作者要感谢的合作与下面的科研资助机构的财政捐款:艾伯塔牲畜和肉类代理有限公司，猪肉CRC，艾伯塔猪肉，海波尔一个汉德克斯公司与NSERC CRSNG。

按照加拿大议会关于动物饲养的指导方针，并与学院动物政策及福利委员会批准 - 阿尔伯塔大学的畜牧，妊娠母猪由经过培训的人员大约一天收集妊娠和胚胎30安乐死。在过程中使用检查手套在所有时间。 1.样品采集和样品储存<ol><li>使用紧固螺栓接着放血安乐死母猪。戴上手套检查从每个安乐死收集母猪生殖道和胚胎。 注:样品和移植胚胎的快速收集到干冰或液氮，将导致更高质量的组织中其他分子的作品，如芯片和qPCR。 </li><li>解剖子宫，轻轻的胚胎盘胎膜内所有的胚胎从底层的子宫壁分离21。确保没有母体组织的污染。 </li><li>回覆冷冻前所有可行的胚胎线的长度和重量。 </li><li>收集每个人的胚胎，并立即与捕捉液氮冷冻前的铝箔包起来。 </li><li>转自液氮冷冻胚胎直接到-80℃的冰箱中。 </li></ol> 2.磨胚胎<ol><li>标记所有样品管（15ml）中与所需的样本数的样本ID加以分析。 </li><li>填充干冰热容器半满，并在干冰插入预标记的样品管中。 </li><li>戴上手套，冬天在上面另一对检查手套从-80转移预冷迫击炮和杵℃冰箱干冰的容器。 </li><li>放置在冷冻胚胎的干冰砂浆内。 </li><li>倾足够液氮覆盖胚胎和用杵成细粉研磨它。 </li><li>胚胎粉末转移到预标记的样品管与MICRospatula（图1），并放置在管中-80℃冷冻机中。 </li><li>使用干净预冷研钵每个胚胎和研磨每个胚胎，以避免样品间的交叉污染后改变考试手套。 </li></ol>采用改良氢氧化钠方法3基因组DNA制备方法<ol><li>标签所需的所有的样本数的离心管（1.5毫升），以进行分析。 </li><li>从之前的传递包含胚粉样品管-80℃冷冻到容器用干冰首发。 </li><li>吸管180微升的50mM的NaOH（氢氧化钠）到每个预标记的微量离心管中。 </li><li>打开培养箱和预热至95℃。 </li><li>用牙签胚胎粉末的正确量（约5-10毫克）（图2）从样品管转移到含有50mM的NaOH溶液的预标记的微量离心管中。 </li><li>拉üP上的同一牙签慢慢可视化所述DNA裂解物作为粘糊糊，白色透明状物质（图3）。 </li><li>用DNA裂解物转移离心管到预加热的培养箱中于95℃五分钟。紧随管转移到装满冰保丽龙箱。 </li><li>加入20μl的1M Tris-HCl中直接进入的离心管，并轻轻拍打该管混合。使用pH试纸，以确保pH在大约8.0（图4）之前，离心。 注意:当有大量样品制备的处理，是可以接受的随机挑选几样检查pH值。 </li><li>离心与DNA裂解液的管中，在2000 xg离心在微量两分钟，在室温下以除去未溶解的组织碎片。 </li><li>转移150微升顶部澄清上清液的进新管或96孔板中。 注:明确DNA裂解液准备在PC模板使用ř反应，这是在4℃下稳定两周，或者也可以保持在-20℃下一年。 </li></ol> 4.设计性别特异性PCR引物<ol><li>获得NCBI网站http://www.ncbi.nlm.nih.gov猪的性别决定区Y（SRY）登录号（NM_214452.3）和锌指蛋白X连锁（ZFX）基因（XM_005673501.1） /。 </li><li>复制这些NCBI登录号粘贴到一个在线的引物设计工具。 注意:最好的引物信息，包括长度，Tm和GC％以及PCR产物大小（bp）的将在计算机屏幕上产生的（图5）。 </li><li>验证的使用核苷酸BLAST程序（BLASTN）逆水猪基因组数据库104，以确保这些序列仅位于X和Y染色体为SRY和ZFX分别（图6）这些引物的特异性。 </li></ol> 5.基因组DNA PCR直接从DNA裂解液<ol><li>使用仅1微升的DNA裂解物作为15微升的PCR反应的模板的。 注意:可以在用多道移液器类似的方式来执行对96孔板的PCR的高通量筛选方法。 </li><li>请执行以下操作只是第一次PCR:通过添加购得已知性别的猪的基因组DNA 1微升（0.5纳克）准备一个PCR管无模板阴性对照和阳性对照两个附加管。以后，包括来自上一次成功的性别鉴定分析作为阳性对照的样品，并与用于性别确定的新PCR反应一起运行。 </li><li>使用任何热启动预拌的PCR酶，并通过添加引物制备主混合物并根据PCR反应的总数无核酸酶的水。加入1微升的DNA裂解成现有预PCR管与PCR主混合物的14微升。添加的引物，使得由两个性别特异性基因的引物的终浓度是在共有15微升的PCR为r的0.3微米反应的影响。 </li><li>设置以下PCR程序在热循环仪:95℃，3分钟，35个循环，每个为20秒熔化步骤98℃，接着在65℃15秒的退火步骤，接着是15秒延伸步骤在72℃。在最后的步骤，孵育1分钟的反应在72℃下，继续在4℃下孵育直至除去PCR管的用于凝胶电泳验证，以确定的性别。 注意:PCR条件和优化，根据在材料表中提到的PCR试 ​​剂盒的说明书。 </li><li>制备2％TBE琼脂糖凝胶时添加无毒绿光荧光SYBR DNA凝胶染色的适当的量。 注意:溴化乙锭可以代替绿光荧光染色，如果它是不可用的。 </li><li>添加1.5微升样染料（10倍）到PCR管中，并通过移液在PCR反应缓冲液向上和向下拌匀。 </li><li>负载10微升样品放入井并且r联合国琼脂糖凝胶具有适当的电压设置（在100伏的小胶设备，在150V 96孔凝胶装置上，直到染料带贯穿凝胶一半）。 </li><li>观察和利用激光扫描仪下的荧光设置调整频带的强度，并捕获凝胶的图像。注意:普通的凝胶成像仪可以取代的溴化乙锭的凝胶。 </li><li>通过识别与一个频带作为女性和两个频带作为男性（图7）的胚胎确定猪胚胎的性别。 </li></ol>

