Name: isolation and cultivation of neural progenitors followed by chromatin-immunoprecipitation of histone 3 lysine 79 dimethylation mark
Uploaded: 2018-01-26T13:00:00
Description: Watch this Scientific Journal Video about Isolation and Cultivation of Neural Progenitors Followed by Chromatin-Immunoprecipitation of Histone 3 Lysine 79 Dimethylation Mark at JoVE.com

We thank Henriette Bertemes for helping to establish the CGN cultivation protocol within the lab. This method paper was supported by the DFG-funded CRC992 Medical Epigenetics by funding to TV. The authors acknowledge the support of the Freiburg Galaxy Team: Pavankumar Videm, Bj&#246;rn Gr&#252;ning and Prof. Rolf Backofen, Bioinformatics, University of Freiburg, Germany funded by Collaborative Research Centre 992 Medical Epigenetics (DFG grant SFB 992/1 2012) and German Federal Ministry of Education and Research (BMBF grant 031 A538A RBC (de.NBI)).

Animal welfare committees of the University of Freiburg and local authorities approved all animal experiments (G12/13, G16/11) mentioned in the following protocol.
1. Preparations
<ol>
	<li>Preparations for CPCs isolation
	<ol>
		<li>Set up timed mating to obtain embryos at different stages of cortex development (between E11.5 and E14.5). Use mice of the strain NMRI (Naval Medical Research Institute) which are at least 8 weeks old. After mating, consider a positive vaginal p...

<ol>
	<li>Molyneaux, B. J., Arlotta, P., Menezes, J. R. L., Macklis, J. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neuronal+subtype+specification+in+the+cerebral+cortex.">Neuronal subtype specification in the cerebral cortex.</a> Nat. Rev. Neurosci. 8, 427-437 (2007).</li><li>Kandel, E. R., Squire, L. 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There are two major ways to perform chromatin immunoprecipitation to detect the genomic occupancy of histone modifications, transcription factors, histone code readers, writers, or erasers. One is the native ChIP method using nuclease digested, native chromatin for immunoprecipitation, and the other is the presented method using PFA-fixed, sheared chromatin, in which the nucleosomes and other DNA-attached proteins are covalently bound to the DNA39. Native ChIP with its high antibody detection rate...

The sensory, motor, and cognitive functions of the brain are highly complex and susceptible to physical and environmental changes. The brain consists of three general parts the hind-, mid-, and forebrain, which are deeply connected. Within the forebrain, the telencephalon can be divided into a dorsal telencephalon (DT) and a ventral telencephalon (VT). The DT of mice consists of six cortical layers which are formed between E11.5 and E18.5 in an &#34;inside-out&#34; manner1. The VT includes the ganglionic eminences in development, which later form the basal ganglia2,3. Several cell types can be classified in the mammalian central nervous system such as neurons, astrocytes, or oligodendrocytes4, which develop in a temporo-spatial manner5. First, the neural progenitor cells (NPCs) give rise to different kinds of neurons, interneurons in the VT, and projection neurons in the DT, and later on to glial cells (e.g., astrocytes6). During cortical development, the most superficial layer (layer I), which contains Cajal-Retzius cells, is formed first. Then, between E12.5 and E14.5, NPCs generate deeper neuronal layers (VI, V) while between 14.5 and 16.5, progenitors give rise to upper layer (IV-II) neurons7,8. Neuronal identity is specified by different morphogen-induced temporo-spatial transcriptional programs and additionally by epigenetic programs2.
The cerebellum, which is implicated in motor coordination, is located in the hindbrain and develops between E10 and roughly P20 in mice9. It contains the cerebellar cortex and the cerebellar nuclei10. The adult cerebellar cortex consists of three layers, the outermost molecular layer, the Purkinje cell layer, and the innermost granular layer containing granular neurons10. The cerebellar granule cells are the smallest neurons and represent about 80% of all neurons in the vertebrate brain11. They develop from precursors located in the external germinal zone and migrate through the Purkinje cell layer to their destination12. Like in the telencephalon, the development of the cerebellum is regulated by several important morphogens, which have specific time- and space-dependent functions and initiate defined transcriptional programs10.
The development of cortical and cerebellar layers is controlled by transcriptional expression of specific morphogens and, thus, by the chromatin state of the DNA. In a simplified view, chromatin states can be divided into euchromatin as transcriptionally active and heterochromatin as transcriptionally silent regions. The nucleosome as the basic unit of chromatin contains two copies of each core histone H2A, H2B, H3, and H4, surrounded by 147 base pairs of DNA13. Histones are highly post-translationally modified by methylation, acetylation, phosphorylation, ubiquitination, sumoylation, ADP-ribosylation, deamination, and proline isomerization14,15. Histone lysine methylation is considered to be the most stable histone modification that controls transcription, replication, recombination16, DNA-damage response17, and genomic imprinting18. Lysines can be mono-, di-, or tri-methylated19 and appear not only on the accessible histone tails but also within the globular domain of histones20. Specific methylations at H3K4 and H3K36 are mainly associated with euchromatin, specific methylations at H3K9, H3K27, or H4K20 are mainly found in heterochromatic regions, although all residues are located within the histone tail14,19,21. H3K79 methylation is located within the histone globular domain and has been associated with transcriptional activity, but also with transcriptionally inert genomic regions22. The modification is evolutionarily conserved since it has been observed in yeast, calf thymus, chicken, and human23. H3K79 mono, di, and trimethylation (H3K79me1, me2, me3) are catalyzed by the histone methyltransferases DOT1L24,25 and the Nuclear SET Domain-Containing Protein 2 (Nsd2)26. DOT1L is implicated in proliferation, DNA repair, and cellular reprograming27. Loss of Dot1l in mice leads to a prenatal death around the developmental stage E10.528,29. During heart development and in myocardiocyte differentiation, DOT1L is essential for gene expression regulation30. In the central nervous system, DOT1L function might be implicated in neural tube development31, it is involved in suppressing Tbr1-expression during forebrain development32, and may function in the regulation of ER-stress response genes33. The context-dependent activating or repressing action of H3K79me, especially with in vivo situations like the development of the central nervous system, is to date only partially understood32. Since H3K79 methylation is located in the globular domain of histone 3, it is sterically less accessible in comparison to modifications on the flexible histone tails23. To understand the function of H3K79 methylation, reliable and reproducible analysis methods to determine its location and genomic environment are needed. In this methods paper, we present isolation methods of different neural progenitors (CPCs for the cortex and CGNPs for the cerebellum), effective DOT1L inhibitor treatment, and a ChIP method to analyze H3K79 methylation via qPCR or sequencing at different time points during cortical and cerebellar development. For an overview of the protocol and its possibilities, see Figure 1.

