A method for analysis of gene expression in isolated mousephotoreceptor and Müller cells

[摘要] Purpose: Molecular analysis of complex phenomena, such as selectivedeath of photoreceptors and their rescue by neuro-protective agents, hasbeen hindered by limitations of techniques for investigating geneexpression in individual cells within a heterogeneous tissue such as theretina. The purpose of this study was to develop methods to assess geneexpression in single retinal cells.Methods: Individual cells from papain-dissociated mouse retinae werecaptured with micropipettes and identified by morphology and byimmunocytochemistry. Single cell cDNA libraries were generated bypoly-d(T)-primed reverse transcription, poly-d(A) tailing of firststrand cDNA, and en masse PCR-amplification using a custom madeoligo-d(T). PCR was used to investigate gene expression in cDNAs fromindividual cells.Results: Dissociated rod and Müller glia cells maintained theirmorphology, which correlated with their immunocytochemical properties.RPE cells were recognized by their pigmentation. With the exception ofbipolar cells, non-photoreceptor neurons were only identifiable byimmunocytochemistry. Abundant cDNA could be synthesized from eachindividual cell. Cell-specific "markers" were detected by PCR almostexclusively in the predicted cell types. The expression of neurotrophicfactor receptors was consistent with previous biological studies.Conclusions: These studies establish a method to compare,investigate, and analyze gene expression in individual cells of theretina.

[学科分类] 生物化学/生物物理