eGFP-proα2(I)
(Plasmid #119826)

• Purpose: Expresses mouse Type I procollagen α2 chain (Col1a2) with GFP between signal sequence and exon 6
• Depositing Lab: Sergey Leikin
• Publication: Omari et al Proc Natl Acad Sci U S A. 2018 Oct 23;115(43):E10099-E10108. doi: 10.1073/pnas.1814552115. Epub 2018 Oct 4.

Backbone

• Vector backbone: pcDNA3.1
• Backbone size w/o insert (bp): 5400
• Total vector size (bp): 10153
• Vector type: Mammalian Expression
• Selectable markers: Neomycin (select with G418)

Growth in Bacteria

• Bacterial Resistance(s): Ampicillin, 100 μg/mL
• Growth Temperature: 37°C
• Growth Strain(s): DH5alpha
• Growth instructions: Originally grown in One Shot TOP10 Chemically competent E. coli
• Copy number: Unknown

Gene/Insert

• Gene/Insert name: Type I procollagen α2 chain
• Alt name: Col1a2
• Species: M. musculus (mouse)
• Insert Size (bp): 4737
• Mutation: Col1a2 exons 2-5 replaced by fluorescent tag
• GenBank ID: NM_007743
• Entrez Gene: Col1a2 (a.k.a. Col1a-2, Cola-2, Cola2, oim)
• Promoter: CMV
• Tag / Fusion Protein:
  • eGFP (N terminal on insert)

Cloning Information

• Cloning method: Restriction Enzyme
• 5′ cloning site: HindIII, BamHI (not destroyed)
• 3′ cloning site: NotI (not destroyed)
• 5′ sequencing primer: CGC AAA TGG GCG GTA GGC GTG
• 3′ sequencing primer: TAG AAG GCA CAG TCG AGG

Resource Information

• Supplemental Documents:
  • eGFP-Col1a2.gbk

Terms and Licenses

• Academic/Nonprofit Terms:
  • UBMTA
• Industry Terms:
  • Not Available to Industry

Depositor 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 () Sergey Leikin ()