Name: targeted knockdown of genes in the choroid plexus
Uploaded: 2023-06-16T14:10:00.000+00:00
Duration: 4 min 43 s
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.</li>
	<li>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.</li>
	<li>Provide the mice with sufficient food and water and maintain them at a constant temperature at 25 &#176;C.</li>
</ol>
2. ChP-specific knockdown of A2ARs with AAV2/5-shRNA
<ol>
	<li>Weigh each mouse and write down the values.</li>
	<li>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&#39;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.</li>
	<li>Use a specialized mouse shaver to trim the top hair of the anesthetized mice.</li>
	<li>Apply&#160;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.</li>
	<li>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.</li>
	<li>Prepare two 10 &#181;L syringes: one with purchased AAV2/5-A2AR-shRNA (titer: 6.27 &#215; 109 vg/&#181;L) and the other with purchased AAV2/5-Scramble (titer: 6.21 &#215; 109 vg/&#181;L). 
	NOTE: When using the syringe, the front end needs to be connected to a glass capillary, and a range of 10 &#181;L can be obtained by controlling the length of the glass capillary.</li>
	<li>Use the microscope to locate the bregma and lambda and set the coordinate point (AP: -0.58; ML: &#177;1.10; DL: -2.20) on the 10 &#181;L syringe (Figure 1). The coordinate point is based on the mouse brain stereotaxic atlas15.</li>
	<li>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.</li>
	<li>Administer 2 &#181;L 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.</li>
	<li>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.</li>
	<li>To facilitate recovery, place the mouse on a heating pad to maintain the body temperature at&#160;37.0 &#177; 0.5 &#176;C, and apply 1% lidocaine topically to the mouse&#39;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.</li>
</ol>
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.
<ol>
	<li>Inject 2 &#181;L of CRE-TAT recombinase into each lateral ventricle of a Rosa-LSL (Lox-StoP-Lox)-tdTomato mouse as the experimental group and 2 &#181;L of sterile phosphate-buffered saline (PBS) as the control group. Using the same protocol as above (section 2), inject 2 &#181;L of CRE-TAT recombinase into each of the lateral ventricles of A2ARflox/flox mice along with 2 &#181;L of sterile PBS as a control group.</li>
	<li>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.</li>
</ol>
4. Transcardial perfusion in mice
<ol>
	<li>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.</li>
	<li>Quickly extract the mouse brain, ensuring minimal protein degradation.</li>
	<li>Submerge the mouse brain in 4% PFA/PBS overnight for post-fixation, followed by replacement with 30% sucrose PB solution for 72 h. 
	&#8203;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.</li>
</ol>
5. Frozen tissue sectioning and staining
<ol>
	<li>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 &#181;m.</li>
	<li>Place the brain sections on the glass slide. Once the brain sections are dry, store them in a -20 &#176;C refrigerator for subsequent experiments.</li>
	<li>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.</li>
	<li>Stain the brain slides with 4&#8242;,6-diamidino-2-phenylindole (DAPI) solution for 10 min.</li>
	<li>Rinse the brain slides with PBS solution for 5 min.</li>
	<li>Apply one drop of antifade mounting medium to the brain sections.</li>
	<li>Place a coverslip on the brain sections, seal the coverslip with nail polish, and analyze the sections using a conventional fluorescence microscope.</li>
</ol>
6. EAE induction
NOTE: Perform EAE induction after 2 weeks of the shRNA or CRE-TAT recombinase injection11.
<ol>
	<li>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).</li>
	<li>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.</li>
	<li>Use a high-speed homogenizer to make the MOG antigen emulsion for the EAE model under ice bath conditions.</li>
	<li>Anesthetize the mice intraperitoneally with 1% pentobarbital sodium at a dosage of 6-8 mL/kg equivalent to 60-80 mg/kg pentobarbital.</li>
	<li>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.</li>
	<li>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.</li>
	<li>After 48 h, intraperitoneally inject the PT solution at the same volume.</li>
</ol>
7. Neurological deficit score
<ol>
	<li>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.</li>
	<li>Assign a quadriplegic animal having complete paralysis&#160;a score of 12, and assign mortality a score of 15.</li>
</ol>
8. Hematoxylin-eosin (H&#38;E) staining
<ol>
	<li>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.</li>
	<li>Cut the mouse brain paraffin block into 5 &#181;m thick slides. Place the paraffin mouse brain section on a glass slide and dry it in a 60 &#176;C oven for 3 h.</li>
	<li>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.</li>
</ol>
9. Quantitative polymerase chain reaction (qPCR) analysis
<ol>
	<li>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.</li>
	<li>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.</li>
</ol>

