Summary

Targeted Knockdown of Genes in the Choroid Plexus

Published: June 16, 2023
doi:

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 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.

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 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'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.

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 A2ARflox/flox mice.
  3. Randomly assign the mice to two groups and house in cages, with a maximum of five mice per cage, under a standard 12 h light/12 h dark cycle for 1 week.
  4. Provide the mice with sufficient food and water and maintain them at a constant temperature at 25 °C.

2. ChP-specific knockdown of A2ARs with AAV2/5-shRNA

  1. Weigh each mouse and write down the values.
  2. Anesthetize the mice intraperitoneally with 1% pentobarbital sodium at a dosage of 6-8 mL/kg equivalent to 60-80 mg/kg pentobarbital, and place the mouse on a heating pad to keep it warm.
    NOTE: It is crucial to administer the correct dosage of pentobarbital sodium according to the mouse's weight to ensure successful surgery. The anesthesia should not be too deep, as it may result in mortality, but should not be too shallow, as the mouse may wake up during the procedure, potentially affecting the effectiveness of the virus injection. Check for the proper anesthesia dose by toe pinch and ensuring that mice do not respond.
  3. Use a specialized mouse shaver to trim the top hair of the anesthetized mice.
  4. Apply eye ointment on both eyes to prevent from drying, and fix the mice onto a stereotaxic apparatus to immobilize the brain. Cover the animal with a sterile drape.
  5. Thoroughly sterilize the skin of the head and neck of the mice with three rounds of iodophor disinfectant and 75% alcohol to reduce postoperative infection. Then apply 1% lidocaine topically to the scalp of mice and make a small incision to fully expose the skull under a microscope.
    NOTE: Use sterile instruments throughout the procedure.
  6. Prepare two 10 µL syringes: one with purchased AAV2/5-A2AR-shRNA (titer: 6.27 × 109 vg/µL) and the other with purchased AAV2/5-Scramble (titer: 6.21 × 109 vg/µL).
    NOTE: When using the syringe, the front end needs to be connected to a glass capillary, and a range of 10 µL can be obtained by controlling the length of the glass capillary.
  7. Use the microscope to locate the bregma and lambda and set the coordinate point (AP: -0.58; ML: ±1.10; DL: -2.20) on the 10 µL syringe (Figure 1). The coordinate point is based on the mouse brain stereotaxic atlas15.
  8. Drill a small hole in the skull at the adjusted coordinate point.
    NOTE: Under the microscope, drilling is performed using a sterile microdrill against the surface of the skull. After drilling, by removing the meninges covering the brain and exposing the underlying brain parenchyma, injury to the blood vessels is prevented. This step prevents the tip of the glass capillary from being broken off by the meninges. The diameter of the drilled hole should be sufficient to allow the needle tip to enter the brain tissue.
  9. Administer 2 µL of AAV2/5-A2AR-shRNA virus into the brain ventricle via lateral injection at a steady rate of 100 nL/min. Keep the needle in place for 10 min before withdrawing. Note that the virus was administered with unilateral injection.
  10. Seal the injured skin using medical biofibrin glue promptly to prevent virus loss, which can be rinsed out with CSF after removing the needle if the glue is not applied quickly. Also, do not use cotton buds to wipe the CSF, as it may also cause virus loss.
  11. To facilitate recovery, place the mouse on a heating pad to maintain the body temperature at 37.0 ± 0.5 °C, and apply 1% lidocaine topically to the mouse's scalp. Allow the mice to recuperate for 2 weeks before inducing the EAE model, as this period is necessary for AAV2/5-A2AR-shRNA virus expression and subsequent A2AR knockdown.

3. ChP-specific knockdown of A2AR with a Cre/locus of X-overP1 (Cre/LoxP) system

NOTE: The following procedures can be achieved using the method described previously. Refer to steps 2.1-2.11 for detailed injection methods.

  1. Inject 2 µL of CRE-TAT recombinase into each lateral ventricle of a Rosa-LSL (Lox-StoP-Lox)-tdTomato mouse as the experimental group and 2 µL of sterile phosphate-buffered saline (PBS) as the control group. Using the same protocol as above (section 2), inject 2 µL of CRE-TAT recombinase into each of the lateral ventricles of A2ARflox/flox mice along with 2 µL of sterile PBS as a control group.
  2. Prepare frozen tissue sections and perform nuclear staining 2 weeks after the CRE-TAT recombinase injection. See steps 4.1-4.3 for more information.

