Stable, neuron-specific gene expression in the mouse brain - PubMed (original) (raw)
Stable, neuron-specific gene expression in the mouse brain
Osama Ahmed et al. J Biol Eng. 2024.
Abstract
Gene delivery to, and expression in, the mouse brain is important for understanding gene functions in brain development and disease, or testing gene therapies. Here, we describe an approach to express a transgene in the mouse brain in a cell-type-specific manner. We use stereotaxic injection of a transgene-expressing adeno-associated virus into the mouse brain via the intracerebroventricular route. We demonstrate stable and sustained expression of the transgene in neurons of adult mouse brain, using a reporter gene driven by a neuron-specific promoter. This approach represents a rapid, simple, and cost-effective method for global gene expression in the mouse brain, in a cell-type-specific manner, without major surgical interventions. The described method represents a helpful resource for genetically engineering mice to express a therapeutic gene, for gene therapy studies, or to deliver genetic material for genome editing and developing knockout animal models.
Keywords: Brain; Brain disease; Gene delivery; Gene expression; Gene therapy; Genetic engineering.
© 2024. The Author(s).
Conflict of interest statement
The authors declare no competing interests.
Figures
Fig. 1
Graphical summary outlining the entire procedure for gene delivery and validating its expression. A pup injection with the transgene-expressing AAV9 using stereotaxic ICV coordinates at P0. B Molecular and cellular approaches to validate the gene expression in the adult mouse brain and spinal cord. “Created with BioRender.com”
Fig. 2
Designed plasmid used to generate the AAV9 for this study. AAV9 expressing the construct encoding NLS-eGFP driven by the human synapsin1 (Syn1) promoter. ITR: inverted terminal repeats; Syn1: Synapsin 1 promoter; Kozak: Kozak sequence; NLS-eGFP: nuclear localization signal-enhanced Green Fluorescent Protein; WPRE: Woodchuck Hepatitis Virus (WHP) Posttranscriptional Regulatory Element; BGH pA: bovine growth hormone polyadenylation signal; Ampicillin: ampicillin resistance gene; pUC ori: pUC origin of replication
Fig. 3
Illustration of the steps involving cryoanesthesia and brain injection. A Stereotaxic instrument used for viral injection. B For cryoanesthesia, the pup is kept on aluminum foil, and not directly in contact with the ice to avoid skin damage. C At P0, lambda (white arrow) and bregma (opposite site) are clearly visible on the surface of the skull. D The ICV injection is performed using the following coordinates ML = ± 1.2, AP = + 0.8, DV = -1.7 mm from the lambda
Fig. 4
Step-by-step depiction of mouse perfusion, brain and spinal cord extraction and tissue processing. A The perfusion apparatus used to perfuse the mouse. (A-i) The perfusion is performed intracardially with ice-cold PBS-Heparin at a constant speed of ~ 200 µl/second. (A-ii and iii) Spinal cord extraction. B The brain is cut into two hemispheres. Each hemisphere follows different processing according to the analysis to be performed: one hemisphere is transferred from PFA/PBS 4% to sucrose 10% and 30% (B-i to iii), with every step taking 24 h. The tissue is then embedded in optimal cutting temperature compound (OCT compound) and stored at –80 °C (B-iv); the other hemisphere is transferred to TRIzol and homogenized for mRNA and protein isolation (B-v to vii)
Fig. 5
eGFP mRNA and protein are highly expressed in the mouse brain. A eGFP mRNA expression levels were determined by qPCR and expressed as % of GAPDH levels; bars represent mean ± SEM, n = 3 mice. B eGFP protein expression was confirmed using immunoblotting on protein samples from mouse brain lysates. Immunoblotting was performed using anti-eGFP (dilution 1:1000) as the primary antibody, and IRDye-800 CW (dilution 1:2000) as the secondary antibody, with TUBB3 (beta 3 tubulin) employed as the internal loading control. CNTL: Control
Fig. 6
Robust and stable eGFP expression throughout the mouse brain. A Immunohistochemistry of P30 mouse brain showing the expression of eGFP in the periventricular regions of the CNS, such as cortex (i) and hippocampus (ii), as well as distant subcortical areas (iii). B Quantification of GFP positive neurons in different brain regions (n = 3 mice), values are represented as mean ± SEM (C) Higher magnification (20x) images of the indicated regions i, ii, and iii in panel (B). D Fluorescence microscopical image of a P60 mouse brain. E qPCR analysis on a P60 mouse brain mRNA; bars represent mean ± SEM, n = 3 mice. CNTL: Control, P30: Postnatal day 30, P60: Postnatal day 60
Fig. 7
eGFP expression in the spinal cord. A Fluorescence microscopical image of a P30 mouse spinal cord; three areas (i, ii, and iii) were arbitrarily chosen for higher magnification images in panel (C). D: Dorsal, V: Ventral, R: Rostral, and C: Caudal. B Quantification of eGFP positive neurons in cervical, thoracic, and lumbar spinal cord regions (n = 3 mice). Values are represented as mean ± SEM. C Higher-magnification (20 x) fluorescence microscopical images of the indicated areas in panel (A). CNTL: Control, P30: Postnatal day 30, P60: Postnatal day 60
References
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