<ol>
	<li>Seidel, G. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Economics+of+selecting+for+sex:+the+most+important+genetic+trait.">Economics of selecting for sex: the most important genetic trait.</a> Theriogenology. 59, 585-598 (2003).</li><li>Guti&#233;rrez-Ad&#225;n, 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=Development+consequences+of+sexual+dimorphism+during+pre-implantation+embryonic+development.">Development consequences of sexual dimorphism during pre-implantation embryonic development.</a> Reprod. Domest. Anim. 41, 54-62 (2006).</li><li>Rosenfeld, C. S., Roberts, R. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Maternal+diet+and+other+factors+affecting+offspring+sex+ratio:+a+review.">Maternal diet and other factors affecting offspring sex ratio: a review.</a> Biol Reprod. 71, 1063-1070 (2004).</li><li>Gorecki, 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=Sex+ratio+in+litters+of+domestic+pigs+(Sus+scrofa+f.+domestica+Linnaeus,+1758).">Sex ratio in litters of domestic pigs (Sus scrofa f. domestica Linnaeus, 1758).</a> Biol. Lett. 40, 111-118 (2003).</li><li>Chen, Z. Y., Dziuk, P. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Influence+of+initial+length+of+uterus+per+embryo+and+gestation+stage+on+prenatal+survival,+development,+and+sex+ratio+in+the+pig.">Influence of initial length of uterus per embryo and gestation stage on prenatal survival, development, and sex ratio in the pig.</a> J. Anim. Sci. 71, 1895-1901 (1993).</li><li>Vinsky, M. D., Novak, S., Dixon, W. T., Dyck, M. K., Foxcroft, G. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nutritional+restriction+in+lactating+primiparous+sows+selectively+affects+female+embryo+survival+and+overall+litter.">Nutritional restriction in lactating primiparous sows selectively affects female embryo survival and overall litter.</a> Reprod. Fertil. Dev. 18, 347-355 (2006).</li><li>Cassar, G., King, W. A., King, G. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Influence+of+sex+on+early+growth+of+pig+conceptuses.">Influence of sex on early growth of pig conceptuses.</a> J. Reprod. Fertil. 101, 317-320 (1994).</li><li>Zudova, D., Rezacova, O., Kubickova, S., Rubes, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Aneuploidy+detection+in+porcine+embryos+using+fluorescence+in+situ+hybridization.">Aneuploidy detection in porcine embryos using fluorescence in situ hybridization.</a> Cytogenet Genome Res. 102, 179-183 (2003).</li><li>Hornak, M., Oracova, E., Hulinska, P., Urbankova, L., Rubes, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Aneuploidy+detection+in+pigs+using+comparative+genomic+hybridization:+from+the+oocytes+to+blastocysts.">Aneuploidy detection in pigs using comparative genomic hybridization: from the oocytes to blastocysts.</a> PLoS One. 7, (2012).</li><li>Handyside, A. H., Pattinson, J. K., Penketh, R. J., Delhanty, J. D., Winston, R. M., Tuddenham, E. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Biopsy+of+human+preimplantation+embryos+and+sexing+by+DNA+amplification.">Biopsy of human preimplantation embryos and sexing by DNA amplification.</a> Lancet. 1, 347-349 (1989).</li><li>Wilton, L. J., Shaw, J. M., Trounson, A. O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Successful+single-cell+biopsy+and+cryopreservation+of+preimplantation+mouse+embryos.">Successful single-cell biopsy and cryopreservation of preimplantation mouse embryos.</a> Feri. Steril. 51, 513-517 (1989).</li><li>Peura, J. M., Turunen, M., Janne, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+reliable+sex+determination+assay+for+bovine+preimplantation+embryos+using+the+polymerase+chain+reaction.">A reliable sex determination assay for bovine preimplantation embryos using the polymerase chain reaction.