<table>
	<tbody>
		<tr>
			<td>Anti-GAPDH</td>
			<td>Abcam</td>
			<td>ab8245</td>
			<td>Category: Antibody 
			Abbreviation/Comment: Immunoblot dilution 1:5000</td>
		</tr>
		<tr>
			<td>Anti-H3</td>
			<td>Abcam</td>
			<td>ab1791</td>
			<td>Category: Antibody 
			Abbreviation/Comment: Immunoblot dilution 1:3000</td>
		</tr>
		<tr>
			<td>Anti-H3K79me2</td>
			<td>Diagenode</td>
			<td>pAb-051-050</td>
			<td>Category: Antibody 
			Abbreviation/Comment: ChIP antibody</td>
		</tr>
		<tr>
			<td>Anti-H3K79me2</td>
			<td>Abcam</td>
			<td>ab-051-050</td>
			<td>Category: Antibody 
			Abbreviation/Comment: Immunoblot dilution 1:1000</td>
		</tr>
		<tr>
			<td>Anti-rabbit-IgG</td>
			<td>Diagenode</td>
			<td>C15410206</td>
			<td>Category: Antibody 
			Abbreviation/Comment: ChIP Ctrl antibody</td>
		</tr>
		<tr>
			<td>Anti-Tubulin alpha</td>
			<td>Abcam</td>
			<td>ab108629</td>
			<td>Category: Antibody 
			Abbreviation/Comment: Immunoblot dilution 1:3000</td>
		</tr>
		<tr>
			<td>Apo-Transferrin (1 mg/ml)</td>
			<td>Sigma-Aldrich</td>
			<td>T1147</td>
			<td>Category: Cell culture 
			Abbreviation/Comment: For CCM</td>
		</tr>
		<tr>
			<td>B27 Supplement (50x)</td>
			<td>Life Technologies</td>
			<td>17504044</td>
			<td>Category: Cell culture 
			Abbreviation/Comment: For CCM</td>
		</tr>
		<tr>
			<td>Bioanalyzer</td>
			<td>Agilent technologies</td>
			<td>G2940CA</td>
			<td>Category: ChIP 
			Abbreviation/Comment: For analysis of sheared chromatin</td>
		</tr>
		<tr>
			<td>Bioruptor NextGen</td>
			<td>Diagenode</td>
			<td>B01020001</td>
			<td>Category: ChIP 
			Abbreviation/Comment: Ultrasonicator</td>
		</tr>
		<tr>
			<td>Boric acid pH 8.4</td>
			<td>Sigma Aldrich</td>
			<td>B6768</td>
			<td>Category: Cell culture 
			Abbreviation/Comment: For CPC culturing</td>
		</tr>
		<tr>
			<td>CFX Connect RT PCR Detection System</td>
			<td>Bio-Rad</td>
			<td>1855201</td>
			<td>Category: ChIP Analysis 
			Abbreviation/Comment: Detection system for qPCR</td>
		</tr>
		<tr>
			<td>DMEM-F12</td>
			<td>Life Technologies</td>
			<td>11320-033</td>
			<td>Category: Cell culture 
			Abbreviation/Comment: For CGM</td>
		</tr>
		<tr>
			<td>Dynabeads Protein A</td>
			<td>Invitrogen</td>
			<td>10001D</td>
			<td>Category: ChIP 
			Abbreviation/Comment: Magnetic beads, for ChIP</td>
		</tr>
		<tr>
			<td>EPZ-5676</td>
			<td>Selleckchem</td>
			<td>S7062</td>
			<td>Category: DOT1L inhibition 
			Abbreviation/Comment: For DOT1L inhibition in cell culture</td>
		</tr>
		<tr>
			<td>Ethylenediamine tetraacetic acid</td>
			<td>SERVA</td>
			<td>39760.01</td>
			<td>Category: ChIP 
			Abbreviation/Comment: EDTA</td>
		</tr>
		<tr>
			<td>Fetal Bovine Serum 10% (v/v)</td>
			<td>Gibco</td>
			<td>10082147</td>
			<td>Category: Cell culture 
			Abbreviation/Comment: For CPC isolation and CGM</td>
		</tr>
		<tr>
			<td>Glucose</td>
			<td>Sigma-Aldrich</td>
			<td>G5767</td>
			<td>Category: Cell culture 
			Abbreviation/Comment: For CGNP isolation</td>
		</tr>
		<tr>
			<td>Glutathione (1.25 mg/ml)</td>
			<td>Sigma-Aldrich</td>
			<td>G4251</td>
			<td>Category: Cell culture 
			Abbreviation/Comment: For CCM</td>
		</tr>
		<tr>
			<td>Glycine</td>
			<td>Carl Roth</td>
			<td>3187</td>
			<td>Category: ChIP 
			Abbreviation/Comment: For cell fixation</td>
		</tr>
		<tr>
			<td>GoTaq mastermix</td>
			<td>Promega</td>
			<td>A6002</td>
			<td>Category: ChIP Analysis 
			Abbreviation/Comment: DNA polymerase master mix for qPCR</td>
		</tr>
		<tr>
			<td>Hank&#8217;s Balanced Salt Solution</td>
			<td>Life Technologies</td>
			<td>14025-100</td>
			<td>Category: Cell culture 
			Abbreviation/Comment: HBSS</td>
		</tr>
		<tr>
			<td>L-glutamine (200 mM)</td>
			<td>Life Technologies</td>
			<td>25030081</td>
			<td>Category: Cell culture 
			Abbreviation/Comment: For CCM</td>
		</tr>
		<tr>
			<td>Laminin</td>
			<td>Sigma-Aldrich</td>
			<td>L2020</td>
			<td>Category: Cell culture 
			Abbreviation/Comment: For CPC culturing</td>
		</tr>
		<tr>
			<td>Lithium chloride</td>
			<td>Sigma-Aldrich</td>
			<td>L4408</td>
			<td>Category: ChIP 
			Abbreviation/Comment: LiCl</td>
		</tr>
		<tr>
			<td>N2 supplement</td>
			<td>Life Technologies</td>
			<td>17502048</td>
			<td>Category: Cell culture 
			Abbreviation/Comment: For CGM</td>
		</tr>
		<tr>
			<td>NanoDrop 3300</td>
			<td>Thermo Fisher</td>
			<td>3300</td>
			<td>Category: ChIP 
			Abbreviation/Comment: Fluorospectrometer for DNA quantification</td>
		</tr>
		<tr>
			<td>NEB Next Ultra DNA Library Prep Kit for Illumina</td>
			<td>NEB</td>
