Stable transfection of human fetal chondrocytes with a type II procollagen minigene: expression of the mutant protein and alterations in the structure of the extracellular matrix in vitro.
OBJECTIVE
To perform stable transfections of human chondrocytes under conditions that allow the maintenance of the chondrocyte-specific phenotype, and to examine the effects of the stable transfection of a mutated type II procollagen gene (COL2A1) on the structure of the cartilaginous extracellular matrix produced in vitro.


METHODS
A type II procollagen minigene that lacks exons 16-27 was stably transfected into human fetal epiphyseal chondrocytes in vitro. Expression of the minigene was detected by reverse transcriptase-polymerase chain reaction, and the encoded protein was identified by Western blot with a human type II collagen-specific antibody. The cartilaginous extracellular matrix produced by the cultured transfected chondrocytes was characterized using histochemical staining, polarized light microscopy analysis, and transmission electron microscopy.


RESULTS
A shortened type II collagen encoded by the transfected minigene was biosynthesized and produced in the cultures of transfected cells. Histologic analyses demonstrated a more dense, negatively charged cartilaginous matrix in control cultures. In contrast, COL2A1 minigene-transfected cultures were more cellular, were populated with cells of irregular shape and less-chondrocytic appearance, contained abundant intracellular dense granules, and were surrounded by a less-dense matrix. Polarized light microscopy and transmission electron microscopy revealed a well-organized collagen fibrillar matrix in untransfected, control chondrocyte cultures, while the matrix in the transfected cultures was less birefringent and contained numerous truncated collagen fibrils.


CONCLUSIONS
The findings illustrate the feasibility of gene transfer into human fetal chondrocytes under conditions that allow the preservation of their specific phenotype, and also shed light on the function of type II collagen in the maintenance of the structural integrity of articular cartilage matrix.
Dharmavaram
R M
RM
Jefferson Medical College, Thomas Jefferson University, Philadelphia, Pennsylvania 19107-5541, USA.
Liu
G
G
Tuan
R S
RS
Stokes
D G
DG
Jiménez
S A
SA
D016475:3T3 Cells / Q000378:metabolism / Q000473:pathology / Q000648:ultrastructure; D000818:Animals; D015153:Blotting, Western; D002358:Cartilage, Articular / Q000166:cytology* / Q000196:embryology*; D005109:Extracellular Matrix / Q000737:chemistry / Q000648:ultrastructure; D005333:Fetus / Q000378:metabolism; D006801:Humans; D051379:Mice; D009154:Mutation; D011347:Procollagen / Q000235:genetics*; D012313:RNA / Q000302:isolation & purification; D020133:Reverse Transcriptase Polymerase Chain Reaction; D014162:Transfection
D011347:Procollagen; D012313:RNA