroviral; Bacterial Resistance:Ampicillin
Defining Citation: PMID:30078555
Proper citation: RRID:Addgene_118825 Copy
Species: Mus musculus
Genetic Insert: Borcs7-Q87X cDNA
Vector Backbone Description: Backbone Marker:Clontech; Backbone Size:4710; Vector Backbone:Clontech C9; Vector Types:Mammalian Expression; Bacterial Resistance:Kanamycin
Proper citation: RRID:Addgene_118750 Copy
Species: Mus musculus
Genetic Insert: Snapin cDNA
Vector Backbone Description: Backbone Marker:Clontech; Backbone Size:4731; Vector Backbone:Clontech C1; Vector Types:Mammalian Expression; Bacterial Resistance:Kanamycin
Proper citation: RRID:Addgene_118742 Copy
Species: Mus musculus
Genetic Insert: Borcs7 cDNA
Vector Backbone Description: Backbone Marker:Clontech; Backbone Size:4710; Vector Backbone:Clontech C7; Vector Types:Mammalian Expression; Bacterial Resistance:Kanamycin
Proper citation: RRID:Addgene_118748 Copy
Species: Mus musculus
Genetic Insert: Borcs7 cDNA
Vector Backbone Description: Backbone Marker:Clontech; Backbone Size:5309; Vector Backbone:Clontech C6; Vector Types:Mammalian Expression; Bacterial Resistance:Kanamycin
Proper citation: RRID:Addgene_118747 Copy
Species: Mus musculus
Genetic Insert: OptoTGFBRs
Vector Backbone Description: Backbone Marker:Clontech; Backbone Size:6132; Vector Backbone:pLNCX2; Vector Types:Retroviral; Bacterial Resistance:Ampicillin
Defining Citation: PMID:29241005
Proper citation: RRID:Addgene_118965 Copy
Species: Mus musculus
Genetic Insert: Ptch1
Vector Backbone Description: Backbone Size:4723; Vector Backbone:mCherry2-N1; Vector Types:Mammalian Expression; Bacterial Resistance:Kanamycin
Comments: Please note that mutation F600L in mouse Ptch1 was found during Addgene's quality control process. The depositing lab noted this mutation should not impact plasmid function.
Proper citation: RRID:Addgene_118982 Copy
Species: Mus musculus
Genetic Insert: mPLD1-PX (71-210)
Vector Backbone Description: Vector Backbone:pGEX4T2; Vector Types:Bacterial Expression; Bacterial Resistance:Ampicillin
Defining Citation: PMID:30948714
Comments: Amino Acid Sequence: NIQTYLSGCPIKAQVLEVERFTSTSRVPSINLYTIELTHGEFTWQVKRKFKHFQEFHRELLKYKAFIRIPIPTKRHTFRRQNVKEEPREMPSLPRSSENAIQEEQFFGRRKQLEDYLTKILKMPMYRNYHATTEFLDVSQ
Proper citation: RRID:Addgene_119127 Copy
Species: Mus musculus
Genetic Insert: Tie1
Vector Backbone Description: Vector Backbone:pMXs; Vector Types:Retroviral; Bacterial Resistance:Ampicillin
Proper citation: RRID:Addgene_119237 Copy
Species: Mus musculus
Genetic Insert: Tie1
Vector Backbone Description: Vector Backbone:FUW; Vector Types:Lentiviral; Bacterial Resistance:Ampicillin
Comments: mTie1 carries a G741D difference compared to the NCBI reference NM_011587.2. This difference is carried in numerous strains found in the Ensembl database and is expected to be functional.
Proper citation: RRID:Addgene_119234 Copy
Species: Mus musculus
Genetic Insert: Vegfd-dN-dC
Vector Backbone Description: Vector Backbone:psubCMV-WPRE; Vector Types:AAV; Bacterial Resistance:Ampicillin
Proper citation: RRID:Addgene_119228 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 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_119826 Copy
Species: Mus musculus
Genetic Insert: RIP
Vector Backbone Description: Backbone Size:4900; Vector Backbone:pGEX4T1; Vector Types:Bacterial Expression; Bacterial Resistance:Ampicillin
Defining Citation: PMID:16547522
Proper citation: RRID:Addgene_11972 Copy
Species: Mus musculus
Genetic Insert: c-jun promoter
Vector Backbone Description: Backbone Marker:Promega; Backbone Size:5000; Vector Backbone:pGL3; Vector Types:Mammalian Expression, Luciferase; Bacterial Resistance:Ampicillin
Defining Citation: PMID:9448004
Comments: Plasmids pJC6GL3, pJSXGL3, pJTXGL3, and pJSTXGL3 contain positions -225 to +150 of the murine c-jun promoter.
Proper citation: RRID:Addgene_11979 Copy
Species: Mus musculus
Genetic Insert: c-fos promoter
Vector Backbone Description: Backbone Size:5000; Vector Backbone:pGL3-promoter modified; Vector Types:Mammalian Expression; Bacterial Resistance:Ampicillin
Defining Citation: PMID:12944485
Comments: See author's map for cloning details.
Proper citation: RRID:Addgene_11983 Copy
Species: Mus musculus
Genetic Insert: c-jun promoter
Vector Backbone Description: Backbone Marker:Promega; Backbone Size:5000; Vector Backbone:pGL3; Vector Types:Mammalian Expression, Luciferase; Bacterial Resistance:Ampicillin
Defining Citation: PMID:9448004
Comments: Plasmids pJC6GL3, pJSXGL3, pJTXGL3, and pJSTXGL3 contain positions -225 to +150 of the murine c-jun promoter. pJSXGL3, pJTXGL3, and pJSTXGL3 contain mutations at the MEF2 site, ATF (AP1-like) site, and both sites, respectively.
Proper citation: RRID:Addgene_11980 Copy
Species: Mus musculus
Genetic Insert: c-jun promoter
Vector Backbone Description: Backbone Marker:Promega; Backbone Size:5000; Vector Backbone:pGL3; Vector Types:Mammalian Expression, Luciferase; Bacterial Resistance:Ampicillin
Defining Citation: PMID:9448004
Comments: Plasmids pJC6GL3, pJSXGL3, pJTXGL3, and pJSTXGL3 contain positions -225 to +150 of the murine c-jun promoter. pJSXGL3, pJTXGL3, and pJSTXGL3 contain mutations at the MEF2 site, ATF (AP1-like) site, and both sites, respectively.
Proper citation: RRID:Addgene_11982 Copy
Species: Mus musculus
Genetic Insert: c-jun promoter
Vector Backbone Description: Backbone Marker:Promega; Backbone Size:5000; Vector Backbone:pGL3; Vector Types:Mammalian Expression, Luciferase; Bacterial Resistance:Ampicillin
Defining Citation: PMID:9448004
Comments: Plasmids pJC6GL3, pJSXGL3, pJTXGL3, and pJSTXGL3 contain positions -225 to +150 of the murine c-jun promoter. pJSXGL3, pJTXGL3, and pJSTXGL3 contain mutations at the MEF2 site, ATF (AP1-like) site, and both sites, respectively.
Proper citation: RRID:Addgene_11981 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 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_119836 Copy
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