Published: June 16th, 2023
Here, we describe a method to selectively alter gene expressions in the choroid plexus while avoiding any impact in other brain areas.
The choroid plexus (ChP) serves as a critical gateway for immune cell infiltration into the central nervous system (CNS) under both physiological and pathological conditions. Recent research has shown that regulating ChP activity may offer protection against CNS disorders. However, studying the biological function of the ChP without affecting other brain regions is challenging due to its delicate structure. This study presents a novel method for gene knockdown in ChP tissue using adeno-associated viruses (AAVs) or cyclization recombination enzyme (Cre) recombinase protein consisting of TAT sequence (CRE-TAT). The results demonstrate that after injecting AAV or CRE-TAT into the lateral ventricle, the fluorescence was exclusively concentrated in the ChP. Using this approach, the study successfully knocked down the adenosine A2A receptor (A2AR) in the ChP using RNA interference (RNAi) or Cre/locus of X-overP1 (Cre/LoxP) systems, and showed that this knockdown could alleviate the pathology of experimental autoimmune encephalomyelitis (EAE). This technique may have important implications for future research on the ChP's role in CNS disorders.
The choroid plexus (ChP) was often thought to help maintain brain functional homeostasis by secreting cerebrospinal fluid (CSF) and brain-derived neurotrophic factor (BDNF)1,2. Increasing research over the last three decades has revealed that the ChP represents a distinct pathway for immune cell infiltration into the central nervous system (CNS).
The tight junctions (TJs) of the ChP, composed of a monolayer ChP epithelium, maintain immunological homeostasis by preventing macromolecules and immune cells from entering the brain3. However, under certain patholog....
All animal procedures described in this study were conducted in accordance with the guidelines outlined in the NIH Guide for the Care and Use of Laboratory Animals and approved by the Institutional Animal Care and Use Committee at Wenzhou Medical University.
ChP-specific A2AR knockdown by ICV injection of AAV2/5-shRNA or CRE-TAT
The role of A2AR in the ChP as a powerful regulator of neural information in EAE pathogenesis remains unclear. Knocking down ChP-specific A2AR expression could shed light on the A2AR regulatory effects on the central immune system in EAE and other nervous system inflammations. This study used ICV injection of CRE-TAT to decrease A2AR expression in the ChP of A2AR.......
The research presented two distinct approaches for the targeted knockdown of ChP genes. The first approach involved the ICV injection of CRE-TAT, which contains Cre recombinase, into A2ARflox/flox mice. The second approach entailed ICV injection of AAV2/5 carrying shRNA of A2AR. By utilizing these two strategies, the work achieved the selective knockdown of A2AR within the ChP and was able to demonstrate the protective effects of inhibiting A2AR signaling in the ChP .......
We gratefully acknowledge the support of the National Natural Science Foundation of China (Grant No. 31800903, awarded to W. Zheng) and the Wenzhou Science and Technology Project (no. Y2020426, awarded to Y. Y. Weng) for this work.....
|State Key Laboratory of Ophthalmology, Optometry and Visual Science, Wenzhou Medical University
|Shanghai Heyuan Biotechnology Co. LTD
|antifade mounting medium
|borosilicate glass capillary
|Beijing Meiyaxian Technology Co. Ltd
|brain stereotaxic apparatus
|Beijing Vital Charles River Laboratory Animal Technology Company
|frozen slicing machine
|Becton Dickinson and company
|Incomplete Freunds adjuvant
|Laser confocal microscope
|Suzhou Qiangyao Biotechnology Co., LTD
|Chengdu Kelong Chemical Reagent Company
|PrimeScript 1st Strand cDNA Synthesis Kit
|Real- Time PCR System
|Rosa-LSL (Lox-StoP-Lox)-tdTomato mice
|super high speed homogenizer
|Chengdu Kelong Chemical Reagent Company
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