Name: crispr-mediated loss of function analysis in cerebellar granule cells using in utero electroporation-based gene transfer
Uploaded: 2018-06-09T13:30:00
Description: Watch this Scientific Journal Video about CRISPR-mediated Loss of Function Analysis in Cerebellar Granule Cells Using In Utero Electroporation-based Gene Transfer at JoVE.com

We appreciate Laura Sieber, Anna Neuerburg, Yassin Harim, and Petra Schroeter for technical assistance. We also thank Drs. K. Reifenberg, K. Dell and P. Pr&#252;ckl&#160;for helpful assistance for animal experiments at DKFZ; the Imaging Core Facilities of the DKFZ and the Carl Zeiss Imaging Center in the DKFZ for confocal microscopy imaging. This work was supported by the Deutsche Forschungsgemeinschaft, KA 4472/1-1 (to D.K.).

All animal experiments were conducted according to animal welfare regulations and have been approved by the responsible authorities (Regierungspr&#228;sidium Karlsruhe, approval numbers: G90/13, G176/13, G32/14, G48/14, and G133/14).
1. Generate pU6-sgRNA-Cbh-Cre Plasmids
<ol>
	<li>Design the sgRNA to target a gene-of-interest according to the previously published protocol14. 
	NOTE: In this experiment, two sgRNAs are designed to target the mouse Top2b g...

<ol>
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Using exo utero electroporation, we have previously reported siRNA-based in vivo functional analyses of Atoh1 at an early stage of cerebellar granule cell differentiation8. Due to siRNA dilution/degradation and exposure of embryos outside the uterine wall, phenotypic analysis of the electroporated granule cells was limited to embryonic stages. However, the current method enabled analysis of the phenotype of postnatal animals.
Our previous study demonst...

Brain diseases are one of the most dreadful mortal diseases. They often result from genetic mutations and subsequent dysregulation. To understand molecular mechanisms of brain diseases, ever-lasting efforts to decipher the genomes of human patients have discovered a number of potential causative genes. So far, germline genetically engineered animal models have been utilized for in vivo gain-of-function (GOF) and loss-of-function (LOF) analyses of such candidate genes. Due to the accelerated development of functional validation studies, a more feasible and flexible in vivo gene assay system for studying gene function is desirable.
The application of an in vivo electroporation-based gene transfer system to the developing mouse brain is suitable for this purpose. In fact, several studies using in utero electroporation have shown their potential to conduct functional analyses in the developing brain1,2,3. Actually, several regions of the mouse brain, such as the cerebral cortex4, retina5, diencephalon6, hindbrain7, cerebellum8, and spinal cord9 have been targeted by somatic gene delivery approaches, so far.
Indeed, transient gene expression by in vivo electroporation on embryonic mouse brains has long been used for GOF analysis. Recent transposon-based genomic integration technologies further enabled long-term and/or conditional expression of genes of interest10,11, which is advantageous to dissect gene function in a spatial and temporal manner during development. In contrast to GOF analysis, LOF analysis has been more challenging. While transient transfection of siRNAs and shRNA-carrying plasmids was performed, long-term effects of LOF of genes are not guaranteed due to eventual degradation of exogenously introduced nucleic acids, such as plasmids and dsRNAs. However, the CRISPR/Cas technology provides a break-through in LOF analyses. Genes encoding fluorescent proteins (e.g., GFP) or bioluminescent proteins (e.g., firefly luciferase) have been co-transfected with CRISPR-Cas9 and sgRNAs to label the cells exposed to CRISPR-Cas9-mediated somatic mutations. Nevertheless, this approach might have limitations in functional studies on proliferating cells, since exogenous marker genes are diluted and degraded after long-term proliferation. While the transfected cells and their daughter cells undergo CRISPR-induced mutations in their genomes, their footprints might get lost over time. Thus, genetic labeling approaches would be suitable to overcome this issue.
We recently developed a CRISPR-based LOF method in cerebellar granule cells that undergo long-term proliferation during their differentiation12. To genetically label the transfected cells, we constructed a plasmid carrying a sgRNA together with Cre and introduced the plasmid into the cerebella of Rosa26-CAG-LSL-Cas9-P2A-EGFP mice13 using in utero electroporation. Unlike regular plasmid vectors encoding EGFP, this approach successfully labeled transfected granule neuron precursors (GNPs) and their daughter cells. This method provides great support in understanding in vivo function of genes of interest in proliferating cells in normal brain development and a tumor-prone background.

