Production of Viral Constructs for Neuroanatomy, Calcium Imaging, and Optogenetics

Abstract

Advances in design and use of light-sensitive and light-emitting sensors have facilitated observation, measurement, and control of neuronal activities. Viruses are effective vectors for delivery of these valuable research tools to mammalian brains. Recombinant viruses are optimized to mediate regulatable, long-term, and cell-specific gene expression. Here, we describe production methods for three of the most commonly used types of recombinant viruses in neurobiology research: adeno-associated virus (AAV), retrovirus/lentivirus, and glycoprotein-deleted rabies virus. These viral constructs are frequently used for calcium imaging or to deliver neural tracers and optogenetic tools. Popular constructs are readily obtained commercially; however, customized virus production through commercial sources is time consuming and costly. This article aims to provide readers with detailed technical information for rapid production and validation of high-quality viral particles in a laboratory setting while highlighting advantages and limitations of each viral type. © 2019 by John Wiley & Sons, Inc.

Keywords: AAVs; lentivirus; rabies delta-G; recombinant virus; retrovirus; viral production.

Figure 1. Reconstitution of recombinant viruses from plasmids.

AAVs are reconstituted from transfer vector, Rep/Cap, and Helper plasmids. Serotypes are determined by the Cap genes. 2^nd generation packaging plasmids for a VSV-G pseudotyped lentivirus include transfer vectors, psPax2, and pMD2.G. Rabies dG transfer vector and viral proteins N, P, L, and G are necessary for formation of initial viral particles.

Figure 2A. Workflow of recombinant virus production.

General steps and length of time for production of Adeno-associated viruses.

Figure 2B. Workflow of recombinant virus production.

General steps and length of time for production of Lentiviruses.

Figure 2C. Workflow of recombinant virus production.

General steps and length of time for production of Pseudotyped rabies dG viruses.

Figure 3. Validation of an AAV preparation for transduction efficiency and purity.

A. Organotypic mouse brain slice cultures were prepared as described previously from 6–8 days postnatal C57BL/6J mice (Bastrikova et al., 2008; Gu et al., 2012; Stoppini et al., 1991). Virus AAV9-hSyn-GFP was added on top of the culture 24 hours post dissection. GFP expression was observed 7 days after transduction and fluorescence images were captured on a Zeiss LSM710 (Carl Zeiss Inc, Oberkochen, Germany) using an EC Plan-Neofluar 10x/0.3 objective. AAV9-hSyn-GFP preparation was validated by ex vivo transduction. B. AAV samples were harvested from culture media (packaging cells were discarded) as described in Protocol 1. Samples were boiled in NuPage sample buffer, loaded onto a 4–12% NuPage Bis-Tris gels (Thermo Fisher Scientific, Rockford, IL, USA) and resolved via polyacrylamide gel electrophoresis (PAGE). Pierce Silver Staining kit (Thermo Fisher Scientific, Rockford, IL, USA) was used according to manufacturer’s protocols to stain the gel and reveal VP1, VP2, VP3 (AAV proteins) and impurities. AAV serotype 1, 5, 8, and 9 samples produced with small-scale protocol have suitable titers and comparable purities to commercially obtained samples. AAV packaged from the same vector backbones for serotypes 2 and 9 were purchased from Addgene and UPenn Vector Core and run on the gel as positive controls.

Figure 4. Transduction of primary rat cortical neurons with a concentrated lentivirus.

Lentiviral transfer vector FCK-ChR2-GFP delivering Channelrhodopsin-2 tethered to eGFP expressed from a cytomegalovirus (CMV) promoter was prepared as described in Protocol 2. The preparation had a yield of 1.2e8 TU/ml. 5 ul of virus was used to infect 1e6 cultured mouse primary neurons. eGFP expression was observed weakly at 24 hours and robustly after 48 hours.

Figure 5. Env A pseudotyped SADB19 rabies dG retrograde transmission.

AAV9-Camk2a-T2A-TVA-E2A-B19G was delivered to deep layers of entorhinal cortex via stereotactic injection to mouse brain. The GFP expression marks the AAV tranduced cells. Env A pseudotyped SADB19 rabies dG expressing mCherry was injected into the same region after 2 weeks. Retrograde transmission of Env A-rabies dG to CA1 pyramidal neurons is indicated by mCherry expression.