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Species: Homo sapiens
Genetic Insert: Dronpa3
Vector Backbone Description: Backbone Size:2412; Vector Backbone:pFA6a; Vector Types:PCR Template; Bacterial Resistance:Ampicillin
Defining Citation: PMID:32406907
Comments: Template plasmid for C-terminal PCR tagging of mammalian genes. For M1 and M2 tagging oligo design please visit www.pcr-tagging.com Please visit https://www.biorxiv.org/content/early/2018/11/20/473876 for BioRxiv preprint
Proper citation: RRID:Addgene_120059 Copy
Species: Homo sapiens
Genetic Insert: N21 3'UTR
Vector Backbone Description: Backbone Marker:pIS2 derived from pRL-SV40 (Promega); Backbone Size:3700; Vector Backbone:pIS2; Vector Types:Mammalian Expression, Luciferase; Bacterial Resistance:Ampicillin
Defining Citation: PMID:16308420
Comments: CASC2 3'UTR in renilla luciferase reporter plasmid.
Proper citation: RRID:Addgene_12017 Copy
Species: Homo sapiens
Genetic Insert: N19 3'UTR
Vector Backbone Description: Backbone Marker:pIS2 derived from pRL-SV40 (Promega); Backbone Size:3700; Vector Backbone:pIS2; Vector Types:Mammalian Expression, Luciferase; Bacterial Resistance:Ampicillin
Defining Citation: PMID:16308420
Comments: NID1 3'UTR in renilla luciferase reporter plasmid.
Proper citation: RRID:Addgene_12016 Copy
Species: Homo sapiens
Genetic Insert: N18 3'UTR
Vector Backbone Description: Backbone Marker:pIS2 derived from pRL-SV40 (Promega); Backbone Size:3700; Vector Backbone:pIS2; Vector Types:Mammalian Expression, Luciferase; Bacterial Resistance:Ampicillin
Defining Citation: PMID:16308420
Comments: PDLIM5 3'UTR in renilla luciferase reporter plasmid.
Proper citation: RRID:Addgene_12015 Copy
Species: Homo sapiens
Genetic Insert: N17 3'UTR
Vector Backbone Description: Backbone Marker:pIS2 derived from pRL-SV40 (Promega); Backbone Size:3700; Vector Backbone:pIS2; Vector Types:Mammalian Expression, Luciferase; Bacterial Resistance:Ampicillin
Defining Citation: PMID:16308420
Comments: NEK6 3'UTR in renilla luciferase reporter plasmid.
Proper citation: RRID:Addgene_12014 Copy
Species: Homo sapiens
Genetic Insert: QKI 3'UTR
Vector Backbone Description: Backbone Marker:pIS2 derived from pRL-SV40 (Promega); Backbone Size:3700; Vector Backbone:pIS2; Vector Types:Mammalian Expression, Luciferase; Bacterial Resistance:Ampicillin
Defining Citation: PMID:16308420
Comments: QKI 3'UTR in renilla luciferase reporter plasmid.
Proper citation: RRID:Addgene_12011 Copy
Species: Homo sapiens
Genetic Insert: PRKD1 3'UTR
Vector Backbone Description: Backbone Marker:pIS2 derived from pRL-SV40 (Promega); Backbone Size:3700; Vector Backbone:pIS2; Vector Types:Mammalian Expression, Luciferase; Bacterial Resistance:Ampicillin
Defining Citation: PMID:16308420
Comments: PRKD1 3'UTR in renilla luciferase reporter plasmid.
