Studying Tuftelin gene function using RNAi

Tuftelin has been suggested to play an important role during the development and mineralization of enamel. Recently, the mouse genome was published, revealing that unlike in the human genome, in the mouse genome the tuftelin gene was duplicated. The duplicated gene, mapped about 100Kb distal to the full size tuftelin gene, was characterized. It contains the first 6 exons (out of 13 in the full length gene), including 6 base changes. We used the RNA interference (RNAi) methodology for down regulation of tuftelin expression in the ameloblast cell line LS8 (Chen et al, 1992) (kindly supplied by Dr. Snead, USC). Four different RNAi targeting probes for: (a) both tuftelin full gene and duplicate (pSuperTuft1), (b) tuftelin full gene only (pSuperTuft2), (c) duplicate gene only (pSuperTuft3) and (d) scrambled negative control plasmid (pSuperTuftSc) were designed and ligated into the pSuper-neo plasmid. The plasmids were characterized by sequencing and were transfected into LS8 ameloblast-lineage cell line. Objectives: of the present project is to study the effect of tuftelin down regulation on LS8 ameloblast cell line, which we previously found to express tuftelin. Methods: Tuftelin RNAi plasmids (pSuperTuft1,2,3 and Sc) were transfected into LS8 cells, and positive clones were selected using G418. Quantification of tuftelin mRNA was established using Real-Time quantitative PCR. Results: Intial results showed maximum tuftelin down regulation of 84% using pSuperTuft1 relative to pSuperTuftSc. Conclusions: Tuftelin down regulation was established in LS8 ameloblast cell line. Future results will provide a better basis for understanding the function(s) of tuftelin.

This work was supported by Bi-national USA-Israel Science Foundation (BSF) grant no. 2003239