Name: preparation of meiotic chromosome spreads from zebrafish spermatocytes
Uploaded: 2020-03-03T12:00:00
Description: Watch this Scientific Journal Video about Preparation of Meiotic Chromosome Spreads from Zebrafish Spermatocytes at JoVE.com

我们感谢特伦特·纽曼和马苏达·沙里对手稿和An Nguyen的评论，感谢他们帮助优化了从斑马鱼染色体中传播和染色的方法。这项工作得到了NIH R01 GM079115授予S.M.B.的支持。

所有涉及斑马鱼的方法都是使用加州大学戴维斯分校机构动物护理和使用委员会批准的伦理标准进行的。
1. 染色体传播程序
注： 以下协议旨在创建 4-6 张幻灯片，每张幻灯片有数百张扩散的中导核。使用的睾丸数量取决于鱼的大小。预计在受精后60天使用20只动物，在受精后6个月使用15只动物（mpf）。对于大型斑马鱼（例如，12 mpf）10只动物应该足够。请?...

<ol>
	<li>Nagaoka, S. I., Hassold, T. J., Hunt, P. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Human+aneuploidy:+mechanisms+and+new+insights+into+an+age-old+problem.">Human aneuploidy: mechanisms and new insights into an age-old problem.</a> Nature Reviews Genetics. 13, 493-504 (2012).</li><li>Zickler, D., Kleckner, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Recombination,+Pairing,+and+Synapsis+of+Homologs+during+Meiosis.">Recombination, Pairing, and Synapsis of Homologs during Meiosis.</a> Cold Spring Harbor Perspectives in Biology. 7, (2015).</li><li>Blokhina, Y. P., Nguyen, A. D., Draper, B. W., Burgess, S. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+telomere+bouquet+is+a+hub+where+meiotic+double-strand+breaks,+synapsis,+and+stable+homolog+juxtaposition+are+coordinated+in+the+zebrafish,+Danio+rerio.">The telomere bouquet is a hub where meiotic double-strand breaks, synapsis, and stable homolog juxtaposition are coordinated in the zebrafish, Danio rerio.</a> PLoS Genetics. 15, e1007730 (2019).</li><li>Saito, K., Sakai, C., Kawasaki, T., Sakai, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Telomere+distribution+pattern+and+synapsis+initiation+during+spermatogenesis+in+zebrafish.">Telomere distribution pattern and synapsis initiation during spermatogenesis in zebrafish.</a> Developmental Dynamics. 243, 1448-1456 (2014).</li><li>Oliver-Bonet, M., Turek, P. J., Sun, F., Ko, E., Martin, R. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Temporal+progression+of+recombination+in+human+males.">Temporal progression of recombination in human males.</a> Molecular Human Reproduction. 11, 517-522 (2005).</li><li>Gruhn, J. R., Rubio, C., Broman, K. W., Hunt, P. A., Hassold, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cytological+studies+of+human+meiosis:+sex-specific+differences+in+recombination+originate+at,+or+prior+to,+establishment+of+double-strand+breaks.">Cytological studies of human meiosis: sex-specific differences in recombination originate at, or prior to, establishment of double-strand breaks.</a> PLoS One. 8, e85075 (2013).</li><li>Pratto, F., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Recombination+initiation+maps+of+individual+human+genomes.">Recombination initiation maps of individual human genomes.</a> Science. 346, 1256442 (2014).