</a> Theriogenology. 35, 547-555 (1991).</li><li>Rao, K. B., Pawshe, C. H., Totey, 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=Sex+determination+of+in+vitro+developed+buffalo+(Buhalus+buhalis.)+embryos+by+DNA+amplification.">Sex determination of in vitro developed buffalo (Buhalus buhalis.) embryos by DNA amplification.</a> Mol. Reprod. Dev. 36, 291-296 (1993).</li><li>Herr, C. M., Matthaei, K. I., Petrzak, U., Reed, K. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+rapid+Y-chromosome-detecting+ovine+embryo+sexing+assay.">A rapid Y-chromosome-detecting ovine embryo sexing assay.</a> Theriogenology. 33, 245 (1990).</li><li>Fajfar-Whetstone, C. J., LaneRayburn, A., Schook, L. B., Wheeler, M. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Sex+determination+of+porcine+during+preimplantation+embryos+via+y-chromosome+specific+DNA+sequences.">Sex determination of porcine during preimplantation embryos via y-chromosome specific DNA sequences.</a> Animal Biotechnology. 4, 183-193 (1993).</li><li>Pomp, D., Good, B. A., Geisert, R. D., Corbin, C. J., Conley, A. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Sex+identification+in+mammals+with+polymerase+chain+reaction+and+its+use+to+examine+sex+effects+on+diameter+of+day-10+or+-11+pig+embryos.">Sex identification in mammals with polymerase chain reaction and its use to examine sex effects on diameter of day-10 or -11 pig embryos.</a> J. Anim. Sci. 73, 1408-1415 (1995).</li><li>Aasen, E., Medrano, J. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Amplification+of+the+ZFY+and+ZFX+genes+for+sex+identification+in+humans,+cattle,+sheep+and+goats.">Amplification of the ZFY and ZFX genes for sex identification in humans, cattle, sheep and goats.</a> Biotechnology (N Y). 8, 1279-1281 (1990).</li><li>Oliver, 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=Restricted+feed+intake+in+lactating+primiparous+sows.+II.+Effects+on+subsequent+litter+sex+ratio+and+embryonic+gene+expression.">Restricted feed intake in lactating primiparous sows. II. Effects on subsequent litter sex ratio and embryonic gene expression.</a> Reprod Fertil. Dev. 23, 899-911 (2011).</li><li>Truett, G. E., Heeger, P., Mynatt, R. L., Truett, A. A., Walker, J. A., Warman, M. L. <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+PCR-quality+mouse+genomic+DNA+with+hot+sodium+hydroxide+and+tris+(HotSHOT).">Preparation of PCR-quality mouse genomic DNA with hot sodium hydroxide and tris (HotSHOT).</a> Biotechniques. 29, 52-54 (2000).</li><li>Meeker, N. D., Hutchinson, S. A., Ho, L., Trede, N. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Method+for+isolation+of+PCR-ready+genomic+DNA+from+zebrafish+embryos.">Method for isolation of PCR-ready genomic DNA from zebrafish embryos.</a> Biotechniques. 43, 610-614 (2007).</li><li>Almeida, F. C. R. L., Machado, G. S., Borges, A. L. C. C., Rosa, B. O., Fontes, D. O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Consequences+of+different+dietary+energy+sources+during+folliculardevelopment+on+subsequent+fertility+of+cyclic+gilts.">Consequences of different dietary energy sources during folliculardevelopment on subsequent fertility of cyclic gilts.</a> Animal. 8, 293-299 (2014).</li><li>Foxcroft, G. R., Dixon, W. T., Dyck, M. K., Novak, S., Harding, J. C. S., Almeida, F. C. R. L., Rodriguez-Martinez, <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Prenatal+programming+of+postnatal+development+in+the+pig.">Prenatal programming of postnatal development in the pig.</a> Control of Pig Reproduction VIII. , 212-213 (2009).</li></ol>

