Name: the detection of 5-hydroxymethylcytosine in neural stem cells and brains of mice
Uploaded: 2019-09-19T14:00:00
Description: Watch this Scientific Journal Video about The Detection of 5-Hydroxymethylcytosine in Neural Stem Cells and Brains of Mice at JoVE.com

XL was supported in part by the National Key R&#38;D Program of China (2017YFE0196600), and the National Natural Science Foundation of China (Grant Nos. 31771395, 31571518). Q.S. was supported by the National Key Research and Development Program of China (2017YFC1001703) and the Key Research and Development Program of Zhejiang Province (2017C03009). W.X. was supported by the Natural Science Foundation of Zhejiang province (LY18H020002) and Science Technology Department of Zhejiang Province (2017C37057).

All the animal procedures have been approved by the Animal Ethics Committee of Zhejiang University.
1. The Culture of Adult Neural Stem Cells and Neurons
<ol>
	<li>Isolate adult neural stem cells from the forebrain of an adult (8-10 week old) C57/BL6 male mouse as described previously31,32.</li>
	<li>Culture adult neural stem cells in DMEM/F-12 medium containing 20 ng/mL FGF-2, 20 ng/mL EGF, 2% B27 supplement, 1% antibiotic-antimycot...

<ol>
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Epigenetic modifications play essential roles during brain development, maturation, and function. As a stable marker for DNA modification, dynamic 5-hmC responds to behavioral adaptation, neuronal activity, and is positively correlated with gene expression; thus, it is involved in the normal function of the brain and neurological disorder4. To explore its function in cells and tissues, it is necessary to detect the existence of 5-hmC and compare the level before and after treatment. Here, we demon...

Epigenetic modifications, including DNA modification, histone modification and RNA modification, have been shown to play essential functions in diverse biological processes and diseases1,2,3,4,5,6,7. For a long time, DNA methylation (i.e., 5-methylcytosine (5-mC)) has been viewed as a highly stable epigenetic marker and can not be further modified in the genome. Recently, it has been found that 5-mC could be oxidized to 5-hydroxymethylcytosine(5-hmC) by TET (Ten-eleven translocations) family proteins including TET1, TET2, and TET38,9. Further studies show that 5-hmC could serve as a stable marker and play biological roles through regulating gene expression4,10,11,12.
The present evidences indicate&#160;that 5-hmC is highly enriched in neuronal tissues/cells relative to other types of tissues in mammals, and exhibits dynamic features during neuronal development13,14. In the&#160;neuronal system, 5-hmC mediated epigenetic modifications play an important role in regulating neural stem cells, neuronal activity, learning and memory, and is involved in multiple neurological disorders including Rett syndrome, autism, Alzheimer&#8217;s disease, Huntington&#8217;s disease, etc.2,13,15,16,17,18,19,20.
There are several approaches for detecting 5-hmC in cells and tissues14,21,22,23,24. Here, we describe two methods to detect the existence of 5-hmC and quantify the global level of 5-hmC: immunofluorescence staining and DNA dot-blot. These two methods are convenient and sensitive, and have been successfully used in previous studies25,26,27,28,29,30. The key steps of these two methods are DNA denaturation. For immunofluorescence staining of 5-hmC, pre-treatment of samples with 1 M HCl is required. For 5-hmC dot-blot, DNA denaturation is performed with&#160;NaOH solution. These two methods together with next-generation sequencing are very useful tools for investigating the function of 5-hmC.

