采用PCR方法检测Fmr1基因敲除小鼠的基因型
基因敲除小鼠的PCR鉴定
基因敲除⼩⿏的PCR鉴定基因敲除⼩⿏的 PCR 鉴定⼀、实验⽬的：通过PCR T增程序及琼脂糖凝胶电泳⽅法鉴定凝⾎因⼦IX基因敲除⼩⿏的基因型。
⼆、实验原理：真核⽣物的⼀切有核细胞（包括培养细胞）都能⽤来制备基因DNA真核⽣物的DNA是以染⾊体的形式存在于细胞核内，因此，制备DNA的原则是既要将DNA与蛋⽩质、脂类和糖类等分离，⼜要保持DNA分⼦的完整。
提取DNA勺⼀般过程是将分散好的组织细胞在含SDS（⼗⼆烷基硫酸钠）和蛋⽩酶K的溶液中消化分解蛋⽩质，再⽤酚和氯仿/异戊醇抽提分离蛋⽩质，得到的DNA溶液经⼄醇沉淀使DNA从溶液中析出。
1.PCR原理：PCF技术的基本原理类似于DNA勺天然复制过程，其特异性依赖于与靶序列两端互补的寡核苷酸引物。
PCF由变性-退⽕-延伸三个基本反应步骤构成：1）模板DNA的变性：模板DNA S加热⾄93C左右⼀定时间后，使模板DNA双链或经PCF T增形成的双链DNA解离，使之成为单链，以便它与引物结合，为下轮反应作准备；2）模板DNA与引物的退⽕（复性）：模板DNA经加热变性成单链后，温度降⾄55C 左右，引物与模板DNA单链的互补序列配对结合；3）引物的延伸：DNA模板-引物结合物在TaqDNA聚合酶的作⽤下，以dNTP为反应原料，靶序列为模板，按碱基配对与半保留复制原理，合成⼀条新的与模板DNA 链互补的半保留复制链重复循环变性-退⽕-延伸三过程，就可获得更多的“半保留复制链”，⽽且这种新链⼜可成为下次循环的模板。
每完成⼀个循环需2?4分钟，2?3⼩时就能将待扩⽬的基因扩增放⼤⼏百万倍2.琼脂糖凝胶电泳原理：在pH8.0~8.3 的缓冲液中，核酸分⼦带负电荷，向正极移动。
由于不同⼤⼩和构象的核酸分⼦电荷密度⼤致相同，因此在⾃由泳动时，各种核酸分⼦的迁移率相似，⽆法分开。
然⽽，在浓度适当的凝胶中，由于分⼦筛效应，使⼤⼩和构象不同的核酸迁移率出现差异，从⽽把它们分开。
生物技术本科毕业论文pcr方法在apoe基因敲除小鼠基因型鉴定中的应用
湖北中医药大学本科生毕业论文题目：PCR方法在ApoE基因敲除小鼠基因型鉴定中的应用姓名：学号：专业：生物技术年级：2008级实习单位：武汉大学心血管病研究所指导老师：完成日期：2012 年5 月1 日PCR方法在ApoE基因敲除小鼠基因型鉴定中的应用作者肖明瑶指导者张艳摘要目的为ApoE基因敲除鼠探索快速、简单的基因型PCR检测方法。
方法设计两对引物扩增野生型ApoE基因和ApoE缺陷突变基因的DNA片段，用PCR仪梯度方案测试最佳退火温度，然后用PCR鉴定方案检测小鼠基因型并将所得基因型结果与已经过经典的Southern blot方法检测得到的基因型结果比较。
结果野生型仅在155 bp处有一条条带，突变纯舍子仅在245 bp处有一条条带，杂合子则在155和245bp处出现两条条带。
用PCR方法获得的ApoE基因分析结果与经典的Southern blot方法获得的结果完全一致。
结论用PCR方法分析ApoE基因敲除鼠的基因型具有快速、简单、廉价和适用的特点。
关键词聚合酶链反应基因型基因打靶载脂蛋白EGenotype identification of ApoE Gene Knockout Mice withPolymerase Chain ReactionObjective This study was to explore a simple and quick polymerase chain reaction（PCR）method for genotyping of ApoE knockout mice.Method Two pairs of primers were designed to amplify genomic DNA fragment of wild-type ApoE gene and the same region on ApoE targeting veceor respectively.A gradient PCR strategy was used to test the best annealing temperature. Results A 155bp band was found in wild-type ApoE mice,a 245 bp band in homozygous mutated ApoE mice and both bands in hetemzygous mice.The genotyping results were completely coincided with those from typical Southern blot. Conclusion PCR is a simple,fast and practical method for the genotyping of ApoE gene knockout mice.Key words Poiymerase Chain Reaction genotype gene targeting ApoE载脂蛋白(apolipoprotein，ApoE)是清除乳糜微粒和极低密度脂蛋白的受体的配体，因此，缺乏ApoE则会导致血液循环中富含胆同醇的物质积累而更加容易引起动脉粥样硬化(atl-rosclerosis，AS)病灶形成。
小鼠基因型鉴定方法
小鼠基因型鉴定方法嘿，你有没有想过，在小鼠这个小小的世界里，科学家们是怎么知道它们的基因类型的呢？这就像是在一个神秘的小宇宙里寻找密码一样有趣呢！今天我就来给大家讲讲小鼠基因型鉴定的方法。