Selective Gene Silencing in the Choroid Plexus

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The authors state that they have no competing financial interests or other disclosures to declare.

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><tbody><tr><td>A&lt;sub&gt;2A&lt;/sub&gt;R&lt;sup&gt;flox/flox&lt;/sup&gt; 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-A&lt;sub&gt;2A&lt;/sub&gt;R-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:&amp;nbsp;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>MOG&lt;sub&gt;35-55&lt;/sub&gt;</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>&amp;nbsp;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.

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

Author Spotlight: Insights and Innovations in Gene Expression Manipulation Techniques for Choroid Plexus Research 

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

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 ...

targeted-knockdown-of-genes-in-the-choroid-plexus

Research

JoVE Journal

Neuroscience

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

Video: Author Spotlight: Insights and Innovations in Gene Expression Manipulation Techniques for Choroid Plexus Research 

Micromanipulation of Gene Expression in the Adult Zebrafish Brain Using Cerebroventricular Microinjection of Morpholino Oligonucleotides

Manipulation of gene expression in tissues is required to perform functional studies. In this paper, we demonstrate the cerebroventricular microinjection (CVMI) technique as a means to modulate gene expression in the adult zebrafish brain. By using CVMI, substances can be administered into the cerebroventricular fluid and be thoroughly distributed along the rostrocaudal axis of the brain. We particularly focus on the use of antisense morpholino oligonucleotides, which are potent tools for knocking down gene expression in vivo. In our method, when applied, morpholino molecules are taken up by the cells lining the ventricular surface. These cells include the radial glial cells, which act as neurogenic progenitors. Therefore, knocking down gene expression in the radial glial cells is of utmost importance to analyze the widespread neurogenesis response in zebrafish, and also would provide insight into how vertebrates could sustain adult neurogenesis response. Such an understanding would also help the efforts for clinical applications in human neurodegenerative disorders and central nervous system regeneration. Thus, we present the cerebroventricular microinjection method as a quick and efficient way to alter gene expression and neurogenesis response in the adult zebrafish forebrain. We also provide troubleshooting tips and other useful information on how to carry out the CVMI procedure.

In this article, we demonstrate a method for manipulation of gene expression in the ventricular cells of the adult zebrafish telencephalon using antisense morpholino oligonucleotides. We present this method as an efficient and quick protocol that can be used for functional studies in the adult vertebrate brain.

Manipulation of gene expression in tissues is  required to perform functional studies. In this paper, we demonstrate the  cerebroventricular ...

Regioselective Biolistic Targeting in Organotypic Brain Slices Using a Modified Gene Gun

Transfection of DNA has been invaluable for biological sciences and with recent advances to organotypic brain slice preparations, the effect of various heterologous genes could thus be investigated easily while maintaining many aspects of in vivo biology. There has been increasing interest to transfect terminally differentiated neurons for which conventional transfection methods have been fraught with difficulties such as low yields and significant losses in viability. Biolistic transfection can circumvent many of these difficulties yet only recently has this technique been modified so that it is amenable for use in mammalian tissues.
New modifications to the accelerator chamber have enhanced the gene gun&#39;s firing accuracy and increased its depths of penetration while also allowing the use of lower gas pressure (50 psi) without loss of transfection efficiency as well as permitting a focused regioselective spread of the particles to within 3 mm. In addition, this technique is straight forward and faster to perform than tedious microinjections. Both transient and stable expression are possible with nanoparticle bombardment where episomal expression can be detected within 24 hr and the cell survival was shown to be better than, or at least equal to, conventional methods. This technique has however one crucial advantage: it permits the transfection to be localized within a single restrained radius thus enabling the user to anatomically isolate the heterologous gene&#39;s effects. Here we present an in-depth protocol to prepare viable adult organotypic slices and submit them to regioselective transfection using an improved gene gun.

