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Related Experiment Videos

Efficient and stable expression of recombinant fibronectin polypeptides.

J E Schwarzbauer, R C Mulligan, R O Hynes

    Proceedings of the National Academy of Sciences of the United States of America
    |February 1, 1987
    PubMed
    Summary

    Researchers developed retroviral vectors to create cell lines producing functional fibronectin fragments (deminectins). These fragments integrate into the extracellular matrix, aiding fibronectin structure-function studies.

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    Area of Science:

    • Molecular Biology
    • Cell Biology
    • Biochemistry

    Background:

    • Fibronectin is a crucial extracellular matrix glycoprotein involved in cell adhesion, migration, and differentiation.
    • Understanding fibronectin structure-function relationships is essential for various biological processes and disease mechanisms.

    Purpose of the Study:

    • To develop a novel system for producing functionally active fibronectin fragments (deminectins) using retroviral expression vectors.
    • To generate and characterize NIH 3T3 cell lines stably producing variant forms of deminectins.
    • To facilitate the analysis of fibronectin structure-function relationships through recombinant protein expression.

    Main Methods:

    • Construction of retroviral expression vectors encoding fibronectin fragments (deminectins) fused with parathyroid hormone signal and pro-sequences.

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  • Generation of NIH 3T3 cell lines stably expressing these recombinant retroviruses.
  • Analysis of deminectin processing, secretion, dimerization, and incorporation into the extracellular matrix.
  • Creation of cDNA constructs for six variant fibronectin forms using retroviral vector-mediated splicing of genomic segments.

    • Main Results:

    • Stable NIH 3T3 cell lines producing functionally active fibronectin fragments (deminectins) were successfully generated.
    • Recombinant deminectins were processed, secreted, and formed disulfide-bonded dimers with themselves and endogenous fibronectin.
    • Fibronectin-deminectin heterodimers were incorporated into the extracellular matrix.
    • Six distinct variant forms of deminectin, reflecting alternatively spliced fibronectin variants, were produced.

    Conclusions:

    • Retroviral expression vectors provide an effective means to generate cell lines producing functional fibronectin fragments.
    • The developed deminectins are processed and secreted similarly to endogenous fibronectin, integrating into the extracellular matrix.
    • These recombinant fibronectin constructs offer a valuable tool for investigating fibronectin structure-function relationships and the role of alternative splicing.