PURPOSE: Molecular analysis of complex phenomena, such as selective death of photoreceptors and their rescue by neuro-protective agents, has been hindered by limitations of techniques for investigating gene expression in individual cells within a heterogeneous tissue such as the retina. The purpose of this study was to develop methods to assess gene expression in single retinal cells. METHODS: Individual cells from papain-dissociated mouse retinae were captured with micropipettes and identified by morphology and by immunocytochemistry. Single cell cDNA libraries were generated by poly-d(T)-primed reverse transcription, poly-d(A) tailing of first strand cDNA, and en masse PCR-amplification using a custom made oligo-d(T). PCR was used to investigate gene expression in cDNAs from individual cells. RESULTS: Dissociated rod and Müller glia cells maintained their morphology, which correlated with their immunocytochemical properties. RPE cells were recognized by their pigmentation. With the exception of bipolar cells, non-photoreceptor neurons were only identifiable by immunocytochemistry. Abundant cDNA could be synthesized from each individual cell. Cell-specific "markers" were detected by PCR almost exclusively in the predicted cell types. The expression of neurotrophic factor receptors was consistent with previous biological studies. CONCLUSIONS: These studies establish a method to compare, investigate, and analyze gene expression in individual cells of the retina.
PURPOSE: Molecular analysis of complex phenomena, such as selective death of photoreceptors and their rescue by neuro-protective agents, has been hindered by limitations of techniques for investigating gene expression in individual cells within a heterogeneous tissue such as the retina. The purpose of this study was to develop methods to assess gene expression in single retinal cells. METHODS: Individual cells from papain-dissociated mouse retinae were captured with micropipettes and identified by morphology and by immunocytochemistry. Single cell cDNA libraries were generated by poly-d(T)-primed reverse transcription, poly-d(A) tailing of first strand cDNA, and en masse PCR-amplification using a custom made oligo-d(T). PCR was used to investigate gene expression in cDNAs from individual cells. RESULTS: Dissociated rod and Müller glia cells maintained their morphology, which correlated with their immunocytochemical properties. RPE cells were recognized by their pigmentation. With the exception of bipolar cells, non-photoreceptor neurons were only identifiable by immunocytochemistry. Abundant cDNA could be synthesized from each individual cell. Cell-specific "markers" were detected by PCR almost exclusively in the predicted cell types. The expression of neurotrophic factor receptors was consistent with previous biological studies. CONCLUSIONS: These studies establish a method to compare, investigate, and analyze gene expression in individual cells of the retina.
Authors: A Germanà; C Sánchez-Ramos; M C Guerrera; M G Calavia; M Navarro; R Zichichi; O García-Suárez; P Pérez-Piñera; Jose A Vega Journal: J Anat Date: 2010-07-21 Impact factor: 2.610
Authors: Ruslan N Grishanin; Haidong Yang; Xiaorong Liu; Kate Donohue-Rolfe; George C Nune; Keling Zang; Baoji Xu; Jacque L Duncan; Matthew M Lavail; David R Copenhagen; Louis F Reichardt Journal: Mol Cell Neurosci Date: 2008-04-22 Impact factor: 4.314
Authors: Joseph G Laird; Yuan Pan; Modestos Modestou; David M Yamaguchi; Hongman Song; Maxim Sokolov; Sheila A Baker Journal: Traffic Date: 2015-10-13 Impact factor: 6.215