BACKGROUND: Translating Ribosome Affinity Purification (TRAP), a method recently developed to generate cell type-specific translational profiles, relies on creating transgenic lines of animals in which a tagged ribosomal protein is placed under regulatory control of a cell type-specific promoter. An antibody is then used to affinity purify the tagged ribosomes so that cell type-specific mRNAs can be isolated from whole tissue lysates. RESULTS: Here, cell type-specific 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 type-specific 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 type-specific 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.
BACKGROUND: Translating Ribosome Affinity Purification (TRAP), a method recently developed to generate cell type-specific translational profiles, relies on creating transgenic lines of animals in which a tagged ribosomal protein is placed under regulatory control of a cell type-specific promoter. An antibody is then used to affinity purify the tagged ribosomes so that cell type-specific mRNAs can be isolated from whole tissue lysates. RESULTS: Here, cell type-specific 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 type-specific 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 type-specific 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.
Authors: Andrea S Viczian; Robert Vignali; Michael E Zuber; Giuseppina Barsacchi; William A Harris Journal: Development Date: 2003-04 Impact factor: 6.868
Authors: Elizabeth A Mills; Chung-ha O Davis; Eric A Bushong; Daniela Boassa; Keun-Young Kim; Mark H Ellisman; Nicholas Marsh-Armstrong Journal: Proc Natl Acad Sci U S A Date: 2015-08-03 Impact factor: 11.205
Authors: Jing Liu; A Michaela Krautzberger; Shannan H Sui; Oliver M Hofmann; Ying Chen; Manfred Baetscher; Ivica Grgic; Sanjeev Kumar; Benjamin D Humphreys; Benjamin Humphreys; Winston A Hide; Andrew P McMahon Journal: J Clin Invest Date: 2014-02-24 Impact factor: 14.808
Authors: Ivica Grgic; A Michaela Krautzberger; Andreas Hofmeister; Matthew Lalli; Derek P DiRocco; Susanne V Fleig; Jing Liu; Jeremy S Duffield; Andrew P McMahon; Bruce Aronow; Benjamin D Humphreys Journal: J Am Soc Nephrol Date: 2014-03-20 Impact factor: 10.121