CYIY is supported by FAPESP (2012/14421-5) and FMV is supported by a fellowship from FAPESP (2009/53695-0). We thank the DNA Technologies Core at University of California, Davis for preparation and sequencing of the RNA-Seq libraries used in this work, the Galaxy team for providing an excellent and free interface for high-throughput sequencing data analysis tools and Dr. Marianne Bronner for allowing us to film in her laboratory space.

1. Electroporate the Construct of Interest in the Neural Tubes of HH12-13 Chicken Embryos In ovo
Use a fluorescent reporter, preferably in a bicistronic transcript or fused to the protein of interest with an intercalating viral 2a peptide11, to enable quick identification of individuals with satisfactory levels of transfection. NOTE: Typical incubation time for this stage is around 48 hr at 37.8 &#176;C.
<ol>
	<li>Open a small window in the eggs after removing 3 ml of albumin with a syringe coupled to a 18 G x 1 1/2 needle. To enhance embryo visualization, inject a small amount of Indian ink diluted 1:50 in sterile Ringer&#8217;s solution underneath the embryo1-4.</li>
	<li>After covering the embryo with sterile Ringer&#8217;s solution, inject a 10 mM Tris-HCl, pH 8.2 and 0.1% Food Dye (F, D &#38; C) solution containing 2.5 &#181;g/&#181;l DNA through the posterior end of the neural tube until it fills up to the hindbrain region. To electroporate, position platinum electrodes (0.5 mm in diameter) separated by 4 mm1 along the flank region and deliver 5 pulses of 50 msec and 20 V with an interval of 100 msec between them4.</li>
	<li>After electroporation, incubate manipulated embryos for 48 hr more until they reach stage HH23 and identify individuals presenting high level of transfection at the flank region under a fluorescent stereomicroscope. 
	NOTE: The duration of incubation after electroporation depends on the aim of the experiment. The electroporated plasmids can generate significant expression levels after 2 hr2. Thus, if the interest lies in early onset genes the post-electroporation incubation period can be reduced.</li>
</ol>
2. Harvest Successfully Transfected Embryos and Dissect the Electroporated Spinal Cord Halves
NOTE: This procedure lasts from 2-3 hr for each 10 embryos and yields 1-2 &#181;g total RNA per embryo. The dissection of neural tubes requires fine movements and some practice sessions may be necessary before applying it to experimental samples.
<ol>
	<li>Decontaminate metallic dissection tools with RNase decontamination solution or by baking at 150 &#176;C for 4 hr12 and prepare DEPC-treated PBS. Use only new certified RNase-free plasticware. 
	NOTE: After harvesting, it is important to follow standard care to prevent RNase contamination during dissection.</li>
	<li>Harvest the embryos using a pair of fine dissecting scissors and a sieved collection spoon. Keep the embryos in cold DEPC-treated PBS in a 100 mm Petri dish.</li>
	<li>With a pair of fine tweezers, cut out the whole flank region (between the posterior limit of the anterior limb bud and the anterior limit of the posterior limb bud) and transfer this section to a new dish containing cold DEPC-treated PBS.</li>
	<li>Use two fine glass needles to perforate at three different points on each dorsolateral side of the embryo between the neural tube and the paraxial mesoderm.</li>
	<li>Introduce the two glass needles in the central perforation and slowly move one of them away along the neural tube anterior-posterior axis. Repeat this for the perforations that were not reached by the first movements. Remove the detached paraxial mesoderm with a pair of fine tweezers.</li>
	<li>Turn the embryo sideways, insert the tips of the tweezers between the neural tube and the notochord and slide them in opposite directions along the anterior-posterior axis to separate the two structures. 
	NOTE: This should also remove most of the mesoderm still attached to the neural tube.</li>
	<li>If necessary, remove any larger pieces of mesodermic tissue that remain attached by pulling with a pair of tweezers while lightly holding the neural tube with another pair of tweezers. When finished, gently transfer the neural tube to another dish containing cold DEPC-treated PBS using a RNase-free glass Pasteur pipette coupled to a manual pipette pump and keep on ice until all the tubes are dissected.</li>
	<li>Divide the neural tubes into two halves. For this, insert the sharp tip of a closed pair of tweezers in the lumen and open the roof plate by moving the tweezers out through it. After this, separate the halves by cutting the floor plate with the tweezers tips.</li>
	<li>Sort electroporated from non-electroporated halves under a fluorescent dissecting scope and transfer to a 1.5 ml tube filled with ice-cold DEPC-treated PBS. If necessary, flick the microtubes gently to release any tissue attached to the plastic walls and to promote sedimentation of the tubes.</li>
	<li>Spin at 100 x g for 3 sec and remove very slowly as much PBS as possible with a 1 ml micropipette. Do this while looking at a light source that shines through the microtube to monitor if any embryonic material is sucked to the pipette tip; if this happens, slowly discharge and repeat the centrifugation.</li>
	<li>Add 70 &#181;l of lysis solution for RNA extraction per neural tube half and flick the tube to mix contents. Incubate at room temperature for 5 min and vortex vigorously. Repeat the 5 min incubation and vortexing two more times. Store at -80 &#176;C.</li>
	<li>Collect at least eight neural tube halves per sample to ensure an adequate yield for the next step. Pool smaller samples if several electroporation/dissection sessions are required to amass sufficient number of neural tubes. Store sufficient samples for duplicates or triplicates for each condition.</li>
</ol>
3. Purify RNA and Prepare Libraries for Illumina Sequencing
<ol>
	<li>Remove samples from -80 &#176;C storage and place on ice for 15-30 min. Resume thawing at room temperature until complete.</li>
