Adeno-associated viruses (AAVs) are commonly used for in vivo gene transfer. Nevertheless, AAVs that provide efficient transduction across specific organs or cell populations are needed. Here, we describe AAV-PHP.eB and AAV-PHP.S, capsids that efficiently transduce the central and peripheral nervous systems, respectively. In the adult mouse, intravenous administration of 1×10 11 vector genomes (vg) of AAV-PHP.eB transduced 69% of cortical and 55% of striatal neurons, while 1×10 12 vg AAV-PHP.S transduced 82% of dorsal root ganglion neurons, as well as cardiac and enteric neurons. The efficiency of these vectors facilitates robust co-transduction and stochastic, multicolor labeling for individual cell morphology studies. To support such efforts, we provide methods for labeling a tunable fraction of cells without compromising color diversity. Furthermore, when used with cell type-specific promoters, these AAVs provide targeted gene expression across the nervous system and enable efficient and versatile gene manipulation throughout the nervous system of transgenic and non-transgenic animals.Users may view, print, copy, and download text and data-mine the content in such documents, for the purposes of academic research, subject always to the full Conditions of use:
Neuromodulatory systems exert profound influences on brain function. Understanding how these systems modify the operating mode of target circuits requires measuring spatiotemporally precise neuromodulator release. We developed dLight1, an intensity-based genetically encoded dopamine indicator, to enable optical recording of dopamine dynamics with high spatiotemporal resolution in behaving mice. We demonstrated the utility of dLight1 by imaging dopamine dynamics simultaneously with pharmacological manipulation, electrophysiological or optogenetic stimulation, and calcium imaging of local neuronal activity. dLight1 enabled chronic tracking of learning-induced changes in millisecond dopamine transients in striatum. Further, we used dLight1 to image spatially distinct, functionally heterogeneous dopamine transients relevant to learning and motor control in cortex. We also validated our sensor design platform for developing norepinephrine, serotonin, melatonin, and opioid neuropeptide indicators.
We recently developed novel AAV capsids for efficient and noninvasive gene transfer across the central and peripheral nervous systems. In this protocol, we describe how to produce and systemically administer AAV-PHP viruses to label and/or genetically manipulate cells in the mouse nervous system and organs including the heart. The procedure comprises three separate stages: AAV production, intravenous delivery, and evaluation of transgene expression. The protocol spans eight days, excluding the time required to assess gene expression, and can be readily adopted by laboratories with standard molecular and cell culture capabilities. We provide guidelines for experimental design and choosing the capsid, cargo, and viral dose appropriate for the experimental aims. The procedures outlined here are adaptable to diverse biomedical applications, from anatomical and functional mapping to gene expression, silencing, and editing. 1). The recombinant AAV (rAAV) genome contains the components required for gene expression including promoters, transgenes, protein trafficking signals, and recombinasedependent expression schemes. Hence, different capsid-cargo combinations create a versatile AAV toolbox for genetic manipulation of diverse cell populations in wild-type and transgenic animals. Here, we provide researchers, especially those new to working with AAVs or systemic delivery, with resources to utilize AAV-PHP viruses in their own research. Overview of the protocol We provide an instruction manual for users of AAV-PHP variants. The procedure includes three main stages (Fig. 1): AAV production (Steps 1-42), intravenous delivery (Steps 43-49), and evaluation of transgene expression (Step 50). The AAV production protocol is adapted from established methods. First, HEK293T cells are transfected with three plasmids 4-6 (Steps 1-3) (Figs. 1 and 6): (1) pAAV, which contains the rAAV genome of interest (Fig. 5 and Table 1); (2) AAV-PHP Rep-Cap, which encodes the viral replication and capsid proteins; and (3) pHelper, which encodes adenoviral proteins necessary for replication. Using this triple transfection approach, the rAAV genome is packaged into an AAV-PHP capsid in HEK293T cells. AAV-PHP viruses are then harvested 7 (Steps 4-14), purified 8,9 (Steps 15-31), and titered 10 (Steps 32-42) (Fig. 6). Purified viruses are intravenously delivered to mice via retro-orbital injection 11 (Steps 43-49) and gene expression is later assessed using molecular, histological, or functional methods relevant to the experimental aims (Step 50). This protocol is optimized to produce AAVs at high titer (over 10 13 vector genomes/ml) and with high transduction efficiency in vivo 2,3. Experimental design Before proceeding with the protocol, a number of factors should be considered, namely the expertise and resources available in the lab; the capsid and rAAV genome to be used; the dose for intravenous administration; and the method(s) available for assessing transgene expression. Each of these topics is discussed below and intended to guide users in de...
Recombinant adeno-associated viruses (rAAVs) are efficient, non-invasive gene delivery vectors via intravenous delivery, however, natural serotypes display a finite set of tropisms. To expand their utility, we evolved AAV capsids to efficiently transduce specific cell types in adult mouse brains. Building upon our previous Cre recombination-based AAV targeted evolution (CREATE) platform, we developed Multiplexed-CREATE (M-CREATE) to quickly and accurately identify variants of interest in a given selection landscape through multiple positive and negative selection criteria by incorporating next-generation sequencing, synthetic library generation, and a novel analysis pipeline. In vivo selections for brain endothelial cell-, astrocyte-, and neuron-transducing capsids have identified variants that can transduce the central nervous system broadly, exhibit bias toward vascular cells and astrocytes, target neurons with greater specificity, or cross the blood-brain barrier across diverse murine strains. Collectively, M-CREATE methodology accelerates the discovery of novel capsids for use in neuroscience and gene therapy applications.
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.
hi@scite.ai
10624 S. Eastern Ave., Ste. A-614
Henderson, NV 89052, USA
Copyright © 2024 scite LLC. All rights reserved.
Made with 💙 for researchers
Part of the Research Solutions Family.