</li><li>Brown, P. W., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Meiotic+synapsis+proceeds+from+a+limited+number+of+subtelomeric+sites+in+the+human+male.">Meiotic synapsis proceeds from a limited number of subtelomeric sites in the human male.</a> American Journal of Human Genetics. 77, 556-566 (2005).</li><li>Poss, K. D., Nechiporuk, A., Stringer, K. F., Lee, C., Keating, M. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Germ+cell+aneuploidy+in+zebrafish+with+mutations+in+the+mitotic+checkpoint+gene+mps1.">Germ cell aneuploidy in zebrafish with mutations in the mitotic checkpoint gene mps1.</a> Genes and Development. 18, 1527-1532 (2004).</li><li>Saito, K., Siegfried, K. R., N&#252;sslein-Volhard, C., Sakai, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isolation+and+cytogenetic+characterization+of+zebrafish+meiotic+prophase+I+mutants.">Isolation and cytogenetic characterization of zebrafish meiotic prophase I mutants.</a> Developmental Dynamics. 240, 1779-1792 (2011).</li><li>Sansam, C. L., Pezza, R. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Connecting+by+breaking+and+repairing:+mechanisms+of+DNA+strand+exchange+in+meiotic+recombination.">Connecting by breaking and repairing: mechanisms of DNA strand exchange in meiotic recombination.</a> Febs Journal. 282, 2444-2457 (2015).</li><li>Feitsma, H., Leal, M. C., Moens, P. B., Cuppen, E., Schulz, R. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mlh1+Deficiency+in+Zebrafish+Results+in+Male+Sterility+and+Aneuploid+as+Well+as+Triploid+Progeny+in+Females.">Mlh1 Deficiency in Zebrafish Results in Male Sterility and Aneuploid as Well as Triploid Progeny in Females.</a> Genetics. 175, 1561-1569 (2007).</li><li>Dia, F., Strange, T., Liang, J., Hamilton, J., Berkowitz, K. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Preparation+of+Meiotic+Chromosome+Spreads+from+Mouse+Spermatocytes.">Preparation of Meiotic Chromosome Spreads from Mouse Spermatocytes.</a> Journal of Visualized Experiments. (129), e55378 (2017).</li><li>Moens, P. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Zebrafish:+chiasmata+and+interference.">Zebrafish: chiasmata and interference.</a> Genome. 49, 205-208 (2006).</li><li>Lisachov, A. P., Zadesenets, K. S., Rubtsov, N. B., Borodin, P. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Sex+chromosome+synapsis+and+recombination+in+male+guppies.">Sex chromosome synapsis and recombination in male guppies.</a> Zebrafish. 12 (2), 174-180 (2015).</li><li>Ocalewicz, K., Mota-Velasco, J. C., Campos-Ramos, R., Penman, D. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=FISH+and+DAPI+staining+of+the+synaptonemal+complex+of+the+Nile+tilapia+(Oreochromis+niloticus)+allow+orientation+of+the+unpaired+region+of+bivalent+1+observed+during+early+pachytene.">FISH and DAPI staining of the synaptonemal complex of the Nile tilapia (Oreochromis niloticus) allow orientation of the unpaired region of bivalent 1 observed during early pachytene.</a> Chromosome Research. 17 (6), 773 (2009).</li></ol>