<table>
	<tbody>
		<tr>
			<td>Alexa 488 Goat anti-Chicken</td>
			<td>ThermoFisher</td>
			<td>A11039</td>
			<td>1:400 dilution</td>
		</tr>
		<tr>
			<td>Alexa 568 Donkey anti-Mouse</td>
			<td>Life Technologies</td>
			<td>A-10037</td>
			<td>1:400 dilution</td>
		</tr>
		<tr>
			<td>Alexa 594 Donkey anti-Rabbit</td>
			<td>ThermoFisher</td>
			<td>A21207</td>
			<td>1:400 dilution</td>
		</tr>
		<tr>
			<td>Alexa 647 Donkey anti-Rabbit</td>
			<td>Life Technologies</td>
			<td>A31573</td>
			<td>1:400 dilution</td>
		</tr>
		<tr>
			<td>Alkaline Phosphatase (FastAP)</td>
			<td>ThermoFisher</td>
			<td>EF0654</td>
			<td></td>
		</tr>
		<tr>
			<td>Autoclave band</td>
			<td>Kisker Biotech</td>
			<td>150262</td>
			<td></td>
		</tr>
		<tr>
			<td>BamHI (HF)</td>
			<td>NEB</td>
			<td>R3136S</td>
			<td></td>
		</tr>
		<tr>
			<td>BbsI (FastDigest)</td>
			<td>ThermoFisher</td>
			<td>FD1014</td>
			<td></td>
		</tr>
		<tr>
			<td>Cellulose Filter Paper (Whatman)</td>
			<td>Sigma-Aldrich</td>
			<td>WHA10347525</td>
			<td></td>
		</tr>
		<tr>
			<td>Cloth</td>
			<td>Tork</td>
			<td>530378</td>
			<td></td>
		</tr>
		<tr>
			<td>Confocal laser scanning microscope</td>
			<td>Zeiss</td>
			<td>LSM800</td>
			<td></td>
		</tr>
		<tr>
			<td>D-Luciferin</td>
			<td>biovision</td>
			<td>7903-1</td>
			<td></td>
		</tr>
		<tr>
			<td>DAPI</td>
			<td>Sigma-Aldrich</td>
			<td>D9542</td>
			<td>1:1000 dilution</td>
		</tr>
		<tr>
			<td>Disposable plastic molds (Tissue-Tek Cyromold)</td>
			<td>VWR</td>
			<td>4566</td>
			<td></td>
		</tr>
		<tr>
			<td>DMEM Glutamax</td>
			<td>ThermoFisher</td>
			<td>31966047</td>
			<td></td>
		</tr>
		<tr>
			<td>Donkey serum</td>
			<td>Sigma-Aldrich</td>
			<td>D9663</td>
			<td></td>
		</tr>
		<tr>
			<td>EcoRI (HF)</td>
			<td>NEB</td>
			<td>R3101S</td>
			<td></td>
		</tr>
		<tr>
			<td>Electro Square Porator</td>
			<td>BTX</td>
			<td>ECM830</td>
			<td></td>
		</tr>
		<tr>
			<td>Endofree Maxi Kit</td>
			<td>Qiagen</td>
			<td>12362</td>
			<td></td>
		</tr>
		<tr>
			<td>Ethanol</td>
			<td>Merck</td>
			<td>107017</td>
			<td></td>
		</tr>
		<tr>
			<td>Eye ointment (Bepanthen)</td>
			<td>Bayer</td>
			<td>81552983</td>
			<td></td>
		</tr>
		<tr>
			<td>Fast Green</td>
			<td>Merck</td>
			<td>104022</td>
			<td></td>
		</tr>
		<tr>
			<td>FBS</td>
			<td>ThermoFisher</td>
			<td>10270-016</td>
			<td></td>
		</tr>
		<tr>
			<td>Filter (0.22 &#181;m)</td>
			<td>Merck</td>
			<td>F8148</td>
			<td></td>
		</tr>
		<tr>
			<td>Fluorescent cell imager (ZOE)</td>
			<td>Biorad</td>
			<td>1450031</td>
			<td></td>
		</tr>
		<tr>
			<td>Forceps straight</td>
			<td>Fine Science Tools</td>
			<td>91150-20</td>
			<td></td>
		</tr>
		<tr>
			<td>Gauze (X100 ES-pads 8f 10 x 10 cm)</td>
			<td>Fisher Scientific</td>
			<td>15387311</td>
			<td></td>
		</tr>
		<tr>
			<td>GFP antibody</td>
			<td>Abcam</td>
			<td>ab13970</td>
			<td>1:1000 dilution</td>
		</tr>
		<tr>
			<td>Gibson Assembly Master Mix</td>
			<td>NEB</td>
			<td>E2611S</td>
			<td></td>
		</tr>
		<tr>
			<td>Glass Capillary with Filament</td>
			<td>Narishige</td>
			<td>GD1-2</td>
			<td></td>
		</tr>
		<tr>
			<td>Heating Pad</td>
			<td>ThermoLux</td>
			<td>463265 / -67</td>
			<td></td>
		</tr>
		<tr>
			<td>Image Processing software (ImageJ and Fiji)</td>
			<td>NIH</td>
			<td>-</td>
			<td></td>
		</tr>
		<tr>
			<td>Insulin syringe (B. Braun OMNICAN U-100)</td>
			<td>Carl Roth</td>
			<td>AKP0.1</td>
			<td></td>
		</tr>
		<tr>
			<td>Isoflurane</td>
			<td>Zoetis</td>
			<td>TU061219</td>
			<td></td>
		</tr>
		<tr>
			<td>IVIS Lumina LT Series III Caliper</td>
			<td>Perkin Elmer</td>
			<td>CLS136331</td>
			<td></td>
		</tr>
		<tr>
			<td>Kalt Suture Needles</td>
			<td>Fine Science Tools</td>
