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Species: Homo sapiens
Genetic Insert: gRNA1+scaffold+H1promoter+gRNA2
Vector Backbone Description: Vector Backbone:pDECKO_mCherry; Vector Types:Lentiviral; Bacterial Resistance:Ampicillin
Defining Citation: PMID:35619054
Proper citation: RRID:Addgene_140107 Copy
Species: Homo sapiens
Genetic Insert: Type I procollagen α1
Vector Backbone Description: Vector Backbone:pcDNA3.1; Vector Types:Mammalian Expression; Bacterial Resistance:Ampicillin
Comments: These human type I procollagen plasmids were designed and validated for human osteoblasts and fibroblasts similar to their previously published/shared murine counterparts e.g. eGFP-Proα1 (Plasmid #119843) https://www.addgene.org/Sergey_Leikin/. Their expression in other cells might disrupt assembly, folding and trafficking of type I procollagen, resulting in artifacts. Even in human osteoblasts and fibroblasts, overexpression of exogenous proα2 might cause intracellular trafficking and degradation of some transfected chains as monomers without integration into normal heterotrimeric (proα1)2proα2 procollagen molecules,. Overexpression of exogenous proα1 might result in excessive formation of homotrimeric (proα1)3 molecules, some of which might contain fluorescent tags on two or even all three proα1 chains, potentially causing severe disruptions in cellular function.
In our experience with the murine plasmids expressed in primary osteoblasts or the MC3T3 osteoblastic cell line, the best evidence of normal behavior of the transfected chains is the appearance of fluorescent extracellular collagen fibers ~ 12-24 h after transfection. These were harder to observe in human Saos2 osteoblast cell line transfected with the human type I procollagen plasmids (likely due to slower fibrillogenesis and more complete cleavage of the fluorescently-tagged N-propeptide prior to collagen fibril formation).
We strongly recommend following these guidelines when using the plasmids
1) Only human cells that have high expression of endogenous type I procollagen and that express fewer transfected than endogenous chains 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) Stable transfection causes excessive accumulation of tagged chains in the ER over time, disruption of ER homeostasis, and disruptions and changes in cellular function. Therefore, low to moderate transient transfection is recommended for studies of physiologically relevant processes. Optimization of transfection efficiency is needed for all cell types.
4) Experiments beyond 24h after transfection are not recommended, to avoid the excessive accumulation of tagged chains in the ER and cell malfunction caused by increased procollagen synthesis.
5) 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_140111 Copy
Species: Homo sapiens
Genetic Insert: Type I procollagen α1
Vector Backbone Description: Vector Backbone:pcDNA3.1; Vector Types:Mammalian Expression; Bacterial Resistance:Ampicillin
Comments: These human type I procollagen plasmids were designed and validated for human osteoblasts and fibroblasts similar to their previously published/shared murine counterparts e.g. eGFP-Proα1 (Plasmid #119843) https://www.addgene.org/Sergey_Leikin/. Their expression in other cells might disrupt assembly, folding and trafficking of type I procollagen, resulting in artifacts. Even in human osteoblasts and fibroblasts, overexpression of exogenous proα2 might cause intracellular trafficking and degradation of some transfected chains as monomers without integration into normal heterotrimeric (proα1)2proα2 procollagen molecules,. Overexpression of exogenous proα1 might result in excessive formation of homotrimeric (proα1)3 molecules, some of which might contain fluorescent tags on two or even all three proα1 chains, potentially causing severe disruptions in cellular function.
In our experience with the murine plasmids expressed in primary osteoblasts or the MC3T3 osteoblastic cell line, the best evidence of normal behavior of the transfected chains is the appearance of fluorescent extracellular collagen fibers ~ 12-24 h after transfection. These were harder to observe in human Saos2 osteoblast cell line transfected with the human type I procollagen plasmids (likely due to slower fibrillogenesis and more complete cleavage of the fluorescently-tagged N-propeptide prior to collagen fibril formation).
