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Updated: Apr 17, 2026
Intracerebroventricular Viral Injection of the Neonatal Mouse Brain for Persistent and Widespread Neuronal Transduction
Published on: September 15, 2014
Recombinant adeno-associated viral (rAAV) vectors mediate efficient gene transduction in cultured neonatal and adult
Wei Su1, John Kang1, Bryce Sopher1
1Department of Neurology, University of Washington, Seattle, Washington, USA.
Abstract:
Microglia are a specialized population of myeloid cells that mediate CNS innate immune responses. Efforts to identify the cellular and molecular mechanisms that regulate microglia behaviors have been hampered by the lack of effective tools for manipulating gene expression. Cultured microglia are refractory to most chemical and electrical transfection methods, yielding little or no gene delivery and causing toxicity and/or inflammatory activation. Recombinant adeno-associated viral (rAAVs) vectors are non-enveloped, single-stranded DNA vectors commonly used to transduce many primary cell types and tissues. In this study, we evaluated the feasibility and efficiency of utilizing rAAV serotype 2 (rAAV2) to modulate gene expression in cultured microglia. rAAV2 yields high transduction and causes minimal toxicity or inflammatory response in both neonatal and adult microglia. To demonstrate that rAAV transduction can induce functional protein expression, we used rAAV2 expressing Cre recombinase to successfully excise a LoxP-flanked miR155 gene in cultured microglia. We further evaluated rAAV serotypes 5, 6, 8, and 9, and observed that all efficiently transduced cultured microglia to varying degrees of success and caused little or no alteration in inflammatory gene expression. These results provide strong encouragement for the application of rAAV-mediated gene expression in microglia for mechanistic and therapeutic purposes. Neonatal microglia are functionally distinct from adult microglia, although the majority of in vitro studies utilize rodent neonatal microglia cultures because of difficulties of culturing adult cells. In addition, cultured microglia are refractory to most methods for modifying gene expression. Here, we developed a novel protocol for culturing adult microglia and evaluated the feasibility and efficiency of utilizing Recombinant Adeno-Associated Virus (rAAV) to modulate gene expression in cultured microglia.
Insights
Recombinant adeno-associated virus (rAAV) vectors efficiently deliver genes into cultured microglia, enabling functional studies and therapeutic development. This method overcomes previous limitations in microglia gene manipulation.
Area of Science:
- Neuroscience
- Immunology
- Molecular Biology
Background:
- Microglia are key immune cells in the central nervous system (CNS).
- Existing methods for manipulating microglia gene expression are inefficient and cause inflammation.
- Culturing adult microglia presents significant challenges.
Purpose of the Study:
- To evaluate the efficiency of recombinant adeno-associated virus (rAAV) for gene delivery in cultured microglia.
- To assess the toxicity and inflammatory response of rAAV transduction in microglia.
- To establish a protocol for culturing adult microglia and test rAAV efficacy.
Main Methods:
- Utilized various rAAV serotypes (2, 5, 6, 8, 9) for gene delivery to cultured neonatal and adult microglia.
- Assessed transduction efficiency, cell viability, and inflammatory gene expression.
- Demonstrated functional gene modulation by excising a miR155 gene using rAAV-Cre.
Main Results:
- rAAV serotype 2 demonstrated high transduction efficiency with minimal toxicity and inflammatory response in both neonatal and adult microglia.
- All tested rAAV serotypes transduced cultured microglia effectively to varying degrees.
- rAAV-mediated gene expression allowed for successful gene knockout (miR155).
Conclusions:
- rAAV vectors are a feasible and efficient tool for modulating gene expression in cultured microglia.
- This approach overcomes limitations of previous gene delivery methods, enabling mechanistic and therapeutic research.
- The study provides a foundation for advanced microglia research using gene editing techniques.