作者宣称，他们没有竞争的经济利益。

最相关的猪胚胎的DNA性别打字现有协议的仅适合于早期预注入阶段15,16。我们已经成功开发出在妊娠后期适用于猪胚胎筛选的协议。基于与从先前的研究6,18胚胎相似发育阶段的研究中，本协议被认为是安全的，成本低。 该协议也适于用于使用PCR通过DNA裂解物转移入96孔板性打字大量样品。板格式允许高通量筛选通过允许转印1μl的DNA的裂解物用于使用多道移液?...

国内生猪已成为发展，遗传学与营养在人类和畜牧部门的基本研究课题。猪作为生物体模型为人类研究的潜在可以归因于他们的生理相似之处。在家畜，性别比例操纵，提高选择和遗传改良计划1的有效性。性别鉴定单个胚胎是许多实验研究，包括但早期胚胎发育过程中2不限于基因，表观遗传学和性别二态性的X染色体失活使用的基本工具。 在小鼠的研究表明，产妇的饮食和其他因素可能导致男女比例失调3。在猪，性别比例失衡的原因包括父系品种4，子宫容量5，母猪的代谢状况6。因为在胚胎和窝中观察到的差异可以通过本身的影响xual异形，研究人员应借鉴有关他们的研究结论之前，了解胎儿的性别和性别比率。高效的工具和协议，在发展30日性别鉴定胚胎养猪的发展，将在这里讨论。 性分型的各种方法已经被开发用于在模式生物和家畜遗传研究。特别是在家畜，识别雌雄早期胚胎是一个非常普遍的做法，以提高遗传选择用于育种计划。早期胚胎用姬姆萨7或强烈的荧光8,9技术猪核型已被用于性别特征。然而，这些方法是耗时，不适合快速，准确地筛选大量的胚胎。 最有效的性别分型方法是使用热稳定DNA聚合酶和一对引物DNA扩增。通过PCR方法DNA性别鉴定是比较具体的，快速，灵敏，邻NLY需要接入蜂窝材料的微量。是对人类进行的10首基于PCR的胚胎性别鉴定，后来在小鼠11，12牛，水牛13和 14只羊植入前胚胎。在猪，通过单一对Y染色体特异性DNA引物15，建立了植入前胚胎早期的DNA性别分型方法。然而，从雄性特异性SRY基因16和位于X和Y染色体17上的锌指基因的非性判别区域的Y染色体选择用于性别确定的最常见的PCR引物。随后，这些引物已经被应用，以确定在这项研究中具有改进的引物特异性日30的胚胎的性别来检测仅一个锌指基因的X染色体。 从猪植入前胚胎基因组DNA可以通过将一个完整的胚泡与proteinas缓冲提取Ëķ16或通过采取一些细胞活检从个体早期卵裂胚胎15并将它们用于直接PCR。然而，从猪血液，毛发，组织或大小超过几厘米大孕的DNA释放不能有效地使用蛋白酶K法进行。使用任一耗时苯酚/氯仿协议6或昂贵基于列的试剂盒18已经建立了这些材料的DNA提取方法。为了避免使用可能有毒的化学品，有开发价格低廉，容易和自由酚DNA提取方法的趋势。这对PCR的质量的基因组DNA，从鼠标 19和斑马鱼20组织中的隔离型协议的已使用热氢氧化钠和三（能手）成立。这项研究提供了一个协议来获取DNA与PCR性爱能手修改和重新设计的复式引物对直接从日细胞裂解液30猪胚胎打字精度高。

<table><tbody><tr><td>KAPA HiFi HotStart Ready Mix PCR kit&amp;nbsp;</td><td>KAPABiosystems</td><td>KR0370</td><td>other Hot Start Taq polymerase can be used after optimization</td></tr><tr><td>SYBR Safe DNA Gel Stain</td><td>Life Technologies</td><td>S33102</td><td>Ethidium bromide can be substituted for SYBR Safe</td></tr><tr><td>Pig female and male genomic DNA</td><td>Zyagen</td><td>GP-160-F1 &amp;amp; GP-160-M5</td><td>Postive controls from the tissues of known sex DNA can be used.</td></tr><tr><td>Typhoon FLA 9500 laser scanner</td><td>GE Healthcare Life Sciences</td><td>28-9969-43</td><td>other imaging system can be used</td></tr><tr><td>Free Soft Nitrile Examination Gloves</td><td>WWR</td><td>89038-270</td><td>any other examination glove can be used</td></tr><tr><td>Sodium hydroxide, solid&amp;nbsp;</td><td>Fisher</td><td>BP 359 -212</td><td>Molecular Biology Grade</td></tr><tr><td>Eppendorff DNA LoBind Tubes, 1.5 ml, PCR clean</td><td>Eppendorff</td><td>0030 108.051</td><td>heat resistant</td></tr><tr><td>ThermoStat plus</td><td>Eppendorff</td><td>22670204</td><td>Use as a incubator for 95 &amp;#176;C , don&#039;t need to use the heater</td></tr><tr><td>Toothpick</td><td>Bunzl Plc</td><td>75200815</td><td>Any round wooden toothpicks can be used - quality wood</td></tr><tr><td>Microcentrifuge 5417R/5417C</td><td>Eppendorff</td><td>22621807</td><td>This model was discontinued. But another newer model can be used</td></tr><tr><td>Microspatula</td><td>Fisher</td><td>SDI28540115</td><td>Autoclaved before use each time.</td></tr></tbody></table>

