Targeted Knockdown of Genes in the Choroid Plexus

Summary

Here, we describe a method to selectively alter gene expressions in the choroid plexus while avoiding any impact in other brain areas.

Abstract

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 A_2A receptor (A_2AR) 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.

Introduction

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....

Protocol

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.

1. Animals

1. Purchase male C57BL/6 mice aged 8-12 weeks and weighing 20-22 g.
2. Obtain the transgenic Rosa-LSL (Lox-StoP-Lox)-tdTomato (Ai9) mouse line, and male A_2AR^flox/flox mice.<.......

Discussion

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 A_2AR^flox/flox mice. The second approach entailed ICV injection of AAV2/5 carrying shRNA of A_2AR. By utilizing these two strategies, the work achieved the selective knockdown of A_2AR within the ChP and was able to demonstrate the protective effects of inhibiting A_2AR signaling in the ChP .......

Materials

Name	Company	Catalog Number	Comments
A2ARflox/flox mice	State Key Laboratory of Ophthalmology, Optometry and Visual Science, Wenzhou Medical University
AAV2/5-A2AR-ShRNA virus	Shanghai Heyuan Biotechnology Co. LTD	pt-4828
antifade mounting medium	Beyotime Biotechnology	0100-01
borosilicate glass capillary	Beijing Meiyaxian Technology Co. Ltd	B100-50-10
brain stereotaxic apparatus	RWD, Shenzhen	69100
C57BL/6 mice	Beijing Vital Charles River Laboratory Animal Technology Company
CRE-TAT recombinase	Millipore	SCR508
DAPI	Absin	B25A031
frozen slicing machine	Leica	CM1950
H37Ra	Becton Dickinson and company	231141
Hamilton syringe	Hamilton, American	P/N: 86259
Incomplete Freunds adjuvant	Sigma	F5506
Laser confocal microscope	Zeiss	LSM900
MOG35-55	Suzhou Qiangyao Biotechnology Co., LTD	4010006243
OCT glue	Epredia	6502p
paraformaldehyde	Chengdu Kelong Chemical Reagent Company	30525-89-4
pentobarbital sodium	Boyun Biotech	PC13003
Pipette gun	Eppendorf	N45014F
PrimeScript 1st Strand cDNA Synthesis Kit	Takara	6110A
Real- Time PCR System	BioRad	CFX96
Rosa-LSL (Lox-StoP-Lox)-tdTomato mice	Jackson Laboratory
sucrose	Sangon Biotech	A502792-0500
super high speed homogenizer	IKA	3737025
Trizol	Invitrogen	15596026
xylene solution	Chengdu Kelong Chemical Reagent Company	1330-20-7