我们要感谢恰克的指导, 约翰·多德帮助制定这一育种战略, 马西埃沃克为她的工作, 完善的骨骼染色, Charline 沃克和邦尼乌尔曼为有益的斑马鱼的建议。这项工作得到了 K99/R00 DE024190 对 JTN 的支持。

本协议中描述的所有实验都是按照科罗拉多大学和俄勒冈大学机构动物保育和使用委员会 (IACUC) 的要求完成的. 
 1. 准备未选定的起始库存 <ol> <li> 通过鳍片剪辑 11 , 通过选择的方法对 full-sibling 动物的基因型基因进行分型, 以确定杂携带者的兴趣KASP 12 , 13 , 14 。此协议是使用斑马鱼 mef2ca b1086 应变执行的. </li>
	<li> KASP 基因分型 " 
	注: KASP 的等位基因特异性底漆是由提供试剂的公司设计的 (请参见 材料表 )。要获得适当的 6-羧基-X-罗丹明 (火箭) 染料浓度为特定的 real-time PCR 机访问公司网站。
	<ol> <li> 将斑马鱼的尾部组织放置在50和 #181; 溶解缓冲液 (10 毫米三 (pH 8.3), 50 毫米氯化钾, 1.5 毫米氯化镁 2 , 0.3% 非离子表面活性剂, 0.3% 苯基-聚乙二醇)。添加10和 #181; 10 毫克/毫升蛋白酶 K 每96井 PCR 板和消化在热循环在55和 #176; c 为 2-5 h, 后跟 20 min 94 和 #176; c 失活步骤。消化后将裂解产物冷藏. #160; 注: 裂解产品可在制备后几个月内成功使用。用50毫米氢氧化钠裂解组织时, KASP 反应不成功。这些发现表明, 氢氧化钠方法的基因组 DNA 制备是不兼容的 KASP. </li>
		<li> 使用分光光度计对基因组 DNA 进行量化。使用分子生物学等级水稀释基因组 DNA 模板, 使每个反应包含约 20-30 ng. 
		注: 量化4或5样本, 将它们汇集, 然后根据平均值为所有样品准备稀释。当使用这个协议为成人尾巴组织时, 1:100 稀释通常是充足的。不需要精确定量的 DNA 浓度. </li>
		<li> 添加0.14 和 #181; l KASP 底漆混合和5和 #181; l KASP 主混合每样品在冰到1.5 毫升离心管和混合好之前简要离心收集的内容在底部的管. 
		注: KASP 主混合是热和轻敏感, 保持在冰上的库存和避免直接光. </li>
		<li> 吸管5和 #181; 将底漆/主混合液的 L 置于96阱光学 pcr 板的井中, 适用于 real-time pcr 机的使用. </li>
		<li> 吸管5和 #181; 稀释 (约 5-7 ng/和 #181; l) DNA 模板样本进入每一个井和吸气吸管混合. </li>
		<li> 添加5和 #181; 核酸3口井为 no-template 控制 (NTC), 并添加5和 #181; 经稀释证实的纯合型野生类型、杂和纯合性突变 DNA 模板用于阳性对照. </li>
		<li> 在板上放置一个光学清晰的胶片密封, 确保胶片完全密封在每个井上. </li>
		<li> 简要地离心板材在 600 x g 收集底部的内容和从混合物中除去气泡. 
		注意: 把盘子放在冰上, 然后从光罩上, 直到准备继续. </li>
		<li> 使用显示在 表1中的循环程序 执行主 KASP 反应. </li>
		<li> 查看由分析计算机生成的结果散点图。一个成功的反应将显示4不同的点分组的情节 ( 图 3 ): 三样本组对应的基因型, 和一个分组的非贸易附近的起源。正控制应与适当的样本分组分隔. </li>
		<li> 如果计算机程序不识别与特定基因型相对应的绘图分组, 则可能需要附加的循环集 ( 表 2 )。重复该附加程序, 直到计算机通过基因型识别紧密分组. </li>
		<li> 将基因分型结果导出到电子表格文件中, 以便更方便地使用和分析. </li>
	</ol> </li> <li> 将已识别的杂动物放在主水系统上. </li> </ol>
 2. 第一轮子代评分 <ol> <li> 配对交叉同级子 <ol> 在动物从鳍片中恢复后, 设置成对的 intercrosses。使用分隔线确保胚胎是同步的 16 . </li>
		<li> 收集胚胎并将双亲对隔离在饲养笼中. </li>
		<li> 监视隔离的成人, 使攻击性鱼类可以被分离或提供覆盖 (鱼网丝很好地工作)。在静态水中, 鱼应该被喂食, 它们的水会随 IACUC 的指引而改变. </li>
	</ol> </li> <li> 胚胎饲养 <ol> <li> 阶段, 并在标准条件下将斑马鱼胚胎提升为幼虫 17 。
		<li> 收集型野生型动物使用玻璃吸管在5或6天, 当游泳膀胱在75% 的离合器充气。在育儿记录中放置表型的野生类型, 父母将其产生. 
