Department of Cell Biology and Human Anatomy, School of Medicine, University of California, Davis
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Sadrian, B., Chen, H., Gong, Q. Lentivirus-mediated Genetic Manipulation and Visualization of Olfactory Sensory Neurons in vivo. J. Vis. Exp. (51), e2951, doi:10.3791/2951 (2011).
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.
This procedure is biosafety level 2, therefore all the following preparations are made in a biohazard hood where the microinjection procedure is performed.
2. Microinjection of lentiviral particles into the olfactory epithelium
All animal procedures performed in this method are done according to IACUC approved guidelines. The Mus musculus strain C57BL/6J is the animal model used in all experiments demonstrated here.
3. Immunohistochemistry of floating olfactory bulb sections
4. Representative Results
With the methods described here, we can transduce olfactory sensory neurons by lentivirus in vivo. We have thus far visualized infected OSNs with GFP and mCherry fluorescent reporters, as well with a myc-tagged fusion protein via immunocytochemistry. This technique provides an abundance of infected OSNs. Still, transduction occurs sporadically enough to provide individually labeled OSNs, thus allowing for analysis of single cell morphology. Axons and axon terminals are imaged within OB tissue sections amidst their native environment by confocal microscopy (Figure 1). The entire OSN axon terminal arbor can typically be captivated within a 40-50μm optical section. High magnification of infected OSN axon terminals allows detailed analysis of axonal arbor morphology at the glomerular layer.
Figure 1. Trajectory of single olfactory sensory neuron axon and its terminal arbor. A) A Coronal section of the olfactory bulb of a P7 mouse showing the olfactory nerve layer (ONL) immunostained for OMP (red) and the glomerular layer (GL) immunostained with both OMP and Vglut2 (grey). A single olfactory sensory neuron axon expressing GFP (green) travels within the ONL and penetrates into the glomerular structure. B) The axon terminal arbor morphology within the glomerulus clearly highlighted by GFP expression. Vglut2 co-staining in grey illuminates the total glomular area within which the GFP-positive axon elaborates a terminal abor. C) Lentiviral infected OSNs expressing GFP (green) in the OE. The cytostructure of the olfactory epithelium is shown with DAPI staining (blue). GFP expression allows visualization of OSN cell bodies and their cellular processes in the OE. D) An OSN axon and its terminal morphology with Rap1GAP2 expression knock-down by shRNA. Lentiviruses carrying a dual expression cassette with Rap1GAP2 shRNA under the mouse U6 promoter and GFP under a CMV promoter were applied to the OE. Infected olfactory axon terminals exhibited a more simplified terminal arbor when compared to that of the GFP only control. Bar = 20μm.
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 to transduce OSNs without any success. We therefore perform microinjections through the dorsal nose surface in order to deliver virus to inner layers of the OE where OSN cell bodies lie.
There are many advantages to using lentivirus over the established adenoviral manipulation. Lentivral transduction integrates into the genome allowing long term studies, while adenovirus gives only transient transfection with a limited timecourse of gene manipulation (Doi et al., 2005). We have detected lentivirally transduced OSNs as long as three months after microinjection. The life span of OSNs in adult rodents is typically between 1-3 months. Lentiviral infected OSNs can thus be visualized through the majority of their lifetime, allowing investigation of OSN axon growth, synaptic formation and maintenance, as well as odorant-stimulated responses and the turnover process. In addition, lentiviruses can infect progenitor cells as well and allow visualization of OSNs for longer periods of time. Adenovirus readily transduces OSNs when infused into the nasal cavity (Zhao et al., 1996). However, adenoviral infection is often widespread from the point of exposure, and does not yield isolated single cell transductions as reliably as lentivirus. Lentiviral-mediated genetic manipulation in OSNs is therefore a powerful and efficient technique for characterization of gene function in single OSNs in vivo.
This protocol is bio-safety level 2 compliant. All appropriate workspace, personal protection, and viral waste containment measures have been established accordingly. All animal procedures were performed according to IACUC guidelines.
This study is supported by NIH DC052256 and DC006015, and NSF 0324769 to QG and T32-DC008072 to BS.
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.