在这里，我们描述了从斑马鱼睾丸分离的精子细胞中探测核表面端粒和染色体相关蛋白质的位置的方法。我们期望这些方法将适用于分析其他远程物种的精子细胞，并调整睾号的大小。
虽然只有几种抗体被提升到斑马鱼的中度蛋白，但我们已经成功地使用以下抗体，这些抗体被提升到人类（h）或小鼠（m）蛋白质。我们的实验室在鸡体内提出了对斑马鱼Sycp1蛋白（zfSycp1）的抗...

性生殖通过两个单倍体配子的组合进行，每个配子携带一半体细胞的染色体补充。美西斯是一种专门的细胞分裂，通过一轮DNA复制和连续两轮染色体分离产生单倍体配子。在前期I中，同源染色体（同源性）必须经过配对、重组和突触，后者的特点是形成由横丝桥接的两个同源轴组成的突触复合体（图1A，B）。未能正确执行这些过程可能导致非倍体配子的产生，这是导致人类流产的主要原因1。我们对配对、重组和突触之间协调的知识，已经通过研究广泛的生物，如酵母，C.elegans，小鼠和果蝇，等等，促进了我们的认识。虽然同源染色体配对和分离的一般过程得到了很好的保存，但其对重组和突触的依赖程度以及这些事件的顺序各不相同。
在leptotene和突触发生后不久，在麻风素和突触发生后不久，在花束中聚集的端粒附近发生，启动同源重组的美因双链断裂（DSB）形成。DSB形成和突触启动的这种配置也是人类雄性梅氏菌病的特征，但不是在小鼠5，6，7，8，表明斑马鱼可以作为人类精子的模型。研究斑马鱼美虫病也有若干实际优点。男性和女性在整个成年期间都经历游戏生成，他们的性腺很容易获得，并且数百个后代是由一个单一的十字架产生的。此外，胚胎是透明的，并在外部发育，这有利于早期发现胚胎发育中的畸变，由于非倍体配子3，9。使用斑马鱼的缺点是，它们缓慢地达到性成熟（约60天），核表面传播所需的材料量必须从10-20只成年动物身上收集，这取决于它们的大小。
由于可以探测染色体动力学的关键特征，因此染色体传播制剂是研究所有模型生物体染色体动力学的重要工具。在斑马鱼中，通过探测核表面扩散（这里称为染色体传播）对中显方案和核组织进展的关键方面进行了剖析，通过FISH3、4、9、10、11、12检测蛋白质和/或核酸的抗体。事实上，在花束中聚集端粒的极化定位可以在传播准备中保留（图1C）。最近，我们使用斑马鱼精子细胞染色体扩散与荧光检测方法和超分辨率显微镜一起，阐明斑马鱼端粒动力学、同源染色体配对、双链断裂定位和关键美感过渡3的突触的详细进展。在这里，我们介绍从斑马鱼睾丸的精子细胞制备染色体扩散的方法，然后用荧光肽核酸（PNA）探针将其染色，以重复端粒序列和染色体相关蛋白质的免疫荧光检测。