Recent improvements in organotypic brain slice preparations have permitted their exploitation for biotechnological applications. Organotypic slices maintain local structural characteristics of in vivo biology, including functional synaptic connections. Here we present a regioselective biolistic delivery method to label and genetically manipulate these slices.

Transfection of DNA has been invaluable for biological sciences and with recent advances to organotypic brain slice preparations, the effect of various ...

A Novel Strategy Combining Array-CGH, Whole-exome Sequencing and In Utero Electroporation in Rodents to Identify Causative Genes for Brain Malformations

Birth defects that involve the cerebral cortex &#8212; also known as malformations of cortical development (MCD) &#8212; are important causes of intellectual disability and account for 20-40% of drug-resistant epilepsy in childhood. High-resolution brain imaging has facilitated in vivo identification of a large group of MCD phenotypes. Despite the advances in brain imaging, genomic analysis and generation of animal models, a straightforward workflow to systematically prioritize candidate genes and to test functional effects of putative mutations is missing. To overcome this problem, an experimental strategy enabling the identification of novel causative genes for MCD was developed and validated. This strategy is based on identifying candidate genomic regions or genes via array-CGH or whole-exome sequencing and characterizing the effects of their inactivation or of overexpression of specific mutations in developing rodent brains via in utero electroporation. This approach led to the identification of the C6orf70 gene, encoding for a putative vesicular protein, to the pathogenesis of periventricular nodular heterotopia, a MCD caused by defective neuronal migration.

A Novel Strategy Combining Array-CGH, Whole-exome Sequencing and In Utero Electroporation in Rodents to Identify Causative Genes for Brain Malformations

Periventricular nodular heterotopia (PNH) is the most common form of malformation of cortical development (MCD) in adulthood but its genetic basis remains unknown in most sporadic cases. We have recently developed a strategy to identify novel candidate genes for MCDs and to directly confirm their causative role in vivo.

Birth defects that involve the cerebral cortex &#8212; also known as malformations of cortical development (MCD) &#8212; are important causes of ...

Genetic Modification of Primate Cerebral Organoids

Targeted Microinjection and Electroporation of Primate Cerebral Organoids for Genetic Modification

The cerebral cortex is the outermost brain structure and is responsible for the processing of sensory input and motor output; it is seen as the seat of higher-order cognitive abilities in mammals, in particular, primates. Studying gene functions in primate brains is challenging due to technical and ethical reasons, but the establishment of the brain organoid technology has enabled the study of brain development in traditional primate models (e.g., rhesus macaque and common marmoset), as well as in previously experimentally inaccessible primate species (e.g., great apes), in an ethically justifiable and less technically demanding system. Moreover, human brain organoids allow the advanced investigation of neurodevelopmental and neurological disorders.
As brain organoids recapitulate many processes of brain development, they also represent a powerful tool to identify differences in, and to functionally compare, the genetic determinants underlying the brain development of various species in an evolutionary context. A great advantage of using organoids is the possibility to introduce genetic modifications, which permits the testing of gene functions. However, the introduction of such modifications is laborious and expensive. This paper describes a fast and cost-efficient approach to genetically modify cell populations within the ventricle-like structures of primate cerebral organoids, a subtype of brain organoids. This method combines a modified protocol for the reliable generation of cerebral organoids from human-, chimpanzee-, rhesus macaque-, and common marmoset-derived induced pluripotent stem cells (iPSCs) with a microinjection and electroporation approach. This provides an effective tool for the study of neurodevelopmental and evolutionary processes that can also be applied for disease modeling.

Author Spotlight: Targeted Microinjection and Electroporation of Primate Cerebral Organoids for Genetic Modification  

The electroporation of primate cerebral organoids provides a precise and efficient approach to introduce transient genetic modification(s) into different progenitor types and neurons in a model system close to primate (patho)physiological neocortex development. This allows the study of neurodevelopmental and evolutionary processes and can also be applied for disease modeling.

