Establishment of an EB keratinocyte collection using Rho kinase chemical transformation (McLean-McGrath 1)Completed
|Project lead||Prof Irwin McLean & Prof John McGrath|
|Organisation||University of Dundee School of Medicine, Ninewells Hospital and Medical School|
|Project budget||GBP 67,007.00|
|Start date / Duration||01. Oct 2011 / 12 months|
|Funder(s) / Co-Funder(s)||DEBRA UK, MSAP/EBEP Recommended|
|Research area||Molecular therapy|
Short lay summary
Current methods to immortalize keratinocytes lead to genomic instability which is obviously undesirable. The Rho kinase (ROCK) inhibitor Y-27632 has been shown to extend the lifespan of keratinocytes essentially indefinitely, with genomic stability and retention of differentiation potential. The drawback is the cost (~€1200 per litre of culture medium). Dundee Drug Discovery Unit has developed a range of new, cheap ROCK inhibitors, such as DDD00033325. Here, we will test the best of these with comparison to Y-27632 for their ability to immortalize keratinocytes from EB patients and controls. These ROCK Inhibitor Transformed (ROCKIT) keratinocytes will be subjected to tests of genomic stability and differentiation potential and a protocol developed for routine use. A bank of ROCKIT cell lines will be developed from EB patients with a range of different mutations in the 14 EB causative genes, which will be distributed to researchers in the field. This work will provide a useful resource for the future study of EB pathomechanisms as well as therapy development.
The overall aim of this 24 month grant proposal was to develop a routine and cost effective method for producing long-term keratinocyte cell lines using a novel Rho kinase inhibitor (also known as ROCK inhibitor), ensure these were genetically “normal”, able to form 3D cultures and ultimately, to exploit this technology to produce banks of cultures able to support basic physiological studies and therapeutic developments in EB.