			<td>E7645S</td>
			<td>Category: ChIP Analysis 
			Abbreviation/Comment: Kit for Library preparation</td>
		</tr>
		<tr>
			<td>NEBNext Multiplex Oligos for Illumina</td>
			<td>NEB</td>
			<td>E7335</td>
			<td>Category: ChIP Analysis 
			Abbreviation/Comment: Oligos for Library preparation</td>
		</tr>
		<tr>
			<td>Neurobasal medium</td>
			<td>Gibco</td>
			<td>21103049</td>
			<td>Category: Cell culture 
			Abbreviation/Comment: For CCM</td>
		</tr>
		<tr>
			<td>NP-40 Alternative</td>
			<td>Calbiochem</td>
			<td>492016</td>
			<td>Category: ChIP 
			Abbreviation/Comment: For ChIP buffer</td>
		</tr>
		<tr>
			<td>Paraformaldehyde</td>
			<td>Carl Roth</td>
			<td>335</td>
			<td>Category: ChIP 
			Abbreviation/Comment: PFA, for cell fixation</td>
		</tr>
		<tr>
			<td>Penicillin-Streptomycin-Neomycin 1% (v/v)</td>
			<td>Life Technologies</td>
			<td>15640055</td>
			<td>Category: Cell culture 
			Abbreviation/Comment: PSN, for CCM and CGM</td>
		</tr>
		<tr>
			<td>Phosphate buffered saline</td>
			<td>Life Technologies</td>
			<td>10010023</td>
			<td>Category: Cell culture 
			Abbreviation/Comment: PBS, for CPC isolation</td>
		</tr>
		<tr>
			<td>PicoGreen Kit</td>
			<td>Thermo Fisher</td>
			<td>P11496</td>
			<td>Category: ChIP Analysis 
			Abbreviation/Comment: Visualizing dye for DNA quantification</td>
		</tr>
		<tr>
			<td>Poly-D-lysine</td>
			<td>Sigma-Aldrich</td>
			<td>P6407</td>
			<td>Category: Cell culture 
			Abbreviation/Comment: For CGNP isolation</td>
		</tr>
		<tr>
			<td>Poly-L-ornithine hydrobromide</td>
			<td>Sigma Aldrich</td>
			<td>P3655</td>
			<td>Category: Cell culture 
			Abbreviation/Comment: For CPC culturing</td>
		</tr>
		<tr>
			<td>Potassium chloride</td>
			<td>Thermo Fisher</td>
			<td>AM9640G</td>
			<td>Category: Cell culture 
			Abbreviation/Comment: KCl, for CGM</td>
		</tr>
		<tr>
			<td>Protease inhibitor</td>
			<td>Roche</td>
			<td>4693159001</td>
			<td>Category: ChIP 
			Abbreviation/Comment: For ChIP</td>
		</tr>
		<tr>
			<td>Proteinase K</td>
			<td>Sigma-Aldrich</td>
			<td>3115879001</td>
			<td>Category: ChIP 
			Abbreviation/Comment: For ChIP</td>
		</tr>
		<tr>
			<td>Qiagen MinElute</td>
			<td>Qiagen</td>
			<td>28004</td>
			<td>Category: ChIP 
			Abbreviation/Comment: Kit for DNA purification</td>
		</tr>
		<tr>
			<td>RNAse</td>
			<td>Sigma-Aldrich</td>
			<td>R6513</td>
			<td>Category: ChIP 
			Abbreviation/Comment: For ChIP</td>
		</tr>
		<tr>
			<td>SGC0946</td>
			<td>Selleckchem</td>
			<td>S7079</td>
			<td>Category: DOT1L inhibition 
			Abbreviation/Comment: For DOT1L inhibition in cell culture</td>
		</tr>
		<tr>
			<td>Sodium bicarbonate</td>
			<td>Carl Roth</td>
			<td>8551.1</td>
			<td>Category: ChIP 
			Abbreviation/Comment: for Elution buffer</td>
		</tr>
		<tr>
			<td>Sodium chloride</td>
			<td>Carl Roth</td>
			<td>9265</td>
			<td>Category: ChIP 
			Abbreviation/Comment: NaCl, for ChIP buffer</td>
		</tr>
		<tr>
			<td>Sodium deoxycholate</td>
			<td>Sigma-Aldrich</td>
			<td>30970</td>
			<td>Category: ChIP 
			Abbreviation/Comment: For ChIP</td>
		</tr>
		<tr>
			<td>Sodium dodecylsulfate</td>
			<td>Carl Roth</td>
			<td>183</td>
			<td>Category: ChIP 
			Abbreviation/Comment: SDS, for ChIP</td>
		</tr>
		<tr>
			<td>Sonic hedgehock (SHH)</td>
			<td>Sigma-Aldrich</td>
			<td>SRP6004</td>
			<td>Category: Cell culture 
			Abbreviation/Comment: For CGNP isolation</td>
		</tr>
		<tr>
			<td>Superoxide dismutase (1mg/ml)</td>
			<td>Sigma-Aldrich</td>
			<td>S7571</td>
			<td>Category: Cell culture 
			Abbreviation/Comment: For CCM</td>
		</tr>
		<tr>
			<td>Tris(hydroxymethyl)aminomethane</td>
			<td>Carl Roth</td>
			<td>9090</td>
			<td>Category: ChIP 
			Abbreviation/Comment: TRIS, for ChIP buffer</td>
		</tr>
		<tr>
			<td>Triton X-100</td>
			<td>Carl Roth</td>
			<td>X100</td>
			<td>Category: ChIP 
			Abbreviation/Comment: For ChIP buffer</td>
		</tr>
		<tr>
			<td>Trypsin-EDTA 0,05% (w/v)</td>
			<td>Sigma Aldrich</td>
			<td>59417C</td>
			<td>Category: Cell culture 
			Abbreviation/Comment: For CPC isolation</td>
		</tr>
		<tr>
			<td>Tween20</td>
			<td>Carl Roth</td>
			<td>28320</td>
			<td>Category: ChIP 
			Abbreviation/Comment: For bead preparation</td>
		</tr>
		<tr>
			<td colspan="4">Other Lab devices: Neubauer counting chamber, Incubator, Rotator, Shaker, Disection set, Water bath</td>
		</tr>
		<tr>
			<td>CCM: Cortical cell medium</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>CGM: CGNP cell culture medium</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
	</tbody>
</table>