We strongly recommend following these guidelines when using the plasmids
1) Only human cells that have high expression of endogenous type I procollagen and that express fewer transfected than endogenous chains 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) Stable transfection causes excessive accumulation of tagged chains in the ER over time, disruption of ER homeostasis, and disruptions and changes in cellular function. Therefore, low to moderate transient transfection is recommended for studies of physiologically relevant processes. Optimization of transfection efficiency is needed for all cell types.
4) Experiments beyond 24h after transfection are not recommended, to avoid the excessive accumulation of tagged chains in the ER and cell malfunction caused by increased procollagen synthesis.
5) 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_140112 Copy
Species: Homo sapiens
Genetic Insert: gRNA1+scaffold+H1promoter+gRNA2
Vector Backbone Description: Vector Backbone:pDECKO_mCherry; Vector Types:Lentiviral; Bacterial Resistance:Ampicillin
Defining Citation: PMID:35619054
Proper citation: RRID:Addgene_140099 Copy
Species: Homo sapiens
Genetic Insert: Wnt10B
Vector Backbone Description: Backbone Marker:Invitrogen; Backbone Size:2580; Vector Backbone:pENTR/D-TOPO; Vector Types:Gateway Cloning; Bacterial Resistance:Kanamycin
Defining Citation: PMID:22784633
Comments: Please note that the ORFs in this collection were cloned open-ended (without a stop codon).
Proper citation: RRID:Addgene_34748 Copy
Species: Homo sapiens
Genetic Insert: PRMT7
Vector Backbone Description: Backbone Marker:Amersham; Backbone Size:5000; Vector Backbone:pGEX(SN); Vector Types:Bacterial Expression; Bacterial Resistance:Ampicillin
Defining Citation: PMID:15044439
Proper citation: RRID:Addgene_34695 Copy
Species: Homo sapiens
Genetic Insert: PRMT7
Vector Backbone Description: Backbone Marker:Amersham; Backbone Size:5000; Vector Backbone:pGEX(SN); Vector Types:Bacterial Expression; Bacterial Resistance:Ampicillin
Defining Citation: PMID:15044439
Proper citation: RRID:Addgene_34694 Copy
Species: Homo sapiens
Genetic Insert: PRMT7
Vector Backbone Description: Backbone Marker:Amersham; Backbone Size:5000; Vector Backbone:pGEX(SN); Vector Types:Bacterial Expression; Bacterial Resistance:Ampicillin
Defining Citation: PMID:15044439
Proper citation: RRID:Addgene_34693 Copy
Species: Homo sapiens
Genetic Insert: TNKS2-M1054V
Vector Backbone Description: Backbone Marker:Sigma; Backbone Size:4700; Vector Backbone:pFLAG-CMV2; Vector Types:Mammalian Expression; Bacterial Resistance:Ampicillin
Defining Citation: PMID:11802774
Proper citation: RRID:Addgene_34692 Copy
Species: Homo sapiens
Genetic Insert: Sorting nexin 9
Vector Backbone Description: Backbone Marker:Novagen; Vector Backbone:pET15b; Vector Types:Bacterial Expression; Bacterial Resistance:Ampicillin
Defining Citation: PMID:17988218
Proper citation: RRID:Addgene_34690 Copy
Species: Homo sapiens
Genetic Insert: dynamin 1ba
Vector Backbone Description: Vector Backbone:pcDNA3.1; Vector Types:Mammalian Expression; Bacterial Resistance:Ampicillin
Proper citation: RRID:Addgene_34683 Copy
Species: Homo sapiens
Genetic Insert: dynamin 1
Vector Backbone Description: Vector Backbone:pUHD10-3; Vector Types:Mammalian Expression; Bacterial Resistance:Ampicillin
Defining Citation: PMID:7559787
Proper citation: RRID:Addgene_34688 Copy
Species: Homo sapiens
Genetic Insert: dynamin 1ba
Vector Backbone Description: Vector Backbone:pcDNA3.1; Vector Types:Mammalian Expression; Bacterial Resistance:Ampicillin
Proper citation: RRID:Addgene_34682 Copy
Species: Homo sapiens
Genetic Insert: dynamin 1ba
Vector Backbone Description: Vector Backbone:pEGFP-N1; Vector Types:Mammalian Expression; Bacterial Resistance:Kanamycin
Defining Citation: PMID:15004222
Proper citation: RRID:Addgene_34681 Copy
Species: Homo sapiens
Genetic Insert: PTD012
Vector Backbone Description: Backbone Size:4700; Vector Backbone:pQTEV; Vector Types:Bacterial Expression; Bacterial Resistance:Ampicillin
Defining Citation: PMID:15998469
Comments: The vector pQTEV is derived from Qiagen pQE-2 and contains an inactive chloramphenicol resistance gene sequence.