	<li>Vortex well and leave at room temperature for 5 min. Vortex again and centrifuge at 15,000 x g for 1 min to pellet insoluble material.</li>
	<li>Transfer the supernatant to a new tube and proceed to RNA extraction according to the total RNA isolation kit manual instructions. Elute in the minimum recommended volume to increase concentration. 
	NOTE: DNase treatment after clean-up is recommended in order to avoid potential contamination of genomic DNA sequences in RNA-Seq datasets.</li>
	<li>Flick the tube to mix contents and remove 5 &#181;l for quality control analysis. Store the remaining sample at -80 &#176;C until library preparation; these samples should be thawed only once to avoid RNA degradation.</li>
	<li>Run purified RNA in a Bioanalyzer instrument using the RNA 6000 Nano kit; RIN value must be at least above 7 to proceed.</li>
	<li>Generate libraries using an Illumina compatible RNA-Seq library preparation kit and multiplex up to 4 libraries in one lane of a HiSeq instrument set to generate 100 bp single reads. If possible, include all samples in the same run to minimize potential technical artifacts. 
	NOTE: This should yield up to 50 million reads per sample, which is more than enough to obtain coverage for most genes13.</li>
</ol>
4. Compare RNA-Seq Datasets in the Galaxy Public Server10.
NOTE: Server storage space is limited and experiments comparing many samples may require deletion of intermediate files generated in the filtering steps.
<ol>
	<li>Create a user account in the Galaxy public server (<a href="https://usegalaxy.org/">https://usegalaxy.org</a>) and FTP upload the Illumina datasets in fastq format.</li>
	<li>Prepare uploaded files with the FastqGroomer14 tool under the NGS:QC and manipulation menu; if the quality scores are already encoded in Phred+33 (Sanger), skip this step by simply changing the file type to fastqsanger under the Attributes for each dataset.</li>
	<li>Analyze the general quality of each dataset using the FastQC tool under the NGS:QC and manipulation menu. 
	NOTE: Special attention should be given to the resulting graphics for per base sequence quality and per base sequence content, as these will provide estimates for the parameters used for trimming the ends of the reads.</li>
	<li>Trim reads to discard 3&#8217; ends with low quality data using the tool FASTQ Quality Trimmer14 under the NGS:QC and manipulation menu. Set the tool to process only 3&#8217; ends for quality score below 20.</li>
	<li>Remove adapter sequences from reads using the Clip tool (<a href="http://hannonlab.cshl.edu/fastx_toolkit/">http://hannonlab.cshl.edu/fastx_toolkit/</a>) under the NGS:QC and manipulation menu. Set the tool to discard reads with less than 45 bp left, input the sequence of the adapter used and choose the output to include both clipped and non-clipped sequences. Additionally, opt not to discard sequences with unknown bases since most low quality bases were already removed in the last step.</li>
	<li>Using the tool Trim sequences (<a href="http://hannonlab.cshl.edu/fastx_toolkit/">http://hannonlab.cshl.edu/fastx_toolkit/</a>) under the NGS:QC and manipulation menu, trim the first bases of the reads to remove 5&#8217; sequence bias detected by FastQC (typically 10). 
	NOTE: At this point, it is a good idea to run FastQC again and compare with the previous analysis to determine if the dataset quality was improved.</li>
	<li>Download and unpack the latest chicken genome assembly for the UCSC genome browser from Illumina iGenomes (<a href="http://support.illumina.com/sequencing/sequencing_software/igenome.ilmn">http://support.illumina.com/sequencing/sequencing_software/igenome.ilmn</a>) and upload the whole genome fasta format file and the genes annotation gtf format file to the Galaxy history using the tool Upload File under the Get Data menu.</li>
	<li>Map reads for each sample to the chicken genome using the aligner TopHat215 under the NGS: RNA Analysis menu. Choose to use a genome from History (select the fasta file uploaded in step 4.7).</li>
	<li>Build a predicted transcriptome for each sample using Cufflinks16 under the NGS: RNA Analysis menu. Choose to use reference annotation as a guide (select the gtf file uploaded in step 4.7) and turn on the options Perform Bias Correction (from History, fasta file from step 4.7) and Use multi-read correct.</li>
	<li>Merge predicted transcriptomes for all samples using Cuffmerge16 under the NGS: RNA Analysis menu. Choose to use reference annotation (gtf file from step 4.7) and sequence data (from History, fasta file from step 4.7).</li>
	<li>Compare BAM files generated by TopHat2 for each sample using Cuffdiff16 under the NGS: RNA Analysis menu. In the field Transcripts select the merged transcriptome generated by Cuffmerge and turn on the options Perform Bias Correction (from History, fasta file from step 4.7) and Use multi-read correct.</li>
	<li>Sort differential expression testing tables numerically ascending in column 13 (q value) using the tool Sort under the Filter and Sort menu. 
	NOTE: Genes/transcripts showing significant differential expression between experimental conditions will present the lowest q values.</li>
	<li>To check results visually with coverage graphs, first convert BAM files to bedGraph format using the tool Create a BedGraph of genome coverage17 under the BEDTools menu. Disable reporting of regions without coverage, choose to treat spliced entries as distinct intervals and scale the coverage by a factor that normalizes sequencing depth for all the datasets being compared.</li>
	<li>Convert the resulting bedGraph file to bigWig format using the tool Wig/BedGraph-to-bigWig (<a href="http://www.genome.ucsc.edu/">http://www.genome.ucsc.edu/</a>) under the Convert Formats menu. Upload the bigwig files as custom tracks in the UCSC genome browser (<a href="http://www.genome.ucsc.edu/">http://www.genome.ucsc.edu/</a>) for the galGal4 assembly and search a gene of interest to visualize the coverage in the datasets.</li>
</ol>

