Name: mutagenesis and analysis of genetic mutations in the gc-rich kiss1 receptor sequence identified in humans with reproductive disorders
Uploaded: 2011-09-04T14:00:00
Description: Watch this Scientific Journal Video about Mutagenesis and Analysis of Genetic Mutations in the GC-rich KISS1 Receptor Sequence Identified in Humans with Reproductive Disorders at JoVE.com

这项工作是部分资金由国家儿童健康和人类发展研究所（NICHD的 - R21 HD059015）的生殖科和查尔斯H.胡德基金会青年研究者儿童健康研究奖（马萨诸塞州波士顿）。

1。定点突变的基因序列高度富含GC KISS1R <ol><li>模板：Myc基因标记与融合其N -末端的人类KISS1R完整的cDNA序列。这个序列克隆到的pCS2 +的表达载体，这是其后用于转染哺乳动物细胞线兼容。这表达载体被称为本pCS2 + Myc基因KISS1R。 </li><li>引物设计：引物设计进行所需的突变，根据Quikchange定点突变试剂盒（Stratagene公司）的说明。总结： <ul><li>两者（正向和反向引物）必须包含?...

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定点突变已被用于研究蛋白质的功能，通过引进超过三十年的靶基因的编码序列的核苷酸变化。原来的技术是于1978年由英国加拿大化学家和诺贝尔经济学奖得主迈克尔史密斯10。迈克尔史密斯分享1993年诺贝尔化学奖与雨霏，美国生物化学家穆利斯是谁发明的聚合酶链式反应（PCR）11。迈克尔史密斯所描述的原始方法多年来已改善。此过程中的高精确度和成功率，以及在一个相对较...

<table border="1"><tbody><tr><td> 试剂名称 </td><td> 公司 </td><td> 目录编号 </td></tr><tr><td> 10X PCRx增强解决方案</td><td> Invitrogen公司</td><td> 52391 </td></tr><tr><td> PfuUltra高保真DNA聚合酶替代洗涤剂</td><td> Stratagene公司</td><td> 600385 </td></tr><tr><td> DPN -我</td><td> New England Biolabs公司</td><td> R0176 </td></tr><tr><td> XL10金Ultracompetent 大肠杆菌大肠杆菌细胞</td><td> Stratagene公司</td><td> 200314 </td></tr><tr><td> DMEM培养液</td><td> Cellgro </td><td> 10 - 013 - CV </td></tr><tr><td>胎牛血清</td><td>亚特兰大生物</td><td> S11550 </td></tr><tr><td> Geneporter转染试剂</td><td> Genlantis </td><td> T201007 </td></tr><tr><td>亮肽素</td><td> Calbiochem </td><td> 108975 </td></tr><tr><td> MG132 </td><td> Calbiochem </td><td> 47491 </td></tr><tr><td> 10xPBS </td><td> Ambion公司</td><td> AM9625 </td></tr><tr><td>蛋白酶抑制剂的鸡尾酒和PMSF </td><td>圣克鲁斯生物技术</td><td> SC - 24948 </td></tr><tr><td>皮尔斯BCA蛋白检测试剂盒</td><td> Thermo Scientific的</td><td> 23225 </td></tr><tr><td>抗- Myc的标签（克隆4A6）琼脂糖共轭</td><td> Millipore公司</td><td> 16-219 </td></tr><tr><td> 2X加载缓冲区</td><td> BioRad公司</td><td> 161-0737 </td></tr><tr><td>标准的Tris - HCl，4-15％的坡度预制凝胶</td><td> BioRad公司</td><td> 345-0028 </td></tr><tr><td>的Immobilon - FL PVDF膜</td><td> Millipore公司</td><td> IPFL00010 </td></tr><tr><td>奥德赛封闭液</td><td> COR Biosciences公司</td><td> 927-40000 </td></tr><tr><td> 10xTBS </td><td> BioRad公司</td><td> 170-6435 </td></tr><tr><td>兔抗Myc抗体</td><td>细胞信号转导</td><td> 2272 </td></tr><tr><td>羊抗兔IRDye 800CW </td><td> COR Biosciences公司</td><td> 926-32211 </td></tr><tr><td>奥德赛成像红外系统</td><td> COR Biosciences公司</td><td></td></tr><tr><td>暂停蛋白酶抑制剂（100X） </td><td> Thermo Scientific的</td><td> 78430 </td></tr></tbody></table>

mutagenesis and analysis of genetic mutations in the gc-rich kiss1 receptor sequence identified in humans with reproductive disorders

kisspeptin受体（KISS1R）确认为青春期的触发和生殖能力的成年 1,2,3调节G蛋白偶联受体。已与iodiopathic hypogonadotropic性腺功能低下症（IHH）1,2和性早熟4的患者确定KISS1R失活的突变。这些突变体的功能研究为我们理解再生产的这种受体的调节，以及背后的机制是至关重要的那些塑造的疾病结果，导致异常KISS1R信令和功能。然而，的高度富含GC序列的KISS1R基因，使得它相当困难的引入突变或扩增PCR这种受体的基因编码。 在这里，我们描述了一种方法，引入利益的突变，这种高度富含GC的序列，已成功地用于产生超过十几KISS1R突变体在我们的实验室。我们已经优化了PCR反应条件，以方便KISS1R包括在KISS1R序列的替换，删除或插入的突变体，放大。的另外一个PCR增强剂解决方案，以及二甲基亚砜的一个很小的比例，尤其是有利于提高放大。这种优化过程可能是有用的，以及其他富含GC的模板。 表达载体编码KISS1R是被用来描述这种受体的信号和功能，以了解如何突变可能会改变KISS1R功能，导致生殖相关的表型。因此，定点突变产生KISS1R的突变体的潜在应用1,4,5,6,7,8许多研究可以说明。作为一个例子，在KISS1R（Arg386Pro），这是与性早熟有关，获得性功能突变已被证明延长受体的反应配体刺激4以及改变的降解率KISS1R 9 。有趣的是，我们的研究表明，KISS1R被蛋白酶体降解，而不是大多数G蛋白偶联受体 9所描述的经典的溶酶体降解。在这里介绍的例子，KISS1R退化是在人胚胎肾细胞（HEK - 293）瞬时表达Myc基因标记KISS1R（MycKISS1R）调查和与蛋白酶或溶酶体抑制剂治疗。使用琼脂糖标记的抗myc抗体免疫印迹分析免疫沉淀的细胞裂解液。使用LI - COR的奥德赛红外线系统进行检测和定量上的污点MycKISS1R。这种方法可能是有用的，以及其他感兴趣的蛋白质降解的研究。

Watch this Scientific Journal Video about Mutagenesis and Analysis of Genetic Mutations in the GC-rich KISS1 Receptor Sequence Identified in Humans with Reproductive Disorders at JoVE.com

Mutagenesis and Analysis of Genetic Mutations in the GC-rich KISS1 Receptor Sequence Identified in Humans with Reproductive Disorders

kisspeptin受体（KISS1R）的突变与患者的生殖系统紊乱。在这里，我们描述如何引入利益的突变的KISS1R富含GC的序列以及使用KISS1R结构特点免疫沉淀和免疫印迹的受体的降解途径

在富含GC KISS1受体序列与人类生殖系统紊乱中确定的诱变和基因突变分析

The kisspeptin receptor (KISS1R) is a G protein-coupled receptor recognized as the trigger of puberty and a regulator of reproductive competence in ...

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