4. Transcardial perfusion in mice

  1. Administer 60-80 mg/kg pentobarbital sodium to deeply anesthetize the mice. Confirm the plane of anesthesia by a toe pinch and perform transcardial perfusion using 40 mL of sterile PBS solution followed by 20 mL of 4% paraformaldehyde (PFA).
    NOTE: Successful perfusion is indicated by a white color in the liver, while the presence of enlarged lungs or PBS outflow from the mouth during perfusion suggests a failure of the procedure.
  2. Quickly extract the mouse brain, ensuring minimal protein degradation.
  3. Submerge the mouse brain in 4% PFA/PBS overnight for post-fixation, followed by replacement with 30% sucrose PB solution for 72 h.
    ​NOTE: It is critical to avoid excessive dehydration of the brain tissue, so the brain should not be left in the sucrose PB solution for too long.

5. Frozen tissue sectioning and staining

  1. Embed the previously dehydrated mouse brain in optimal cutting temperature (OCT) glue, freeze the embedded brain, and use a sliding microtome to cut coronal sections with a thickness of 20 µm.
  2. Place the brain sections on the glass slide. Once the brain sections are dry, store them in a -20 °C refrigerator for subsequent experiments.
  3. Place each glass slide into a frame case and submerge the frame into a container filled with PBS to thoroughly rinse the slide. Gently rinse the brain slides with PBS solution three times, for 10 min each time.
    NOTE: Caution should be exercised to prevent the brain sections from falling off the glass slide.
  4. Stain the brain slides with 4′,6-diamidino-2-phenylindole (DAPI) solution for 10 min.
  5. Rinse the brain slides with PBS solution for 5 min.
  6. Apply one drop of antifade mounting medium to the brain sections.
  7. Place a coverslip on the brain sections, seal the coverslip with nail polish, and analyze the sections using a conventional fluorescence microscope.

6. EAE induction

NOTE: Perform EAE induction after 2 weeks of the shRNA or CRE-TAT recombinase injection11.

  1. Create an aqueous solution by mixing 2.5 mg myelin oligodendrocyte glycoprotein (MOG35-55) with 2 mL of PBS. Prepare a complete Freunds adjuvant (CFA) oil solution by mixing M. tuberculosis (H37Ra) with incomplete Freunds adjuvant (IFA).
  2. Mix the aqueous and oil solutions in a 1:1 ratio. Use a tee pipe to whip the mixture into an oil-in-water state.
    NOTE: The tee pipe is also called a three-way pipe. It is a plastic tube that can control the direction of flowing solution to fully mix the MOG35-55 and CFA.
  3. Use a high-speed homogenizer to make the MOG antigen emulsion for the EAE model under ice bath conditions.
  4. Anesthetize the mice intraperitoneally with 1% pentobarbital sodium at a dosage of 6-8 mL/kg equivalent to 60-80 mg/kg pentobarbital.
  5. Subcutaneously inject the MOG antigen emulsion into four different points (neck, back, left and right hips) at a volume of 10 mL/kg for a total of four injections each.
    NOTE: It is important to carefully select the injection site, as different sites may have varying effects on the morbidity and mortality of mice. Additionally, repeated MOG injections can lead to immune tolerance, so the research team chose a single injection method to prevent this potential issue.
  6. Immediately inject 500 ng/mL of pertussis toxin (PT) intraperitoneally at a dosage of 5 mg/kg after MOG injection.
    NOTE: PT increases the permeability of the blood-brain barrier (BBB) and facilitates the infiltration of T cells into the brain.
  7. After 48 h, intraperitoneally inject the PT solution at the same volume.

7. Neurological deficit score

  1. Evaluate and grade the mice daily using a rating scale16 ranging from 0 to 15 to assess the incidence and severity of EAE according to the following neurological deficits:
    Tail: 0 indicates no signs, 1 represents a half-paralyzed tail, and 2 indicates a fully paralyzed tail.
    Limbs: 0 indicates no signs, 1 represents a weak or altered gait, 2 indicates paresis, and 3 represents a fully paralyzed limb.
  2. Assign a quadriplegic animal having complete paralysis a score of 12, and assign mortality a score of 15.

8. Hematoxylin-eosin (H&E) staining

  1. Take the dehydrated mouse brain and embed it in molten paraffin. Allow the paraffin block to cool and solidify for later use.
    NOTE: The paraffin block should be completely dry and cool to avoid tissue damage.
  2. Cut the mouse brain paraffin block into 5 µm thick slides. Place the paraffin mouse brain section on a glass slide and dry it in a 60 °C oven for 3 h.
  3. Submerge the slides sequentially in xylene solution I for 10 min, xylene solution II for 10 min, 100% alcohol I for 3 min, 100% alcohol II for 3 min, 95% alcohol for 3 min, 90% alcohol for 3 min, 80% alcohol for 3 min, 70% alcohol for 3 min, and distilled water for 1 min.