isolation and cultivation of neural progenitors followed by chromatin-immunoprecipitation of histone 3 lysine 79 dimethylation mark

Brain development is a complex process, which is controlled in a temporo-spatial manner by gradients of morphogens and different transcriptional programs. Additionally, epigenetic chromatin modifications, like histone methylation, have an important role for establishing and maintaining specific cell fates within this process. The vast majority of histone methylation occurs on the flexible histone tail, which is accessible to histone modifiers, erasers, and histone reader proteins. In contrast, H3K79 methylation is located in the globular domain of histone 3 and is implicated in different developmental functions. H3K79 methylation is evolutionarily conserved and can be found in a wide range of species from Homo sapiens to Saccharomyces cerevisiae. The modification occurs in different cell populations within organisms, including neural progenitors. The location of H3K79 methylation in the globular domain of histone 3 makes it difficult to assess. Here, we present methods to isolate and culture cortical progenitor cells (CPCs) from embryonic cortical brain tissue (E11.5-E14.5) or cerebellar granular neuron progenitors (CGNPs) from postnatal tissue (P5-P7), and to efficiently immunoprecipitate H3K79me2 for quantitative PCR (qPCR) and genome-wide sequencing.

General scheme of neural progenitor isolation, cultivation, H3K79me2 ChIP and ChIP analysis methods: Figure 1 shows a flowchart to perform H3K79me2 ChIP of cortical progenitor cells at different time points during embryonic brain development or of cerebellar granular neuron progenitors in postnatal stages. As a first step, the brain has to be isolated and the telencephalon (between E11.5 and E14.5) or the cerebellum (P5-P7) has to be retrieve...

Watch this Scientific Journal Video about Isolation and Cultivation of Neural Progenitors Followed by Chromatin-Immunoprecipitation of Histone 3 Lysine 79 Dimethylation Mark at JoVE.com

Isolation and Cultivation of Neural Progenitors Followed by Chromatin-Immunoprecipitation of Histone 3 Lysine 79 Dimethylation Mark

We present an effective and reproducible method to isolate and culture neural progenitor cells from embryonic and postnatal brain tissue for chromatin immunoprecipitation (ChIP) of histone 3 lysine 79 dimethylation (H3K79me2) - a histone mark located within the globular domain of histone 3.

Brain development is a complex process, which is controlled in a temporo-spatial manner by gradients of morphogens and different transcriptional programs. ...