		注: 与突变等位基因的隐性致死, 这是真实的许多骨骼突变, 合合突变体无法形成鱼鳔 18 , 19 。因此, 表型野生类型可以很容易地从他们的变种兄弟姐妹根据充气鱼鳔. </li>
	</ol> </li> <li> 新蓝/茜素红软骨和骨染色 <ol> <li> 麻醉幼虫在胚胎介质中使用0.17 克/升 Tricaine, 不充气其鱼鳔。收集动物到1.5 毫升离心管与宽口径玻璃巴斯德吸管。每管增加不超过100只幼虫. </li>
		<li> 从每管中取出胚胎水, 并在每管1毫升的2% 甲醛中修复动物. 
		 警告: 水中的粉煤灰是易燃的, 会引起严重的皮肤和眼部刺激。穿戴适当的个人防护设备, 如手套和眼睛保护, 使用后彻底洗手. </li>
		<li> 岩石为1小时, 较长的固定会损害骨染色。检查管定期在这和所有随后的摇摆步骤, 以确保没有幼虫已成为粘在一侧或成群在底部的管. </li>
		<li> 用玻璃吸管将定影液从试管中取出, 加入1毫升的50% 乙醇, 岩石10分钟. </li>
		<li> 准备新鲜染色液, 加入20和 #181; 每1毫升的新预混料 (0.04% 新 Blue/10 mM 氯化镁 2 /80% 乙醇) 的0.5% 茜素红。去除50% 乙醇, 加入1毫升的染色液, 每管, 和摇滚过夜。幼虫可在污渍中停留5天. </li>
		<li> 从管道中取出着色液, 并在每个管中添加1毫升 80% ethanol/10 mM 氯化镁 2 解决方案。岩石的管至少1小时;隔夜冲洗可以产生更清晰的新蓝色污渍. </li>
		<li> 从管中卸下 80% ethanol/10 mM 氯化镁 2 解决方案, 并添加1毫升的50% 乙醇; 岩石为 5 min. </li>
		<li> 从试管中取出50% 乙醇溶液, 并在5分钟内加入1毫升的25% 乙醇和岩石. </li>
		<li> 从管道中取出50% 乙醇溶液并添加1毫升新鲜准备的 3% H 2 O 2/0.5% KOH 漂白溶液。把瓶盖打开, 让管子在微管架中站立大约10分钟, 或者直到溶液上部可以看到微弱的褐色. 
		注: 溶液顶部形成一层气泡。在这一步需要仔细的定时, 因为过度漂白会导致一个糟糕的双重污点. </li>
		<li> 除去漂白溶液, 并添加1毫升25% 甘油/0.1% KOH; 岩石为 10 min 到 1 h. </li>
		<li> 从试管中取出25% 甘油/0.1% koh 溶液, 在每根管子上加1毫升的50% 甘油/0.1% koh 溶液, 在一夜之间将岩石移动10分钟. </li>
		<li> 从试管中取出50% 甘油/0.1% koh 溶液, 再在每根管子上加入50% 甘油/0.1% koh 溶液。隔夜摇摆将有助于消除气泡, 可以积累在幼虫。在未使用时, 将染色的骨骼准备工作保存在4和 #176; C. </li>
	</ol> </li> <li> 表型评分 <ol> <li> 为所选表型的显染色突变后代。在尼科尔斯 et al. 1 突变体对任何异位骨的发生都有评分. 
		注: 由于斑马鱼的父母是在静态水和茜素红染色可以褪色的时间, 这是重要的是要在几天内完成骨骼准备的骨骼. </li>
		<li> 显是具有表型的基因型的比例。按以下公式计算显百分比: % 显 = (具有表型的突变体)/(变种人总数) 和 #215; 100 </li> </ol> </li> </ol> 3。家庭选择 <ol> <li> 为下一代选择两个高显和两个低显家庭。除了显, 在选择繁殖的家庭时, 重要的是要考虑生育力和活力;有关此问题的详细信息, 请参见 讨论 . </li>
	<li> 给每个双亲配对一个子股票标识符:01,. 02, 等 </li>
	<li> 在主系统上的家庭双亲对有几个混合性和 #39; 同伴和 #39; 鱼。同伴鱼可以是任何斑马鱼线具有永久性的, 明显的区别性特征, 如改变色素沉着或鳍结构. 
	注: 虽然许多研究人员成功地只饲养了对在坦克, 有利结果被看见由几个伴生的鱼的住房对。这一可选步骤允许饲养的动物对在较大的学校饲养, 并容易检索, 以便他们可以反复交叉的需要。父母对可以反复交叉, 产生大的 full-sibling 家庭;请至少等待一周, 然后再重复过同一家长对. </li>
	<li> 从步骤2.2.2 中剔除幼虫系列. 这不是为下一代选择的. </li>
	<li> 从选定的家族中抽取含有野生型同胞幼虫的显, 从他们的变种兄弟姐妹中挑选, 并将其提升为正常的成年. </li>
	<li> 下一代将至少有四 full-sibling 家庭, 两个为上行线, 两个为下行线. </li>
	<li> 当幼虫达到性成熟度时, 从第一步开始与新的世代重复该协议. </li>
</ol>