The cerebral cortex is the outermost brain structure and is responsible for the processing of sensory input and motor output; it is seen as the seat of ...

Developmental Biology

CRISPR/Cas9 Gene Knockout in Sliced Human Cortical Organoids via Electroporation

Electroporation of Sliced Human Cortical Organoids for Studies of Gene Function

Human cortical organoids have become important tools for studying human brain development, neurodevelopmental disorders, and human brain evolution. Studies analyzing gene function by overexpression or knockout have been instrumental in animal models to provide mechanistic insights into the regulation of neocortex development. Here, we present a detailed protocol for CRISPR/Cas9-mediated acute gene knockout by electroporation of sliced human cortical organoids. The slicing of cortical organoids aids the identification of ventricle-like structures for injection and subsequent electroporation, making this a particularly well-suited model for acute genetic manipulation during human cortical development. We describe the design of guide RNAs and the validation of targeting efficiency in vitro and in cortical organoids. Electroporation of cortical organoids is performed at mid-neurogenic stages, enabling the targeting of most major cell classes in the developing neocortex, including apical radial glia, basal progenitor cells, and neurons. Taken together, the electroporation of sliced human cortical organoids represents a powerful technique to investigate gene function, gene regulation, and cell morphology during cortical development.

Here, we present the acute genetic manipulation of sliced human cortical organoids by electroporation. These cortical organoid models are particularly amenable to injection as ventricle-like structures can be readily identified after slicing, enabling the functional investigation of human cortical development, neurodevelopmental disorders, and cortical evolution.

Human cortical organoids have become important tools for studying human brain development, neurodevelopmental disorders, and human brain evolution. ...

In vivo Electroporation of Morpholinos into the Adult Zebrafish Retina

Many devastating inherited eye diseases result in progressive and irreversible blindness because humans cannot regenerate dying or diseased retinal neurons. In contrast, the adult zebrafish retina possesses the robust ability to spontaneously regenerate any neuronal class that is lost in a variety of different retinal damage models, including retinal puncture, chemical ablation, concentrated high temperature, and intense light treatment 1-8. Our lab extensively characterized regeneration of photoreceptors following constant intense light treatment and inner retinal neurons after intravitreal ouabain injection 2, 5, 9. In all cases, resident M&uuml;ller glia re-enter the cell cycle to produce neuronal progenitors, which continue to proliferate and migrate to the proper retinal layer, where they differentiate into the deficient neurons. 
	We characterized five different stages during regeneration of the light-damaged retina that were highlighted by specific cellular responses. We identified several differentially expressed genes at each stage of retinal regeneration by mRNA microarray analysis 10. Many of these genes are also critical for ocular development. To test the role of each candidate gene/protein during retinal regeneration, we needed to develop a method to conditionally limit the expression of a candidate protein only at times during regeneration of the adult retina. 
	Morpholino oligos are widely used to study loss of function of specific proteins during the development of zebrafish, Xenopus, chick, mouse, and tumors in human xenografts 11-14. These modified oligos basepair with complementary RNA sequence to either block the splicing or translation of the target RNA. Morpholinos are stable in the cell and can eliminate or "knockdown" protein expression for three to five days 12. 
	Here, we describe a method to efficiently knockdown target protein expression in the adult zebrafish retina. This method employs lissamine-tagged antisense morpholinos that are injected into the vitreous of the adult zebrafish eye. Using electrode forceps, the morpholino is then electroporated into all the cell types of the dorsal and central retina. Lissamine provides the charge on the morpholino for electroporation and can be visualized to assess the presence of the morpholino in the retinal cells. 
	Conditional knockdown in the retina can be used to examine the role of specific proteins at different times during regeneration. Additionally, this approach can be used to study the role of specific proteins in the undamaged retina, in such processes as visual transduction and visual processing in second order neurons.

In vivo Electroporation of Morpholinos into the Adult Zebrafish Retina

A method to conditionally knockdown a target protein&#x2019;s expression in the adult zebrafish retina is described, which involves intravitreally injecting antisense morpholinos and electroporating them into the retina. The resulting protein is knocked down for several days, which allows testing the protein&#x2019;s role in the regenerating or intact retina.