			<td>12050-02</td>
			<td></td>
		</tr>
		<tr>
			<td>KAPA HIFI HOTSTART READY mix</td>
			<td>Kapa Biosystems</td>
			<td>KK2601</td>
			<td></td>
		</tr>
		<tr>
			<td>Ki67 antibody</td>
			<td>Abcam</td>
			<td>ab15580</td>
			<td>1:500 dilution</td>
		</tr>
		<tr>
			<td>Light Pointer</td>
			<td>Photonic</td>
			<td>PL3000</td>
			<td></td>
		</tr>
		<tr>
			<td>Liquid blocker pen</td>
			<td>Kisker Biotech</td>
			<td>MKP-1</td>
			<td></td>
		</tr>
		<tr>
			<td>Metamizol</td>
			<td>WDT</td>
			<td>-</td>
			<td></td>
		</tr>
		<tr>
			<td>Microgrinder</td>
			<td>Narishige</td>
			<td>EG-45</td>
			<td></td>
		</tr>
		<tr>
			<td>Microinjector</td>
			<td>Narishige</td>
			<td>IM300</td>
			<td></td>
		</tr>
		<tr>
			<td>Micropipette Puller</td>
			<td>Sutter Instrument Co.</td>
			<td>P-97</td>
			<td></td>
		</tr>
		<tr>
			<td>Microscope software ZEN</td>
			<td>Zeiss</td>
			<td>-</td>
			<td></td>
		</tr>
		<tr>
			<td>Non-sterile Silk Suture Thread (0.12 mm)</td>
			<td>Fine Science Tools</td>
			<td>18020-50</td>
			<td></td>
		</tr>
		<tr>
			<td>O.C.T. Compound (Tissue-Tek)</td>
			<td>VWR</td>
			<td>4583</td>
			<td></td>
		</tr>
		<tr>
			<td>p27 antibody</td>
			<td>BD bioscience</td>
			<td>610241</td>
			<td>1:200 dilution</td>
		</tr>
		<tr>
			<td>Paraformaldehyde</td>
			<td>Roth</td>
			<td>335.3</td>
			<td></td>
		</tr>
		<tr>
			<td>PBS (1x)</td>
			<td>Life Technologies</td>
			<td>14190169</td>
			<td></td>
		</tr>
		<tr>
			<td>pCAG-EGxxFP</td>
			<td>Addgene</td>
			<td>50716</td>
			<td></td>
		</tr>
		<tr>
			<td>Polyethylenimine</td>
			<td>Sigma-Aldrich</td>
			<td>408727</td>
			<td></td>
		</tr>
		<tr>
			<td>pX330 plasmid</td>
			<td>Addgene</td>
			<td>42230</td>
			<td></td>
		</tr>
		<tr>
			<td>QIAprep Spin Miniprep Kit</td>
			<td>Qiagen</td>
			<td>27104</td>
			<td></td>
		</tr>
		<tr>
			<td>QIAquick Gel Extraction Kit</td>
			<td>Qiagen</td>
			<td>28704</td>
			<td></td>
		</tr>
		<tr>
			<td>Quick Ligation Kit</td>
			<td>NEB</td>
			<td>M2200S</td>
			<td></td>
		</tr>
		<tr>
			<td>Ring Forceps</td>
			<td>Fine Science Tools</td>
			<td>11103-09</td>
			<td></td>
		</tr>
		<tr>
			<td>Slides (SuperFrost)</td>
			<td>ThermoFisher</td>
			<td>10417002</td>
			<td></td>
		</tr>
		<tr>
			<td>Software for biostatistics (Prism 7)</td>
			<td>GraphPad Software, Inc</td>
			<td>-</td>
			<td></td>
		</tr>
		<tr>
			<td>Spitacid</td>
			<td>EcoLab</td>
			<td>3003840</td>
			<td></td>
		</tr>
		<tr>
			<td>Stereomicroscope</td>
			<td>Nikon</td>
			<td>C-PS</td>
			<td></td>
		</tr>
		<tr>
			<td>Sucrose</td>
			<td>Sigma-Aldrich</td>
			<td>S5016</td>
			<td></td>
		</tr>
		<tr>
			<td>Surgical scissors</td>
			<td>Fine Science Tools</td>
			<td>91460-11</td>
			<td></td>
		</tr>
		<tr>
			<td>Surgical scissors with blunt tip</td>
			<td>Fine Science Tools</td>
			<td>14072-10</td>
			<td></td>
		</tr>
		<tr>
			<td>Suture (Supramid schwarz DS 16, 1.5 (4/0))</td>
			<td>SMI</td>
			<td>220340</td>
			<td></td>
		</tr>
		<tr>
			<td>T4 DNA Ligation Buffer</td>
			<td>NEB</td>
			<td>B0202S</td>
			<td></td>
		</tr>
		<tr>
			<td>T4 PNK</td>
			<td>NEB</td>
			<td>M0201S</td>
			<td></td>
		</tr>
		<tr>
			<td>Tissue scissors Blunt (11.5 cm)</td>
			<td>Fine Science Tools</td>
			<td>14072-10</td>
			<td></td>
		</tr>
		<tr>
			<td>TOP2B antibody</td>
			<td>Santa Cruz</td>
			<td>sc13059</td>
			<td>1:200 dilution</td>
		</tr>
		<tr>
			<td>Trypsin (2.5 %)</td>
			<td>ThermoFisher</td>
			<td>15090046</td>
			<td></td>
		</tr>
		<tr>
			<td>Tweezers w/5mm &#216; disk electrodes Platinum</td>
			<td>Xceltis GmbH</td>
			<td>CUY650P5</td>
			<td></td>
		</tr>
		<tr>
			<td>Vaporizer</td>
			<td>Dr&#228;gerwerk AG</td>
			<td>GS186</td>
			<td></td>
		</tr>
	</tbody>
</table>

