Dharmavaram R M, Baldwin C T, Reginato A M, Jimenez S A
Department of Medicine, Jefferson Medical College, Thomas Jefferson University, Philadelphia, PA 19107.
Matrix. 1993 Mar;13(2):125-33. doi: 10.1016/s0934-8832(11)80071-5.
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.
在遗传性骨关节炎（OA）和其他遗传性软骨疾病中，由于难以获取足够的组织来分离RNA，以及体外软骨细胞培养过程中软骨表型的丧失，使得鉴定软骨特异性胶原基因中的突变受到阻碍。为了克服这些限制，我们采用RNA聚合酶链反应（PCR）从少量软骨细胞获得的mRNA中扩增软骨特异性II型、IX型和XI型胶原的cDNA。我们还从爱泼斯坦-巴尔病毒（EBV）转化的淋巴细胞的“非法转录本”中扩增了这些胶原的cDNA。总RNA从新鲜分离的人胎儿和成人软骨细胞以及长期（90天）软骨细胞培养物中获得。RNA被逆转录为cDNA，并在同一管中用针对II型、IX型和XI型胶原的寡核苷酸引物扩增cDNA。扩增的双链产物被克隆并测序。从新鲜分离的人胎儿软骨细胞中低至6 ng的RNA成功扩增出了完整的4.4 kb II型前胶原cDNA。还从这些细胞中扩增出了780个碱基对的α1（IX）胶原cDNA和已发表的α2（XI）胶原cDNA的完整序列。我们还能够从EBV转化的淋巴细胞的“非法转录本”中扩增出三种软骨特异性胶原的cDNA。因此，这些方法将有助于从少量软骨或软骨细胞RNA或非软骨来源中鉴定遗传性OA和其他遗传性软骨疾病患者软骨特异性胶原基因中的突变。
