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Species: Mus musculus
Genetic Insert: Evi-1
Vector Backbone Description: Vector Backbone:pLV-EF1a-PGK-BFPT2aPuro; Vector Types:Mammalian Expression, Lentiviral; Bacterial Resistance:Ampicillin
Proper citation: RRID:Addgene_101867 Copy
Species: Mus musculus
Genetic Insert: Mds1-Evi-1
Vector Backbone Description: Vector Backbone:pcDNA3.1; Vector Types:Mammalian Expression; Bacterial Resistance:Ampicillin
Proper citation: RRID:Addgene_101860 Copy
Species: Mus musculus
Genetic Insert: U6_sgRNA(Fgfr3)_U6_sgRNA(Tacc3)_CBh_FLAG-Cas9
Vector Backbone Description: Backbone Marker:Gene Transfer Vector Core University of Iowa; Backbone Size:5679; Vector Backbone:pacAd5; Vector Types:Adenoviral; Bacterial Resistance:Ampicillin
Defining Citation: PMID:28695888
Proper citation: RRID:Addgene_101824 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: Vrk2
Vector Backbone Description: Vector Backbone:pLV-EF1a-IRES-Puro; Vector Types:Lentiviral; Bacterial Resistance:Ampicillin
Defining Citation: PMID:31142202
Proper citation: RRID:Addgene_120240 Copy
Species: Mus musculus
Genetic Insert: Sec61B
Vector Backbone Description: Vector Backbone:pLV-EF1a-IRES-Puro; Vector Types:Lentiviral; Bacterial Resistance:Ampicillin
Defining Citation: PMID:31142202
Proper citation: RRID:Addgene_120244 Copy
Species: Mus musculus
Genetic Insert: Lem2
Vector Backbone Description: Vector Backbone:pLV-EF1a-IRES-Puro; Vector Types:Lentiviral; Bacterial Resistance:Ampicillin
Defining Citation: PMID:31142202
Proper citation: RRID:Addgene_120245 Copy
Species: Mus musculus
Genetic Insert: Mfsd10
Vector Backbone Description: Vector Backbone:pLV-EF1a-IRES-Puro; Vector Types:Lentiviral; Bacterial Resistance:Ampicillin
Defining Citation: PMID:31142202
Proper citation: RRID:Addgene_120238 Copy
Species: Mus musculus
Genetic Insert: Tmem128
Vector Backbone Description: Vector Backbone:pLV-EF1a-IRES-Puro; Vector Types:Lentiviral; Bacterial Resistance:Ampicillin
Proper citation: RRID:Addgene_120239 Copy
Species: Mus musculus
Genetic Insert: Arl6ip6
Vector Backbone Description: Vector Backbone:pLV-EF1a-IRES-Puro; Vector Types:Lentiviral; Bacterial Resistance:Ampicillin
Defining Citation: PMID:31142202
Proper citation: RRID:Addgene_120235 Copy
Species: Mus musculus
Genetic Insert: Smpd4
Vector Backbone Description: Vector Backbone:pLV-EF1a-IRES-Puro; Vector Types:Lentiviral; Bacterial Resistance:Ampicillin
Defining Citation: PMID:31142202
Proper citation: RRID:Addgene_120237 Copy
Species: Mus musculus
Genetic Insert: CAG:mCherry:P2A:HA_NLGN
Vector Backbone Description: Vector Backbone:paavCAG-Jx; Vector Types:AAV; Bacterial Resistance:Ampicillin
Proper citation: RRID:Addgene_120177 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: Lipin-1
Vector Backbone Description: Vector Backbone:pRK5; Vector Types:Mammalian Expression; Bacterial Resistance:Ampicillin
Defining Citation: PMID:30718857
Proper citation: RRID:Addgene_120278 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: sox11
Vector Backbone Description: Backbone Size:7424; Vector Backbone:pLenti CMV GFP; Vector Types:Mammalian Expression, Lentiviral; Bacterial Resistance:Ampicillin
Defining Citation: PMID:30401432
Proper citation: RRID:Addgene_120387 Copy
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