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J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+ovo+electroporations+of+HH+stage+10+chicken+embryos.">In ovo electroporations of HH stage 10 chicken embryos.</a> J. Vis. Exp. (9), 10-3791 (2007).</li><li>Sauka-Spengler, T., Barembaum, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Gain-+and+loss-of-function+approaches+in+the+chick+embryo.">Gain- and loss-of-function approaches in the chick embryo.</a> Methods Cell Biol. 87, 237-256 (2008).</li><li>Ghadge, G. 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The authors have nothing to disclose.

Here we provide guidelines for analyzing effects after electroporation of the chicken spinal cord. Although electroporation of DNA vectors is more frequently used to overexpress a gene of interest, one can also use constructs encoding dominant negatives, quimeric proteins or precursors for siRNAs to generate gene function knock-down conditions19-21. In fact, comparison of transcriptome profiles resulting from both gain- and loss-of-function analysis may point out genes differentially expressed in opposite ways...

Genetic studies on live organisms frequently use the chicken embryo as a model because in ovo electroporation represents a fast and inexpensive way to partially transfect embryonic structures in vivo with DNA constructs1-5. Bicistronic expression vectors that encode one transcript containing a fluorescent reporter together with the gene of interest, such as pCIG6 or pMES7, allow quick verification of transfection quality in the desired region under a stereomicroscope before processing embryos for downstream analysis.
With recent advances in DNA sequencing, it is now possible to obtain digital whole transcriptome expression profiles from RNA samples using RNA-Seq8,9. Therefore, instead of time-consuming methods that enable analysis of only a few gene products in parallel, such as in situ hybridization, immunohistochemistry and qPCR, preparation of cDNA libraries for high-throughput sequencing can provide information of the whole transcriptome. Observation of the experimental effects on the expression levels of every single gene may in turn provide powerful insights on the pathways altered by the construct used. This is particularly easy if a genomic assembly for the organism used is available, thus the chick embryo again is a suitable model.
If the user has access to a reliable genome sequencing center, the main issue is obtaining high quality RNA samples. This work demonstrates a method for obtaining RNA samples from transfected neural tubes suitable for RNA-Seq, as well as the downstream steps for in silico analysis of the resulting datasets. After electroporation at HH12-13 followed by 48 hr incubation, transfected trunk spinal cord halves were dissected in ribonuclease-free conditions, immediately lysed and further protected from degradation by ultra-low freezing until RNA purification and library preparation.
The Galaxy public server10 provides access to free resources for analyzing high-throughput sequencing data. The amount of space offered for registered users is enough for small experiments, thus eliminating the need for local computational infrastructure. RNA-Seq data analysis includes filtering, alignment and quantitation/comparison of gene expression. Although there are general guidelines for RNA-Seq data analysis, filtering parameters will largely depend on the quality of sequencing and should be determined after quality control analysis of the raw data.
This protocol describes the steps to obtain and compare RNA-Seq profiles from chicken embryonic spinal cords transfected in vivo. As a representative result, comparison of datasets obtained from two independent control samples transfected with an empty vector showed that the method is reproducible and should allow identification of differentially expressed transcripts in experimental samples. Therefore, application of this method has the potential to greatly increase the number of discoveries after one single experiment and thus provide a large set of data for subsequent investigation.

