Expression of extracellular matrix genes by cultured human cells: localization of messenger RNAs and antigenic epitopes.
Simplified and expedient methodologies for examination of cellular gene expression at the mRNA and protein levels, utilizing in situ hybridization and peroxidase-anti-peroxidase immunodetection, were developed. These techniques were first optimized for the detection of extracellular matrix genes expressed by cultured human skin fibroblasts and keratinocytes, the two principal cell types of human skin. In situ hybridizations and Northern transfer analyses with human-sequence-specific cDNAs encoding collagenous and noncollagenous protein sequences demonstrated selective expression of different matrix genes by these two cell types, indicating different biosynthetic capacities of these cells and attesting to the specificity of the hybridizations. The utility of in situ hybridization was also demonstrated in mixed primary cell cultures established from cutaneous neurofibromas consisting of Schwann cells, perineurial cells, and fibroblasts. The methodologies developed here were further utilized for simultaneous detection of fibronectin mRNA and immunoreactive protein in fibroblast cultures. This procedure allowed detection of grains representative of radioactively labeled cDNA-mRNA hybrids and protein epitopes, as visualized by peroxidase-anti-peroxidase immunodetection on the same cells. This methodology, with appropriate modifications, may be applicable to other cell types as well as tissue specimens.
Peltonen
J
J
Department of Dermatology, Jefferson Medical College, Thomas Jefferson University, Philadelphia, Pennsylvania 19107.
Jaakkola
S
S
Gay
K
K
Olsen
D R
DR
Chu
M L
ML
Uitto
J
J
D002478:Cells, Cultured; D015342:DNA Probes; D000078404:Epidermal Cells; D000939:Epitopes / Q000032:analysis*; D005109:Extracellular Matrix / Q000032:analysis*; D005347:Fibroblasts / Q000032:analysis; D005353:Fibronectins / Q000032:analysis; D005786:Gene Expression Regulation; D006801:Humans; D007124:Immunoenzyme Techniques; D007633:Keratins / Q000235:genetics; D007797:Laminin / Q000032:analysis; D009693:Nucleic Acid Hybridization; D011347:Procollagen / Q000032:analysis; D012333:RNA, Messenger / Q000032:analysis*
D015342:DNA Probes; D000939:Epitopes; D005353:Fibronectins; D007797:Laminin; D011347:Procollagen; D012333:RNA, Messenger; D007633:Keratins