The overall goal of this procedure is to analyze epigenetic modifications during brain development, especially in the cerebral cortex and the cerebellum. This method can be used to answer key questions in the neurobiology field, such as how histone modifications like H3K79 methylation can affect the development of cortical and cerebellar layering. The main advantage of this technique is that we can study in vivo the histone modifications, locus-specific or genome-wide, in distinct brain regions at specific developmental time points.Begin with brains from P5 to P7 and MRI mice. Remove all meninges and blood vessels. Then transfer three to five cerebella per chip into 15-milliliter tubes.Wash cerebellar with HBSS glucose before centrifugation. Centrifuge the tubes to collect the tissue. After the final wash, homogenize the cerebella by gently pipetting up and down two to three times with a one-milliliter pipette until the fragments are 0.5 to one-cubed millimeters in size.Gently remove all liquid until 2.5 milliliters are left. Then add 0.05%trypsin into the tube with HBSS glucose containing the cerebella. And incubate the tissue in a 37 degrees Celsius water bath for 15 minutes.Invert the tissue every one to three minutes. Following the incubation, stop the digestion by adding five milliliters of CGNP cell culture medium, or CGM, and collect the tissue by centrifugation as before. After centrifuging, remove the supernatant and add one milliliter CGM.Triturate the tissue with a one-milliliter pipette tip, avoiding the formation of air bubbles. Then add five milliliters of CGM and incubate the mixture for two minutes on ice to settle tissue remnants. Once the tissue has settled, transfer the supernatant into a new 15-milliliter tube.Then add two milliliters of CGM to the residual tissue and repeat the trituration procedure before harvesting the supernatant and discarding the tissue remnants. Pool the supernatants from each 15-milliliter tube of tissue and centrifuge to collect the cerebellar cells. Resuspend the pellet in 10 milliliters of CGM.Since astrocytes adhere faster and stronger to poly-D-lysine than CGNPs, add up to four milliliters of cell suspension to the wells of poly-D-lysine-coated 6-well plates and incubate for 20 minutes at 37 degrees Celsius to remove astrocytes. Shake the plate, collect the supernatant in a 15-milliliter tube and then centrifuge the supernatant as before. Resuspend the pellet in 10 milliliters of CGM and count the cells with a Neubauer counting chamber.After four to six hours, the adhered CGNPs appear round and are proliferative. Seed the cells in CGM on poly-L-ornithine-coated plates and incubate at 37 degrees Celsius, 5%CO2 and 100%relative humidity. After one day in vitro, or DIV1, most CGNPs are round and proliferate.Some CGNPs will start to differentiate and begin outgrowth of neuronal protrusions. To fix the cultured cells, add up to one milliliter of 1%PFA directly to the wells containing CGNPs. After a five-minute incubation at 22 degrees Celsius, add 53 microliters of glycine and wash twice with three milliliter of ice-cold PBS.Use a cell scraper to harvest the cells in one milliliter of PBS. Transfer the cells into 1.5-milliliter tubes and centrifuge for five minutes at 1, 000 g and four degrees Celsius. After removing the supernatant, add 700 microliters of lysis buffer containing protease inhibitor and incubate the samples for 15 minutes at four degrees Celsius.Vortex the samples every five minutes during this time. Following the incubation, divide the sample tubes to ensure that the volume does not exceed 400 microliters per tube. Then sonicate for 10 minutes at maximum power to shear the chromatin of the lyse cells.After 10 minutes, vortex the samples and then repeat the sonication and the vortexing two more times. Check the lysate for remaining nuclei with a phase contrast microscope or fluorescent microscope using DAPI. Pellet the cell remnants by centrifuging for 10 minutes at 13, 000 g and four degrees Celsius.When finished, use the supernatant for the preclearing step. To preclear the samples, first wash 20 micro liters of unconjugated magnetic beads per sample in ice-cold PBS Tween. Remove the washes with aid of a magnetic stand.Then add 600 microliters of dilution buffer and 20 microliters of washed magnetic beads to each sample for preclearing and incubate for two hours at four degrees Celsius on a rotator. Following the incubation, remove the beads using a magnetic stand. Then take 5%of each lysate as input samples and freeze them at minus 20 degrees Celsius.Antibody-coupling can be performed while the samples are being precleared. Wash 45 microliters of Protein A Magnetic Beads per chip with ice-cold PBS Tween, as before. After the final wash, add one milliliter of ice-cold PBS to the beads.Then add three micrograms of antibody per 45 microliter aliquot of beads. Incubate for two hours at four degrees Celsius on a rotator to bind the antibodies to the beads. When the incubation time has elapsed, resuspend washed antibody-coupled beads in one bead volume of ice-cold PBS.Divide each precleared extract into two tubes of approximately 643.5 microliters. Add the same volume of dilution buffer and antibody-bound beads and incubate the samples at four degrees Celsius overnight on a rotator. The next day, wash the beads with ice-cold chip buffers one through three for 10 minutes each on a rotator at four degrees Celsius, follow with three five-minute washes with TE buffer.Add elution buffer and dilute for one hour at 1, 400 RPM in a shaker at room temperature. Once the hour has elapsed, add 10 micrograms of Rnase to each chip sample and five micrograms to the input samples. Incubate the samples on the shaker for 30 minutes at 37 degrees Celsius.Finally, add 100 micrograms of Proteinase K per chip sample and 50 micrograms per input. Incubate overnight at 65 degrees Celsius at 1, 400 RPM. H3K79 dimethylation levels are decreased after three days of CPC culturing and DOT1L inhibition by SGC 0946.QCPR after chip analysis revealed that genes with high histone-3-lysine-79 methylation levels at baseline were most responsive to the inhibitor treatment. Genes with a lower H3K79 dimethylation coverage showed no significant change in methylation levels. This heat map shows that histone-3-lysine-79 methylation levels peak around the transcription start site and decrease toward the transcription end site.Once mastered, this technique can be done in about one week, if it is performed properly. After watching this video, you should have a good understanding in how to perform chromatin immunoprecipitation on histone modifications in isolated brain cells.

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Molecular Biology

Neuroscience

Unit 2

Analyzing Gene Expression and Function

SCIENTISTS IN ACTION

Production of Chick Embryo Extract for the Cultivation of Murine Neural Crest Stem Cells

The neural crest arises from the neuro-ectoderm during embryogenesis and persists only temporarily. Early experiments already proofed pluripotent progenitor cells to be an integral part of the neural crest1. Phenotypically, neural crest stem cells (NCSC) are defined by simultaneously expressing p75 (low-affine nerve growth factor receptor, LNGFR) and SOX10 during their migration from the neural crest2,3,4,5. These progenitor cells can differentiate into smooth muscle cells, chromaffin cells, neurons and glial cells, as well as melanocytes, cartilage and bone6,7,8,9. To cultivate NCSC in vitro, a special neural crest stem cell medium (NCSCM) is required10. The most complex part of the NCSCM is the preparation of chick embryo extract (CEE) representing an essential source of growth factors for the NCSC as well as for other types of neural explants. Other NCSCM ingredients beside CEE are commercially available. Producing CCE using laboratory standard equipment it is of high importance to know about the challenging details as the isolation, maceration, centrifugation, and filtration processes. In this protocol we describe accurate techniques to produce a maximized amount of pure and high quality CEE.

To cultivate neural crest stem cells (NCSC) in vitro, a special medium (NCSCM) is required. Essential part of NCSCM is chick embryo extract (CEE). We here describe accurate techniques to produce a maximized amount of pure and high quality CEE, including details as the isolation, maceration, centrifugation, and filtration processes.

The neural crest arises from the neuro-ectoderm during embryogenesis and persists only temporarily. Early experiments already proofed pluripotent ...