The Rosetta bacterial strain contains an additional plasmid. The second plasmid, pRARE, provides rare tRNAs for overexpression of recombinant proteins. It has 4694 bp and is chloramphenicol resistence.
Proper citation: RRID:Addgene_34797 Copy
Species: Homo sapiens
Genetic Insert: CD68 promoter/enhancer -680/+140
Vector Backbone Description: Backbone Marker:Invitrogen; Vector Backbone:pcDNA3; Vector Types:Mammalian Expression; Bacterial Resistance:Ampicillin
Defining Citation: PMID:11907098
Comments: The CD68 promoter/enhancer in this construct consists of ~680bp 5' of the ATG and the 83bp first intron of human CD68.
Please note that Addgene's sequencing results found a single nucleotide deletion at bp# 440 when compared to the full plasmid sequence provided by the depositor. The depositing scientist states that it is probably a polymorphism in the promoter and is not a concern for the function of the plasmid.
Proper citation: RRID:Addgene_34837 Copy
Species: Homo sapiens
Genetic Insert: HORMA domain containing protein
Vector Backbone Description: Backbone Size:4700; Vector Backbone:pQTEV; Vector Types:Bacterial Expression; Bacterial Resistance:Ampicillin
Defining Citation: PMID:15998469
Comments: The vector pQTEV is derived from Qiagen pQE-2 and contains an inactive chloramphenicol resistance gene sequence.
The Rosetta bacterial strain contains an additional plasmid. The second plasmid, pRARE, provides rare tRNAs for overexpression of recombinant proteins. It has 4694 bp and is chloramphenicol resistence.
Proper citation: RRID:Addgene_34821 Copy
Species: Homo sapiens
Genetic Insert: TFAM
Vector Backbone Description: Backbone Marker:Novagen; Backbone Size:5369; Vector Backbone:pET28a; Vector Types:Bacterial Expression; Bacterial Resistance:Kanamycin
Defining Citation: PMID:22037171
Proper citation: RRID:Addgene_34705 Copy
Species: Homo sapiens
Genetic Insert: epithelial stromal interaction 1
Vector Backbone Description: Backbone Size:4700; Vector Backbone:pQTEV; Vector Types:Bacterial Expression; Bacterial Resistance:Ampicillin
Defining Citation: PMID:15998469
Comments: The vector pQTEV is derived from Qiagen pQE-2 and contains an inactive chloramphenicol resistance gene sequence.
The Rosetta bacterial strain contains an additional plasmid. The second plasmid, pRARE, provides rare tRNAs for overexpression of recombinant proteins. It has 4694 bp and is chloramphenicol resistence.
Proper citation: RRID:Addgene_34826 Copy
Species: Homo sapiens
Genetic Insert: phytanoyl-CoA hydroxylase interacting protein-like
Vector Backbone Description: Backbone Size:4700; Vector Backbone:pQTEV; Vector Types:Bacterial Expression; Bacterial Resistance:Ampicillin
Defining Citation: PMID:15998469
Comments: The vector pQTEV is derived from Qiagen pQE-2 and contains an inactive chloramphenicol resistance gene sequence.
The Rosetta bacterial strain contains an additional plasmid. The second plasmid, pRARE, provides rare tRNAs for overexpression of recombinant proteins. It has 4694 bp and is chloramphenicol resistence.
Proper citation: RRID:Addgene_34823 Copy
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