Name: Специфический нокдаун A2AR с помощью AAV2/5-shRNA и системы Cre/LoxP
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

Селективный сайленсинг гена в сосудистом сплетении

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.

В центре внимания автора: Идеи и инновации в методах манипулирования экспрессией генов для исследования сосудистого сплетения 

Направленный нокдаун генов в сосудистом сплетении

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

Специфический нокдаун A2AR с помощью AAV2/5-shRNA и системы Cre/LoxP  

Специфический нокдаун A2ARs с помощью AAV2/5-shRNA и системы Cre/LoxP

Для начала поместите мышей под наркозом на операционную платформу. После стрижки волос нанесите глазную мазь на оба глаза, чтобы предотвратить их высыхание. Затем закрепите мышей на стереотаксическом аппарате для иммобилизации.Сделайте небольшой разрез под микроскопом, чтобы полностью обнажить череп. Подготовьте два шприца объемом 10 микролитров, один из которых содержит шРНК рецептора AAV2/5-аденозина 2А, а другой — скрембл AAV2/5. Затем с помощью микроскопа определите местоположение брегмы и лямбды и установите координатную точку на шприце объемом 10 микролитров на основе стереотаксического атласа мозга мыши.Просверлите небольшое отверстие в черепе в установленной координатной точке. Введите два микролитра вируса шРНК рецептора AAV2/5-аденозина 2А в желудочек головного мозга путем боковой инъекции с постоянной скоростью 100 нанолитров в минуту. Удерживайте иглу на месте в течение 10 минут перед извлечением.Своевременно запечатайте поврежденную кожу с помощью медицинского биофибринового клея, чтобы предотвратить потерю вируса. Поместите мышь на грелку, поддерживая температуру ее тела примерно на уровне 37 градусов по Цельсию, чтобы облегчить восстановление. Введите два микролитра рекомбиназы CRE-TAT в каждый боковой желудочек мыши томата Arosa L-D-L-T-D для экспериментальной группы и два микролитра стерильной PBS для контрольной группы.Через две недели после инъекции рекомбиназы CRE-TAT подготовьте замороженные срезы тканей, выполните ядерное окрашивание и подготовьте предметное стекло для визуализации Сбивание рецептора аденозина 2A в сосудистом сплетении у гомозиготных аденозиновых рецепторов 2A мыши с использованием CRE-TAT привело к снижению клинических показателей EAE. Аналогичным образом, сбивание сосудистого сплетения у мышей дикого типа с рецептором аденозина 2А с использованием шРНК AAV2/5 снижало клинические баллы EAE. Анализ QPCR показал, что уровни мРНК рецептора аденозина 2А снижались в группах AAV2/5 shRNA и CRE-TAT по сравнению с каждым контролем.Сайленсинг рецептора аденозина 2А, особенно в сосудистом сплетении, снижает инфильтрацию иммунных клеток в спинной мозг.

chp-specific-knockdown-of-a2ars-with-aav25-shrna-and-creloxp-system

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 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.

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 ...

To begin, place the anesthetized mice on the surgical platform. After trimming the hair, apply eye ointment down both eyes to prevent them from drying. Then fix the mice onto a stereotaxic apparatus for immobilization.Make a small incision under a microscope to fully expose the skull. Prepare two 10 microliter syringes, one containing AAV2/5-adenosine 2A receptor shRNA, and the other containing AAV2/5 scramble. Then use the microscope to locate the bregma and lambda and set the coordinate point on the 10 microliter syringe based on the mouse brain stereotaxic atlas.Drill a small hole in the skull at the established coordinate point. Inject two microliters of AAV2/5-Adenosine 2A receptor shRNA virus into the brain ventricle via lateral injection at a steady rate of 100 nanoliters per minute. Keep the needle in place for 10 minutes before withdrawing.Seal the injured skin promptly using medical bio fibrin glue to prevent virus loss. Place the mouse on a heating pad, maintaining its body temperature approximately at 37 degrees Celsius to facilitate recovery. Inject two microliters of CRE-TAT Recombinase into each lateral ventricle of Arosa L-D-L-T-D tomato mouse for the experimental group, and two microliters of sterile PBS for the control group.Two weeks after the CRE-TAT Recombinase injection, prepare frozen tissue sections, perform nuclear staining and prepare the slide for imaging Knocking down adenosine 2A receptor in the choroid plexus in homozygous adenosine 2A receptor fluxed mouse, using CRE-TAT, led to decreased EAE clinical scores. Similarly, knocking down choroid plexus in adenosine 2A receptor wild-type mice, using AAV2/5 shRNA decreased EAE clinical scores. The QPCR analysis showed that the mRNA levels of adenosine 2A receptor decreased in the AAV2/5 shRNA and CRE-TAT groups, compared to each control.Silencing adenosine 2A receptor, specifically in the choroid plexus, reduced immune cell infiltration into the spinal cord.