Name: ChP Specific Knockdown of A2ARs with AAV2/5-shRNA and Cre/LoxP System
Uploaded: 2023-06-16T14:10:00
Description: Watch this Scientific Journal Video about ChP Specific Knockdown of A2ARs with AAV2/5-shRNA and Cre/LoxP System at JoVE.com

chp specific knockdown of a2ars with aav2/5-shrna and cre/loxp system

Selective Gene Silencing in the Choroid Plexus

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 pathological conditions, the ChP tissue detects and responds to danger-associated molecular patterns (DAMPs) in the CSF and blood, leading to abnormal immune infiltration and brain dysfunction4,5. Despite its critical role, the ChP&#39;s small size and unique location in the brain make it difficult to study its function without affecting other brain regions. Therefore, manipulating gene expression specifically in the ChP is an ideal approach to understanding its function.
Initially, cyclization recombination enzyme (Cre) transgenic lines, which express Cre under the control of promoters specific to genes expressed in the ChP, were commonly used to delete target genes by breeding with floxed candidate genes6,7,8. For example, the transcription factor Forkhead box J1 (FoxJ1) is exclusively expressed in the ChP epithelium of the prenatal mouse brain7. Thus, the FoxJ1-Cre line was often used to delete genes located in the ChP6,9. However, the success of this strategy relies heavily on the specificity of the promoter. It was gradually discovered that the FoxJ1 expression pattern was not distinctive enough, as FoxJ1 was also present in ciliated epithelial cells in other parts of the brain and peripheral system7. To overcome this limitation, intra-cerebroventricular (ICV) injection of Cre recombinase was performed to deliver recombinase into the ventricles of floxed transgenic lines. This strategy showed high specificity, as evidenced by the presence of tdTomato fluorescence solely in the ChP tissue10,11. However, this method is still limited by the availability of floxed transgenic mouse lines. To address this issue, researchers have employed ICV injection of adeno-associated virus (AAV) to achieve ChP-specific knockdown or the overexpression of target genes12,13. A comprehensive evaluation of different AAV serotypes for ChP infection revealed that AAV2/5 and AAV2/8 exhibit strong infection abilities in the ChP, while not infecting other brain regions. However, AAV2/8 was found to infect the ependyma surrounding ventricles, whereas the AAV2/5 group showed no infection14. This method has the advantage of overcoming the limitations of acquiring floxed transgenic animals.
This article describes a step-by-step protocol for gene knockdown in the ChP using two methods: ICV of AAV2/5 carrying shRNA of the adenosine A2A receptor (A2AR) and Cre recombinase protein consisting of TAT sequence (CRE-TAT) recombinase to achieve ChP-specific knockdown of A2AR. The study findings suggest that knocking down A2AR in the ChP can alleviate experimental autoimmune encephalomyelitis (EAE). This detailed protocol provides useful guidance for ChP function studies and the specific knockdown of genes in the ChP.

저자 스포트라이트: 맥락총(Choroid Plexus) 연구를 위한 유전자 발현 조작 기법의 통찰력과 혁신 

맥락총(Choroid Plexus)에서 유전자의 표적 녹다운(Targeted knockdown of Genes in the Choroid Plexus)

The objective of this research is to investigate the techniques for manipulating gene expression in the choroid plexus. Our study has introduced the two methods that allow for technique exclusively in the choroid plexus without any discernible impact on other regions. Our findings can potentially enhance future function studies of the choroid plexus in neuroscience.Our research has reviewed that inhibiting T-cell infiltration across the choroid plexus through selective lockdown of adenosine to a receptor is sufficient to protect mice from EAE pathology. This finding supports the notion that detecting gateway activity in the choroid plexus could present a promising new strategy for treating multiple sclerosis. This study presents two alternative methods for gene expression manipulation in the choroid plexus.ICV injection of Cre recombinase into those transgenic mice or ICV injection of AAV2/5, or it's targeted SHI in the WT mice. These methods overcome issues with promoting specificity and transgenetic mouse breeding.

Watch this Scientific Journal Video about ChP Specific Knockdown of A2ARs with AAV2/5-shRNA and Cre/LoxP System at JoVE.com

ChP Specific Knockdown of A2ARs with AAV2/5-shRNA and Cre/LoxP System  

AAV2/5-shRNA 및 Cre/LoxP 시스템을 사용한A 2AR의 ChP 특이적 knockdown

시작하려면 마취된 쥐를 수술 플랫폼에 놓습니다. 머리카락을 다듬은 후 양쪽 눈 아래에 눈 연고를 발라 건조하지 않도록 합니다. 그런 다음 고정을 위해 쥐를 입체 장치에 고정합니다.두개골이 완전히 노출되도록 현미경으로 작게 절개합니다. AAV2/5-아데노신 2A 수용체 shRNA를 포함하는 주사기 2개와 AAV2/5 스크램블을 포함하는 주사기 2개를 준비합니다. 그런 다음 현미경을 사용하여 브레그마와 람다를 찾고 쥐 뇌 입체 아틀라스를 기반으로 10마이크로리터 주사기의 좌표 점을 설정합니다.설정된 좌표 지점에서 두개골에 작은 구멍을 뚫습니다. AAV2/5-아데노신 2A 수용체 shRNA 바이러스 2마이크로리터를 분당 100나노리터의 일정한 속도로 측면 주입을 통해 뇌실에 주입합니다. 빼기 전에 바늘을 10분 동안 제자리에 두십시오.바이러스 손실을 방지하기 위해 의료용 바이오 피브린 접착제를 사용하여 손상된 피부를 즉시 밀봉하십시오. 마우스를 가열 패드에 놓고 체온을 약 섭씨 37도로 유지하여 회복을 용이하게 합니다. 실험군의 경우 Arosa L-D-L-T-D 토마토 마우스의 각 외측 심실에 2마이크로리터의 CRE-TAT 재조합효소를 주입하고 대조군의 경우 2마이크로리터의 멸균 PBS를 주입합니다.CRE-TAT 재조합효소 주입 2주 후, 냉동 조직 절편을 준비하고, 핵 염색을 수행하고, 이미징을 위한 슬라이드를 준비합니다 CRE-TAT를 사용하여 동형접합 아데노신 2A 수용체 플럭스 마우스의 맥락총에서 아데노신 2A 수용체를 녹다운하면 EAE 임상 점수가 감소했습니다. 마찬가지로, AAV2/5 shRNA를 사용하여 아데노신 2A 수용체 야생형 마우스에서 맥락총(choroid plexus)을 녹다운하면 EAE 임상 점수가 감소했습니다. QPCR 분석 결과, AAV2/5 shRNA 및 CRE-TAT 투여군에서 아데노신 2A 수용체의 mRNA 수치가 각 대조군에 비해 감소한 것으로 나타났다.특히 맥락총에 있는 아데노신 2A 수용체를 침묵시키면 척수로의 면역 세포 침투가 감소합니다.

Research

JoVE Journal

Neuroscience

ChP Specific Knockdown of A2ARs with AAV2/5-shRNA and Cre/LoxP System

Targeted Knockdown of Genes in the Choroid Plexus

The choroid plexus (ChP) serves as a critical gateway for immune cell infiltration into the central nervous system (CNS) under both physiological and ...