Many devastating inherited eye diseases result in progressive and irreversible blindness because humans cannot regenerate dying or diseased retinal ...

Biology

Combining Optogenetics with Artificial microRNAs to Characterize the Effects of Gene Knockdown on Presynaptic Function within Intact Neuronal Circuits

The purpose of this protocol is to characterize the effect of gene knockdown on presynaptic function within intact neuronal circuits. We describe a workflow on how to combine artificial microRNA (miR)-mediated RNA interference with optogenetics to achieve selective stimulation of manipulated presynaptic boutons in acute brain slices. The experimental approach involves the use of a single viral construct and a single neuron-specific promoter to drive the expression of both an optogenetic probe and artificial miR(s) against presynaptic gene(s). When stereotactically injected in the brain region of interest, the expressed construct makes it possible to stimulate with light exclusively the neurons with reduced expression of the gene(s) under investigation. This strategy does not require the development and maintenance of genetically modified mouse lines and can in principle be applied to other organisms and to any neuronal gene of choice. We have recently applied it to investigate how the knockdown of alternative splice isoforms of presynaptic P/Q-type voltage-gated calcium channels (VGCCs) regulates short-term synaptic plasticity at CA3 to CA1 excitatory synapses in acute hippocampal slices. A similar approach could also be used to manipulate and probe the neuronal circuitry in vivo.

This protocol provides a workflow on how to combine artificial microRNA-mediated RNA interference with optogenetics to stimulate specifically presynaptic boutons with reduced expression of selective gene(s) within intact neuronal circuits.

The purpose of this protocol is to characterize the effect of gene knockdown on presynaptic function within intact neuronal circuits. We describe a ...

Molecular Modulation by Lentivirus-Delivered Specific shRNAs in Endoplasmic Reticulum Stressed Neurons

The accumulation of unfolded proteins within the endoplasmic reticulum (ER), caused by any stress condition, triggers the unfolded protein response (UPR) through the activation of specialized sensors. UPR attempts first to restore homeostasis; but if damage persists the signaling induces apoptosis.
There is increasing evidence that sustained and unresolved ER stress contributes to many pathological conditions including neurodegenerative diseases. Because the UPR controls cell fate by switching between cytoprotective and apoptotic processes, it is essential to understand the events defining this transition, as well as the elements involved in its modulation.
Recently, we demonstrated that abnormal GM2 ganglioside accumulation causes depletion of ER Ca2+ content, which in turn activates PERK (PKR-like-ER kinase), one of the UPR sensors. Furthermore, PERK signaling participates in the neurite atrophy and apoptosis induced by GM2 accumulation. In this respect, we have established an experimental system that allows us to molecularly modulate the expression of downstream PERK components and thus change vulnerability of neurons to undergo neuritic atrophy.
We performed knockdown of calcineurin (cytoprotective) and CHOP (pro-apoptotic) expression in rat cortical neuronal cultures. Cells were infected with lentivirus-delivered specific shRNA and then treated with GM2 at different times, fixed and immunostained with anti-MAP2 (microtube-associated protein 2) antibody. Later, cell images were recorded using a fluorescence microscope and total neurite outgrowth was evaluated by using the public domain image processing software ImageJ. The inhibition of expression of those PERK signaling components clearly made it possible to either accelerate or delay the neuritic atrophy induced by ER stress.
This approach might be used in cell system models of ER stress to evaluate the vulnerability of neurons to neurite atrophy.

In the present study, the expression is knocked down of two downstream signaling components of the PERK pathway, the cytoprotective calcineurin and the pro-apoptotic CHOP, by using specific shRNAs. In opposite ways, these modulate the susceptibility of primary cortical neurons to neurite atrophy after induction of endoplasmic reticulum stress.

The accumulation of unfolded proteins within the endoplasmic reticulum (ER), caused by any stress condition, triggers the unfolded protein response (UPR) ...

