Cell Type-Specific Translational Profiling in the Xenopus laevis Retina

Background: Translating Ribosome Affinity Purification (TRAP), a method recently developed to generate cell typespecific translational profiles, relies on creating transgenic lines of animals in which a tagged ribosomal protein is placed under regulatory control of a cell typespecific promoter. An antibody is then used to affinity purify the tagged ribosomes so that cell typespecific mRNAs can be isolated from whole tissue lysates. Results: Here, cell typespecific transgenic lines were generated to enable TRAP studies for retinal ganglion cells and rod photoreceptors in the Xenopus laevis retina. Using real time quantitative PCR for assessing expression levels of cell typespecific mRNAs, the TRAP method was shown to selectively isolate mRNAs expressed in the targeted cell and was efficient at purifying mRNAs expressed at both high and low levels. Statistical measures used to distinguish cell typespecific RNAs from low level background and non-specific RNAs showed TRAP to be highly effective in Xenopus. Conclusions: TRAP can be used to purify mRNAs expressed in rod photoreceptors and retinal ganglion cells in X. laevis. The generated transgenic lines will enable numerous studies into the development, disease, and injury of the X. laevis retina. Developmental Dynamics, 2012. (C) 2012 Wiley Periodicals, Inc.