<ol>
	<li>Nichols, J. T., 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=Ligament+versus+bone+cell+identity+in+the+zebrafish+hyoid+skeleton+is+regulated+by+mef2ca.">Ligament versus bone cell identity in the zebrafish hyoid skeleton is regulated by mef2ca.</a> Development. 143 (23), 4430-4440 (2016).</li><li>Sheehan-Rooney, K., Swartz, M. E., Zhao, F., Liu, D., Eberhart, J. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ahsa1+and+Hsp90+activity+confers+more+severe+craniofacial+phenotypes+in+a+zebrafish+model+of+hypoparathyroidism,+sensorineural+deafness+and+renal+dysplasia+(HDR).">Ahsa1 and Hsp90 activity confers more severe craniofacial phenotypes in a zebrafish model of hypoparathyroidism, sensorineural deafness and renal dysplasia (HDR).</a> Dis Model Mech. 6 (5), 1285-1291 (2013).</li><li>Cox, S. G., 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=An+essential+role+of+variant+histone+H3.3+for+ectomesenchyme+potential+of+the+cranial+neural+crest.">An essential role of variant histone H3.3 for ectomesenchyme potential of the cranial neural crest.</a> PLoS Genet. 8 (9), e1002938 (2012).</li><li>DeLaurier, A., 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=Role+of+mef2ca+in+developmental+buffering+of+the+zebrafish+larval+hyoid+dermal+skeleton.">Role of mef2ca in developmental buffering of the zebrafish larval hyoid dermal skeleton.</a> Dev Biol. 385 (2), 189-199 (2014).</li><li>McGurk, P. D., Ben Lovely, C., Eberhart, J. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Analyzing+Craniofacial+Morphogenesis+in+Zebrafish+Using+4D+Confocal+Microscopy.">Analyzing Craniofacial Morphogenesis in Zebrafish Using 4D Confocal Microscopy.</a> J Vis Exp. (83), e51190 (2014).</li><li>Kemp, H. A., Carmany-Rampey, A., Moens, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Generating+chimeric+zebrafish+embryos+by+transplantation.">Generating chimeric zebrafish embryos by transplantation.</a> J Vis Exp. (29), e1394 (2009).</li><li>Lush, J. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Progeny+test+and+individual+performance+as+indicators+of+an+animal's+breeding+value.">Progeny test and individual performance as indicators of an animal's breeding value.</a> J Dairy Science. 18 (1), 1-19 (1935).</li><li>Lerner, I. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Population Genetics and Animal Improvement. , (1950).</li><li>Shinya, M., 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=Generation+of+Highly+Homogeneous+Strains+of+Zebrafish+Through+Full+Sib-Pair+Mating.">Generation of Highly Homogeneous Strains of Zebrafish Through Full Sib-Pair Mating.</a> G3. 1 (5), 377-386 (2011).</li><li>Neff, M. M., Neff, J. D., Chory, J., Pepper, A. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=dCAPS,+a+simple+technique+for+the+genetic+analysis+of+single+nucleotide+polymorphisms:+experimental+applications+in+Arabidopsis+thaliana+genetics.">dCAPS, a simple technique for the genetic analysis of single nucleotide polymorphisms: experimental applications in Arabidopsis thaliana genetics.</a> Plant J. 14 (3), 387-392 (1998).</li><li>Xing, L. Y., Quist, T. S., Stevenson, T. J., Dahlem, T. J., Bonkowsky, J. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rapid+and+Efficient+Zebrafish+Genotyping+Using+PCR+with+High-resolution+Melt+Analysis.">Rapid and Efficient Zebrafish Genotyping Using PCR with High-resolution Melt Analysis.</a> Jove-Journal of Visualized Experiments. (84), e51138 (2014).</li><li>He, C., Holme, J., Anthony, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=SNP+genotyping:+the+KASP+assay.">SNP genotyping: the KASP assay.</a> Methods Mol Biol. 1145, 75-86 (2014).</li><li>Semagn, K., Babu, R., Hearne, S., Olsen, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Single+nucleotide+polymorphism+genotyping+using+Kompetitive+Allele+Specific+PCR+(KASP):+overview+of+the+technology+and+its+application+in+crop+improvement.">Single nucleotide polymorphism genotyping using Kompetitive Allele Specific PCR (KASP): overview of the technology and its application in crop improvement.</a> Molecular Breeding. 33 (1), 1-14 (2014).</li><li>Yuan, J., Wen, Z., Gu, C., Wang, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Introduction+of+high+throughput+and+cost+effective+SNP+genotyping+platforms+in+soybean.">Introduction of high throughput and cost effective SNP genotyping platforms in soybean.</a> Plant Genet Genomics Biotech. 2 (1), 90-94 (2014).</li><li>Walker, M. B., Kimmel, C. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+two-color+acid-free+cartilage+and+bone+stain+for+zebrafish+larvae.">A two-color acid-free cartilage and bone stain for zebrafish larvae.</a> Biotech Histochem. 82 (1), 23-28 (2007).</li><li>Nasiadka, A., Clark, M. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Zebrafish+breeding+in+the+laboratory+environment.">Zebrafish breeding in the laboratory environment.</a> ILAR J. 53 (2), 161-168 (2012).</li><li>Kimmel, C. B., Ballard, W. W., Kimmel, S. R., Ullmann, B., Schilling, T. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Stages+of+embryonic+development+of+the+zebrafish.">Stages of embryonic development of the zebrafish.</a> Dev dyn. 203 (3), 253-310 (1995).</li><li>Schilling, T. 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=Jaw+and+branchial+arch+mutants+in+zebrafish+I:+branchial+arches.">Jaw and branchial arch mutants in zebrafish I: branchial arches.</a> Development. 123, 329-344 (1996).</li><li>McCune, A. R., Carlson, R. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Twenty+ways+to+lose+your+bladder:+common+natural+mutants+in+zebrafish+and+widespread+convergence+of+swim+bladder+loss+among+teleost+fishes.">Twenty ways to lose your bladder: common natural mutants in zebrafish and widespread convergence of swim bladder loss among teleost fishes.</a> Evol Dev. 6 (4), 246-259 (2004).</li><li>Mrakov&#269;i&#269;, M., Haley, L. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Inbreeding+depression+in+the+Zebra+fish+Brachydanio+rerio+(Hamilton+Buchanan).">Inbreeding depression in the Zebra fish Brachydanio rerio (Hamilton Buchanan).</a> J Fish Biol. 15 (3), 323-327 (1979).</li><li>Charlesworth, D., Willis, J. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+genetics+of+inbreeding+depression.">The genetics of inbreeding depression.</a> Nat Rev Genet. 10 (11), 783-796 (2009).</li><li>McCune, A. R., 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=A+low+genomic+number+of+recessive+lethals+in+natural+populations+of+bluefin+killifish+and+zebrafish.">A low genomic number of recessive lethals in natural populations of bluefin killifish and zebrafish.</a> Science. 296 (5577), 2398-2401 (2002).</li><li>Streisinger, G., Walker, C., Dower, N., Knauber, D., Singer, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Production+of+clones+of+homozygous+diploid+zebra+fish+(Brachydanio+rerio).">Production of clones of homozygous diploid zebra fish (Brachydanio rerio).</a> Nature. 291 (5813), 293-296 (1981).</li><li>Dreosti, E., Lopes, G., Kampff, A. R., Wilson, S. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Development+of+social+behavior+in+young+zebrafish.">Development of social behavior in young zebrafish.</a> Front Neural Circuits. 9, 39 (2015).</li><li>Eames, B. 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=FishFace:+interactive+atlas+of+zebrafish+craniofacial+development+at+cellular+resolution.">FishFace: interactive atlas of zebrafish craniofacial development at cellular resolution.</a> BMC Dev Bio. 13 (1), 23 (2013).</li><li>Nichols, J. T., Pan, L., Moens, C. B., Kimmel, C. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=barx1+represses+joints+and+promotes+cartilage+in+the+craniofacial+skeleton.">barx1 represses joints and promotes cartilage in the craniofacial skeleton.</a> Development. 140 (13), 2765-2775 (2013).</li><li>Sasaki, M. M., Nichols, J. T., Kimmel, C. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=edn1+and+hand2+Interact+in+early+regulation+of+pharyngeal+arch+outgrowth+during+zebrafish+development.">edn1 and hand2 Interact in early regulation of pharyngeal arch outgrowth during zebrafish development.</a> PLoS One. 8 (6), e67522 (2013).</li></ol>