accurate and phenol free dna sexing of day 30 porcine embryos by pcr

研究中的猪产前编程已经表明，发育中的胚胎或胎儿的性别可以影响发育结果。因此，确定胚胎的性别的能力是必要的，许多实验特别是关于早期发展。本协议表明猪基因组DNA用PCR使用一种廉价的，快速的和无毒​​的制剂。第30天的胚胎必须按照机构动物政策及福利委员会在本协议确定的方针得到人道收集。整个胚胎此基于PCR性别鉴定技术制备简单地涉及使用预冷研钵和杵研磨冷冻的胚胎成细粉。 PCR的质量DNA从胚胎粉末少量通过在碱性裂解试剂施加热孵育释放。接着，将DNA溶液与中和缓冲液混合，并直接用于PCR。两个引物对产生的DETEC吨特定性别确定Y-染色体（SRY）和在X染色体以高精度和特异性的ZFX区域的区域。相同的协议可以被应用到其它细长胚胎（第10天至14天）比日早30.此外，此协议可与96涌出板筛选大量的胚胎时，使之成为自动化和高可行进行吞吐量性别特征。

从345的DNA裂解物通过PCR筛选性别决定的代表性结果示于图7和表 1中总结。 如在图7中可以看出 ，引物退火温度在65℃是在不同样品之间的这种PCR方案产生类似的强度和谓词的扩增子的大小（图5）的最佳条件。 SRY（400?...

Watch this Scientific Journal Video about Accurate and Phenol Free DNA Sexing of Day 30 Porcine Embryos by PCR at JoVE.com

Accurate and Phenol Free DNA Sexing of Day 30 Porcine Embryos by PCR

This protocol describes an accurate, inexpensive, rapid and non-toxic method to determine the sex of Day 30 porcine embryo using PCR method after grinding an embryo into powder without phenol chloroform extraction and DNA column purification.

30日猪胚胎的准确和无酚DNA性别鉴定的PCR

Research into prenatal programming in the pig has shown that the sex of the developing embryo or fetus can influence the developmental outcome. Therefore, ...

accurate-and-phenol-free-dna-sexing-of-day-30-porcine-embryos-by-pcr

Research

JoVE Journal

Developmental Biology

10.0K 次观看.  University of Alberta. This protocol describes an accurate, inexpensive, rapid and non-toxic method to determine the sex of Day 30 porcine embryo using PCR method after grinding an embryo into powder without phenol chloroform extraction and DNA column purification.

视频: Accurate and Phenol Free DNA Sexing of Day 30 Porcine Embryos by PCR

Quantitative Analysis of Protein Expression to Study Lineage Specification in Mouse Preimplantation Embryos

This protocol presents a method to perform quantitative, single-cell in situ analyses of protein expression to study lineage specification in mouse preimplantation embryos. The procedures necessary for embryo collection, immunofluorescence, imaging on a confocal microscope, and image segmentation and analysis are described. This method allows quantitation of the expression of multiple nuclear markers and the spatial (XYZ) coordinates of all cells in the embryo. It takes advantage of MINS, an image segmentation software tool specifically developed for the analysis of confocal images of preimplantation embryos and embryonic stem cell (ESC) colonies. MINS carries out unsupervised nuclear segmentation across the X, Y and Z dimensions, and produces information on cell position in three-dimensional space, as well as nuclear fluorescence levels for all channels with minimal user input. While this protocol has been optimized for the analysis of images of preimplantation stage mouse embryos, it can easily be adapted to the analysis of any other samples exhibiting a good signal-to-noise ratio and where high nuclear density poses a hurdle to image segmentation (e.g., expression analysis of embryonic stem cell (ESC) colonies, differentiating cells in culture, embryos of other species or stages, etc.).

This protocol presents a method to perform quantitative, single-cell in situ analysis of protein expression to study lineage specification in mouse preimplantation embryos. The procedures necessary for collection of blastocysts, whole-mount immunofluorescent detection of proteins, imaging of samples on a confocal microscope, and nuclear segmentation and image analysis are described.

蛋白表达的定量分析来研究宗族规范小鼠植入前胚胎

This protocol presents a method to perform quantitative, single-cell in situ analyses of protein expression to study lineage specification in mouse ...