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>1.5 mL centrifuge tubes</td>
			<td>Several commercial brands available</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>1.5 mL microcentrifuge tube rack</td>
			<td>Several commercial brands available</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>16% formaldehyde, methanol-free</td>
			<td>ThermoFisher Scientific</td>
			<td>28908</td>
			<td></td>
		</tr>
		<tr>
			<td>2 mL</td>
			<td>Several commercial brands available</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>24 x 50 mm glass coverslips</td>
			<td>Corning</td>
			<td>2980-245</td>
			<td></td>
		</tr>
		<tr>
			<td>24 x 60 mm glasscoverslips</td>
			<td>VWR International</td>
			<td>16004-312</td>
			<td></td>
		</tr>
		<tr>
			<td>50 mL conical centrifuge tubes</td>
			<td>ThermoFisher Scientific</td>
			<td>363696</td>
			<td></td>
		</tr>
		<tr>
			<td>Autoclave bag</td>
			<td>Several commercial brands available</td>
			<td></td>
			<td>Used to make plastic coverslips.</td>
		</tr>
		<tr>
			<td>Bovine Serum Albumin (BSA)</td>
			<td>Fisher Scientific</td>
			<td>BP1605-100</td>
			<td>Prepare a 100 mg/ml stock solution in sterile distilled water.</td>
		</tr>
		<tr>
			<td>Cell Strainer, 100 &#181;m</td>
			<td>Fisher Scientific</td>
			<td>08-771-19</td>
			<td></td>
		</tr>
		<tr>
			<td>CF405M goat anti-chicken IgY (H+L), highly cross-adsorbed</td>
			<td>Biotium</td>
			<td>203775-500uL</td>
			<td>Use at 1:1000</td>
		</tr>
		<tr>
			<td>Chicken anti-zfSycp1</td>
			<td>Generated by Burgess lab</td>
			<td>N/A</td>
			<td>Use at 1:100</td>
		</tr>
		<tr>
			<td>Collagenase from Clostridium histolyticum</td>
			<td>Sigma-Aldrich</td>
			<td>C0130-500MG</td>
			<td></td>
		</tr>
		<tr>
			<td>Coplin jar</td>
			<td>Several commercial brands available</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>DNase I, grade II from bovine pancreas</td>
			<td>Roche Diagnostics</td>
			<td>10104159001</td>
			<td></td>
		</tr>
		<tr>
			<td>Dulbecco&#39;s Modified Eagle Medium (DMEM)</td>
			<td>Fisher Scientific</td>
			<td>MT10014CV</td>
			<td></td>
		</tr>
		<tr>
			<td>Dumont No. 5 Forceps</td>
			<td>Fine Science Tools</td>
			<td>11252-30</td>
			<td>Two are required for dissecting the testes.</td>
		</tr>
		<tr>
			<td>Eppendorf Tubes, 5 mL</td>
			<td>VWR International</td>
			<td>89429-308</td>
			<td></td>
		</tr>
		<tr>
			<td>Formamide</td>
			<td>Fisher Scientific</td>
			<td>BP228-100</td>
			<td></td>
		</tr>
		<tr>
			<td>Goat anti-chicken IgY (H+L) secondary antibody, Alexa Fluor 488</td>
			<td>ThermoFisher Scientific</td>
			<td>A-11039</td>
			<td>Use at 1:1000</td>
		</tr>
		<tr>
			<td>Goat anti-chicken IgY (H+L) secondary antibody, Alexa Fluor 594</td>
			<td>ThermoFisher Scientific</td>
			<td>A-11042</td>
			<td>Use at 1:1000</td>
		</tr>
		<tr>
			<td>Goat anti-hDMC1</td>
			<td>Santa Cruz Biotechnology</td>
			<td>sc-8973</td>
			<td>Does not work in our hands</td>
		</tr>
		<tr>
			<td>Goat anti-rabbit IgG (H+L) cross-adsorbed secondary antibody, Alexa Fluor 488</td>
			<td>ThermoFisher Scientific</td>
			<td>A-11008</td>
			<td>Use at 1:1000</td>
		</tr>
		<tr>
			<td>Goat anti-rabbit IgG (H+L) cross-adsorbed secondary antibody, Alexa Fluor 594</td>
			<td>ThermoFisher Scientific</td>
			<td>A-11012</td>
			<td>Use at 1:1000</td>
		</tr>
		<tr>
			<td>Goat serum</td>
			<td>Sigma-Aldrich</td>
			<td>G9023-10mL</td>
			<td></td>
		</tr>
		<tr>
			<td>Heparin sodium salt</td>
			<td>Sigma-Aldrich</td>
			<td>H3393-100KU</td>
			<td></td>
		</tr>
		<tr>
			<td>Humidity chamber</td>
			<td>Fisher Scientific</td>
			<td>50-112-3683</td>
			<td></td>
		</tr>
		<tr>
			<td>Hybridization Oven</td>
			<td>VWR International</td>
			<td>230401V (Model 5420)</td>
			<td></td>
		</tr>
		<tr>
			<td>Incubator Shaker</td>
