A brief guide for gene delivery to the brain using adeno-associated viral vectors

Abstract

The advent of recombinant adeno-associated viral (rAAV) vector-mediated gene delivery has accelerated the comprehensive analysis and manipulation of the nervous system owing to its ability to regulate gene expression in a spatiotemporal manner, thereby facilitating the study of brain physiology and the investigation of the pathophysiology of neurological disorders. Here, we provide a concise guide to stereotaxic gene delivery into the mouse brain using rAAV vectors. Key considerations for designing a customized rAAV vector are discussed, along with an overview of the surgical procedures of intracranial stereotaxic injection. This article aims to assist neuroscientists in establishing experimental setups for genetic manipulation in the mouse brain.

Keywords: Gene delivery; Intracranial injection; Recombinant adeno-associated viral vector; Stereotaxic surgery.

Fig. 1

Engineering gene expression cassette for recombinant adeno-associated viral (rAAV) vector. (A) An example of a typical rAAV vector gene expression cassette for protein expression. Expression of the transgene can be induced either by a ubiquitous promoter or a cell-/tissue-specific promoter. Gene expression can be traced by fusing an epitope tag to the transgene or by incorporating a reporter protein through a multicistronic element, such as internal ribosome entry sites or 2A peptides. (B) An example of a gene knockdown vector. Noncoding RNAs (ncRNAs), such as short hairpin RNA, guide RNA, or primary artificial microRNA, can be delivered using an rAAV vector. To drive the expression of ncRNAs, RNA polymerase (Pol) III promoters (eg, U6 or H1) and ubiquitous or cell-type-specific Pol II promoters can be used. An additional promoter may be used for the expression of a reporter protein. CMV, cytomegalovirus enhancer and promoter; CBA, CMV enhancer/chicken β-actin promoter hybrid; CAG, CBA/rabbit β-globin intron hybrid; EF1α, elongation factor 1--alpha; hSyn, human synapsin 1; hGFAP, human glial fibrillary acidic protein; mMBP, mouse myelin basic protein; DREADD, designer receptors exclusively activated by designer drug; CRISPR, clustered regularly interspaced short palindromic repeats; ITR, inverted terminal repeat.

Fig. 2

Mouse brain atlas and stereotaxic system. (A) A modified image of mouse brain atlas from the online tool (https://labs.gaidi.ca/mouse-brain-atlas/) at the level of AP: −3.1. (B) An image of an animal mounted on the stereotaxic apparatus with the 3 stereotaxic axes labeled (AP, ML, and DV). (C) A typical stereotaxic apparatus equipped with an injector. The position of the injector is controlled in 3 axes by manipulators. Inset, enlarged image of the dotted box. Ear bars and a tooth bar are used to secure the animal’s head to the stereotaxic apparatus. (D) An image of a shaved scalp of the mouse secured on the stereotaxic apparatus. The points bregma and lambda are denoted by magenta and cyan dots, respectively. (E) A diagrammatic representation of a rodent skull showing the bones and suture lines. Best-fit lines of the suture lines (magenta and cyan lines) and the midline (black line) define the stereotaxic references, bregma (magenta dot) and lambda (cyan dot). (F-H) Adjustments to the flat-skull position. (F) Dorsal, (G) lateral, and (H) posterior views in the flat-skull position. Bidirectional orange dashed arrows show possible adjustments: (F) moving the ear bars to modify the ML values, (G) adjusting the tooth bar to modify the DV values, (H) and making additional adjustments to correct rotation around the AP axis. AP, anteroposterior; ML, mediolateral; DV, dorsoventral.

Fig. 3

Evaluating transgene expression induced by recombinant adeno-associated viral (rAAV) vectors after stereotaxic injection. (A) An example of a coronal (top) and a sagittal (bottom) section modified from the online mouse brain atlas (https://labs.gaidi.ca/mouse-brain-atlas/). Stereotaxic coordinates for the substantia nigra (Jeong et al., 2018) are shown as an example. (B) An image of a midbrain coronal section showing viral transduction in the target region, as evidenced by green fluorescent protein (GFP) expression observed a few weeks after rAAV injection. Scale bar, 1 mm. (C) Multichannel images after immunohistochemistry. Shown is a brain section injected with rAAVs exhibiting typical neuronal tropism, as evidenced by GFP expression in NeuN⁺ cells. Scale bar, 50 µm. AP, anteroposterior; ML, mediolateral; DV, dorsoventral; NeuN, neuronal nuclei; GFAP, glial fibrillary acidic protein.