作者没有什么可透露的。

选择性育种揭开基因功能的微妙之处
通过选择性育种, 改变突变型的表现形式或多或少是严重的, 这是获得基因功能新见解的直接途径。与未选定育种的标准方法相比, 本文所提出的协议可以更全面地了解突变表型。具体来说, 通过产生严重的菌株, 突变型的全广度可能会被揭示, 包括一些观测与未选定的股票。因此, 研究中的基因的新的发展作用可以被发现。相反, 产生轻度菌株可以揭示最关键的作用基因的兴趣。例如, 在轻度突变体中, 只有单一的表型存在。在这里很可能, 在轻度菌株中仍然被破坏的发育过程, 最重要的是需要基因的功能。因此, 通过选择性育种, 可以更充分地理解给定突变的全表型序列。
避免近亲繁殖抑郁症
相关个体的后代在许多动物和植物系统中显示了减少的生存和繁殖力, 包括斑马鱼20。众所周知的近亲繁殖抑郁症, 大多数模型假设, 增加纯在基因座, 无论是隐性有害等位基因或等位基因, 是有益的, 作为子的基础这一现象21。研究与野生捕捉斑马鱼揭示了低频率的有害等位基因在自然种群22。此外, 像 AB 这样的实验室菌株进一步清除了隐性致死突变23。因此, 仔细的饲养似乎可以允许无限期的近亲繁殖选择斑马鱼线。事实上, 最近的一份报告显示, 斑马鱼可以保持 full-sibling 近亲繁殖至少16代9。
这项议定书强调了一些简单但关键的步骤, 以确保斑马鱼自交系继续生产足够的后代, 以有助于发展研究。这一选择育种战略最关键的方面是甄选过程本身。首先, 必须对发展中的动物进行仔细的监测, 以便有发育缺陷或畸形的家庭, 与兴趣的突变没有任何链接, 可以严格挑选。其次, 应考虑离合器的大小, 因为选择性育种需要大家庭。除了选择产生大离合器的对外, 还可以通过同一双亲对的重复交叉生成大家族。保持父母对在坦克与许多其他伴侣鱼, 很容易辨别从育种对, 如色素沉着或鳍突变体, 允许长期住房的育种对大型同种群体, 同时允许他们仍然容易标识和检索。这种做法促进了一个健康的社会环境, 其中父母对浅滩与其他斑马鱼24。然而, 研究员可以很容易地检索父母对, 以便他们可以反复交叉, 使非常大的 full-sibling 家庭的世代。
选择一个可选的表型
这个协议涉及大量的表型评分。因此, 一个限制是, 选择需要适用于一个表型, 是容易可视化和快速得分。同样重要的是决定在什么阶段动物将被固定和染色的评分。对于骨表型, 这是有利的等待, 直到 6 dpf, 因为骨骼是更发达的25 , 将更容易得分。对于软骨模式突变表型, 4 dpf 通常适合表现评分26,27。在决定何时修复动物时, 重要的是要考虑是否有兴趣的基因在其他组织中导致发育突变的表型效的作用, 这可能会导致二次缺陷的混淆。例如, 与骨骼突变体也显示心脏水肿重要的是要修复 4 dpf 之前的次要缺陷, 如一般延迟在软骨清单26。
为了简单起见, 我们选择了一个二进制评分系统的重点是显为我们的选择育种。然而, 突变的表型也可以有可变的表现。在未来的研究, 这将是有趣的测试, 如果像显, 表现是敏感的选择性育种。即, 特定异位骨的频率是否可以通过选择性育种来改变？
该协议描述了经典的选择育种策略的后裔选育和应用于斑马鱼发展遗传学研究。这里所描述的直接饲养方法在任何实验室都是可能的, 即使在小型设施中也是如此。通过选择性育种, 斑马鱼系统的最大优势之一, 可驯服的遗传学, 可以用来了解基因功能在新发展的突变体以及变种人已经传播了几十年。

斑马鱼是一个强大的模型系统, 了解骨骼的发展。与变种斑马鱼菌株, 生物学家可以破译基因功能在骨化。然而, 斑马鱼骨骼突变表型可以出现可变显1,2,3,4 , 这可能会阻碍发展和遗传分析。这种方法的目的是三重的。首先, 生成斑马鱼突变线, 持续产生严重的表型, 使下游的发展研究, 如延时记录5和移植6。这类研究可能会因为试图研究表型而被削弱, 而这只是表现出不一致。其次, 近亲繁殖的斑马鱼菌株可以减少遗传背景的变化, 从而促进实验的一致性和重现性。例如, 对一个选择性的近亲繁殖菌株进行所有的原位杂交分析, 可以减少混杂的变异性并加强结论。第三, 产生严重的和轻度的菌株将揭示出整个表型系列, 可能产生的一个特定的突变。
乍一看, 选择性繁殖致命突变体似乎是不可能的。当被选入的动物死了的时候, 你怎么能为显繁殖呢？幸运的是, 选择育种的方法, 特别是子代试验, 在家畜育种项目中已经证明了许多年的有效性7,8。这些程序主要用于选择性育种的性状, 只存在于一个性别, 如牛奶生产的奶牛或鸡蛋生产母鸡。这些物种的雄性不能直接得分, 但他们的后代得分, 然后将价值分配给父母。从这一策略中, 这里所提出的协议包括对斑马鱼的固定和染色的突变后代进行评分, 这两个杂是为了一个感兴趣的突变基因。在决定哪些个体会产生下一代的时候, 显的一个表型会被分配给父母。我们发现这种方法是一种有效的方式转移显在斑马鱼致死性骨骼突变体1。
与其他研究类似, 这一选择育种协议考虑的标准, 如离合器大小, 后代的生存, 胚胎的正常发育和性别比例9。然而, 这些因素都是在一个突变背景下考虑的, 目的是转移突变显。因此, 该协议扩展了以往的选择育种范式, 提供了一种加强发育突变体分析和增加背景均匀性的方法。
这项协议需要广泛的基因分型, 所以必须事先制定一个可靠的快速基因分型协议。有许多基因分型协议可用10,11, 但是我们发现 KASP 基因分型12,13,14的速度更快, 成本效率更高, 比方法更可靠基于限制性酶裂解的扩增序列10。因此, 我们在这项工作中包括了一个 KASP 协议。此外, 我们的重点是在这个协议的骨骼突变表型, 并包括一个过程的新蓝/茜素红染色修改以前的协议15。
这里描述的方法是一个简单的策略, 转移致死突变显向上或向下。虽然这个协议的重点是骨骼突变的表型, 我们相信这将是一个有用的战略, 畜牧业的所有变种斑马鱼线。事实上, 这种育种策略的效用可能超越斑马鱼。我们预测, 这一协议可以修改, 以转移显在一个广泛的有机体范围。通过子代测试转移致死显可以帮助推动任何发展遗传学家的进步。

