Analysis of Gene Expression in Genetically Labeled Single Cells

Abstract

A combination of transgenic technology and single-cell reverse transcription polymerase chain reaction (RT-PCR)has been used to study gene expression in dopaminergic amacrine (DA)cells of the mouse retina. Because there are only 900 DA cells, and they cannot be distinguished from neighboring neurons on the basis of their morphology, we labeled them with human placental alkaline phosphatase (PLAP)by introducing into the mouse genome PLAP cDNA under the control of the promoter of the gene for tyrosine hydroxylase (TH), the rate-limiting enzyme for dopamine biosynthesis. Because PLAP is an enzyme that resides on the outer surface of the cell membrane, we can identify DA cells after dissociation of the retina by immunocytochemistry in the living state. Cells are then patch clamped and harvested for single-cell RT-PCR analysis of gene expression. Here, we describe the preparation of the fluorescent antibody E6-Cy3 to specifically detect PLAP-expressing cells, methods to obtain short-term cultures of solitary neurons from mouse retinas, and techniques to detect gene expression in individual neurons. Properties and pitfalls of single-cell RT-PCR are described and discussed.