Physiological, Morphological and Neurochemical Characterization of Neurons Modulated by Movement

The role of individual neurons and their function in neuronal circuits is fundamental to understanding the neuronal mechanisms of sensory and motor functions. Most investigations of sensorimotor mechanisms rely on either examination of neurons while an animal is static1,2 or record extracellular neuronal activity during a movement.3,4 While these studies have provided the fundamental background for sensorimotor function, they either do not evaluate functional information which occurs during a movement or are limited in their ability to fully characterize the anatomy, physiology and neurochemical phenotype of the neuron. A technique is shown here which allows extensive characterization of individual neurons during an in vivo movement. This technique can be used not only to study primary afferent neurons but also to characterize motoneurons and sensorimotor interneurons. Initially the response of a single neuron is recorded using electrophysiological methods during various movements of the mandible followed by determination of the receptive field for the neuron. A neuronal tracer is then intracellularly injected into the neuron and the brain is processed so that the neuron can be visualized with light, electron or confocal microscopy (Fig. 1). The detailed morphology of the characterized neuron is then reconstructed so that neuronal morphology can be correlated with the physiological response of the neuron (Figs. 2,3). In this communication important key details and tips for successful implementation of this technique are provided. Valuable additional information can be determined for the neuron under study by combining this method with other techniques. Retrograde neuronal labeling can be used to determine neurons with which the labeled neuron synapses; thus allowing detailed determination of neuronal circuitry. Immunocytochemistry can be combined with this method to examine neurotransmitters within the labeled neuron and to determine the chemical phenotypes of neurons with which the labeled neuron synapses. The labeled neuron can also be processed for electron microscopy to determine the ultrastructural features and microcircuitry of the labeled neuron. Overall this technique is a powerful method to thoroughly characterize neurons during in vivo movement thus allowing substantial insight into the role of the neuron in sensorimotor function.

A technique is described to quantify the in vivo physiological response of mammalian neurons during movement and correlate the physiology of the neuron with neuronal morphology, neurochemical phenotype and synaptic microcircuitry.

The role of individual neurons and their function in neuronal circuits is fundamental to understanding the neuronal mechanisms of sensory and motor ...

Chromatin Immunoprecipitation from Dorsal Root Ganglia Tissue following Axonal Injury

Axons in the central nervous system (CNS) do not regenerate while those in the peripheral nervous system (PNS) do regenerate 
to a limited extent after injury (Teng et al., 2006). It is recognized that transcriptional programs essential for neurite and axonal outgrowth are 
reactivated upon injury in the PNS (Makwana et al., 2005). However the tools available to analyze neuronal gene regulation in vivo are limited and 
often challenging.
The dorsal root ganglia (DRG) offer an excellent injury model system because both the CNS and PNS are innervated by a 
bifurcated axon originating from the same soma. The ganglia represent a discrete collection of cell bodies where all transcriptional events occur, 
and thus provide a clearly defined region of transcriptional activity that can be easily and reproducibly removed from the animal. Injury of nerve 
fibers in the PNS (e.g. sciatic nerve), where axonal regeneration does occur, should reveal a set of transcriptional programs that are distinct from 
those responding to a similar injury in the CNS, where regeneration does not take place (e.g. spinal cord). Sites for transcription factor binding, 
histone and DNA modification resulting from injury to either PNS or CNS can be characterized using chromatin immunoprecipitation (ChIP). 
Here, we describe a ChIP protocol using fixed mouse DRG tissue following axonal injury. This powerful combination provides a means for characterizing the pro-regeneration chromatin environment necessary for promoting axonal regeneration.

We present a method for chromatin immunoprecipitation from dorsal root ganglia tissue following axonal injury. The approach can be used to identify specific transcription factor binding sites and epigenetic modification of histone and DNA important for the regeneration of injured axons in both the peripheral and central nervous system.

Axons in the central nervous system (CNS) do not regenerate while those in the peripheral nervous system (PNS) do regenerate 
to a limited extent after ...

Detection of Microregional Hypoxia in Mouse Cerebral Cortex by Two-photon Imaging of Endogenous NADH Fluorescence

The brain's ability to function at high levels of metabolic demand depends on continuous oxygen supply through blood flow and tissue oxygen diffusion. Here we present a visualized experimental and methodological protocol to directly visualize microregional tissue hypoxia and to infer perivascular oxygen gradients in the mouse cortex. It is based on the non-linear relationship between nicotinamide adenine dinucleotide (NADH) endogenous fluorescence intensity and oxygen partial pressure in the tissue, where observed tissue NADH fluorescence abruptly increases at tissue oxygen levels below 10 mmHg1. We use two-photon excitation at 740 nm which allows for concurrent excitation of intrinsic NADH tissue fluorescence and blood plasma contrasted with Texas-Red dextran. The advantages of this method over existing approaches include the following: it takes advantage of an intrinsic tissue signal and can be performed using standard two-photon in vivo imaging equipment; it permits continuous monitoring in the whole field of view with a depth resolution of ~50 &mu;m. We demonstrate that brain tissue areas furthest from cerebral blood vessels correspond to vulnerable watershed areas which are the first to become functionally hypoxic following a decline in vascular oxygen supply. This method allows one to image microregional cortical oxygenation and is therefore useful for examining the role of inadequate or restricted tissue oxygen supply in neurovascular diseases and stroke.

Here we describe a method to directly visualize microregional tissue hypoxia in the mouse cortex in vivo. It is based on concurrent two-photon imaging of nicotinamide adenine dinucleotide (NADH) and the cortical microcirculation. This method is useful for high resolution analysis of tissue oxygen supply.

The brain's ability to function at high levels of metabolic demand depends on continuous oxygen supply through blood flow and tissue oxygen diffusion. ...

Reproducible Mouse Sciatic Nerve Crush and Subsequent Assessment of Regeneration by Whole Mount Muscle Analysis

Regeneration in the peripheral nervous system (PNS) is widely studied both for its relevance to human disease and to understand the robust regenerative response mounted by PNS neurons thereby possibly illuminating the failures of CNS regeneration1. Sciatic nerve crush (axonotmesis) is one of the most common models of peripheral nerve injury in rodents2. Crushing interrupts all axons but Schwann cell basal laminae are preserved so that regeneration is optimal3,4. This allows the investigator to study precisely the ability of a growing axon to interact with both the Schwann cell and basal laminae4. Rats have generally been the preferred animal models for experimental nerve crush. They are widely available and their lesioned sciatic nerve provides a reasonable approximation of human nerve lesions5,4. Though smaller in size than rat nerve, the mouse nerve has many similar qualities. Most importantly though, mouse models are increasingly valuable because of the wide availability of transgenic lines now allows for a detailed dissection of the individual molecules critical for nerve regeneration6, 7. Prior investigators have used multiple methods to produce a nerve crush or injury including simple angled forceps, chilled forceps, hemostatic forceps, vascular clamps, and investigator-designed clamps8,9,10,11,12. Investigators have also used various methods of marking the injury site including suture, carbon particles and fluorescent beads13,14,1. We describe our method to obtain a reproducibly complete sciatic nerve crush with accurate and persistent marking of the crush-site using a fine hemostatic forceps and subsequent carbon crush-site marking. As part of our description of the sciatic nerve crush procedure we have also included a relatively simple method of muscle whole mount we use to subsequently quantify regeneration.