9. Quantitative polymerase chain reaction (qPCR) analysis

  1. After perfusing the mice with PBS, remove the ChP from the ventricles and extract RNA with Trizol. Synthesize the cDNA using the first Strand cDNA Synthesis Kit.
  2. Carry out qPCR analysis using Ex Taq SYBR-green premix and a real-time PCR system. Use the following A2AR primers: forward – GCCATCCCATTCGCCATCA; reverse – GCAATAGCCAAGAGGCTGAAGA.

Representative Results

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 A2ARflox/flox mice. To ensure ChP specificity, we first injected CRE-TAT into the lateral ventricles of Rosa-LSL (Lox-StoP-Lox)-tdTomato (Ai9) mice. The images indicate that the spontaneous tdTomato fluorescence was restricted to the ChP tissue (Figure 2). Similarly, the study administered AAV2/5-CMV-A2AR-shRNA-CMV-enhanced green fluorescent protein (EGFP) into C57BL/6 mice and found that the EGFP fluorescence was limited to the epithelial cell layer of the ChP and did not infect the surrounding parenchymal cells near the lateral ventricles (Figure 3).

EAE induction with MOG35-55
To induce stable EAE, mice were subcutaneously injected with an emulsion consisting of MOG35-55 and CFA, followed by intraperitoneal injection of PT on days 0 and 2 after immunization (Figure 4). The Clinical Symptom Scale was used to evaluate EAE scores daily, based on the tail and limb condition. The onset of EAE was defined as the first day with an EAE score ≥1, while the duration between onset and peak EAE scores was called the progressive stage.

ChP-specific A2AR knockdown alleviates EAE pathology
To investigate the involvement of the A2AR signal in EAE pathology, the study employed a ChP-specific A2AR knockdown. The study specifically knocked down A2AR in the ChP using ICV injection of CRE-TAT or AAV2/5-A2AR-shRNA. At 2 weeks after knockdown, EAE was induced by MOG35-55 immunization. The results showed that, compared to the control group, mice with A2AR knockdown developed milder EAE pathology, as evidenced by lower scores and a reduced infiltration of immune cells in the spinal cord (Figure 5A,B,E,F). In addition, five EAE-induced mice from each group at day 20 following MOG35-55 immunization were randomly selected. After perfusion with PBS, the ChPs were isolated for RNA extraction and qPCR. The qPCR analysis showed that the mRNA levels of A2AR were obviously decreased in the AAV2/5-shRNA (A2AR-Kd) and CRE-TAT groups, compared to each control (Figure 5C,D).

Figure 1
Figure 1: Anatomical localization of the lateral ventricle injection site. (A) A schematic diagram showing the point of virus injection. (B) The injection site of the lateral ventricle is located 0.58 mm below the bregma and 1.1 mm lateral to the sagittal suture, as indicated by the red dot. (C) An image showing the site of virus injection on a mouse. Please click here to view a larger version of this figure.

Figure 2
Figure 2: Localization of tdTomato fluorescence in the ChP. (A,B) Representative image of Rosa-LSL (Lox-StoP-Lox)-tdTomato mice treated with ICV injection of CRETAT. At 2 weeks later, the tdTomato autofluorescence was specifically localized to the ChP tissue (n = 3/group). (C,D) The merged images of the tdTomato autofluorescence and DAPI. Please click here to view a larger version of this figure.

Figure 3
Figure 3: Representative images of C57BL/6 mice taken 2 weeks after ICV injection of AAV2/5-scramble-EGFP or AAV2/5-shRNA-EGFP. (A,B) The representative images of mice injected with AAV2/5-scramble-EGFP (n = 3/group). (C,D) The representative images of mice injected with AAV2/5-shRNA-EGFP (n = 3/group). Please click here to view a larger version of this figure.

Figure 4
Figure 4: Induction of the EAE model. (A) Firstly, the MOG antigen emulsion is subcutaneously injected into four different locations (neck, back, left and right hips), which are denoted by red dots. (B) PT is intraperitoneally injected at the time of immunization and repeated 2 days later. Please click here to view a larger version of this figure.

Figure 5
Figure 5: ChP-specific knockdown of A2AR alleviated EAE pathology. (A) ChP-specific knockdown of A2AR in A2ARflox/flox mice, achieved through ICV injection of CRE-TAT, led to decreased EAE clinical scores (n = 6-7/group). Statistical analysis was performed using twoway RM ANOVA followed by Sidak's multiple comparison test. (B) ChP-specific knockdown of A2AR in wild-type (WT) mice, achieved with ICV injection of AAV2/5-shRNA, decreased EAE clinical scores (n = 7-8/group). Statistical analysis was performed using twoway RM ANOVA followed by Sidak's multiple comparison test. (C,D) Results of the qPCR analysis of A2AR mRNA levels in ChP tissue (n = 5/group). Statistical analysis was performed using an unpaired t-test. (E,F) H&E staining. ChP-specific A2AR knockdown attenuated immune cell infiltration into the spinal cord. Statistical significance is represented as ###p < 0.001, **p < 0.01, and ***p < 0.001. Please click here to view a larger version of this figure.

