Name: Insights and Innovations in Gene Expression Manipulation Techniques for Choroid Plexus Research (Video) | JoVE
Uploaded: 2023-06-16T14:10:00
Description: Watch this Scientific Journal Video about Insights and Innovations in Gene Expression Manipulation Techniques for Choroid Plexus Research at JoVE.com

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.

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
<ol>
	<li>Purchase male C57BL/6 mice aged 8-12 weeks and weighing 20-22 g.</li>
	<li>Obtain the transgenic Rosa-LSL (Lox-StoP-Lox)-tdTomato (Ai9) mouse line, and male A2ARflox/flox mice.<...

Selective Gene Silencing in the Choroid Plexus

<ol>
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B., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cotransporter-mediated+water+transport+underlying+cerebrospinal+fluid+formation.">Cotransporter-mediated water transport underlying cerebrospinal fluid formation.</a> Nature Communications. 9 (1), 2167 (2018).</li><li>Zhu, L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Klotho+controls+the+brain-immune+system+interface+in+the+choroid+plexus.">Klotho controls the brain-immune system interface in the choroid plexus.</a> Proceedings of the National Academy of Sciences. 115 (48), E11388-E11396 (2018).</li><li>Chen, X., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Different+serotypes+of+adeno-associated+virus+vector-+and+lentivirus-mediated+tropism+in+choroid+plexus+by+intracerebroventricular+delivery.">Different serotypes of adeno-associated virus vector- and lentivirus-mediated tropism in choroid plexus by intracerebroventricular delivery.</a> Human Gene Therapy. 31 (7-8), 440-447 (2020).</li><li>Konsman, J. 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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 ...

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.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>A2ARflox/flox mice</td>
			<td>State Key Laboratory of Ophthalmology, Optometry and Visual Science, Wenzhou Medical University</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>AAV2/5-A2AR-ShRNA virus</td>
			<td>Shanghai Heyuan Biotechnology Co. LTD</td>
			<td>pt-4828</td>
			<td></td>
		</tr>
		<tr>
			<td>antifade mounting medium</td>
			<td>Beyotime Biotechnology</td>
			<td>0100-01</td>
			<td></td>
		</tr>
		<tr>
			<td>borosilicate glass capillary</td>
			<td>Beijing Meiyaxian Technology Co. Ltd</td>
			<td>B100-50-10</td>
			<td></td>
		</tr>
		<tr>
			<td>brain stereotaxic apparatus</td>
			<td>RWD, Shenzhen</td>
			<td>69100</td>
			<td></td>
		</tr>
		<tr>
			<td>C57BL/6 mice</td>
			<td>Beijing Vital Charles River Laboratory Animal Technology Company</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>CRE-TAT recombinase</td>
			<td>Millipore</td>
			<td>SCR508</td>
			<td></td>
		</tr>
		<tr>
			<td>DAPI</td>
			<td>Absin</td>
			<td>B25A031</td>
			<td></td>
		</tr>
		<tr>
			<td>frozen slicing machine</td>
			<td>Leica</td>
			<td>CM1950</td>
			<td></td>
		</tr>
		<tr>
			<td>H37Ra</td>
			<td>Becton Dickinson and company</td>
			<td>231141</td>
			<td></td>
		</tr>
		<tr>
			<td>Hamilton syringe</td>
			<td>Hamilton, American</td>
			<td>P/N:&#160;86259</td>
			<td></td>
		</tr>
		<tr>
			<td>Incomplete Freunds adjuvant</td>
			<td>Sigma</td>
			<td>F5506</td>
			<td></td>
		</tr>
		<tr>
			<td>Laser confocal microscope</td>
			<td>Zeiss</td>
			<td>LSM900</td>
			<td></td>
		</tr>
		<tr>
			<td>MOG35-55</td>
			<td>Suzhou Qiangyao Biotechnology Co., LTD</td>
			<td>4010006243</td>
			<td></td>
		</tr>
		<tr>
			<td>OCT glue</td>
			<td>Epredia</td>
			<td>6502p</td>
			<td></td>
		</tr>
		<tr>
			<td>paraformaldehyde</td>
			<td>Chengdu Kelong Chemical Reagent Company</td>
			<td>30525-89-4</td>
			<td></td>
		</tr>
		<tr>
			<td>pentobarbital sodium</td>
			<td>Boyun Biotech</td>
			<td>PC13003</td>
			<td></td>
		</tr>
		<tr>
			<td>Pipette gun</td>
			<td>Eppendorf</td>
			<td>N45014F</td>
			<td></td>
		</tr>
		<tr>
			<td>PrimeScript 1st Strand cDNA Synthesis Kit</td>
			<td>Takara</td>
			<td>&#160;6110A</td>
			<td></td>
		</tr>
		<tr>
			<td>Real- Time PCR System</td>
			<td>BioRad</td>
			<td>CFX96</td>
			<td></td>
		</tr>
		<tr>
			<td>Rosa-LSL (Lox-StoP-Lox)-tdTomato mice</td>
			<td>Jackson Laboratory</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>sucrose</td>
			<td>Sangon Biotech</td>
			<td>A502792-0500</td>
			<td></td>
		</tr>
		<tr>
			<td>super high speed homogenizer</td>
			<td>IKA</td>
			<td>3737025</td>
			<td></td>
		</tr>
		<tr>
			<td>Trizol</td>
			<td>Invitrogen</td>
			<td>15596026</td>
			<td></td>
		</tr>
		<tr>
			<td>xylene solution</td>
			<td>Chengdu Kelong Chemical Reagent Company</td>
			<td>1330-20-7</td>
			<td></td>
		</tr>
	</tbody>
</table>

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.

Targeted Knockdown of Genes in the Choroid Plexus

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...

Watch this Scientific Journal Video about Insights and Innovations in Gene Expression Manipulation Techniques for Choroid Plexus Research at JoVE.com

Insights and Innovations in Gene Expression Manipulation Techniques for Choroid Plexus Research (Video) | JoVE

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 ...