30日猪胚胎的准确和无酚DNA性别鉴定的PCR
Summary
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.
Abstract
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。
Introduction
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。
Protocol
Representative Results
Discussion
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?…
Divulgations
The authors have nothing to disclose.
Acknowledgements
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。
Materials
| KAPA HiFi HotStart Ready Mix PCR kit | KAPABiosystems | KR0370 | other Hot Start Taq polymerase can be used after optimization |
| SYBR Safe DNA Gel Stain | Life Technologies | S33102 | Ethidium bromide can be substituted for SYBR Safe |
| Pig female and male genomic DNA | Zyagen | GP-160-F1 & GP-160-M5 | Postive controls from the tissues of known sex DNA can be used. |
| Typhoon FLA 9500 laser scanner | GE Healthcare Life Sciences | 28-9969-43 | other imaging system can be used |
| Free Soft Nitrile Examination Gloves | WWR | 89038-270 | any other examination glove can be used |
| Sodium hydroxide, solid | Fisher | BP 359 -212 | Molecular Biology Grade |
| Eppendorff DNA LoBind Tubes, 1.5 ml, PCR clean | Eppendorff | 0030 108.051 | heat resistant |
| ThermoStat plus | Eppendorff | 22670204 | Use as a incubator for 95C, don't need to use the heater |
| Toothpick | Bunzl Plc | 75200815 | Any round wooden toothpicks can be used – quality wood |
| Microcentrifuge 5417R/5417C | Eppendorff | 22621807 | This model was discontinued. But another newer model can be used |
| Microspatula | Fisher | SDI28540115 | Autoclaved before use each time. |
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