crispr-mediated loss of function analysis in cerebellar granule cells using in utero electroporation-based gene transfer

Brain malformation is often caused by genetic mutations. Deciphering the mutations in patient-derived tissues has identified potential causative factors of the diseases. To validate the contribution of a dysfunction of the mutated genes to disease development, the generation of animal models carrying the mutations is one obvious approach. While germline genetically engineered mouse models (GEMMs) are popular biological tools and exhibit reproducible results, it is restricted by time and costs. Meanwhile, non-germline GEMMs often enable exploring gene function in a more feasible manner. Since some brain diseases (e.g., brain tumors) appear to result from somatic but not germline mutations, non-germline chimeric mouse models, in which normal and abnormal cells coexist, could be helpful for disease-relevant analysis. In this study, we report a method for the induction of CRISPR-mediated somatic mutations in the cerebellum. Specifically, we utilized conditional knock-in mice, in which Cas9 and GFP are chronically activated by the CAG (CMV enhancer/chicken &#223;-actin) promoter after Cre-mediated recombination of the genome. The self-designed single-guide RNAs (sgRNAs) and the Cre recombinase sequence, both encoded in a single plasmid construct, were delivered into cerebellar stem/progenitor cells at an embryonic stage using in utero electroporation. Consequently, transfected cells and their daughter cells were labeled with green fluorescent protein (GFP), thus facilitating further phenotypic analyses. Hence, this method is not only showing electroporation-based gene delivery into embryonic cerebellar cells but also proposing a novel quantitative approach to assess CRISPR-mediated loss-of-function phenotypes.

For in vivo functional analysis, it is critical to identify the cells into which exogenous gene(s) have been introduced. While the expression of a marker, such as GFP in non-proliferating cells can be followed-up for a long period of time, the signal gets sequentially lost in proliferating cells. An illustration of this effect is demonstrated in Figure 1. To circumvent losing the footprint of transfected cells in LOF analyses, we developed a novel ap...

Watch this Scientific Journal Video about CRISPR-mediated Loss of Function Analysis in Cerebellar Granule Cells Using In Utero Electroporation-based Gene Transfer at JoVE.com

CRISPR-mediated Loss of Function Analysis in Cerebellar Granule Cells Using In Utero Electroporation-based Gene Transfer

Conventional loss-of-function studies of genes using knockout animals have often been costly and time-consuming. Electroporation-based CRISPR-mediated somatic mutagenesis is a powerful tool to understand gene functions in vivo. Here, we report a method to analyze knockout phenotypes in proliferating cells of the cerebellum.

CRISPR-mediated Loss of Function Analysis in Cerebellar Granule Cells Using In Utero Electroporation-based Gene Transfer

Brain malformation is often caused by genetic mutations. Deciphering the mutations in patient-derived tissues has identified potential causative factors ...

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