			<td>New Brunswick Scientific</td>
			<td>Model Classic C25</td>
			<td></td>
		</tr>
		<tr>
			<td>KCl</td>
			<td>Fisher Scientific</td>
			<td>P217-500</td>
			<td></td>
		</tr>
		<tr>
			<td>Kimwipes</td>
			<td>Kimerbly-Clark Professional</td>
			<td>34155</td>
			<td>Used for the humidity chamber</td>
		</tr>
		<tr>
			<td>KH2PO4</td>
			<td>Fisher Scientific</td>
			<td>P285-500</td>
			<td></td>
		</tr>
		<tr>
			<td>Microscope</td>
			<td>Several commercial brands available</td>
			<td></td>
			<td>Any standard microscope capable of at least ~1.65X magnification is sufficient.</td>
		</tr>
		<tr>
			<td>Microscope slides</td>
			<td>Fisher Scientific</td>
			<td>12-544-7</td>
			<td></td>
		</tr>
		<tr>
			<td>Mouse anti-hamsterSCP3</td>
			<td>Abcam</td>
			<td>ab97672</td>
			<td>Does not work in our hands</td>
		</tr>
		<tr>
			<td>Mouse anti-hMLH1</td>
			<td>BD Biosciences</td>
			<td>550838</td>
			<td>Does not work in our hands</td>
		</tr>
		<tr>
			<td>Mouse anti-hRPA</td>
			<td>Sigma-Alrich</td>
			<td>MABE285</td>
			<td>Does not work in our hands</td>
		</tr>
		<tr>
			<td>Na2HPO4 &#183; 7 H2O</td>
			<td>Fisher Scientific</td>
			<td>S373-500</td>
			<td></td>
		</tr>
		<tr>
			<td>NaCl</td>
			<td>Fisher Scientific</td>
			<td>S271-3</td>
			<td></td>
		</tr>
		<tr>
			<td>Photo-Flo 200 solution</td>
			<td>Electron Microscopy Sciences</td>
			<td>74257</td>
			<td></td>
		</tr>
		<tr>
			<td>Plastic transfer pipettes</td>
			<td>Several commercial brands available</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>PNA TelC-Alexa647</td>
			<td>PNA Bio Inc</td>
			<td>F1013</td>
			<td>Prepare as per manufacturer&#39;s instructions.</td>
		</tr>
		<tr>
			<td>PNA TelC-Cy3</td>
			<td>PNA Bio Inc</td>
			<td>F1002</td>
			<td>Prepare as per manufacturer&#39;s instructions.</td>
		</tr>
		<tr>
			<td>ProLong Diamond Antifade Mountant</td>
			<td>ThermoFisher Scientific</td>
			<td>P36970</td>
			<td></td>
		</tr>
		<tr>
			<td>ProLong Diamond Antifade Mountant with DAPI</td>
			<td>ThermoFisher Scientific</td>
			<td>P36971</td>
			<td></td>
		</tr>
		<tr>
			<td>Rabbit anti-hRPA</td>
			<td>Bethyl</td>
			<td>A300-244A</td>
			<td>Use at 1:300</td>
		</tr>
		<tr>
			<td>Rabbit anti-hSCP3</td>
			<td>Abcam</td>
			<td>ab150292</td>
			<td>Use at 1:200</td>
		</tr>
		<tr>
			<td>Rabbit anti-hRad51</td>
			<td>GeneTex</td>
			<td>GTX100469</td>
			<td>Use at 1:300</td>
		</tr>
		<tr>
			<td>Sodium citrate</td>
			<td>Fisher Scientific</td>
			<td>S279-500</td>
			<td></td>
		</tr>
		<tr>
			<td>Sucrose</td>
			<td>Fisher Scientific</td>
			<td>S5-500</td>
			<td></td>
		</tr>
		<tr>
			<td>Supercut Scissors, 30&#176; angle, 10 cm</td>
			<td>Fisher Scientific</td>
			<td>50-822-353</td>
			<td>Can also use any pair of small scissors.</td>
		</tr>
		<tr>
			<td>Sylgard kit</td>
			<td>Fisher Scientific</td>
			<td>NC9897184</td>
			<td>Prepare as per manufacturer&#39;s instructions.</td>
		</tr>
		<tr>
			<td>Triton X-100</td>
			<td>Fisher Scientific</td>
			<td>BP151-100</td>
			<td>Dilute in sterile distilled water to make a 20% working solution. Store at room temperature. Triton X-100 forms a precipitate when diluted in water; precipitate dissolves overnight.</td>
		</tr>
		<tr>
			<td>Trypsin</td>
			<td>Worthington Biochemical</td>
			<td>LS003708</td>
			<td></td>
		</tr>
		<tr>
			<td>Trypsin inhibitor from chicken egg white</td>
			<td>Sigma-Aldrich</td>
			<td>T9253-500MG</td>
			<td></td>
		</tr>
		<tr>
			<td>Tween 20</td>
			<td>Bio-Rad</td>
			<td>170-6531</td>
			<td>Dilute in sterile distilled water to make a 20% working solution. Store at room temperature.</td>
		</tr>
		<tr>
			<td>Vannas Spring Scissors - 4 mm (micro scissors)</td>
			<td>Fine Science Tools</td>
			<td>15018-10</td>
			<td></td>
		</tr>
	</tbody>
</table>

