Differentiation potential of intestinal mesenchyme and its interaction with epithelial cells: a study using beta-galactosidase-expressing fibroblast lines.
Rat intestinal fibroblast lines (F1:G9 and A1:F1) differing in their potential to support intestinal mucosal development were marked with reporter genes to investigate their differentiation potential. The fibroblasts were transfected with plasmids expressing either beta-galactosidase (with or without a nuclear localisation signal) or green fluorescent protein (GFP). Transfection using Tfx50 or Fugene was more efficient than electroporation. The expression of beta-galactosidase was more stable and stronger than GFP. Cells were optimally labelled using the plasmid pL27B-GAL, and sub-clones with a strong and uniform nuclear expression of beta-galactosidase were isolated. These clones expressed beta-galactosidase even after prolonged passage in the absence of selection. The beta-galactosidase tagged lines (F1:G9gal and A1:F1gal) retained the morphological characteristics, viability and differentiation properties of the parental non-transfected lines. In co-culture with a colorectal tumour cell line Caco-2, the F1:G9gal and A1:F1gal cells differed in their morphological organisation but this did not change their expression of smooth muscle alpha-actin.
Subramanian
V
V
Department of Biology and Biochemistry, University of Bath, Claverton Down, Bath, BA2 7AY, UK.
Sneddon
S F
SF
Martin
L
L
Evans
G S
GS
D000818:Animals; D018938:Caco-2 Cells; D002450:Cell Communication; D002454:Cell Differentiation; D002455:Cell Division / Q000187:drug effects; D002460:Cell Line; D018920:Coculture Techniques; D004305:Dose-Response Relationship, Drug; D004815:Epidermal Growth Factor / Q000494:pharmacology; D004847:Epithelial Cells / Q000166:cytology*; D005347:Fibroblasts / Q000166:cytology / Q000378:metabolism; D015971:Gene Expression Regulation, Enzymologic; D049452:Green Fluorescent Proteins; D006493:Heparin / Q000494:pharmacology; D006801:Humans; D007422:Intestines / Q000166:cytology*; D008164:Luminescent Proteins / Q000235:genetics / Q000378:metabolism; D008648:Mesoderm / Q000166:cytology*; D008856:Microscopy, Fluorescence; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D013997:Time Factors; D014162:Transfection; D016212:Transforming Growth Factor beta / Q000494:pharmacology; D001616:beta-Galactosidase / Q000235:genetics / Q000378:metabolism
D008164:Luminescent Proteins; D011993:Recombinant Fusion Proteins; D016212:Transforming Growth Factor beta; D049452:Green Fluorescent Proteins; D004815:Epidermal Growth Factor; D006493:Heparin; D001616:beta-Galactosidase