Generation of full-length cDNA recombinant vectors for the transient expression of human fibronectin in mammalian cell lines.
In order to study the roles of the different alternatively spliced variants of human fibronectin (FN) as well as of its binding sites and structural domains in the processes of extracellular matrix assembly, cell adhesion, and cell migration, we constructed expression vectors coding for different human full-length FN polypeptides and deleted versions of these constructs. We expressed them transiently in mammalian cells by calcium phosphate transfection and microinjection techniques. While the deleted recombinants were expressed by both transfection and microinjection, the full-length recombinants could be only expressed by microinjection. Calcium phosphate transfection leads to the accumulation of recombinant FN in cytoplasmic vesicles of both matrix-forming and nonforming cells. On the contrary, microinjection leads to the accumulation of recombinant FN in cytoplasmic vesicles in cells that do not form a matrix, but to the rapid incorporation into the extracellular matrix of matrix-forming cells in addition to a cytoplasmic localization. Identical results were obtained when the FN signal and propeptides were replaced by those of E-cadherin.
Dufour
S
S
Laboratoire de Physiopathologie du Développement, CNRS URA 1337, Paris, France.
Gutman
A
A
Bois
F
F
Lamb
N
N
Thiery
J P
JP
Kornblihtt
A R
AR
D000818:Animals; D003001:Cloning, Molecular / Q000379:methods; D004247:DNA / Q000235:genetics; D005353:Fibronectins / Q000235:genetics* / Q000378:metabolism; D005455:Fluorescent Antibody Technique; D015870:Gene Expression; D005822:Genetic Vectors; D006801:Humans; D051379:Mice; D008845:Microinjections; D014162:Transfection
D005353:Fibronectins; D004247:DNA