<table border="0" cellpadding="0" cellspacing="0" width="627">
	<tbody>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Paraformaldehyde, pelleted, solid</td>
			<td style="width:125px;">Ted Pella Co.</td>
			<td style="width:119px;">18501</td>
			<td style="width:167px;">Pelleted PFA is a safer alternative to powdered PFA</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Magnesium Chloride, solid</td>
			<td style="width:125px;">Acros Organics</td>
			<td>223210010</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">10x PBS, Aqueous</td>
			<td style="width:125px;">Fisher</td>
			<td style="width:119px;">BP3994</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">190 proof Ethanol</td>
			<td style="width:125px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Alcian Blue, solid</td>
			<td style="width:125px;">Anatech Ltd.</td>
			<td style="width:119px;">867</td>
			<td>Must be from Anatech</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Alizarin Red, solid</td>
			<td style="width:125px;">Sigma</td>
			<td style="width:119px;">A5533-25G</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Glycerol, liquid</td>
			<td style="width:125px;">Fisher</td>
			<td style="width:119px;">BP229 1</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Hydrogen peroxide, liquid</td>
			<td style="width:125px;">Fisher</td>
			<td style="width:119px;">BP263500</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Potassium hydroxide,&#160; solid</td>
			<td style="width:125px;">Fisher</td>
			<td style="width:119px;">P250 500</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">StepOnePlus Real-time PCR Machine</td>
			<td style="width:125px;">Applied Biosystems</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:216px;">MicroAmp Fast Optical 96-well Reaction Plate with Barcode (0.1mL)</td>
			<td style="width:125px;">Applied Biosystems</td>
			<td style="width:119px;">4346906</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Microseal &#39;B&#39; seal</td>
			<td style="width:125px;">BioRad</td>
			<td style="width:119px;">MSB1001</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">KASP Master Mix, High ROX</td>
			<td style="width:125px;">LGC</td>
			<td>KBS-1016-022</td>
			<td>https://www.lgcgroup.com/products/kasp-genotyping-chemistry/#.WOPX41UrKUk</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">KASP By Design Primer Mix</td>
			<td style="width:125px;">LGC</td>
			<td style="width:119px;">KBS-2100-100</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Tris HCl, solid</td>
			<td style="width:125px;">Fisher</td>
			<td style="width:119px;">BP153 500</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">potassium chloride, solid</td>
			<td style="width:125px;">Fisher</td>
			<td style="width:119px;">BP366 500</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Tween-20, liquid</td>
			<td style="width:125px;">Fisher</td>
			<td style="width:119px;">BP337 100</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Nonidet P40</td>
			<td style="width:125px;">ThermoFisher</td>
			<td>28324</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Tricaine-S</td>
			<td style="width:125px;">Western Chemicals</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Proteinase K</td>
			<td style="width:125px;">Fisher</td>
			<td style="width:119px;">BP1700 100</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">T100 Thermal Cycler</td>
			<td style="width:125px;">BioRad</td>
			<td>1861096</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Controlled Drop Pasteur Pipets</td>
			<td style="width:125px;">Fisher</td>
			<td>13-678-30</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Nanodrop</td>
			<td style="width:125px;">ThermoFisher</td>
			<td style="width:119px;"></td>
			<td>for DNA quantitation</td>
		</tr>
	</tbody>
</table>

shifting zebrafish lethal skeletal mutant penetrance by progeny testing

斑马鱼变种的表型往往是不完全渗透, 只是在一些变种的显着。有趣的表型, 不一致的出现可能是很难研究, 并可能导致混淆的结果。这里描述的协议是一个简单的繁殖范例, 以增加和减少显在致命的斑马鱼骨骼突变体。由于致命突变体不能直接繁殖, 因此采用了典型的子代检测育种策略。该方法还包括 Kompetitive 等位基因特异性 PCR (KASP) 基因分型斑马鱼和染色幼虫斑马鱼软骨和骨的协议。应用这里描述的畜牧业战略可以增加一个有趣的骨骼表型的显, 使下游应用更具可重现性的结果。此外, 通过这一选择育种策略减少突变显, 可以揭示最关键的发展过程, 需要突变基因的功能。虽然骨骼是在这里被特别考虑, 我们建议, 这一方法将是有用的所有斑马鱼突变线。

该协议是一种长期饲养技术, 有助于了解斑马鱼骨骼突变体 (图 1)。后代测试的选择育种应该在几代人的显中产生一个整体的向下和向上的转变 (图 2)。在我们以前的工作中, 两轮的选择性育种使平均显向下, 从17% 到 3%1。同样, 在我们的上行线, 我们把平均显从63% 代上升到93% 在三世代。如果在四轮的选择育种后, 显不反应向下或向上, 这是可能的表型, 正在评分是不敏感的选择育种的后代测试。
在成功的 KASP 过程中, 应通过计算机程序识别与基因型相对应的紧密样本组, 并且在执行了足够的循环之后, 非贸易应位于绘图的原点附近 (图 3)。如果 KASP 散射图的数据没有被紧密分组或一个 NTC 的污染检测, 该程序将无法确定基因型。如果大多数样本是由基因型分组识别的, 但仍有一些是不明确的, 从数据集中省略不确定的样本可能允许程序识别基因分簇。不确定的样品调用和不明确的基因型分组可能起因于以下: 被污染的反应混合物在其中非贸易不会编组在起源附近, over-diluted 模板脱氧核糖核酸导致样品定位在起源附近, under-diluted 模板脱氧核糖核酸, 或反应周期的数量不足。如果怀疑有污染, 准备新鲜非贸易。
成功的新蓝/茜素红染色将有活力染色的骨和软骨元素 (图 4A), 不透明或微弱 (图 4B)。软骨元素的边界应该是脆的, 个别的软骨细胞会被辨认出来。茜素红骨染色通常更挑剔的两个污点。扇形盖 (op) 骨和棒状鳃射线 (br) 是一个成功的6天后受精 (dpf) 骨染色 (图 4A, op, br) 的良好指标。漂白步骤不应褪色的污渍, 在骨和软骨, 而充分清除其他组织的颜色。即使在成功的染色和清除后, 眼睛也会有褐色的色调。过于集中的新蓝将无法从其他组织中清除, 无法进行分析 (图 4C)。新蓝色的供应商除了在材料的表中所描述的, 也可以产生不良的软骨污渍。
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/56200/56200fig1.jpg"> 
图 1.通过子代试验对骨骼显的选择性育种进行了概要介绍.(a) 基因型一个 full-sibling 家族从突变线识别杂载体。(B)对交叉确定了杂的兄弟姐妹, 并将父母隔离, 直到能够获得后代。(C)将型的野生型幼虫与充气的鱼鳔放在育儿室中, 并将其固定在没有充气鱼鳔的情况下进行软骨和骨染色。(D)分新蓝/茜素红染色突变动物的显。选择哪些家庭将向上和向下孕育, 并提高从每个家庭到成年的表型野生类型。(E)与同伴的家长对, 以便他们可以反复跨越, 以产生大 full-sibling 家庭。<a href="//ecsource-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/56200/56200fig1large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/56200/56200fig2.jpg"> 
图 2.通过子代检测改变骨骼突变显的例子.本手稿中所描述的选择性育种适用于斑马鱼mef2cab1086 变种。在本实验中, 我们选择了高或低显异位骨之间的盖和鳃射线的致命合纯突变体。显异位骨的变异后代被分配到父母对谁是 color-coded 由显如图所示。此图已从1进行了修改。<a href="//ecsource-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/56200/56200fig2large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/56200/56200fig3.jpg"> 
图 3.屏幕截图从软件用于 KASP 基因分型和结果分析。 在这成功的 KASP 反应, 严密的样品分组被形成了。蓝点是纯合的突变体, 绿点是杂的, 红点是纯合型的, 黑方块是非贸易的. 分组由计算机程序指定。非贸易位于附近的地块的起源表示很少, 没有反应混合物污染。未确定的样本, 尚未分配的基因型将表明与 x。在此成功完成的运行中, 没有待定的示例。<a href="//ecsource-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/56200/56200fig3large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/56200/56200fig4.jpg"> 
图 4.好, 坏和丑陋的新蓝/茜素红色骨骼准备。 
(a)显示了一个新鲜、染色和清除的软骨和骨斑的例子。注意明显的软骨元素和容易形象化的红色盖 (op) 和鳃射线 (br) 骨骼。(B)显示带有褪色的茜素红染色的弱软骨染色。这个样品在4° c 坐了许多月导致退色的软骨并且盖和鳃射线骨头是难区分的。(C)使用过多新蓝色的 over-stained 幼虫。刻度条是200μ m。<a href="//ecsource-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/56200/56200fig4large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>