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			<td height="21" style="height:21px;width:425px;">Name of Material/ Equipment</td>
			<td style="width:175px;">Company</td>
			<td style="width:164px;">Catalog Number</td>
			<td style="width:267px;">Comments/Description</td>
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		<tr height="20">
			<td height="20" style="height:20px;">Indian &#236;nk</td>
			<td>Any supplier</td>
			<td></td>
			<td></td>
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		<tr height="20">
			<td height="20" style="height:20px;">18G x 1 1/2 needle</td>
			<td>BD</td>
			<td>305196</td>
			<td></td>
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		<tr height="20">
			<td height="20" style="height:20px;">3 ml syringe</td>
			<td>BD</td>
			<td>309657</td>
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		<tr height="20">
			<td height="20" style="height:20px;">Tris</td>
			<td>MP Biomedicals</td>
			<td>819620</td>
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			<td height="21" style="height:21px;">F D &#38; C Blue 1</td>
			<td>Spectra Colors Corporation</td>
			<td>5.FC.0010P0</td>
			<td>Food Dye used to visualize DNA injection</td>
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			<td height="21" style="height:21px;">Ringer&#39;s solution (1 L)</td>
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		<tr height="21">
			<td height="21" style="height:21px;">&#160;&#160;&#160;&#160; - 7.2 g NaCl</td>
			<td>Sigma-Aldrich</td>
			<td>S-9888</td>
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			<td height="21" style="height:21px;">&#160;&#160;&#160;&#160; - 0.37 g KCl</td>
			<td>Sigma-Aldrich</td>
			<td>P-4504</td>
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		<tr height="24">
			<td height="24" style="height:24px;">&#160;&#160;&#160;&#160; - 0.225 g CaCl2.2H2O</td>
			<td>Fisher Scientific</td>
			<td>10043-52-4</td>
			<td></td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;">&#160;&#160;&#160;&#160; - 0.217 g Na2HPO4.7H2O</td>
			<td>Sigma-Aldrich</td>
			<td>S-9390</td>
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			<td height="24" style="height:24px;">&#160;&#160;&#160;&#160; - 0.02 g KH2PO4</td>
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			<td height="24" style="height:24px;">&#160;&#160;&#160;&#160; - ddH2O to 800 ml and adjust pH to 7.4</td>
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		</tr>
		<tr height="24">
			<td height="24" style="height:24px;">&#160;&#160;&#160;&#160; - ddH2O to 1 L</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">&#160;&#160;&#160;&#160; - Filter and autoclave</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">PBS (Phosphate Buffered Solution) (1 L)</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">&#160;&#160;&#160;&#160; - 8 g NaCl</td>
			<td>Sigma-Aldrich</td>
			<td>S-9888</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">&#160;&#160;&#160;&#160; - 0.2 g KCl</td>
			<td>Sigma-Aldrich</td>
			<td>P-4504</td>
			<td></td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;">&#160;&#160;&#160;&#160; - 1.15 g Na2HPO4.7H2O</td>
			<td>Sigma-Aldrich</td>
			<td>S-9390</td>
			<td></td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;">&#160;&#160;&#160;&#160; - 0.2 g KH2PO4</td>
			<td>Sigma-Aldrich</td>
			<td>P-5379</td>
			<td></td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;">&#160;&#160;&#160;&#160; - ddH2O to 800 ml and adjust pH to 7.2</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;">&#160;&#160;&#160;&#160; - ddH2O to 1 L and filter</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">&#160;&#160;&#160;&#160; - 1 ml DEPC (Diethylpyrocarbonate)</td>
			<td>Sigma-Aldrich</td>
			<td>D-5758</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">&#160;&#160;&#160;&#160; - Shake vigorously and let stand overnight at room temperature</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">&#160;&#160;&#160;&#160; - Autoclave</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Sieved spoon</td>
			<td>Any supplier</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Sterile 60 x 15 mm polystirene Petri dishes</td>
			<td>Corning Life Sciences</td>
			<td>351007</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">RNaseZap RNase decontamination solution</td>
			<td>Life Technologies</td>
			<td>AM9780</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Fine point surgical scissors</td>
			<td>Any supplier</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Straight fine point tweezers</td>
			<td>Any supplier</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Pulled glass needle made from 1.1 x 75 mm glass capillary tubes</td>
			<td>Kimble Chase</td>
			<td>40A502</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">9&#39;&#39; glass Pasteur pipette</td>
			<td>Any supplier</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Manual pipette pump</td>
			<td>Any supplier</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Clear 1.5 ml microcentrifuge polypropilene tubes</td>
			<td>Corning Life Sciences</td>
			<td>MCT-150-C</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Microcentrifuge</td>
			<td>Any supplier</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">RNAlater solution for RNA stabilization</td>
			<td>Life Technologies</td>
			<td>AM7020</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">RNAqueous total RNA isolation kit</td>
			<td>Life Technologies</td>
			<td>AM1912</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Molecular Biology Grade Ethanol</td>
			<td>Any supplier</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">RNA 6000 Nano Kit</td>
			<td>Agilent Technologies</td>
			<td>5067-1511</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">2100 Electrophoresis Bioanalyzer Instrument</td>
			<td>Agilent Technologies</td>
			<td>G2939AA</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Truseq RNA Sample Preparation Kit v2</td>
			<td>Illumina</td>
			<td>RS-122-2001/RS-122-2002</td>
			<td></td>
		</tr>
	</tbody>
</table>

rna-seq analysis of differential gene expression in electroporated chick embryonic spinal cord