科研

发育生物学

Loss- and Gain-of-function Approach to Investigate Early Cell Fate Determinants in Preimplantation Mouse Embryos

Gene silencing and overexpression techniques are instrumental for the identification of genes involved in embryonic development. Direct target gene modification in preimplantation embryos provides a means to study the underlying mechanisms of genes implicated in, for instance, cellular differentiation into the trophectoderm (TE) and the inner cell mass (ICM). Here, we describe a protocol that examines the role of neogenin as an authentic receptor for initial cell fate determination in preimplantation mouse embryos. First, we discuss the experimental manipulations that were used to produce gain and loss of neogenin function by microinjecting neogenin cDNA and shRNA; the effectiveness of this approach was confirmed by a strong correlation between the pair-wise expression levels of either red fluorescent protein (RFP) or green fluorescent protein (GFP) and the immunocytochemical quantification of neogenin expression. Secondly, overexpression of neogenin in preimplantation mouse embryos leads to normal ICM development while neogenin knockdown causes the ICM to develop abnormally, implying that neogenin could be a receptor that relays extracellular cues to drive blastomeres to early cell fates. Given the success of this detailed protocol in investigating the function of a novel embryonic developmental stage-specific receptor, we propose that it has the potential to aid in exploration and identification of other stage-specific genes during embryogenesis.

The goal of this protocol is to describe a loss- and gain-of function method that is applicable to identify neogenin as a stage-specific receptor that leads to trophectoderm and inner cell mass differentiation in preimplantation mouse embryos.

损失 - 和增益的功能角度进行调查研究早期细胞命运决定在植入前小鼠胚胎

 Gene silencing and overexpression techniques are instrumental for the identification of genes involved in embryonic development. Direct target gene ...

Transcriptome Profiling of In-Vivo Produced Bovine Pre-implantation Embryos Using Two-color Microarray Platform

Early embryonic loss is a large contributor to infertility in cattle. Moreover, bovine becomes an interesting model to study human preimplantation embryo development due to their similar developmental process. Although genetic factors are known to affect early embryonic development, the discovery of such factors has been a serious challenge. Microarray technology allows quantitative measurement and gene expression profiling of transcript levels on a genome-wide basis. One of the main decisions that have to be made when planning a microarray experiment is whether to use a one- or two-color approach. Two-color design increases technical replication, minimizes variability, improves sensitivity and accuracy as well as allows having loop designs, defining the common reference samples. Although microarray is a powerful biological tool, there are potential pitfalls that can attenuate its power. Hence, in this technical paper we demonstrate an optimized protocol for RNA extraction, amplification, labeling, hybridization of the labeled amplified RNA to the array, array scanning and data analysis using the two-color analysis strategy.

Transcriptome Profiling of In-Vivo Produced Bovine Pre-implantation Embryos Using Two-color Microarray Platform

Microarray technology allows quantitative measurement and gene expression profiling of transcripts on a genome-wide basis. Therefore, this protocol provides an optimized technical procedure in a two-color custom made bovine array using Day 7 bovine embryos to demonstrate the feasibility of using low amount of total RNA.

转录组分析]<em&gt;活体</em&gt;生产牛胚胎植入前胚胎采用双色微阵列平台

Early embryonic loss is a large contributor to infertility in cattle. Moreover, bovine becomes an interesting model to study human preimplantation embryo ...

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

Zebrafish (Danio rerio) possess orthologues for 84% of the genes known to be associated with human diseases. In addition, these animals have a short generation time, are easy to handle, display a high reproductive rate, low cost, and are easily amenable to genetic manipulations by microinjection of DNA in embryos. Recent advances in gene editing tools are enabling precise introduction of mutations and transgenes in zebrafish. Disease modeling in zebrafish often leads to larval phenotypes and early death which can be challenging to interpret if genotypes are unknown. This early identification of genotypes is also needed in experiments requiring sample pooling, such as in gene expression or mass spectrometry studies. However, extensive genotypic screening is limited by traditional methods, which in most labs are performed only on adult zebrafish or in postmortem larvae. We addressed this problem by adapting a method for the isolation of PCR-ready genomic DNA from live zebrafish larvae that can be achieved as early as 72 h post-fertilization (hpf). This time and cost-effective technique, improved from a previously published genotyping protocol, allows the identification of genotypes from microscopic fin biopsies. The fins quickly regenerate as the larvae develop. Researchers are then able to select and raise the desired genotypes to adulthood by utilizing this high-throughput PCR-based genotyping procedure.

Zebrafish have been used as reliable genetic model organisms in biomedical research, especially with the advent of gene-editing technologies. When larval phenotypes are expected, DNA extraction and genotype identification can be challenging. Here, we describe an efficient genotyping procedure for zebrafish larvae, by tail clipping, as early as 72-h post-fertilization.

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 ...