Watch this Scientific Journal Video about Shifting Zebrafish Lethal Skeletal Mutant Penetrance by Progeny Testing at JoVE.com

Shifting Zebrafish Lethal Skeletal Mutant Penetrance by Progeny Testing

本议定书的目的是改变显致命的骨骼突变体表型在斑马鱼的选择性育种。致命的突变体不能成长为成年和繁殖, 因此本协议描述了通过后代测试来跟踪和选择显的方法。

通过子代检测改变斑马鱼致死性骨骼突变显

Zebrafish mutant phenotypes are often incompletely penetrant, only manifesting in some mutants. Interesting phenotypes that inconsistently appear can be ...

shifting-zebrafish-lethal-skeletal-mutant-penetrance-progeny

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7.5K 次观看.  University of Colorado Anschutz Medical Campus. 本议定书的目的是改变显致命的骨骼突变体表型在斑马鱼的选择性育种。致命的突变体不能成长为成年和繁殖, 因此本协议描述了通过后代测试来跟踪和选择显的方法。

视频: Shifting Zebrafish Lethal Skeletal Mutant Penetrance by Progeny Testing

Kidney Regeneration in Adult Zebrafish by Gentamicin Induced Injury

The kidney is essential for fluid homeostasis, blood pressure regulation and filtration of waste from the body. The fundamental unit of kidney function is the nephron. Mammals are able to repair existing nephrons after injury, but lose the ability to form new nephrons soon after birth. In contrast to mammals, adult fish produce new nephrons (neonephrogenesis) throughout their lives in response to growth requirements or injury. Recently, lhx1a has been shown to mark nephron progenitor cells in the adult zebrafish kidney, however mechanisms controlling the formation of new nephrons after injury remain unknown. Here we show our method for robust and reproducible injury in the adult zebrafish kidney by intraperitoneal (i.p.) injection of gentamicin, which uses a noninvasive visual screening process to select for fish with strong but nonlethal injury. Using this method, we can determine optimal gentamicin dosages for injury and go on to demonstrate the effect of higher temperatures on kidney regeneration in zebrafish.

Here we present a reliable method to study adult kidney regeneration by inducing acute kidney injury by gentamicin injection. We show that injury is dependent on gentamicin dosage and environmental temperature using in situ hybridization to label lhx1a+ developing new nephrons.

肾再生的成年斑马鱼庆大霉素性损伤

The kidney is essential for fluid homeostasis, blood pressure regulation and filtration of waste from the body. The fundamental unit of kidney function is ...

科研

发育生物学

Mechanical Vessel Injury in Zebrafish Embryos

Zebrafish (Danio rerio) embryos have proven to be a powerful model for studying a variety of developmental and disease processes. External development and optical transparency make these embryos especially amenable to microscopy, and numerous transgenic lines that label specific cell types with fluorescent proteins are available, making the zebrafish embryo an ideal system for visualizing the interaction of vascular, hematopoietic, and other cell types during injury and repair in vivo. Forward and reverse genetics in zebrafish are well developed, and pharmacological manipulation is possible. We describe a mechanical vascular injury model using micromanipulation techniques that exploits several of these features to study responses to vascular injury including hemostasis and blood vessel repair. Using a combination of video and timelapse microscopy, we demonstrate that this method of vascular injury results in measurable and reproducible responses during hemostasis and wound repair. This method provides a system for studying vascular injury and repair in detail in a whole animal model.

This article describes a method for creating a mechanical vessel injury in zebrafish embryos. This injury model provides a platform for studying hemostasis, injury-related inflammation, and wound healing in an organism ideally suited for real-time microscopy.

在斑马鱼胚胎的机械血管损伤

Zebrafish (Danio rerio) embryos have proven to be a powerful model for studying a variety of developmental and disease processes.  External development ...

Analysis of Zebrafish Larvae Skeletal Muscle Integrity with Evans Blue Dye

The zebrafish model is an emerging system for the study of neuromuscular disorders. In the study of neuromuscular diseases, the integrity of the muscle membrane is a critical disease determinant. To date, numerous neuromuscular conditions display degenerating muscle fibers with abnormal membrane integrity; this is most commonly observed in muscular dystrophies. Evans Blue Dye (EBD) is a vital, cell permeable dye that is rapidly taken into degenerating, damaged, or apoptotic cells; in contrast, it is not taken up by cells with an intact membrane. EBD injection is commonly employed to ascertain muscle integrity in mouse models of neuromuscular diseases. However, such EBD experiments require muscle dissection and/or sectioning prior to analysis. In contrast, EBD uptake in zebrafish is visualized in live, intact preparations. Here, we demonstrate a simple and straightforward methodology for performing EBD injections and analysis in live zebrafish. In addition, we demonstrate a co-injection strategy to increase efficacy of EBD analysis. Overall, this video article provides an outline to perform EBD injection and characterization in zebrafish models of neuromuscular disease.