In ovo electroporation of the chick neural tube is a fast and inexpensive method for identification of gene function during neural development. Genome wide analysis of differentially expressed transcripts after such an experimental manipulation has the potential to uncover an almost complete picture of the downstream effects caused by the transfected construct. This work describes a simple method for comparing transcriptomes from samples of transfected embryonic spinal cords comprising all steps between electroporation and identification of differentially expressed transcripts. The first stage consists of guidelines for electroporation and instructions for dissection of transfected spinal cord halves from HH23 embryos in ribonuclease-free environment and extraction of high-quality RNA samples suitable for transcriptome sequencing. The next stage is that of bioinformatic analysis with general guidelines for filtering and comparison of RNA-Seq datasets in the Galaxy public server, which eliminates the need of a local computational structure for small to medium scale experiments. The representative results show that the dissection methods generate high quality RNA samples and that the transcriptomes obtained from two control samples are essentially the same, an important requirement for detection of differential expression genes in experimental samples. Furthermore, one example is provided where experimental overexpression of a DNA construct can be visually verified after comparison with control samples. The application of this method may be a powerful tool to facilitate new discoveries on the function of neural factors involved in spinal cord early development.

To validate the method described, two control samples were generated from embryos electroporated with the empty vector pMES7 and one sample from embryos transfected with the same vector containing the insert construct SCRT2-ZNF, which encodes the zinc-finger domains of chicken SCRT218. Samples yielding 11-22 &#181;g total RNA each were obtained from pools of 8 to 12 embryos. All three samples scored an optimal RIN value of 10 in a quality check performed with the Bioanalyzer instrument (Figu...

Watch this Scientific Journal Video about RNA-Seq Analysis of Differential Gene Expression in Electroporated Chick Embryonic Spinal Cord at JoVE.com

RNA-Seq Analysis of Differential Gene Expression in Electroporated Chick Embryonic Spinal Cord

This video shows the basic steps for performing whole transcriptome analysis on dissected chick embryonic spinal cord samples after transfection with in ovo electroporation.

In ovo electroporation of the chick neural tube is a fast and inexpensive method for identification of gene function during neural development. Genome ...

rna-seq-analysis-differential-gene-expression-electroporated-chick

Research

JoVE Journal

Neuroscience

14.5K vistas.  Universidade de São Paulo. This video shows the basic steps for performing whole transcriptome analysis on dissected chick embryonic spinal cord samples after transfection with in ovo electroporation.

Video: RNA-Seq Analysis of Differential Gene Expression in Electroporated Chick Embryonic Spinal Cord

Dissection and Culture of Commissural Neurons from Embryonic Spinal Cord

Commissural neurons have been widely used to investigate the mechanisms underlying axon guidance during embryonic spinal cord development. The cell bodies of these neurons are located in the dorsal spinal cord and their axons follow stereotyped trajectories during embryonic development. Commissural axons initially project ventrally towards the floorplate. After crossing the midline, these axons turn anteriorly and project towards the brain. Each of these steps is regulated by the action of several guidance cues. Cultures highly enriched in commissural neurons are ideally suited for many experiments addressing the mechanisms of axon pathfinding, including turning assays, immunochemistry and biochemistry. Here, we describe a method to dissect and culture commissural neurons from E13 rat dorsal spinal cord. First, the spinal cord is isolated and dorsal strips are dissected out. The dorsal tissue is then dissociated into a cell suspension by trypsinization and mechanical disruption. Neurons are plated onto poly-L-lysine-coated glass coverslips or tissue-culture dishes. After 30 hours in vitro, most neurons have extended an axon. The purity of the culture (Yam et al. 2009), typically over 90%, can be assessed by immunolabeling with the commissural neuron markers DCC, LH2 and TAG1 (Helms and Johnson, 1998). This neuronal preparation is a useful tool for in vitro studies of the cellular and molecular mechanisms of commissural axon growth and guidance during spinal cord development.

This video demonstrates a method to dissect and culture commissural neurons from E13 rat dorsal spinal cord. Dissociated commissural neurons are useful to study the cellular and molecular mechanisms of axon growth and guidance.

Commissural neurons have been widely used to investigate the mechanisms underlying axon guidance during embryonic spinal cord development. The cell bodies ...

Spinal Cord Electrophysiology II: Extracellular Suction Electrode Fabrication

Development of neural circuitries and locomotion can be studied using neonatal rodent spinal cord central pattern generator (CPG) behavior. We demonstrate a method to fabricate suction electrodes that are used to examine CPG activity, or fictive locomotion, in dissected rodent spinal cords. The rodent spinal cords are placed in artificial cerebrospinal fluid and the ventral roots are drawn into the suction electrode. The electrode is constructed by modifying a commercially available suction electrode. A heavier silver wire is used instead of the standard wire given by the commercially available electrode. The glass tip on the commercial electrode is replaced with a plastic tip for increased durability. We prepare hand drawn electrodes and electrodes made from specific sizes of tubing, allowing consistency and reproducibility. Data is collected using an amplifier and neurogram acquisition software. Recordings are performed on an air table within a Faraday cage to prevent mechanical and electrical interference, respectively.