In this report we describe a method to crush mouse sciatic nerve. This method uses readily available hemostatic forceps and easily and reproducibly produces complete sciatic nerve crush. In addition, we describe a method to prepare muscle whole mounts suitable for analysis of nerve regeneration after sciatic nerve crush.

Regeneration in the peripheral nervous system (PNS) is widely studied both for its relevance to human disease and to understand the robust regenerative ...

Isolation and Culture of Neural Crest Cells from Embryonic Murine Neural Tube

The embryonic neural crest (NC) is a multipotent progenitor population that originates at the dorsal aspect of the neural tube, undergoes an epithelial to mesenchymal transition (EMT) and migrates throughout the embryo, giving rise to diverse cell types 1-3. NC also has the unique ability to influence the differentiation and maturation of target organs4-6. When explanted in vitro, NC progenitors undergo self-renewal, migrate and differentiate into a variety of tissue types including neurons, glia, smooth muscle cells, cartilage and bone. 
NC multipotency was first described from explants of the avian neural tube7-9. In vitro isolation of NC cells facilitates the study of NC dynamics including proliferation, migration, and multipotency. Further work in the avian and rat systems demonstrated that explanted NC cells retain their NC potential when transplanted back into the embryo10-13. Because these inherent cellular properties are preserved in explanted NC progenitors, the neural tube explant assay provides an attractive option for studying the NC in vitro. 
To attain a better understanding of the mammalian NC, many methods have been employed to isolate NC populations. NC-derived progenitors can be cultured from post-migratory locations in both the embryo and adult to study the dynamics of post-migratory NC progenitors11,14-20, however isolation of NC progenitors as they emigrate from the neural tube provides optimal preservation of NC cell potential and migratory properties13,21,22. Some protocols employ fluorescence activated cell sorting (FACS) to isolate a NC population enriched for particular progenitors11,13,14,17. However, when starting with early stage embryos, cell numbers adequate for analyses are difficult to obtain with FACS, complicating the isolation of early NC populations from individual embryos. Here, we describe an approach that does not rely on FACS and results in an approximately 96% pure NC population based on a Wnt1-Cre activated lineage reporter23. 
The method presented here is adapted from protocols optimized for the culture of rat NC11,13. The advantages of this protocol compared to previous methods are that 1) the cells are not grown on a feeder layer, 2) FACS is not required to obtain a relatively pure NC population, 3) premigratory NC cells are isolated and 4) results are easily quantified. Furthermore, this protocol can be used for isolation of NC from any mutant mouse model, facilitating the study of NC characteristics with different genetic manipulations. The limitation of this approach is that the NC is removed from the context of the embryo, which is known to influence the survival, migration and differentiation of the NC2,24-28.

Isolation of embryonic neural crest from the neural tube facilitates the use of in vitro methods for studying migration, self-renewal, and multipotency of neural crest.

The embryonic neural crest (NC) is a multipotent progenitor population that originates at the dorsal aspect of the neural tube, undergoes an epithelial to ...

An Optimized Procedure for Fluorescence-activated Cell Sorting (FACS) Isolation of Autonomic Neural Progenitors from Visceral Organs of Fetal Mice

During development neural crest (NC)-derived neuronal progenitors migrate away from the neural tube to form autonomic ganglia in visceral organs like the intestine and lower urinary tract. Both during development and in mature tissues these cells are often widely dispersed throughout tissues so that isolation of discrete populations using methods like laser capture micro-dissection is difficult. They can however be directly visualized by expression of fluorescent reporters driven from regulatory regions of neuron-specific genes like Tyrosine hydroxylase (TH). We describe a method optimized for high yields of viable TH+ neuronal progenitors from fetal mouse visceral tissues, including intestine and lower urogenital tract (LUT), based on dissociation and fluorescence-activated cell sorting (FACS). 
The Th gene encodes the rate-limiting enzyme for production of catecholamines. Enteric neuronal progenitors begin to express TH during their migration in the fetal intestine1 and TH is also present in a subset of adult pelvic ganglia neurons2-4 . The first appearance of this lineage and the distribution of these neurons in other aspects of the LUT, and their isolation has not been described. Neuronal progenitors expressing TH can be readily visualized by expression of EGFP in mice carrying the transgene construct Tg(Th-EGFP)DJ76Gsat/Mmnc1. We imaged expression of this transgene in fetal mice to document the distribution of TH+ cells in the developing LUT at 15.5 days post coitus (dpc), designating the morning of plug detection as 0.5 dpc, and observed that a subset of neuronal progenitors in the coalescing pelvic ganglia express EGFP. 
To isolate LUT TH+ neuronal progenitors, we optimized methods that were initially used to purify neural crest stem cells from fetal mouse intestine2-6. Prior efforts to isolate NC-derived populations relied upon digestion with a cocktail of collagenase and trypsin to obtain cell suspensions for flow cytometry. In our hands these methods produced cell suspensions from the LUT with relatively low viability. Given the already low incidence of neuronal progenitors in fetal LUT tissues, we set out to optimize dissociation methods such that cell survival in the final dissociates would be increased. We determined that gentle dissociation in Accumax (Innovative Cell Technologies, Inc), manual filtering, and flow sorting at low pressures allowed us to achieve consistently greater survival (&gt;70% of total cells) with subsequent yields of neuronal progenitors sufficient for downstream analysis. The method we describe can be broadly applied to isolate a variety of neuronal populations from either fetal or adult murine tissues.

