cDNA

eGFP-hCOL1A1

140110

eGFP-hCOL1A1

Ampicillin

Depositor confirms K206A point mutation does not appear to impact folding of the GFP, fluorescence or trafficking of the molecule in Saos-2 cells but cell-specific analysis may be conducted if desired. 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-Pro 1 (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 pro 2 might cause intracellular trafficking and degradation of some transfected chains as monomers without integration into normal heterotrimeric (pro 1)2pro 2 procollagen molecules,. Overexpression of exogenous pro 1 might result in excessive formation of homotrimeric (pro 1)3 molecules, some of which might contain fluorescent tags on two or even all three pro 1 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 M 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 (shakib.omari@nih.gov), Sergey Leikin (leikins@mail.nih.gov).

Expresses fluorescently-tagged human Type I procollagen 1 chain with eGFP replacing exon 2-3 retaining N-propeptide minor triple helix and N-terminal cleavage site

37

Origene

Unknown