A demonstration of the fabrication and use of an extracellular suction electrode used to measure electrophysiological recordings of neonatal rodent spinal cords in vitro

Development of neural circuitries and locomotion can be studied using neonatal rodent spinal cord central pattern generator (CPG) behavior. We demonstrate ...

Dissection and Culture of Chick Statoacoustic Ganglion and Spinal Cord Explants in Collagen Gels for Neurite Outgrowth Assays

The sensory organs of the chicken inner ear are innervated by the peripheral processes of statoacoustic ganglion (SAG) neurons. Sensory organ innervation depends on a combination of axon guidance cues1 and survival factors2 located along the trajectory of growing axons and/or within their sensory organ targets. For example, functional interference with a classic axon guidance signaling pathway, semaphorin-neuropilin, generated misrouting of otic axons3. Also, several growth factors expressed in the sensory targets of the inner ear, including Neurotrophin-3 (NT-3) and Brain Derived Neurotrophic Factor (BDNF), have been manipulated in transgenic animals, again leading to misrouting of SAG axons4. These same molecules promote both survival and neurite outgrowth of chick SAG neurons in vitro5,6.
Here, we describe and demonstrate the in vitro method we are currently using to test the responsiveness of chick SAG neurites to soluble proteins, including known morphogens such as the Wnts, as well as growth factors that are important for promoting SAG neurite outgrowth and neuron survival. Using this model system, we hope to draw conclusions about the effects that secreted ligands can exert on SAG neuron survival and neurite outgrowth. 
SAG explants are dissected on embryonic day 4 (E4) and cultured in three-dimensional collagen gels under serum-free conditions for 24 hours. First, neurite responsiveness is tested by culturing explants with protein-supplemented medium. Then, to ask whether point sources of secreted ligands can have directional effects on neurite outgrowth, explants are co-cultured with protein-coated beads and assayed for the ability of the bead to locally promote or inhibit outgrowth. We also include a demonstration of the dissection (modified protocol7) and culture of E6 spinal cord explants. We routinely use spinal cord explants to confirm bioactivity of the proteins and protein-soaked beads, and to verify species cross-reactivity with chick tissue, under the same culture conditions as SAG explants. These in vitro assays are convenient for quickly screening for molecules that exert trophic (survival) or tropic (directional) effects on SAG neurons, especially before performing studies in vivo. Moreover, this method permits the testing of individual molecules under serum-free conditions, with high neuron survival8.

We demonstrate how to dissect and culture chick E4 statoacoustic ganglion and E6 spinal cord explants. Explants are cultured under serum-free conditions in 3D collagen gels for 24 hours. Neurite responsiveness is tested with growth factor-supplemented medium and with protein-coated beads.

The sensory organs of the chicken inner ear are innervated by the peripheral processes of statoacoustic ganglion (SAG) neurons. Sensory organ innervation ...

DiI-Labeling of DRG Neurons to Study Axonal Branching in a Whole Mount Preparation of Mouse Embryonic Spinal Cord

Here we present a technique to label the trajectories of small groups of DRG neurons into the embryonic spinal cord by diffusive staining using the lipophilic tracer 1,1'-dioctadecyl-3,3,3',3'-tetramethylindocarbocyanine perchlorate (DiI)1. The comparison of axonal pathways of wild-type with those of mouse lines in which genes are mutated allows testing for a functional role of candidate proteins in the control of axonal branching which is an essential mechanism in the wiring of the nervous system. Axonal branching enables an individual neuron to connect with multiple targets, thereby providing the physical basis for the parallel processing of information. Ramifications at intermediate target regions of axonal growth may be distinguished from terminal arborization. Furthermore, different modes of axonal branch formation may be classified depending on whether branching results from the activities of the growth cone (splitting or delayed branching) or from the budding of collaterals from the axon shaft in a process called interstitial branching2 (Fig. 1).
The central projections of neurons from the DRG offer a useful experimental system to study both types of axonal branching: when their afferent axons reach the dorsal root entry zone (DREZ) of the spinal cord between embryonic days 10 to 13 (E10 - E13) they display a stereotyped pattern of T- or Y-shaped bifurcation. The two resulting daughter axons then proceed in rostral or caudal directions, respectively, at the dorsolateral margin of the cord and only after a waiting period collaterals sprout from these stem axons to penetrate the gray matter (interstitial branching) and project to relay neurons in specific laminae of the spinal cord where they further arborize (terminal branching)3. DiI tracings have revealed growth cones at the dorsal root entry zone of the spinal cord that appeared to be in the process of splitting suggesting that bifurcation is caused by splitting of the growth cone itself4 (Fig. 2), however, other options have been discussed as well5.
This video demonstrates first how to dissect the spinal cord of E12.5 mice leaving the DRG attached. Following fixation of the specimen tiny amounts of DiI are applied to DRG using glass needles pulled from capillary tubes. After an incubation step, the labeled spinal cord is mounted as an inverted open-book preparation to analyze individual axons using fluorescence microscopy.