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 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 on EAE pathology. Of note, this protocol includes two crucial steps. Firstly, the stereotaxic localization operation is essential, and significant deviation from the suggested coordinates may result in random failures. Secondly, the volume of the injected virus is critical, as it has been found that injecting less than 1 µL of virus (~6 x 1012) also has a certain chance of failure. This is possibly due to the need for an adequate amount of virus particles for sufficient infection.

Using the Cre/LoxP system did not allow observation of the precise distribution of CRE-TAT recombinase in the brain following ICV injection. As a result, the study utilized Rosa-LSL (Lox-StoP-Lox)-tdTomato mice to track the distribution of CRE-TAT using the autofluorescence of the tdTomato protein. The tdTomato fluorescence was exclusively located in ChP tissues 2 weeks after injecting CRE-TAT intracerebroventricularly, indicating that the recombinase was primarily absorbed by the ChP. Next, CRE-TAT was administered to A2ARflox/flox mice, and a decrease in A2AR mRNA levels was observed in the ChP tissue. This targeted knockdown strategy was used to investigate the role of corticosteroid signaling in the ChP in mediating psychological stress17. Alternatively, the research used AAV2/5 to deliver shRNA designed to target ChP genes. Previous studies have evaluated the infection ability of different serotypes of AAVs and lentiviruses in ChP tissue and found that AAV2/5 and AAV2/8 were the most effective14. The study revealed that AAV2/5 was capable of infecting the ChP specifically without causing obvious infections in other brain regions. These two methods are less laborious than classical knockout strategies that require two transgenic lines (Cre and Flox lines)6,18. One limitation of this study is the absence of a gain-of-function experiment with gene overexpression. However, a possible approach to achieve this would be to clone the full cDNA and package it into the AAV2/5 virus, which would be administered via ICV infection. In a previous study, overexpression of NKCC1 in the ChP using AAV2/5 was found to promote CSF clearance and reduce ventriculomegaly in vivo9. It is important to note that either the AAV2/5 or Cre/LoxP strategy have their own advantage. The ICV injection of CRE-TAT into transgenic mice ensures high knockdown efficiency, as the target gene is directly deleted from the DNA of the cells. However, this method depends on floxed mice production and breeding. On the other hand, the ICV injection of AAV2/5 avoids the time-consuming process of breeding transgenic mice. However, the knockdown efficiency of this method largely depends on the performance of the designed shRNA. Therefore, researchers can choose a suitable method based on their experimental conditions.

Multiple sclerosis (MS) is an autoimmune disease that causes inflammation and demyelination of white matter in the CNS19. To study the pathological mechanisms of MS, researchers have used an EAE model that simulates the disease's symptoms, such as demyelination and immune infiltration. This model is considered ideal for understanding MS. In 2009, it was discovered that immune cells infiltrate the CNS through the ChP, which is a key route for immune cell entry in MS pathology. The "first wave" of immune cells enters CSF through the ChP, followed by the "second wave", which enters the brain parenchyma through the BBB20. The "first wave" of immune cells promotes inflammation and accelerates BBB leakage, so inhibiting immune infiltration at the ChP could be useful for early intervention in MS. Chemokines and adhesion molecules regulate the gating activity of ChP, controlling the ability of lymphocytes to infiltrate across it21,22,23.

Previous studies used whole-body knockout or pharmacological technology (such as neutralizing antibodies) to investigate signaling molecules in the ChP, but these methods did not accurately define their biological function in the MS pathological process. In a recent study, researchers knocked down A2AR, located in the ChP of A2ARflox/flox mice, and found that EAE scores and immune infiltration were significantly reduced11. This study confirms the role of A2AR signaling in the ChP during EAE pathology and demonstrates that ChP-specific knockdown methods are useful tools for studying ChP function in CNS pathologies.

In conclusion, the ChP-specific manipulation protocol is an ideal tool to explore the biology function of the ChP involved in degenerative diseases in the CNS, such as Parkinson's disease, Alzheimer's disease, MS, and so on.

Declarações

The authors have nothing to disclose.

Acknowledgements

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.

Materials

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

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Yang, Y., Qi, C., Hu, L., Zheng, C., Li, X., Zheng, W., Weng, Y., Lin, H. Targeted Knockdown of Genes in the Choroid Plexus. J. Vis. Exp. (196), e65555, doi:10.3791/65555 (2023).

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