AVS 46th International Symposium
    Biomaterial Interfaces Group Wednesday Sessions
       Session BI-WeP

Paper BI-WeP4
Gene Expression in Reaction to Micro and Nano-topography

Wednesday, October 27, 1999, 5:30 pm, Room 4C

Session: Poster Session
Presenter: R. Hartley, University of Glasgow, Scotland, UK
Authors: R. Hartley, University of Glasgow, Scotland, UK
A.S.G. Curtis, University of Glasgow, Scotland, UK
Correspondent: Click to Email
Cellular reaction to surfaces has particular relevance to engineering tissue constructs. Advances in microfabrication enabling production of structures with defined surface topography have facilitated our understanding of cell elongation, orientation and movement. The vastly differing cell morphologies and cytoskeletal arrangement on planar and topographical surfaces necessitate an investigation of adhesion, signal transduction and transcriptional regulation. This work investigates gene transcription in reaction to micro and nano-topography. In this study we used two differing methods Differential Display RT-PCR to assess gene transcription. The first relies on large scale total RNA isolation following in-situ cell lysis or transferral to suspension and subsequent lysis. The second follows the Klebe method of RT-PCR without RNA isolation, where although RT-PCR is generally carried out and optimised for 250 cells, the protocol is suitable for four cells. This method has particular significance for analysis of gene expression on small areas where cell number is limited. Methods: Sub-confluent tendon epitenon were trypsinised and seeded directly onto topographic and control substrata. For large scale total RNA isolation directly in-situ, or in suspension, GITC phenol/chloroform was used and RNA equilibrated using 260/280nm absorbance. The Klebe method used a -70@super o@ C freeze/rapid-thaw in the presence of RNase inhibitor allowing time for cDNA library creation. In each case cDNA libraries were amplified by PCR in the presence of @super 32@P dATP, then denatured and run on a 7M urea, 5% acrylamide electrophoresis gel. Separated isotopically labelled ssDNAs were viewed by autoradiography.Phage display systems were also used.