Isolation and purification of Drosophil peripheral neurons by magnetic bead sorting.
The Drosophila peripheral nervous system (PNS) is a powerful model for investigating the complex processes of neuronal development and dendrite morphogenesis at the functional and molecular levels. To aid in these analyses, we have developed a strategy for the isolation of a subclass of PNS neurons called dendritic arborization (da) neurons that have been widely used for studying dendrite morphogenesis. These neurons are very difficult to isolate as a pure population, due in part to their extremely low occurrence and their difficult-to-reach location below the tough chitinous larval cuticle. Our newly developed method overcomes these challenges, and is based on a fast and specific cell enrichment using antibody-coated magnetic beads. For our magnetic bead sorting studies, we have used age-matched third instar larvae expressing a mouse CD8 tagged GFP fusion protein (UAS-mCD8-GFP) under the control of either the class IV dendritic arborization (da) neuron-specific pickpocket (ppk)-GAL4 driver or the control of the pan-da neuron-specific GAL4 driver. Although this protocol has been optimized for isolating PNS cells which are attached to the inner wall of the larval cuticle, by varying a few parameters, the same protocol could be used to isolate many different cell types attached to the cuticle at larval or pupal stages of development (e.g. epithelia, muscle, oenocytes etc.), or other cell types from larval organs depending upon the GAL4-specific driver expression pattern. The RNA isolated by this method is of high quality and can be readily used for downstream genomic analyses such as microarray gene expression profiling studies. This approach offers a powerful new tool to perform studies on isolated Drosophila dendritic arborization (da) neurons thereby providing novel insights into the molecular mechanisms underlying dendrite morphogenesis.
Iyer
Eswar Prasad R
EP
Department of Molecular and Microbiology, George Mason University, USA.
Iyer
Srividya Chandramouli
SC
Sulkowski
Mikolaj J
MJ
Cox
Daniel N
DN
D000818:Animals; D003712:Dendrites / Q000502:physiology; D004330:Drosophila / Q000166:cytology*; D008280:Magnetics / Q000379:methods*; D051379:Mice; D009474:Neurons / Q000166:cytology*; D017933:Peripheral Nervous System / Q000166:cytology*