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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_119844 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_119842 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_119840 Copy
Species: Mus musculus
Genetic Insert: Mouse histone H3-CFP-FHA2-histone H3 peptide (1-14aa) S10A-YFP
Vector Backbone Description: Backbone Marker:Invitrogen; Backbone Size:5368; Vector Backbone:pcDNA3.1; Vector Types:Mammalian Expression; Bacterial Resistance:Ampicillin
Defining Citation: PMID:30478057
Proper citation: RRID:Addgene_120810 Copy
Species: Mus musculus
Genetic Insert: PACT
Vector Backbone Description: Vector Backbone:pSV40_HA; Vector Types:Mammalian Expression; Bacterial Resistance:Ampicillin
Proper citation: RRID:Addgene_120537 Copy
Species: Mus musculus
Genetic Insert: TARBP2
Vector Backbone Description: Vector Backbone:pSV40_HA; Vector Types:Mammalian Expression; Bacterial Resistance:Ampicillin
Proper citation: RRID:Addgene_120539 Copy
Species: Mus musculus
Genetic Insert: Truncated YPet-HP1-EV linker-ECFP-mouse histone H3
Vector Backbone Description: Backbone Size:4717; Vector Backbone:pCAGGS vector; Vector Types:Mammalian Expression; Bacterial Resistance:Ampicillin
Defining Citation: PMID:30478057
Proper citation: RRID:Addgene_120802 Copy
Species: Mus musculus
Genetic Insert: Truncated YPet-HP1-EV linker-ECFP-mouse histone H3(K9L mutation)
Vector Backbone Description: Backbone Size:4717; Vector Backbone:pCAGGS vector; Vector Types:Mammalian Expression; Bacterial Resistance:Ampicillin
Defining Citation: PMID:30478057
Proper citation: RRID:Addgene_120807 Copy
Species: Mus musculus
Genetic Insert: Mouse histone H3-CFP-FHA2-histone H3 peptide (1-14aa)-YFP
Vector Backbone Description: Backbone Marker:Invitrogen; Backbone Size:5368; Vector Backbone:pcDNA3.1; Vector Types:Mammalian Expression; Bacterial Resistance:Ampicillin
Defining Citation: PMID:30478057
Proper citation: RRID:Addgene_120809 Copy
Species: Mus musculus
Genetic Insert: Renilla luciferase
Vector Backbone Description: Vector Backbone:phRL-SV40; Vector Types:Luciferase; Bacterial Resistance:Ampicillin
Comments: luciferase is fused with Elavl2 gene
Proper citation: RRID:Addgene_120521 Copy
Species: Mus musculus
Genetic Insert: OKMS cassette
Vector Backbone Description: Vector Backbone:PB-TAC-Cas9; Vector Types:Mammalian Expression, CRISPR, piggyBac transposon; Bacterial Resistance:Ampicillin
Defining Citation: PMID:30639212
Comments: Please Note: Addgene NGS is unable to fully resolve the CAG promoter sequence. Please refer to depositor's provided sequence for this section of the plasmid.
Proper citation: RRID:Addgene_120353 Copy
Species: Mus musculus
Genetic Insert: OK+9MS cassette
Vector Backbone Description: Vector Backbone:PB-TAC-Cas9; Vector Types:Mammalian Expression, CRISPR, piggyBac transposon; Bacterial Resistance:Ampicillin
Defining Citation: PMID:30639212
Proper citation: RRID:Addgene_120354 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: 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: 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: 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: Mus musculus
Genetic Insert: Lipin-1
Vector Backbone Description: Vector Backbone:pRK5; Vector Types:Mammalian Expression; Bacterial Resistance:Ampicillin
Defining Citation: PMID:30718857
Proper citation: RRID:Addgene_120277 Copy
Species: Mus musculus
Genetic Insert: Dicer
Vector Backbone Description: Vector Backbone:pEF1alpha.Blast; Vector Types:Mammalian Expression; Bacterial Resistance:Ampicillin
Proper citation: RRID:Addgene_120541 Copy
Species: Mus musculus
Genetic Insert: TBC1D15
Vector Backbone Description: Backbone Marker:Invitrogen; Backbone Size:5800; Vector Backbone:pEF6/V5-His; Vector Types:Mammalian Expression; Bacterial Resistance:Ampicillin
Defining Citation: PMID:20363736
Comments: Please note- The TBC1D15 mutation S616N identified during Addgene's quality control process does not affect plasmid function.
Proper citation: RRID:Addgene_79148 Copy
Species: Mus musculus
Genetic Insert: Arf3
Vector Backbone Description: Backbone Marker:Invitrogen; Vector Backbone:pcDNA3; Vector Types:Mammalian Expression; Bacterial Resistance:Ampicillin
Defining Citation: PMID:22971977
Proper citation: RRID:Addgene_79415 Copy
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