Name: shifting zebrafish lethal skeletal mutant penetrance by progeny testing
Uploaded: 2017-09-01T15:00:00
Description: Watch this Scientific Journal Video about Shifting Zebrafish Lethal Skeletal Mutant Penetrance by Progeny Testing at JoVE.com

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?...

<ol>
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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,...

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,...

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 ...

The overall goal of this breeding strategy is to manipulate mutant penetrance upward or downward. This selective breeding method enables a more complete understanding of mutant phenotypes. By generating high and low penetrance mutant strains, the entire phenotypic series of a mutation can be understood.Most zebrafish scale where mutants are recessive lethal, so homozygous mutants die before they can be directly bred. Therefore, we employ the classical selective breeding strategy of progeny testing, which we demonstrate here. We first had the item for this method when attempting to time lapse record ectopic bone cell development.Frustratingly, extra bone cells appeared in it frequently, so we set out to alter mutant penetrants by selective breeding. Although we perform this method on a zebrafish scale with a mutant line, it can also be applied to other zebrafish mutant phenotypes and likely, other organisms. After preparing unselected starting zebrafish stock according to the text protocol, carry out KASP genotyping as follows.Place the zebrafish tail tissue in 50 microliters of lysis buffer in the wells of a 96 well plate. Add 10 microliters of 10 milligrams per milliliter proteinase k per well and digest the tissue samples in a thermocycler at 55 degree celsius for two to five hours. Followed by a 20 minute 94 degree celsius inactivation step.Refrigerate the lysis product after digestion. Use a spectrophotometer to quantify the genomic DNA. Then, using molecular biology grade water, dilute the genomic template so that each reaction contains approximately 20 to 30 nanograms.Add 0.14 microliters of the KASP primer mix and five microliters of the KASP master mix per sample on ice, into a 1.5 milliliter microcentrifuge tube and mix well. Then, briefly centrifuge to collect the contents at the bottom of the tube. Pipette five microliters of primer master mix solution into the wells of a 96 well optical PCR plate suitable for the real time PCR machine being used.Then, pipette five microliters of diluted DNA template samples into each well and aspirate with a pipette to mix. Place an optically clear film seal over the plate, ensuring that the film is fully sealed on each well. Briefly centrifuge the plate at 600 x G to collect the contents at the bottom and to remove bubbles from the mix.After performing the main KASP reaction, view the resulting scatter plot produced by the analysis computer. House the identified heterozygous animals together on the main water system. After the animal recover from the fin clip, set up pairwise intercrosses, use dividers to ensure embryos are synchronized.Collect the embryos, and keep the parental pairs isolated in breeding cages. Monitor isolated adults, so that aggressive fish can be separated or provided with cover. To rear the embryos on day five or six, when the swim bladders is inflated in 75%of a clutch, use a glass pipette to collect phenotypically wild type animals.Place phenotypic wild types in the nursery, recording which parents yielded them. After anesthetizing larvae that do not inflate their swim bladders according to the text protocol, use a wide bore glass pasteur pipette to collect the animals into 1.5 milliliters microcentrifuge tubes. Remove the embryo water from each tube and replace it with one milliliter of 2%paraformaldehyde in 1x PBS.Rock the tubes for one hour to fix the animals. Track the tubes regularly during this and all subsequent rocking steps to ensure no larvae have become stuck to the side or clumped in the bottom of the tube. Use a glass pipette to remove the fixative from the tubes.Then add one milliliter of 50%ethanol. Rock the samples for an additional 10 minutes. Prepare fresh staining solution by adding 20 microliters of 0.5%alizarin red per one milliliter of Alcian premix.Remove the 50%ethanol, and add one milliliter of staining solution to each tube. Then, rock the tubes overnight. Within a few days after bleaching the embryos according to the text protocol, score stained mutant offspring for penetrants.In this example, we score for any occurrence of a topic bone. For the next generation, choose two high penetrants and two low penetrants families. Give each parental pair a sub stock identifier, for example, family 4.01, 02, et cetera.It's also helpful to indicate the or inbred generation number on all labels. House parental pairs on the main system with several mixed sex companion fish. Companion fish can be any zebrafish line with permanent obvious distinguishing features, such as altered pigmentation or fin structure.Label the tanks containing the wild type sibling larvae from selected families with the penetrants from the mutant siblings and raise the fish to adulthood as normal. Selective breeding by progeny testing, should yield a shift in overall penetrants both downward and upward in a few generations. In these examples, apply to the mef2ca b1086 mutant, high or low penetrants of a topic bone between the opercal and the branchiostegal ray in lethal homozygous mutants was selected for.In a successful cast procedure, type groupings of samples corresponding to gene type should be recognized by the computer program and the NTCs should be located near the origin of the plot after sufficient cycles have been performed. As demonstrated here, successful Alcian blue, alizarin red stain will have vibrantly stained bone and cartilage elements that are not transparent or faint. The boundaries of the cartilage elements should appear crisp and individual contracytes will be discernible.Alcian blue that is too concentrated were not clear from other tissues making analysis impossible. Implementing this technique from zebrafish line maintenance will likely alter penetrants in a few generations. Following this procedure, other methods like NC2 hybridization and timelapse imaging studies may yield more consistent results.We use this technique to explore the heritability of phenotype variation. After watching this video, you should have a good understanding of how to selectively breed your mutant strains to alter penetrance, even if the mutation is lethal.

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