preparation of meiotic chromosome spreads from zebrafish spermatocytes

肌病是制造性生殖单倍体配子所需的关键细胞过程。模型生物体在理解在中位前相期间发生的染色体事件，包括配对、突触和重组事件，以确保正确的染色体分离。虽然小鼠是理解这些过程背后的分子机制的重要模型，但并不是这个系统中的所有中态事件都类似于人类的美氏症。我们最近展示了斑马鱼作为人类精子生成模型的令人振奋的潜力。在这里，我们详细描述了我们的方法，可视化染色体和相关蛋白质在染色体传播制剂。这些制剂的优点是允许对染色体结构进行高分辨率分析。首先，我们描述了从成年斑马鱼中解剖睾丸的过程，然后是细胞分离、解压和染色体扩散。接下来，我们描述了通过免疫荧光检测和核酸序列，通过荧光原位杂交（FISH）检测中值染色体蛋白定位的过程。这些技术为斑马鱼系统中的中色质结构的细胞学分析提供了一套有用的工具。斑马鱼群落的研究人员应该能够迅速掌握这些技术，并将其纳入其生殖功能的标准分析中。

我们已经概述了一种制备和可视化斑马鱼精子细胞传播制剂的方法。正确执行时，我们的程序产生良好的传播，非重叠的核。为了恢复这种核，重要的是要有适量的起始材料（即睾度），治疗睾度在胰蛋白酶和足够数量的DNase I治疗足够长的时间。然后，这些传播可以染色端粒和中度蛋白，以研究期I期间的间质进展。图1描述了在期前一阶段的不同阶?...

Watch this Scientific Journal Video about Preparation of Meiotic Chromosome Spreads from Zebrafish Spermatocytes at JoVE.com

Preparation of Meiotic Chromosome Spreads from Zebrafish Spermatocytes

核表面扩散是研究染色体事件的重要工具。在这里，我们演示了一种在斑马鱼精子细胞的前期I期间制备和可视化的中端染色体的方法。

从斑马鱼精子细胞中培养染色体传播

Meiosis is the key cellular process required to create haploid gametes for sexual reproduction. Model organisms have been instrumental in understanding ...

