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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:5957; Vector Backbone:Clontech C8; Vector Types:Mammalian Expression; Bacterial Resistance:Kanamycin
Proper citation: RRID:Addgene_118749 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: Kif5a cDNA
Vector Backbone Description: Backbone Marker:Clontech; Backbone Size:5957; Vector Backbone:Clontech C4; Vector Types:Mammalian Expression; Bacterial Resistance:Kanamycin
Proper citation: RRID:Addgene_118745 Copy
Species: Mus musculus
Genetic Insert: eSOX17FNV
Vector Backbone Description: Vector Backbone:pMX; Vector Types:Mammalian Expression, Retroviral; Bacterial Resistance:Ampicillin
Defining Citation: PMID:30078555
Proper citation: RRID:Addgene_118838 Copy
Species: Mus musculus
Genetic Insert: eSOX17WHC
Vector Backbone Description: Vector Backbone:pMX; Vector Types:Mammalian Expression, Retroviral; Bacterial Resistance:Ampicillin
Defining Citation: PMID:30078555
Proper citation: RRID:Addgene_118839 Copy
Species: Mus musculus
Genetic Insert: eSox2AHK
Vector Backbone Description: Vector Backbone:pMX; Vector Types:Mammalian Expression, Retroviral; Bacterial Resistance:Ampicillin
Defining Citation: PMID:30078555
Proper citation: RRID:Addgene_118826 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: mPLD2-PX (62-192)
Vector Backbone Description: Vector Backbone:pGEX4T2; Vector Types:Bacterial Expression; Bacterial Resistance:Ampicillin
Defining Citation: PMID:30948714
Proper citation: RRID:Addgene_119128 Copy
Species: Mus musculus
Genetic Insert: mSNX13-PX (558-677)
Vector Backbone Description: Vector Backbone:pGEX4T2; Vector Types:Bacterial Expression; Bacterial Resistance:Ampicillin
Defining Citation: PMID:30948714
Comments: Amino Acid Sequence: GVPVIAQVVGTERYTSGSKVGTCTLYSVRLTHGDFTWTTKKKFRHFQELHRDLQRHKVLMSLLPLARFAVTHSPAREAAAEDIPSLPRGGSEGSARHTASKQKYLENYLNRLLTMSFYRNYHAMTEFLEVS
Proper citation: RRID:Addgene_119094 Copy
Species: Mus musculus
Genetic Insert: SNX19-PX (528-664)
Vector Backbone Description: Vector Backbone:pGEX4T2; Vector Types:Bacterial Expression; Bacterial Resistance:Ampicillin
Defining Citation: PMID:30948714
Comments: Amino Acid Sequence: SPDGPVVIQNLRITGTITAREHSGTGFHPYTLYTVKYETVLNGENSSGLQQLAYHTVNRRYREFLNLQTRLEEKPDLRKFIKNVKGPKKLFPDLPFGNMDSDRVEARKSLLESFLKQLCAIPEIGNSEEVQEFLALN
Proper citation: RRID:Addgene_119100 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: Vegfd
Vector Backbone Description: Vector Backbone:psubCMV-WPRE; Vector Types:AAV; Bacterial Resistance:Ampicillin
Proper citation: RRID:Addgene_119226 Copy
Species: Mus musculus
Genetic Insert: IL3SP-Vegfd-dNdC
Vector Backbone Description: Vector Backbone:psubCMV-WPRE; Vector Types:AAV; Bacterial Resistance:Ampicillin
Proper citation: RRID:Addgene_119229 Copy
Species: Mus musculus
Genetic Insert: Id1GFP + homology arms
Vector Backbone Description: Backbone Size:2700; Vector Backbone:pUC19; Vector Types:Reporter; Bacterial Resistance:Ampicillin
Defining Citation: PMID:31582374
Proper citation: RRID:Addgene_119282 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: beta Actin
Vector Backbone Description: Backbone Size:2905; Vector Backbone:PX551; Vector Types:Mouse Targeting, AAV, CRISPR; Bacterial Resistance:Ampicillin
Defining Citation: PMID:29056297
Proper citation: RRID:Addgene_119870 Copy
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