Optimization of a 96-well electroporation assay for postnatal rat CNS neurons suitable for cost effective medium-throughput screening of genes that promote neurite outgrowth

被引：8

作者：

Hutson, Thomas H. ^{[1
]}

Buchser, William J. ^{[2
]}

Bixby, John L. ^{[2
]}

Lemmon, Vance P. ^{[2
]}

Moon, Lawrence D. F. ^{[1
]}

机构：

[1] Kings Coll London, Neurorestorat Grp, Wolfson Ctr Age Related Dis, London SE1 1UL, England

[2] Univ Miami, Miller Sch Med, Miami Project Cure Paralysis, Miami, FL 33136 USA

来源：

FRONTIERS IN MOLECULAR NEUROSCIENCE | 2011年 / 4卷

基金：

英国生物技术与生命科学研究理事会;

关键词：

electroporation; 96-well; neurite outgrowth; transfection; CNS neuron; neuronal regeneration; RAG; inhibitory substrate;

D O I：

10.3389/fnmol.2011.00055

中图分类号：

Q189 [神经科学];

学科分类号：

071006 ;

摘要：

Following an injury, central nervous system (CNS) neurons show a very limited regenerative response which results in their failure to successfully form functional connections with their original target. This is due in part to the reduced intrinsic growth state of CNS neurons, which is characterized by their failure to express key regeneration-associated genes (RAGs) and by the presence of growth inhibitory molecules in CNS environment that form a molecular and physical barrier to regeneration. Here we have optimized a 96-well electroporation and neurite outgrowth assay for postnatal rat cerebellar granule neurons (CGNs) cultured upon an inhibitory cellular substrate expressing myelin-associated glycoprotein or a mixture of growth inhibitory chondroitin sulfate proteoglycans. Optimal electroporation parameters resulted in 28% transfection efficiency and 51% viability for postnatal rat CGNs. The neurite outgrowth of transduced neurons was quantitatively measured using a semi-automated image capture and analysis system. The neurite outgrowth was significantly reduced by the inhibitory substrates which we demonstrated could be partially reversed using a Rho Kinase inhibitor. We are now using this assay to screen large sets of RAGs for their ability to increase neunte outgrowth on a variety of growth inhibitory and permissive substrates.

引用

页数：11