斑马鱼正在成为研究美因病染色体事件的优秀脊椎动物模型，包括同源染色体配对、突触和重组。这种核表面扩散协议使我们能够使用超分辨率显微镜来可视化美的染色体结构的关键特征。研究人员预计，与其他斑马鱼传播协议一样，每张幻灯片上有数百个分布良好的核，碎片更少。这个程序最具技术挑战性的部分是解剖斑马鱼睾丸，因为它靠近皮肤，也连接到其他器官。可视化解剖程序将使研究人员知道当看到嵌入在动物组织中的性腺时会发生什么。对雄性斑马鱼进行安乐死后，用小剪刀一次斩首一条鱼，然后用微型剪刀沿着腹中线切割，露出体腔。将鱼放在硅胶涂层的培养皿中，并覆盖在1X PBS的浅池中。在1.65倍放大的显微镜下，使用钳子解剖睾丸。从睾丸中去除尽可能多的脂肪和周围组织。性腺在解剖范围光下会半透明，而周围的脂肪可能略暗。在不妨碍程序的情况下，一定数量的脂肪可能留在性腺上。将每个解剖的睾丸直接加入一个五毫升的管中，并加入两毫升的DMEM，并留在冰上。为了分离睾丸细胞，将200微升的胶原酶溶液加入五毫升管中。通过反转解决方案多次混合解决方案。在 32 摄氏度的 100 RPM 下，以 100 RPM 水平摇动孵化器摇床中的睾丸。每10分钟快速反转一次管子，以方便分离。一小时后，DMEM 多云，睾丸成小块。当睾丸在胶原酶中孵育时，预热100微升0.1摩尔蔗糖溶液至37摄氏度。要洗掉胶原酶，将DMEM加入五毫升的最终体积，并反转管子几次。在室温下，在200克下将睾丸取出3分钟。取出三毫升的上一液，使只保留两毫升。重复 DMEM 洗涤两次。上次 DMEM 洗涤后，取出四毫升的上经剂，并添加 1 毫升 DMEM、200 微升的 trypsin 溶液和 20 微升的 DNase I 溶液。反转管子几次以混合溶液。在 100 RPM 时，在 32 摄氏度下水平摇动管。每五分钟快速反转一次管子，以方便分离。5 到 15 分钟后，DMEM 解决方案仅包含几团。加入500微升的尝试性抑制剂溶液和50微升的DNase I溶液。反转管子几次混合，然后在室温下以 200 g 短暂旋转三分钟，以去除可能粘附在管盖或管侧的液体或团块。将管子放在冰上，用塑料转移移液器反复上下移液，以方便分离任何剩余的团块，从而重新悬浮细胞悬浮液。确保电池悬架不会进入转移移液器的灯泡。然后用DMEM预湿100微米细胞过滤器，并放在50毫升的冰管上。使用塑料转移移液器一次通过过滤器一滴转移细胞悬浮液。使用塑料移液器将滤液转移到新的五毫升管中。请务必收集连接到过滤器底面的池单元格。将DMEM加入管子，最终体积为5毫升。在室温下，在200克下将细胞取出5分钟。在不干扰颗粒的情况下，尽可能去除上一除更远，并将五升DNase I溶液直接加入到颗粒中。然后，通过沿着空的微离心管机架轻轻刮四到五次管外底，将颗粒与 DNase I 溶液混合。将 DMEM 添加到五毫升的最终体积中，通过多次反转管子混合溶液。在室温下，以200克对细胞悬浮液进行两分钟。重复DNase处理一到三次，直到重新消耗的颗粒在加入DMEM后不会团块。最后一次旋转后，尽可能去除上一除，而不会干扰颗粒。接下来，加入1X PBS的毫升，然后沿着空管架轻轻刮取管子的外底，重新暂停颗粒。在200g下将细胞悬浮液在5分钟内，然后在不干扰颗粒的情况下去除尽可能多的上一液。从 200 微升移液器尖端的末端切割 3 毫米，以扩大孔径。使用切割移液器尖端，通过上下移液，将颗粒重新在 0.1 摩尔蔗糖溶液的 80 微升中重新入库。让电池悬浮液在室温下坐三分钟。接下来，用移液器尖端，用 100 微升 1%甲醛涂覆一张幻灯片，用 0.15% Triton X-100 涂上一张幻灯片。然后，以垂直于长边直线将 18 微升的蔗糖悬浮液添加到幻灯片的中心。来回倾斜幻灯片，以方便将细胞悬浮液扩散到所有角落。将滑梯平放在稍微裂开的打开的湿度室中，以防止甲醛溶液干燥。将湿度室放在黑暗的抽屉中过夜。早晨，从湿度室取下盖子，让滑梯完全干燥。将幻灯片放在一个科普林罐中，然后用蒸馏水填充科普林罐。在室温下轻轻摇晃，孵育五分钟。倒出水，用1至250个润湿剂填满罐子。在室温下轻轻摇晃，每次洗涤两次，每次五分钟。让幻灯片完全干燥并储存在零下 20 摄氏度，直到它们被染色。该协议概述了一种准备和可视化斑马鱼精子细胞传播的方法，该方法产生分布良好的非重叠核。左侧的面板显示了三个阶段的梅蒂克前体，钩端，早期的青霉素，和帕奇滕。每个阶段都用端粒染色品红色。此处显示了由于 DNase I 治疗不足而导致质量差的点差示例。为SYCP3染色的美的染色体表明，由于粘性蔗糖细胞悬浮液，阻止细胞在幻灯片上正确扩散，核重叠。从正确的起始材料量开始，在三叶草中治疗斑马鱼睾丸的时间适当，并进行必要数量的DNase I治疗是非常重要的，因为不这样做会导致很少的核或块核。使用甲醛时，应始终佩戴适当的 PPE。遵循扩散过程，染色体可以染色，以可视化各种染色体结构，以及双链断裂，以分析时间依赖和定位的美的事件。我们的技术为表明斑马鱼可能是人类精子生成更好的模型铺平了道路，因为根据染色体特征和端粒花束，斑马鱼可能是人类精子生成更好的模型。