遗传学

Semiconductor Sequencing for Preimplantation Genetic Testing for Aneuploidy

Chromosomal aneuploidy, one of the main causes leading to embryonic development arrest, implantation failure, or pregnancy loss, has been well documented in human embryos. Preimplantation genetic testing for aneuploidy (PGT-A) is a genetic test that significantly improves reproductive outcomes by detecting chromosomal abnormalities of embryos. Next-generation sequencing (NGS) provides a high-throughput and cost-effective approach for genetic analysis and has shown clinical applicability in PGT-A. Here, we present a rapid and low-cost semiconductor sequencing-based NGS method for screening of aneuploidy in embryos. The first step of the workflow is whole genome amplification (WGA) of the biopsied embryo specimen, followed by construction of sequencing library, and subsequent sequencing on the semiconductor sequencing system. Generally, for a PGT-A application, 24 samples can be loaded and sequenced on each chip generating 60&#8722;80 million reads at an average read length of 150 base pairs. The method provides a refined protocol for performing template amplification and enrichment of sequencing library, making the PGT-A detection reproducible, high-throughput, cost-efficient, and timesaving. The running time of this semiconductor sequencer is only 2&#8722;4 hours, shortening the turnaround time from receiving samples to issuing reports into 5 days. All these advantages make this assay an ideal method to detect chromosomal aneuploidies from embryos and thus, facilitate its wide application in PGT-A.

Here, we introduce a semiconductor sequencing method for preimplantation genetic testing for aneuploidy (PGT-A) with the advantages of short turnaround time, low cost, and high throughput.

用于抗倍体植入前基因测试的半导体测序

Chromosomal aneuploidy, one of the main causes leading to embryonic development arrest, implantation failure, or pregnancy loss, has been well documented ...

Bottlenose Dolphin (Tursiops truncatus) Spermatozoa: Collection, Cryopreservation, and Heterologous In Vitro Fertilization

The use of cryopreserved dolphin spermatozoa facilitates the exchange of genetic material between aquatic parks and makes spermatozoa accessible to laboratories for studies to further our understanding of marine mammal reproduction. Heterologous IVF, a replacement for homologous IVF, could provide a means to test the sperm fertility potential; to study gamete physiology and early embryo development; and to avoid the use of valuable dolphin oocytes, which are difficult to obtain. Here, we present protocols that have been successfully used to collect and cryopreserve dolphin spermatozoa. The collection of semen is performed by manual stimulation on trained dolphins. Cryopreservation is accomplished using a TRIS egg-yolk based extender with glycerol. In addition, we present a protocol that describes heterologous IVF using dolphin spermatozoa and bovine oocytes and that verifies the hybrid nature of the resulting embryo using PCR. Heterologous fertilization raises questions on fertilization and can be used as a tool to study gamete physiology and early embryo development. In addition, the success of heterologous IVF demonstrates the potential of this technique to test dolphin sperm fertilizing capacity, which is worth further examination.

Bottlenose Dolphin Tursiops truncatus Spermatozoa: Collection, Cryopreservation, and Heterologous In Vitro Fertilization

Here, we present protocols that have been successfully used for dolphin spermatozoa collection, cryopreservation, and heterologous IVF performance using bovine oocytes.

宽吻海豚 (海豚截形) 精子: 收集、 冷冻保存和外源体外受精

The use of cryopreserved dolphin spermatozoa facilitates the exchange of genetic material between aquatic parks and makes spermatozoa accessible to ...

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

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 that forms the placenta. This method, however, requires specialized skills, is invasive, and suffers from false positives and negatives because the chromosome numbers in the TE and the inner cell mass (ICM), which develops into the fetus, are not always the same. NICS, a technology requiring sequencing of DNA that released into the culture medium from both TE and ICM, may offer a way out to these problems but has previously been shown to have limited efficacy. The present study reports the full protocol of NICS, which includes culture medium sampling methods, whole genome amplification (WGA) and library preparation, and NGS data analysis by analysis software. Considering the different cryopreservation times in different embryo laboratories, embryologists have two methods for collecting embryo culture medium that can be selected according to the actual conditions of the IVF laboratory.

The present study reports a protocol for chromosome screening of human embryos that uses spent culture medium, which avoids embryo biopsy and enables chromosome ploidy identification using NGS. The present article presents the detailed procedure, including the preparation of culture medium, whole genome amplification (WGA), next-generation sequencing (NGS) library preparation, and data 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 ...

Efficient Genome Editing of Mice by CRISPR Electroporation of Zygotes

With exceptional efficiency, accuracy, and ease, the CRISPR/Cas9 system has significantly improved genome editing in cell culture and lab animal experiments. When generating animal models, the electroporation of zygotes offers higher efficiency, simplicity, cost, and throughput as an alternative to the gold standard method of microinjection. Electroporation is also gentler, with higher viability, and reliably delivers Cas9/single-guide RNA (sgRNA) ribonucleoproteins (RNPs) into the zygotes of common laboratory mouse strains (e.g., C57BL/6J and C57BL/6N) that approaches 100% delivery efficiency. This technique enables insertion/deletion (indels) mutations, point mutations, the deletion of whole genes or exons, and small insertions in the range of 100-200 bp to insert LoxP or short tags like FLAG, HA, or V5. While constantly being improved, here we present the current state of CRISPR-EZ in a protocol that includes sgRNA production through in vitro transcription, embryo processing, RNP assembly, electroporation, and the genotyping of preimplantation embryos. A graduate-level researcher with minimal experience manipulating embryos can obtain genetically edited embryos in less than 1 week using this protocol. Here, we offer a straightforward, low-cost, efficient, high-capacity method that could be used with mouse embryos.

