Amplification of cDNAs for human cartilage-specific types II, IX and XI collagens from chondrocytes and Epstein-Barr virus-transformed lymphocytes.
The identification of mutations in cartilage-specific collagen genes in inherited forms of osteoarthritis (OA) and other heritable cartilage diseases has been hampered by the difficulty in obtaining sufficient tissue to isolate RNA and by the loss of the cartilage phenotype during in vitro chondrocyte culture. To overcome these limitations we employed RNA Polymerase Chain Reaction (PCR) to amplify cDNAs for the cartilage-specific collagens types II, IX and XI from mRNA obtained from small numbers of chondrocytes. We also amplified cDNAs for these collagens from "illegitimate transcripts" from Epstein-Barr virus (EBV)-transformed lymphocytes. Total RNA was obtained from freshly isolated human fetal and adult chondrocytes and from long-term (90 days) chondrocyte cultures. The RNA was reverse transcribed to cDNA and the cDNA amplified in the same tube with oligonucleotide primers specific for types II, IX and XI collagens. The amplified double-stranded products were cloned and sequenced. Successful amplification of the entire 4.4 kb of the type II procollagen cDNA was obtained from as little as 6 ng of RNA from freshly isolated fetal human chondrocytes. Seven hundred and eighty base pairs of the alpha 1 (IX) collagen cDNA and the entire published sequence of alpha 2 (XI) collagen cDNA, were also amplified from these cells. We were also able to amplify cDNAs for the three cartilage-specific collagens from "illegitimate transcripts" from EBV-transformed lymphocytes. Thus, these methods will allow the identification of mutations in cartilage-specific collagen genes in patients with inherited OA and other heritable cartilage diseases from small amounts of cartilage or chondrocyte RNA or from non-cartilaginous sources.
Department of Medicine, Jefferson Medical College, Thomas Jefferson University, Philadelphia, PA 19107.
R M
RM
Dharmavaram
C T
CT
Baldwin
A M
AM
Reginato
S A
SA
Jimenez
D001483:Base Sequence; D002356:Cartilage / Q000166:cytology / Q000378:metabolism*; D002461:Cell Line, Transformed; D003094:Collagen / Q000145:classification / Q000235:genetics / Q000378:metabolism*; D004247:DNA / Q000235:genetics*; D004854:Herpesvirus 4, Human; D006801:Humans; D008214:Lymphocytes / Q000378:metabolism*; D008969:Molecular Sequence Data; D016133:Polymerase Chain Reaction
D003094:Collagen; D004247:DNA