The stereotyped projections of sensory afferents into the rodent spinal cord offer an easily accessible experimental system to study axonal branching through the tracing of single axons.

Here we  present a technique to label the trajectories of small groups of DRG neurons  into the embryonic spinal cord by diffusive staining using the ...

In ovo Expression of MicroRNA in Ventral Chick Midbrain

Non-coding RNAs are additional players in regulating gene expression. Targeted in ovo electroporation of specific areas provides a unique tool for spatial and temporal control of ectopic microRNA expression. However, ventral brain structures like ventral midbrain are rather difficult to reach for any manipulations. Here, we demonstrate an efficient way to electroporate miRNA into ventral midbrain using thin platinum electrodes. This method offers a reliable way to transfect specific areas of the midbrain and a useful tool for in vivo studies.

In ovo Expression of MicroRNA in Ventral Chick Midbrain

Ectopic expression is one technique to elucidate the microRNAs role in brain development. However, targeting specific areas using in ovo electroporation is challenging. Here, we show an efficient way to selectively electroporate ventral and dorsal midbrain regions.

Non-coding RNAs are additional players in regulating gene expression. Targeted in ovo electroporation of specific areas provides a unique tool for spatial ...

Stereotaxic Injection of a Viral Vector for Conditional Gene Manipulation in the Mouse Spinal Cord

Intraparenchymal injection of a viral vector enables conditional gene manipulation in distinct populations of neurons or particular regions of the central nervous system. We demonstrate a stereotaxic injection technique that allows targeted gene expression or silencing in the dorsal horn of the mouse spinal cord. The surgical procedure is brief. It requires laminectomy of a single vertebra, providing for quick recovery of the animal and unimpaired motility of the spine. Controlled injection of a small vector suspension volume at low speed and use of a microsyringe with beveled glass cannula minimize the tissue lesion. The local immune response to the vector depends on the intrinsic properties of the virus employed; in our experience, it is minor and short-lived when a recombinant adeno-associated virus is used. A reporter gene such as enhanced green fluorescent protein facilitates monitoring spatial distribution of the vector, and the efficacy and cellular specificity of the transfection.

Viral vectors allow for targeted gene manipulation. We demonstrate a method for conditional gene expression or ablation in the mouse spinal cord, using stereotaxic injection of a viral vector into the dorsal horn, a prominent site of synaptic contact between primary somatosensory afferents and neurons of the central nervous system.

Intraparenchymal injection of a viral vector enables conditional gene manipulation in distinct populations of neurons or particular regions of the central ...

Dissection and Lateral Mounting of Zebrafish Embryos: Analysis of Spinal Cord Development

The zebrafish spinal cord is an effective investigative model for nervous system research for several reasons. First, genetic, transgenic and gene knockdown approaches can be utilized to examine the molecular mechanisms underlying nervous system development. Second, large clutches of developmentally synchronized embryos provide large experimental sample sizes. Third, the optical clarity of the zebrafish embryo permits researchers to visualize progenitor, glial, and neuronal populations. Although zebrafish embryos are transparent, specimen thickness can impede effective microscopic visualization. One reason for this is the tandem development of the spinal cord and overlying somite tissue. Another reason is the large yolk ball, which is still present during periods of early neurogenesis. In this article, we demonstrate microdissection and removal of the yolk in fixed embryos, which allows microscopic visualization while preserving surrounding somite tissue. We also demonstrate semipermanent mounting of zebrafish embryos. This permits observation of neurodevelopment in the dorso-ventral and anterior-posterior axes, as it preserves the three-dimensionality of the tissue.

Developmental processes such as proliferation, patterning, differentiation, and axon guidance can be readily modeled in the zebrafish spinal cord. In this article, we describe a mounting procedure for zebrafish embryos, which optimizes visualization of these events.

The zebrafish spinal cord is an effective investigative model for nervous system research for several reasons. First, genetic, transgenic and gene ...

Spinal Cord Transection in the Larval Zebrafish

Mammals fail in sensory and motor recovery following spinal cord injury due to lack of axonal regrowth below the level of injury as well as an inability to reinitiate spinal neurogenesis. However, some anamniotes including the zebrafish Danio rerio exhibit both sensory and functional recovery even after complete transection of the spinal cord. The adult zebrafish is an established model organism for studying regeneration following spinal cord injury, with sensory and motor recovery by 6&#160;weeks post-injury. To take advantage of in vivo analysis of the regenerative process available in the transparent larval zebrafish as well as genetic tools not accessible in the adult, we use the larval zebrafish to study regeneration after spinal cord transection. Here we demonstrate a method for reproducibly and verifiably transecting the larval spinal cord. After transection, our data shows sensory recovery beginning at 2&#160;days post-injury (dpi), with the C-bend movement detectable by 3 dpi and resumption of free swimming by 5 dpi. Thus we propose the larval zebrafish as a companion tool to the adult zebrafish for the study of recovery after spinal cord injury.