An Optimized Procedure for Fluorescence-activated Cell Sorting FACS Isolation of Autonomic Neural Progenitors from Visceral Organs of Fetal Mice

An optimized procedure to purify neural crest-derived neuronal progenitors from fetal mouse tissues is described. This method takes advantage of expression from fluorescent reporter alleles to isolate discrete populations by fluorescence-activated cell sorting (FACS). The technique can be applied to isolate neuronal subpopulations throughout development or from adult tissues.

During development neural crest (NC)-derived neuronal progenitors migrate away from the neural tube to form autonomic ganglia in visceral organs like the ...

Promotion of Survival and Differentiation of Neural Stem Cells with Fibrin and Growth Factor Cocktails after Severe Spinal Cord Injury

Neural stem cells (NSCs) can self-renew and differentiate into neurons and glia. Transplanted NSCs can replace lost neurons and glia after spinal cord injury (SCI), and can form functional relays to re-connect spinal cord segments above and below a lesion. Previous studies grafting neural stem cells have been limited by incomplete graft survival within the spinal cord lesion cavity. Further, tracking of graft cell survival, differentiation, and process extension had not been optimized. Finally, in previous studies, cultured rat NSCs were typically reported to differentiate into glia when grafted to the injured spinal cord, rather than neurons, unless fate was driven to a specific cell type. To address these issues, we developed new methods to improve the survival, integration and differentiation of NSCs to sites of even severe SCI. NSCs were freshly isolated from embryonic day 14 spinal cord (E14) from a stable transgenic Fischer 344 rat line expressing green fluorescent protein (GFP) and were embedded into a fibrin matrix containing growth factors; this formulation aimed to retain grafted cells in the lesion cavity and support cell survival. NSCs in the fibrin/growth factor cocktail were implanted two weeks after thoracic level-3 (T3) complete spinal cord transections, thereby avoiding peak periods of inflammation. Resulting grafts completely filled the lesion cavity and differentiated into both neurons, which extended axons into the host spinal cord over remarkably long distances, and glia. Grafts of cultured human NSCs expressing GFP resulted in similar findings. Thus, methods are defined for improving neural stem cell grafting, survival and analysis of in vivo findings.

Fibrin matrices containing growth factors were used to retain grafted neural stem cells into sites of complete spinal cord transection. Grafted cells completely filled the lesion cavity and differentiated into multiple neural cell types, including neurons that extended axons into host spinal cord over long distances.

Neural stem cells (NSCs) can self-renew and differentiate into neurons and glia. Transplanted NSCs can replace lost neurons and glia after spinal cord ...

A Quantitative Cell Migration Assay for Murine Enteric Neural Progenitors

Neural crest cells (NCC) are a transient and multipotent cell population that originates from the dorsal neural tube and migrates extensively throughout the developing vertebrate embryo. In addition to providing peripheral glia and neurons, NCC generate melanocytes as well as most of the cranio-facial skeleton. NCC migration and differentiation is controlled by a combination of their axial origin along the neural tube and their exposure to regionally distinct extracellular cues. Such contribution of extracellular ligands is especially evident during the formation of the enteric nervous system (ENS), a complex interconnected network of neural ganglia that locally controls (among other things) gut muscle movement and intestinal motility. Most of the ENS is derived from a small initial pool of NCC that undertake a long journey in order to colonize - in a rostral to caudal fashion - the entire length of the prospective gut. Among several signaling pathways known to influence enteric NCC colonization, GDNF/RET signaling is recognized as the most important. Indeed, spatiotemporally controlled secretion of the RET ligand GDNF by the gut mesenchyme is chiefly responsible for the attraction and guidance of RET-expressing enteric NCC to and within the embryonic gut. Here, we describe an ex vivo cell migration assay, making use of a transgenic mouse line possessing fluorescently labeled NCC, which allows precise quantification of enteric NCC migration potential in the presence of various growth factors, including GDNF.

We present an ex vivo cell migration assay that allows precise quantification of enteric neural crest cell migration potential in the presence of various growth factors.

Neural crest cells (NCC) are a transient and multipotent cell population that originates from the dorsal neural tube and migrates extensively throughout ...

Isolation and Quantification of Botulinum Neurotoxin From Complex Matrices Using the BoTest Matrix Assays

Accurate detection and quantification of botulinum neurotoxin (BoNT) in complex matrices is required for pharmaceutical, environmental, and food sample testing. Rapid BoNT testing of foodstuffs is needed during outbreak forensics, patient diagnosis, and food safety testing while accurate potency testing is required for BoNT-based drug product manufacturing and patient safety. The widely used mouse bioassay for BoNT testing is highly sensitive but lacks the precision and throughput needed for rapid and routine BoNT testing. Furthermore, the bioassay&#39;s use of animals has resulted in calls by drug product regulatory authorities and animal-rights proponents in the US and abroad to replace the mouse bioassay for BoNT testing. Several in vitro replacement assays have been developed that work well with purified BoNT in simple buffers, but most have not been shown to be applicable to testing in highly complex matrices. Here, a protocol for the detection of BoNT in complex matrices using the BoTest Matrix assays is presented. The assay consists of three parts: The first part involves preparation of the samples for testing, the second part is an immunoprecipitation step using anti-BoNT antibody-coated paramagnetic beads to purify BoNT from the matrix, and the third part quantifies the isolated BoNT&#39;s proteolytic activity using a fluorogenic reporter. The protocol is written for high throughput testing in 96-well plates using both liquid and solid matrices and requires about 2 hr of manual preparation with total assay times of 4-26 hr depending on the sample type, toxin load, and desired sensitivity. Data are presented for BoNT/A testing with phosphate-buffered saline, a drug product, culture supernatant, 2% milk, and fresh tomatoes and includes discussion of critical parameters for assay success.

The BoTest Matrix botulinum neurotoxin (BoNT) detection assays rapidly purify and quantify BoNT from a range of sample matrices.&#160;Here, we present a protocol for the detection and quantification of BoNT from both solid and liquid matrices and demonstrate the assay with BOTOX, tomatoes, and milk.

Accurate detection and quantification of botulinum neurotoxin (BoNT) in complex matrices is required for pharmaceutical, environmental, and food sample ...