Here, we describe a simple technique intended for the efficient generation of genetically modified mice called CRISPR RNP Electroporation of Zygotes (CRISPR-EZ). This method delivers editing reagents by electroporation into embryos at an efficiency approaching 100%. This protocol is effective for point mutations, small genomic insertions, and deletions in mammalian embryos.

通过受精卵的CRISPR电穿孔对小鼠进行高效基因组编辑

With exceptional efficiency, accuracy, and ease, the CRISPR/Cas9 system has significantly improved genome editing in cell culture and lab animal ...

用于单细胞测序的原肠胚胚分离和基因分型

Single-Cell RNA Sequencing of Mutant Whole Mouse Embryos: From the Epiblast to the End of Gastrulation

Over the last decade, single-cell approaches have become the gold standard for studying gene expression dynamics, cell heterogeneity, and cell states within samples. Before single-cell advances, the feasibility of capturing the dynamic cellular landscape and rapid cell transitions during early development was limited. In this paper, a robust pipeline was designed to perform single-cell and nuclei analysis on mouse embryos from embryonic day E6.5 to E8, corresponding to the onset and completion of gastrulation. Gastrulation is a fundamental process during development that establishes the three germinal layers: mesoderm, ectoderm, and endoderm, which are essential for organogenesis. Extensive literature is available on single-cell omics applied to wild-type perigastrulating embryos. However, single-cell analysis of mutant embryos is still scarce and often limited to FACS-sorted populations. This is partially due to the technical constraints associated with the need for genotyping, timed pregnancies, the count of embryos with desired genotypes per pregnancy, and the number of cells per embryo at these stages. Here, a methodology is presented designed to overcome these limitations. This method establishes breeding and timed pregnancy guidelines to achieve a higher chance of synchronized pregnancies with desired genotypes. Optimization steps in the embryo isolation process coupled with a same-day genotyping protocol (3 h) allow for microdroplet-based single-cell to be performed on the same day, ensuring the high viability of cells and robust results. This method further includes guidelines for optimal nuclei isolations from embryos. Thus, these approaches increase the feasibility of single-cell approaches of mutant embryos at the gastrulation stage. We anticipate that this method will facilitate the analysis of how mutations shape the cellular landscape of the gastrula.

作者聚焦：以单细胞分辨率分析谱系特异性突变胚胎的管道

This paper establishes a pipeline for high-quality single-cell and nuclei suspensions of gastrulating mouse embryos for sequencing of single cells and nuclei.

突变全小鼠胚胎的单细胞 RNA 测序：从上胚层到原肠胚形成结束

Over the last decade, single-cell approaches have become the gold standard for studying gene expression dynamics, cell heterogeneity, and cell states ...

30日猪胚胎的准确和无酚DNA性别鉴定的PCR

摘要

探索更多视频

此视频中的章节

蛋白表达的定量分析来研究宗族规范小鼠植入前胚胎

损失 - 和增益的功能角度进行调查研究早期细胞命运决定在植入前小鼠胚胎

转录组分析]

3dpf 斑马鱼幼虫的高通量 DNA 提取及基因分型研究

用于抗倍体植入前基因测试的半导体测序

宽吻海豚 (海豚截形) 精子: 收集、冷冻保存和外源体外受精

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

通过受精卵的CRISPR电穿孔对小鼠进行高效基因组编辑

突变全小鼠胚胎的单细胞 RNA 测序：从上胚层到原肠胚形成结束

突变全小鼠胚胎的单细胞 RNA 测序：从上胚层到原肠胚形成结束

30日猪胚胎的准确和无酚DNA性别鉴定的PCR

摘要

探索更多视频

此视频中的章节

蛋白表达的定量分析来研究宗族规范小鼠植入前胚胎

损失 - 和增益的功能角度进行调查研究早期细胞命运决定在植入前小鼠胚胎

转录组分析]

3dpf 斑马鱼幼虫的高通量 DNA 提取及基因分型研究

用于抗倍体植入前基因测试的半导体测序

宽吻海豚 (海豚截形) 精子: 收集、 冷冻保存和外源体外受精

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

通过受精卵的CRISPR电穿孔对小鼠进行高效基因组编辑

突变全小鼠胚胎的单细胞 RNA 测序：从上胚层到原肠胚形成结束

突变全小鼠胚胎的单细胞 RNA 测序：从上胚层到原肠胚形成结束

宽吻海豚 (海豚截形) 精子: 收集、冷冻保存和外源体外受精