In this study, we describe a straightforward method to perform Evans Blue Dye (EBD) analysis on zebrafish larvae. This technique is a powerful tool for the characterization of skeletal muscle integrity and delineation of zebrafish models of muscular dystrophy, and is a valuable method for the development of novel therapeutics.

斑马鱼幼虫骨骼肌诚信与伊文思蓝分析

The zebrafish model is an emerging system for the study of neuromuscular disorders.  In the study of neuromuscular diseases, the integrity of the muscle ...

Generation of Parabiotic Zebrafish Embryos by Surgical Fusion of Developing Blastulae

Surgical parabiosis of two animals of different genetic backgrounds creates a unique scenario to study cell-intrinsic versus cell-extrinsic roles for candidate genes of interest, migratory behaviors of cells, and secreted signals in distinct genetic settings. Because parabiotic animals share a common circulation, any blood or blood-borne factor from one animal will be exchanged with its partner and vice versa. Thus, cells and molecular factors derived from one genetic background can be studied in the context of a second genetic background. Parabiosis of adult mice has been &#160;used extensively to research aging, cancer, diabetes, obesity, and brain development. More recently, parabiosis of zebrafish embryos has been used to study the developmental biology of hematopoiesis. In contrast to mice, the transparent nature of zebrafish embryos permits the direct visualization of cells in the parabiotic context, making it a uniquely powerful method for investigating fundamental cellular and molecular mechanisms. The utility of this technique, however, is limited by a steep learning curve for generating the parabiotic zebrafish embryos. This protocol provides a step-by-step method on how to surgically fuse the blastulae of two zebrafish embryos of different genetic backgrounds to investigate the role of candidate genes of interest. In addition, the parabiotic zebrafish embryos are tolerant to heat shock, making temporal control of gene expression possible. This method does not require a sophisticated set-up and has broad applications for studying cell migration, fate specification, and differentiation in vivo during embryonic development.

This protocol provides step-by-step instruction on how to generate parabiotic zebrafish embryos of different genetic backgrounds. When combined with the unparalleled imaging capabilities of the zebrafish embryo, this method provides a uniquely powerful means to investigate cell-autonomous versus non-cell-autonomous functions for candidate genes of interest.

联体斑马鱼胚胎一代发展囊胚的手术融合

Surgical parabiosis of two animals of different genetic backgrounds creates a unique scenario to study cell-intrinsic versus cell-extrinsic roles for ...

Induction of Acute Skeletal Muscle Regeneration by Cardiotoxin Injection

Skeletal muscle regeneration is a physiological process that occurs in adult skeletal muscles in response to injury or disease. Acute injury-induced skeletal muscle regeneration is a widely used, powerful model system to study the events involved in muscle regeneration as well as the mechanisms and different players. Indeed, a detailed knowledge of this process is essential for a better understanding of the pathological conditions that lead to skeletal muscle degeneration, and it aids in identifying new targeted therapeutic strategies. The present work describes a detailed and reproducible protocol to induce acute skeletal muscle regeneration in mice through a single intramuscular injection of cardiotoxin (CTX). CTX belongs to the family of snake venom toxins and causes myolysis of myofibers, which eventually triggers the regeneration events. The dynamics of skeletal muscle regeneration is evaluated by histological analysis of muscle sections. The protocol also illustrates the experimental procedures for dissecting, freezing, and cutting the Tibialis Anterior muscle, as well as the routine Hematoxylin &#38; Eosin staining that is widely used for subsequent morphological and morphometric analysis.

This manuscript describes a detailed protocol to induce acute skeletal muscle regeneration in adult mice and subsequent manipulations of the muscles, such as dissection, freezing, cutting, routine staining, and myofiber cross-sectional area analysis.

通过注射心脏毒素急性骨骼肌再生的诱导

Skeletal muscle regeneration is a physiological process that occurs in adult skeletal muscles in response to injury or disease. Acute injury-induced ...

Identification Of Erythromyeloid Progenitors And Their Progeny In The Mouse Embryo By Flow Cytometry

Macrophages are professional phagocytes from the innate arm of the immune system. In steady-state, sessile macrophages are found in adult tissues where they act as front line sentinels of infection and tissue damage. While other immune cells are continuously renewed from hematopoietic stem and progenitor cells (HSPC) located in the bone marrow, a lineage of macrophages, known as resident macrophages, have been shown to be self-maintained in tissues without input from bone marrow HSPCs. This lineage is exemplified by microglia in the brain, Kupffer cells in the liver and Langerhans cells in the epidermis among others. The intestinal and colon lamina propria are the only adult tissues devoid of HSPC-independent resident macrophages. Recent investigations have identified that resident macrophages originate from the extra-embryonic yolk sac hematopoiesis from progenitor(s) distinct from fetal hematopoietic stem cells (HSC). Among yolk sac definitive hematopoiesis, erythromyeloid progenitors (EMP) give rise both to erythroid and myeloid cells, in particular resident macrophages. EMP are only generated within the yolk sac between E8.5 and E10.5 days of development and they migrate to the fetal liver as early as circulation is connected, where they expand and differentiate until at least E16.5. Their progeny includes erythrocytes, macrophages, neutrophils and mast cells but only EMP-derived macrophages persist until adulthood in tissues. The transient nature of EMP emergence and the temporal overlap with HSC generation renders the analysis of these progenitors difficult. We have established a tamoxifen-inducible fate mapping protocol based on expression of the macrophage cytokine receptor Csf1r promoter to characterize EMP and EMP-derived cells in vivo by flow cytometry.

While infiltrating macrophages are continuously recruited to adult tissues from circulating precursors, resident macrophages seed their tissue during development, where they are maintained without further input from progenitors. The progenitors for resident macrophages were recently identified. Here, we present methods for the genetic fate mapping of the resident macrophage progenitors.

通过流式细胞术鉴定小鼠胚胎中的红细胞生成素及其后代

Macrophages are professional phagocytes from the innate arm of the immune system. In steady-state, sessile macrophages are found in adult tissues where ...

