The primary objective of this proposed program is to extend the life-span of normal human chondrocyte cell cultures to develop continuous lines for distribution to the research community and for tissue engineering. Some existing 50 chondrocytic strains are available for study. The consequence(s) of constitutive expression of human telomerase reverse transcriptase (hTERT) will be determined. The human cDNA clone for hTERT has been cloned in the retroviral expression system pLXSN for ready transfection to selected human cell strains. Introduction of this gene has been shown to immortalize cells of some systems. Reports with chondrocyte cell lines are needed. Functionality and doubling potential of chondrocytic lines will be assessed both before and after transfection. Histoenzymological, immunohistochemical, and molecular tests are proposed. The expression of hTERT will be determined using the TRAP (Telomeric Repeat Amplification Protocol) assay. To assess the functional consequences of the constitutive or periodic expression of hTERT, a series of structural and phenotypic tests will be performed. Positive controls will consist of related lines normal or immortalized in similar fashion but with the HPV16 E6/E7 genes (on hand). Thus, our cell line generation and engineering strategy should provide sets of potentially therapeutic populations for further developmental and transplantation research.
