Lentivirus-mediated Genetic Manipulation and Visualization of Olfactory Sensory Neurons in vivo

Summary

We present a lentiviral technique for genetic manipulation and visualization of single olfactory sensory neuron axon and its terminal arborization in vivo.

Abstract

Development of a precise olfactory circuit relies on accurate projection of olfactory sensory neuron (OSN) axons to their synaptic targets in the olfactory bulb (OB). The molecular mechanisms of OSN axon growth and targeting are not well understood. Manipulating gene expression and subsequent visualizing of single OSN axons and their terminal arbor morphology have thus far been challenging. To study gene function at the single cell level within a specified time frame, we developed a lentiviral based technique to manipulate gene expression in OSNs in vivo. Lentiviral particles are delivered to OSNs by microinjection into the olfactory epithelium (OE). Expression cassettes are then permanently integrated into the genome of transduced OSNs. Green fluorescent protein expression identifies infected OSNs and outlines their entire morphology, including the axon terminal arbor. Due to the short turnaround time between microinjection and reporter detection, gene function studies can be focused within a very narrow period of development. With this method, we have detected GFP expression within as few as three days and as long as three months following injection. We have achieved both over-expression and shRNA mediated knock-down by lentiviral microinjection. This method provides detailed morphologies of OSN cell bodies and axons at the single cell level in vivo, and thus allows characterization of candidate gene function during olfactory development.

Protocol

1. Preparation This procedure is biosafety level 2, therefore all the following preparations are made in a biohazard hood where the microinjection procedure is performed. Prepare and preheat a 37°C waterbed: Fill a plastic storage bag with water – seal and set on an emptied heating block incubator. This will allow mice to recover following injection. Fill a biohazard waste container with 10% bleach to a suitable volume for immersion of all waste from …

Discussion

Microinjection of lentivirus results in permanent transfer of gene constructs into OSN genomic DNA. This approach allows us to perform short-term or long-term manipulations of candidate genes via overexpression or shRNA mediated knock-down. In addition, we can fluorescently label single OSNs in an existing transgenic mouse line to observe co-localization of odorant receptor populations. Microinjection is required for lentiviral transduction of OSNs. We have attempted flushing lentivirus suspension into the nasal cavity t…

Materials

Microinjection: Newborn mice were microinjected using a 5μL Hamilton syringe (Hamilton #7647-01) fitted with a 33 gauge needle (Hamilton #7762-06).

Immunocytochemistry Reagents: Primary antibodies: rabbit polyclonal antibody GFP (Molecular Probes# A-6455), chicken polyclonal antibody against OMP (custom) (Chen et al., 2005), guinea pig polyclonal antibody against VGlut2 (Millipore# AB2251). Secondary antibodies: Cy2-conjugated donkey anti-rabbit (Jackson Immunolab# 711-225-152), Cy3-conjugated donkey-anti-chicken (Jackson Immunolab# 703-165-155) and Cy5-conjugated goat anti-guinea pig (Jackson Immunolab# 106-175-008). Sections were mounted on glass slides with Fluoromount G (Southern Biotech# 0010-01) with 50ng/mL DAPI chromatin stain solution.

Confocal Imaging: Z-stack images were taken using an Olympus Flouview FV1000 confocal microscope and images collected and processed into 3D projections using Olympus FV10-ASW 2.01 confocal acquisition and analysis software.