Loss-of-Function Approach in the Embryonic Chick Retina by Using Tol2 Transposon-Mediated Transgenic Expression of Artificial microRNAs

The chick retina has long been an important model system in developmental neurobiology, with advantages including its large size, rapid development, and accessibility for visualization and experimental manipulations. However, its major technical limitation had been the lack of robust loss-of-function approaches for gene function analyses. This protocol describes a methodology of gene silencing in the developing chick retina that involves transgenic expression of artificial microRNAs (miRNAs) by using the Tol2 transposon system. In this approach, a Tol2 transposon plasmid that contains an expression cassette for the EmGFP (emerald green fluorescent protein) marker and artificial pre-miRNA sequences against a target gene is introduced into the embryonic chick retina with a Tol2 transposase expression construct by in ovo electroporation. In the transfected retinal cells, the transposase catalyzes the excision of the expression cassette from the transposon vector and its integration into host chromosomes, leading to the stable expression of miRNAs and the EmGFP protein. In our previous study, we have demonstrated that the expression of Nel, a glycoprotein that exerts multiple functions in neural development, can be significantly suppressed in the developing chick retina by using this technique. Our results indicate that this methodology induces a stable and robust suppression of gene expression and thus provides an efficient loss-of-function approach for studies of retinal development.

We have developed a novel loss-of-function approach that involves the introduction and genomic integration of artificial micro-RNA sequences into chick embryos by using in ovo electroporation and the Tol2 transposon system. This technique provides a robust and stable gene knockdown methodology for studies of gene function during development.

The chick retina has long been an important model system in developmental neurobiology, with advantages including its large size, rapid development, and ...

Zinc-finger Nuclease Enhanced Gene Targeting in Human Embryonic Stem Cells

One major limitation with current human embryonic stem cell (ESC) differentiation protocols is the generation of heterogeneous cell populations.&#160;These cultures contain the cells of interest, but are also contaminated with undifferentiated ESCs, non-neural derivatives and other neuronal subtypes.&#160; This limits their use in in vitro and in vivo applications, such as in vitro modeling for drug discovery or cell replacement therapy. To help overcome this, reporter cell lines, which offer a means to visualize, track and isolate cells of interest, can be engineered.&#160;However, to achieve this in human embryonic stem cells via conventional homologous recombination is extremely inefficient.&#160;This protocol describes targeting of the Pituitary homeobox 3 (PITX3) locus in human embryonic stem cells using custom designed zinc-finger nucleases, which introduce site-specific double-strand DNA breaks, together with a PITX3-EGFP-specific DNA donor vector. Following the generation of the PITX3 reporter cell line, it can then be differentiated using published protocols for use in studies such as in vitro Parkinson&#8217;s disease modeling or cell replacement therapy.

Reporter cell lines offer a means to visualize, track and isolate cells of interest from heterogeneous populations.&#160;However, gene-targeting using conventional homologous recombination in human embryonic stem cells is extremely inefficient. Herein, we describe targeting CNS midbrain specific transcription factor PITX3 locus with EGFP using zinc-finger nuclease enhanced homologous recombination.

Targeted Knockdown of Genes in the Choroid Plexus

Summary

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Chapters in this video

Micromanipulation of Gene Expression in the Adult Zebrafish Brain Using Cerebroventricular Microinjection of Morpholino Oligonucleotides

Regioselective Biolistic Targeting in Organotypic Brain Slices Using a Modified Gene Gun

A Novel Strategy Combining Array-CGH, Whole-exome Sequencing and In Utero Electroporation in Rodents to Identify Causative Genes for Brain Malformations

Targeted Microinjection and Electroporation of Primate Cerebral Organoids for Genetic Modification

Electroporation of Sliced Human Cortical Organoids for Studies of Gene Function

In vivo Electroporation of Morpholinos into the Adult Zebrafish Retina

Combining Optogenetics with Artificial microRNAs to Characterize the Effects of Gene Knockdown on Presynaptic Function within Intact Neuronal Circuits

Molecular Modulation by Lentivirus-Delivered Specific shRNAs in Endoplasmic Reticulum Stressed Neurons

Loss-of-Function Approach in the Embryonic Chick Retina by Using Tol2 Transposon-Mediated Transgenic Expression of Artificial microRNAs

Zinc-finger Nuclease Enhanced Gene Targeting in Human Embryonic Stem Cells