Proper citation: RRID:Addgene_12010 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_120160 Copy
Species: Homo sapiens
Genetic Insert: Strep-II
Vector Backbone Description: Backbone Size:2412; Vector Backbone:pFA6a; Vector Types:PCR Template; Bacterial Resistance:Ampicillin
Defining Citation: PMID:32406907
Comments: Template plasmid for C-terminal PCR tagging of mammalian genes. For M1 and M2 tagging oligo design please visit www.pcr-tagging.com Please visit https://www.biorxiv.org/content/early/2018/11/20/473876 for BioRxiv preprint
Proper citation: RRID:Addgene_120040 Copy
Species: Homo sapiens
Genetic Insert: VSV-G
Vector Backbone Description: Backbone Size:2412; Vector Backbone:pFA6a; Vector Types:PCR Template; Bacterial Resistance:Ampicillin
Defining Citation: PMID:32406907
Comments: Template plasmid for C-terminal PCR tagging of mammalian genes. For M1 and M2 tagging oligo design please visit www.pcr-tagging.com Please visit https://www.biorxiv.org/content/early/2018/11/20/473876 for BioRxiv preprint
Proper citation: RRID:Addgene_120041 Copy
Species: Homo sapiens
Genetic Insert: CENPB poly(A) site
Vector Backbone Description: Vector Backbone:pCMV-GFP; Vector Types:Mammalian Expression; Bacterial Resistance:Ampicillin
Defining Citation: PMID:28525757
Proper citation: RRID:Addgene_120162 Copy
Species: Mus musculus
Genetic Insert: kinesin family member 5A
Vector Backbone Description: Backbone Marker:Clontech; Backbone Size:4727; Vector Backbone:pmCherry-C1; Vector Types:Mammalian Expression; Bacterial Resistance:Kanamycin
Defining Citation: PMID:31100061
Proper citation: RRID:Addgene_120163 Copy
Species: Homo sapiens
Genetic Insert: S-Tag
Vector Backbone Description: Backbone Size:2412; Vector Backbone:pFA6a; Vector Types:PCR Template; Bacterial Resistance:Ampicillin
Defining Citation: PMID:32406907
Comments: Template plasmid for C-terminal PCR tagging of mammalian genes. For M1 and M2 tagging oligo design please visit www.pcr-tagging.com Please visit https://www.biorxiv.org/content/early/2018/11/20/473876 for BioRxiv preprint
Proper citation: RRID:Addgene_120043 Copy
Species: Mus musculus
Genetic Insert: kinesin family member 1A
Vector Backbone Description: Backbone Marker:Clontech; Backbone Size:4727; Vector Backbone:pmCherry-C1; Vector Types:Mammalian Expression; Bacterial Resistance:Kanamycin
Defining Citation: PMID:31100061
Proper citation: RRID:Addgene_120165 Copy
Species: Homo sapiens
Genetic Insert: TagBFP
Vector Backbone Description: Backbone Size:2412; Vector Backbone:pFA6a; Vector Types:PCR Template; Bacterial Resistance:Ampicillin
Defining Citation: PMID:32406907
Comments: Template plasmid for C-terminal PCR tagging of mammalian genes. For M1 and M2 tagging oligo design please visit www.pcr-tagging.com Please visit https://www.biorxiv.org/content/early/2018/11/20/473876 for BioRxiv preprint
Proper citation: RRID:Addgene_120045 Copy
Species: Mus musculus
Genetic Insert: BICD cargo adaptor 2
Vector Backbone Description: Backbone Marker:Clontech; Backbone Size:4713; Vector Backbone:pmCherry-C1; Vector Types:Mammalian Expression; Bacterial Resistance:Kanamycin
Defining Citation: PMID:31100061
Proper citation: RRID:Addgene_120168 Copy
Species: Homo sapiens
Genetic Insert: eGFP
Vector Backbone Description: Backbone Size:2412; Vector Backbone:pFA6a; Vector Types:PCR Template; Bacterial Resistance:Ampicillin
Defining Citation: PMID:32406907
Comments: Template plasmid for C-terminal PCR tagging of mammalian genes. For M1 and M2 tagging oligo design please visit www.pcr-tagging.com Please visit https://www.biorxiv.org/content/early/2018/11/20/473876 for BioRxiv preprint
Proper citation: RRID:Addgene_120048 Copy
Species: Homo sapiens
Genetic Insert: 3xFLAG
Vector Backbone Description: Backbone Size:2412; Vector Backbone:pFA6a; Vector Types:PCR Template; Bacterial Resistance:Ampicillin
Defining Citation: PMID:32406907
Comments: Template plasmid for C-terminal PCR tagging of mammalian genes. For M1 and M2 tagging oligo design please visit www.pcr-tagging.com Please visit https://www.biorxiv.org/content/early/2018/11/20/473876 for BioRxiv preprint
Proper citation: RRID:Addgene_120030 Copy
Species: Homo sapiens
Genetic Insert: SNAP
Vector Backbone Description: Backbone Size:2412; Vector Backbone:pFA6a; Vector Types:PCR Template; Bacterial Resistance:Ampicillin
Defining Citation: PMID:32406907
Comments: Template plasmid for C-terminal PCR tagging of mammalian genes. For M1 and M2 tagging oligo design please visit www.pcr-tagging.com Please visit https://www.biorxiv.org/content/early/2018/11/20/473876 for BioRxiv preprint
Proper citation: RRID:Addgene_120033 Copy
Species: Homo sapiens
Genetic Insert: mAID-Clover
Vector Backbone Description: Backbone Size:2412; Vector Backbone:pFA6a; Vector Types:PCR Template; Bacterial Resistance:Ampicillin
Defining Citation: PMID:32406907
Comments: Template plasmid for C-terminal PCR tagging of mammalian genes. For M1 and M2 tagging oligo design please visit www.pcr-tagging.com Please visit https://www.biorxiv.org/content/early/2018/11/20/473876 for BioRxiv preprint
Proper citation: RRID:Addgene_120037 Copy
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