Research

JoVE Journal

Genetics

Sample Preparation and Analysis of RNASeq-based Gene Expression Data from Zebrafish

斑马鱼 RNASeq-based 基因表达数据的样品制备与分析

The analysis of global gene expression changes is a valuable tool for identifying novel pathways underlying observed phenotypes. The zebrafish is an ...

科研

遗传学

Expedited Radiation Biodosimetry by Automated Dicentric Chromosome Identification (ADCI) and Dose Estimation

Expedited Radiation Biodosimetry by Automated Dicentric Chromosome Identification ADCI and Dose Estimation

自动 Dicentric 染色体识别 (ADCI) 和剂量估计加速辐射剂量

Biological radiation dose can be estimated from dicentric chromosome frequencies in metaphase cells. Performing these cytogenetic dicentric chromosome ...

Pooled shRNA Screen for Reactivation of MeCP2 on the Inactive X Chromosome

在非活动 X 染色体上重新激活 MeCP2 的池 shRNA 屏幕

Forward genetic screens using reporter genes inserted into the heterochromatin have been extensively used to investigate mechanisms of epigenetic control ...

Ultralow Input Genome Sequencing Library Preparation from a Single Tardigrade Specimen

单缓步动物标本的超低输入基因组测序库制备

Tardigrades are microscopic animals that enter an ametabolic state called anhydrobiosis when facing desiccation and can return to their original state ...

High-throughput DNA Extraction and Genotyping of 3dpf Zebrafish Larvae by Fin Clipping

3dpf 斑马鱼幼虫的高通量 DNA 提取及基因分型研究

Zebrafish (Danio rerio) possess orthologues for 84% of the genes known to be associated with human diseases. In addition, these animals have a short ...

High-throughput Identification of Gene Regulatory Sequences Using Next-generation Sequencing of Circular Chromosome Conformation Capture (4C-seq)

High-throughput Identification of Gene Regulatory Sequences Using Next-generation Sequencing of Circular Chromosome Conformation Capture 4C-seq

利用下一代序列染色体构象捕获 (4 c 序列) 的高通量识别基因调控数列

The identification of regulatory elements for a given target gene poses a significant technical challenge owing to the variability in the positioning and ...

A Non-random Mouse Model for Pharmacological Reactivation of Mecp2 on the Inactive X Chromosome

非随机小鼠模型在非活性 X 染色体上对 Mcp2 的药理再激活

X chromosome inactivation (XCI) is the random silencing of one X chromosome in females to achieve gene dosage balance between the sexes. As a result, all ...

Identification of Enhancer-Promoter Contacts in Embryoid Bodies by Quantitative Chromosome Conformation Capture (4C)

Identification of Enhancer-Promoter Contacts in Embryoid Bodies by Quantitative Chromosome Conformation Capture 4C

通过定量染色体形成捕获（4C）鉴定胚胎体增强剂-促进者接触

During mammalian development, cell fates are determined through the establishment of regulatory networks that define the specificity, timing, and spatial ...

Chromosome Screening of Human Preimplantation Embryos by Using Spent Culture Medium: Sample Collection and Chromosomal Ploidy Analysis

使用废培养基对人类植入前胚胎进行染色体筛选：样品采集和染色体倍性分析

In clinical in vitro fertilization (IVF), the prevailing method for PGT-A requires biopsy of a few cells from the trophectoderm (TE). This is the lineage ...

Capturing Chromosome Conformation Across Length Scales

跨长度尺度捕获染色体构象

Chromosome conformation capture (3C) is used to detect three-dimensional chromatin interactions. Typically, chemical crosslinking with formaldehyde (FA) ...