<table>
	<tbody>
		<tr>
			<td>4&#39;-6-diamidino-2-phenylindole (DAPI )</td>
			<td>Sigma-Aldrich</td>
			<td>D8417</td>
		</tr>
		<tr>
			<td>Adobe Photoshop software</td>
			<td>Adobe Inc.</td>
			<td>/</td>
		</tr>
		<tr>
			<td>Alexa Fluor 488 goat anti-rabbit IgG</td>
			<td>Thermo Fisher</td>
			<td>A11008</td>
		</tr>
		<tr>
			<td>Alexa Fluor 568 goat anti-mouse IgG</td>
			<td>Thermo Fisher</td>
			<td>A11001</td>
		</tr>
		<tr>
			<td>anti-5-hydroxymethylcytosine</td>
			<td>Active Motif</td>
			<td>39769</td>
		</tr>
		<tr>
			<td>anti-NeuN</td>
			<td>Millipore</td>
			<td>MAB377</td>
		</tr>
		<tr>
			<td>B27 supplement</td>
			<td>Gibco</td>
			<td>12587-010</td>
		</tr>
		<tr>
			<td>B27 supplement</td>
			<td>Gibco</td>
			<td>12580-010</td>
		</tr>
		<tr>
			<td>B27 supplement</td>
			<td>Gibco</td>
			<td>17504-044</td>
		</tr>
		<tr>
			<td>Cryostat microtome</td>
			<td>Leica</td>
			<td>CM1950</td>
		</tr>
		<tr>
			<td>DMEM/F-12 medium</td>
			<td>OmegaScientific</td>
			<td>DM25</td>
		</tr>
		<tr>
			<td>epidermal growth factor</td>
			<td>PeproTech</td>
			<td>100-15</td>
		</tr>
		<tr>
			<td>Fibroblast growth factor-basic</td>
			<td>PeproTech</td>
			<td>100-18B</td>
		</tr>
		<tr>
			<td>forskolin</td>
			<td>Sigma-Aldrich</td>
			<td>F6886</td>
		</tr>
		<tr>
			<td>GlutaMax</td>
			<td>Thermo</td>
			<td>35050061</td>
		</tr>
		<tr>
			<td>L-Glutamine</td>
			<td>Gibco</td>
			<td>25030-149</td>
		</tr>
		<tr>
			<td>neurobasal medium</td>
			<td>Gibco</td>
			<td>21103-049</td>
		</tr>
		<tr>
			<td>normal goat serum</td>
			<td>Vector Laboratories</td>
			<td>Z0325</td>
		</tr>
		<tr>
			<td>nylon membrane (Hybond&#8482;-N+ )</td>
			<td>Amersham Biosciences</td>
			<td>RPN303B</td>
		</tr>
		<tr>
			<td>OCT</td>
			<td>Leica</td>
			<td>14020108926</td>
		</tr>
		<tr>
			<td>Pen Strep</td>
			<td>Gibco</td>
			<td>15140-122</td>
		</tr>
		<tr>
			<td>phenol: chloroform: isoamyl alcohol (25: 24:1 )</td>
			<td>Sigma-Aldrich</td>
			<td>516726</td>
		</tr>
		<tr>
			<td>Poly-D-Lysine</td>
			<td>Sigma</td>
			<td>P0899-10</td>
		</tr>
		<tr>
			<td>proteinase K</td>
			<td>VVR</td>
			<td>39450-01-6</td>
		</tr>
		<tr>
			<td>retinoic acid</td>
			<td>Sigma-Aldrich</td>
			<td>R2625</td>
		</tr>
		<tr>
			<td>Triton X-100</td>
			<td>Solarbio</td>
			<td>T8210</td>
		</tr>
	</tbody>
</table>

the detection of 5-hydroxymethylcytosine in neural stem cells and brains of mice

Multiple&#160;DNA modifications have been identified in the mammalian genome. Of that, 5-methylcytosine and 5-hydroxymethylcytosine-mediated epigenetic mechanisms have been intensively studied. 5-hydroxymethylcytosine displays dynamic features during embryonic and postnatal development of the brain, plays a regulatory function in gene expression, and is involved in multiple neurological disorders. Here, we describe the detailed methods including immunofluorescence staining and&#160;DNA dot-blot to detect 5-hydroxymethylcytosine in cultured cells and brain tissues of mouse.

To reveal the distribution of 5-hmC in the hippocampus of adult mice, we performed immunofluorescence with antibodies against neuronal cells (NeuN) and 5-hmC. In the hippocampus, 5-hmC co-localized well with neuronal cell marker NeuN (Figure 1A-H), suggesting an enrichment of 5-hmC in neurons.
To determine the dynamics of 5-hmC during neuronal development, a dot-blot was first performed with DNA samples isolated from proliferating and differentiat...

Watch this Scientific Journal Video about The Detection of 5-Hydroxymethylcytosine in Neural Stem Cells and Brains of Mice at JoVE.com

The Detection of 5-Hydroxymethylcytosine in Neural Stem Cells and Brains of Mice

Here, we present a protocol to detect 5-hydroxymethylcytosine in cells and brain tissues, utilizing immunofluorescence staining and DNA dot-blot methods.

Multiple&#160;DNA modifications have been identified in the mammalian genome. Of that, 5-methylcytosine and 5-hydroxymethylcytosine-mediated epigenetic ...

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