After spinal transection, adult zebrafish have functional recovery by six weeks post-injury. To take advantage of larval transparency and faster recovery, we present a method for transecting the larval spinal cord. After transection, we observe sensory recovery beginning at 2&#160;days post-injury, and C-bend movement by 3 days post-injury.

Mammals fail in sensory and motor recovery following spinal cord injury due to lack of axonal regrowth below the level of injury as well as an inability ...

An Ex Vivo Laser-induced Spinal Cord Injury Model to Assess Mechanisms of Axonal Degeneration in Real-time

Injured CNS axons fail to regenerate and often retract away from the injury site. Axons spared from the initial injury may later undergo secondary axonal degeneration. Lack of growth cone formation, regeneration, and loss of additional myelinated axonal projections within the spinal cord greatly limits neurological recovery following injury. To assess how central myelinated axons of the spinal cord respond to injury, we developed an ex vivo living spinal cord model utilizing transgenic mice that express yellow fluorescent protein in axons and a focal and highly reproducible laser-induced spinal cord injury to document the fate of axons and myelin (lipophilic fluorescent dye Nile Red) over time using two-photon excitation time-lapse microscopy. Dynamic processes such as acute axonal injury, axonal retraction, and myelin degeneration are best studied in real-time. However, the non-focal nature of contusion-based injuries and movement artifacts encountered during in vivo spinal cord imaging make differentiating primary and secondary axonal injury responses using high resolution microscopy challenging. The ex vivo spinal cord model described here mimics several aspects of clinically relevant contusion/compression-induced axonal pathologies including axonal swelling, spheroid formation, axonal transection, and peri-axonal swelling providing a useful model to study these dynamic processes in real-time. Major advantages of this model are excellent spatiotemporal resolution that allows differentiation between the primary insult that directly injures axons and secondary injury mechanisms; controlled infusion of reagents directly to the perfusate bathing the cord; precise alterations of the environmental milieu (e.g., calcium, sodium ions, known contributors to axonal injury, but near impossible to manipulate in vivo); and murine models also offer an advantage as they provide an opportunity to visualize and manipulate genetically identified cell populations and subcellular structures. Here, we describe how to isolate and image the living spinal cord from mice to capture dynamics of acute axonal injury.

An Ex Vivo Laser-induced Spinal Cord Injury Model to Assess Mechanisms of Axonal Degeneration in Real-time

We present a protocol utilizing two-photon excitation time-lapse microscopy to simultaneously visualize the dynamics of axon and myelin injuries in real time. This proposed protocol permits studies of both intrinsic and extrinsic factors which can influence central myelinated axon fate after injury and contribute to permanent clinical disability.

Injured CNS axons fail to regenerate and often retract away from the injury site. Axons spared from the initial injury may later undergo secondary axonal ...

Adeno-associated Virus-mediated Transgene Expression in Genetically Defined Neurons of the Spinal Cord

Selective manipulation of spinal neuronal subpopulations has mainly been achieved by two different methods: 1) Intersectional genetics, whereby double or triple transgenic mice are generated in order to achieve selective expression of a reporter or effector gene (e.g., from the Rosa26 locus) in the desired spinal population. 2) Intraspinal injection of Cre-dependent recombinant adeno-associated virus (rAAV); here Cre-dependent AAV vectors coding for the reporter or effector gene of choice are injected into the spinal cord of mice expressing Cre recombinase in the desired neuronal subpopulation. This protocol describes how to generate Cre-dependent rAAV vectors and how to transduce neurons in the dorsal horn of the lumbar spinal cord segments L3-L5 with rAAVs. As the lumbar spinal segments L3-L5 are innervated by those peripheral sensory neurons that transmit sensory information from the hindlimbs, spontaneous behavior and responses to sensory tests applied to the hindlimb ipsilateral to the injection side can be analyzed in order to interrogate the function of the manipulated neurons in sensory processing. We provide examples of how this technique can be used to analyze genetically defined subsets of spinal neurons. The main advantages of virus-mediated transgene expression in Cre transgenic mice compared to classical reporter mouse-induced transgene expression are the following: 1) Different Cre-dependent rAAVs encoding various reporter or effector proteins can be injected into a single Cre transgenic line, thus overcoming the need to create several multiple transgenic mouse lines. 2) Intraspinal injection limits manipulation of Cre-expressing cells to the injection site and to the time after injection. The main disadvantages are: 1) Reporter gene expression from rAAVs is more variable. 2) Surgery is required to transduce the spinal neurons of interest. Which of the two methods is more appropriate depends on the neuron population and research question to be addressed.

Intraspinal injection of recombinase dependent recombinant adeno-associated virus (rAAV) can be used to manipulate any genetically labelled cell type in the spinal cord. Here we describe how to transduce neurons in the dorsal horn of the lumbar spinal cord. This technique enables functional interrogation of the manipulated neuron subtype.

Selective manipulation of spinal neuronal subpopulations has mainly been achieved by two different methods: 1) Intersectional genetics, whereby double or ...