Lentivirus-mediated Genetic Manipulation and Visualization of Olfactory Sensory Neurons <em>in vivo</em>

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 PreparationThis procedure is biosafety level 2, therefore all the following preparations are made in a biohazard hood where the microinjection procedure is performed. 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.Page 1 of 4 Journal of Visualized Experiments www.jove.comCopyright © 2011 Journal of Visualized Experiments 1. 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. 2. Fill a biohazard waste container with 10% bleach to a suitable volume for immersion of all waste from this procedure. 3. Prepare a small open dish of 70% ethanol and a second dish with sterilized ddH2O and place in the biological safety cabinet. 4. Sterilize a 5μL capacity Hamilton syringe with a 33-gauge needle by spraying down the body and needle with 70% ethanol with the plunger pulled out all the way. Repeatedly aspirate ethanol from the dish and eject it all the way out at least 5 times. 5. Rinse the syringe cartridge by aspirating and ejecting sterile water at least 5 times. Place syringe safely aside in the culture hood and allow it to dry. 6. Remove viral stocks from -80°C freezer storage and place on ice to allow thawing. Microinjection requires 2μL of virus stock per mouse at a titre of 10 9 infectious units/mL. Lentiviruses can be obtained...

ACE2-lentiviral transduction enables mouse SARS-CoV-2 infection and mapping of receptor interactions

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N-myc is a key switch regulating the proliferation cycle of postnatal cerebellar granule cell progenitors

“…C57BL/6J mouse pups were anesthetised as previously described 32 at 4.5 days postpartum. The lentivirus-mmu-miR-9 or control lentivirus (Genechem, Shanghai, China) were injected into the developing cerebellum by a 5-μL capacity syringe (Hamilton, Switzerland) with a 33-gauge needle.…”

Olfactory Loss and Dysfunction in Ciliopathies: Molecular Mechanisms and Potential Therapies

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