Culture of Adult Transgenic Zebrafish Retinal Explants for Live-cell Imaging by Multiphoton Microscopy

An endogenous regeneration program is initiated by M&#252;ller glia in the adult zebrafish (Danio rerio) retina following neuronal damage and death. The M&#252;ller glia re-enter the cell cycle and produce neuronal progenitor cells that undergo subsequent rounds of cell divisions and differentiate into the lost neuronal cell types. Both M&#252;ller glia and neuronal progenitor cell nuclei replicate their DNA and undergo mitosis in distinct locations of the retina, i.e. they migrate between the basal Inner Nuclear Layer (INL) and the Outer Nuclear Layer (ONL), respectively, in a process described as Interkinetic Nuclear Migration (INM). INM has predominantly been studied in the developing retina. To examine the dynamics of INM in the adult regenerating zebrafish retina in detail, live-cell imaging of fluorescently-labeled M&#252;ller glia/neuronal progenitor cells is required. Here, we provide the conditions to isolate and culture dorsal retinas from Tg[gfap:nGFP]mi2004 zebrafish that were exposed to constant intense light for 35 h. We also show that these retinal cultures are viable to perform live-cell imaging experiments, continuously acquiring z-stack images throughout the thickness of the retinal explant for up to 8 h using multiphoton microscopy to monitor the migratory behavior of gfap:nGFP-positive cells. In addition, we describe the details to perform post-imaging analysis to determine the velocity of apical and basal INM. To summarize, we established conditions to study the dynamics of INM in an adult model of neuronal regeneration. This will advance our understanding of this crucial cellular process and allow us to determine the mechanisms that control INM.

Zebrafish retinal regeneration has mostly been studied using fixed retinas. However, dynamic processes such as interkinetic nuclear migration occur during the regenerative response and require live-cell imaging to investigate the underlying mechanisms. Here, we describe culture and imaging conditions to monitor Interkinetic Nuclear Migration (INM) in real-time using multiphoton microscopy.

成年转基因斑马鱼视网膜外植体培养由多光子显微镜活细胞成像

An endogenous regeneration program is initiated by M&#252;ller glia in the adult zebrafish (Danio rerio) retina following neuronal damage and death. The ...

Isolation of Type I and Type II Pericytes from Mouse Skeletal Muscles

Pericytes are perivascular multipotent cells that show heterogeneity in different organs or even within the same tissue. In skeletal muscles, there are at least two pericyte subpopulations (called type I and type II), which express different molecular markers and have distinct differentiation capabilities. Using NG2-DsRed and Nestin-GFP double-transgenic mice, type I (NG2-DsRed+Nestin-GFP-) and type II (NG2-DsRed+Nestin-GFP+) pericytes have been successfully isolated. However, the availability of these double-transgenic mice prevents the widespread use of this purification method. This work describes an alternative protocol that allows for the easy and simultaneous isolation of type I and type II pericytes from skeletal muscles. This protocol utilizes the fluorescence-activated cell sorting (FACS) technique and targets PDGFR&#946;, rather than NG2, together with the Nestin-GFP signal. Following isolation, type I and type II pericytes show distinct morphologies. In addition, type I and type II pericytes isolated with this new method, like those isolated from the double-transgenic mice, are adipogenic and myogenic, respectively. These results suggest that this protocol can be used to isolate pericyte subpopulations from skeletal muscles and possibly from other tissues.

This work describes a FACS-based protocol that allows for easy and simultaneous isolation of type I and type II pericytes from skeletal muscles.

从小鼠骨骼肌分离I型和II型周细胞

Pericytes are perivascular multipotent cells that show heterogeneity in different organs or even within the same tissue. In skeletal muscles, there are at ...

Identification of Skeletal Muscle Satellite Cells by Immunofluorescence with Pax7 and Laminin Antibodies

Immunofluorescence is an effective method that helps to identify different cell types on tissue sections. In order to study the desired cell population, antibodies for specific cell markers are applied on tissue sections. In adult skeletal muscle, satellite cells (SCs) are stem cells that contribute to muscle repair and regeneration. Therefore, it is important to visualize and trace the satellite cell population under different physiological conditions. In resting skeletal muscle, SCs reside between the basal lamina and myofiber plasma membrane. A commonly used marker for identifying SCs on the myofibers or in cell culture is the paired box protein Pax7. In this article, an optimized Pax7 immunofluorescence protocol on skeletal muscle sections is presented that minimizes non-specific staining and background. Another antibody that recognizes a protein (laminin) of the basal lamina was also added to help identify SCs. Similar protocols can also be used to perform double or triple labeling with Pax7 and antibodies for additional proteins of interest.

The precise identification of satellite cells is essential for studying their functions under various physiological and pathological conditions. This article presents a protocol to identify satellite cells on adult skeletal muscle sections by immunofluorescence-based staining.

Pax7 和层粘连蛋白抗体免疫荧光法鉴定骨骼肌卫星细胞

Immunofluorescence is an effective method that helps to identify different cell types on tissue sections. In order to study the desired cell population, ...

Performing Human Skeletal Muscle Xenografts in Immunodeficient Mice

Treatment effects observed in animal studies often fail to be recapitulated in clinical trials. While this problem is multifaceted, one reason for this failure is the use of inadequate laboratory models. It is challenging to model complex human diseases in traditional laboratory organisms, but this issue can be circumvented through the study of human xenografts. The surgical method we describe here allows for the creation of human skeletal muscle xenografts, which can be used to model muscle disease and to carry out preclinical therapeutic testing. Under an Institutional Review Board (IRB)-approved protocol, skeletal muscle specimens are acquired from patients and then transplanted into NOD-Rag1null IL2r&gamma;null (NRG) host mice. These mice are ideal hosts for transplantation studies due to their inability to make mature lymphocytes and are thus unable to develop cell-mediated and humoral adaptive immune responses. Host mice are anaesthetized with isoflurane, and the mouse tibialis anterior and extensor digitorum longus muscles are removed. A piece of human muscle is then placed in the empty tibial compartment and sutured to the proximal and distal tendons of the peroneus longus muscle. The xenografted muscle is spontaneously vascularized and innervated by the mouse host, resulting in robustly regenerated human muscle that can serve as a model for preclinical studies.

Complex human diseases can be challenging to model in traditional laboratory model systems. Here, we describe a surgical approach to model human muscle disease through the transplantation of human skeletal muscle biopsies into immunodeficient mice.

在免疫缺陷小鼠中表演人类骨骼肌异种移植物

Treatment effects observed in animal studies often fail to be recapitulated in clinical trials. While this problem is multifaceted, one reason for this ...