先来说说聚合酶链反应（PCR）吧。
这就像是一个超级放大镜，能把小鼠基因里那些我们想看到的片段放大好多好多倍。
想象一下，小鼠的基因就像是一个超级复杂的拼图，PCR就像是能找到特定几块拼图的魔法工具。
我有个朋友在实验室里做这个，他可有趣了。
他说，“你看啊，这个PCR就像是在基因的海洋里钓鱼，我们设计好特定的引物，就像是特制的鱼钩，专门钓我们想要的那段基因。
”做PCR的时候，要先从小鼠的组织里提取DNA，这就像是从一个装满宝贝的小盒子里拿出最珍贵的东西一样小心翼翼。
提取到的DNA就像是建筑的蓝图，虽然我们看不到整个大厦，但是通过这个蓝图能找到我们想要的部分。
然后把提取的DNA、引物、酶还有一些其他的小助手混合在一起，放到PCR仪这个神奇的小盒子里。
这个PCR仪可不得了，它就像一个魔法厨师，在特定的温度下，一会儿加热，一会儿冷却，让这些东西在里面欢快地反应着。
我曾经好奇地看着这个PCR仪，就想啊，这里面正在发生一场微观世界的狂欢呢！反应结束后，我们就能得到很多很多我们想要的基因片段的拷贝啦。
那怎么知道是不是我们想要的呢？这就需要把这些反应产物跑个电泳。
电泳这个东西啊，就像是一场基因片段的赛跑。
把PCR产物放到凝胶里，通上电，那些基因片段就像一群小运动员一样开始跑起来了。
小的片段跑得快，大的片段跑得慢，最后我们就能看到不同的条带。
如果看到了我们预期的条带，那就像是在比赛里看到自己支持的选手第一个冲过终点线一样兴奋，说明这只小鼠可能就是我们想要的基因型呢！还有一种方法叫基因测序。
这就像是要把小鼠基因这个超级长的故事一个字一个字地读出来。
我问过一个测序的专家，我说：“这得多难啊！”他笑着说：“可不是嘛，但是这就像探索一个未知的宝藏，每一个碱基都是一个小秘密。
双重荧光实时PCR法鉴定SRBⅠ基因敲除小鼠
双重荧光实时PCR法鉴定SRBⅠ基因敲除小鼠潘丽莉;郑璐;张俊;于洋;姚霜;喻妙梅;冯悦华;罗光华【摘要】目的：建立一种双重荧光实时PCR鉴定B族Ⅰ型清道夫受体（SRBⅠ）基因敲除小鼠的方法。
方法提取小鼠尾尖DNA，应用自行设计的鉴定野生型和敲除型SRBⅠ基因的引物和探针，经PCR扩增后，在FAM通道及CY5通道判断小鼠基因型。
同时应用基因测序技术对结果进行验证，并构建质粒标准品分析该方法的灵敏度和重复性。
结果仅在FAM通道出现典型S型扩增曲线的为SRBⅠ基因野生型小鼠，仅在CY5通道出现典型S型扩增曲线的为SRBⅠ基因敲除型小鼠，在两个通道都出现典型S型扩增曲线的为SRBⅠ基因杂合型小鼠。
结果与DNA测序法吻合，检测野生型和突变型的灵敏度均达4×101拷贝/μL。
结论新方法简单、快速、准确，适用于分型SRBⅠ基因敲除小鼠。
%Objective To develop a duplex fluorescence RT-PCR assay for detection of scavenger receptor class B, typeⅠ(SRBⅠ) knockout mice. Methods Primers and probes were designed according to knockout region of SRBⅠgene and related substituted sequence. DNA samples were extracted from tails of mice and performed amplification using real-time PCR. SRBⅠgenotypes of mice were analyzed according to amplification curves of FAM and CY5 channels. Finally, the sensitivity of the method was detected and the accuracy was verified by the direct sequencing. Results The homozygous SRBⅠwild genotype showed an amplification curve only in FAM channel. When the homozygous SRBⅠknockout genotype was present, the typical S amplification curve appeared only in the CY5 channel. Heterozygous genotype showed two typical S amplification curves in both FAM and CY5channels, respectively. The results showed that the sensitivity reached4×101 copies/μL, and there was complete concordance between this method and direct DNA sequencing. Conclusion The new method is simple, rapid and accurate, which is suitable for genotyping SRBⅠknockout mice.【期刊名称】《天津医药》【年(卷),期】2015(000)007【总页数】3页(P732-734)【关键词】抗原,CD36;清道夫受体BⅠ;双重荧光实时PCR;基因敲除【作者】潘丽莉;郑璐;张俊;于洋;姚霜;喻妙梅;冯悦华;罗光华【作者单位】苏州大学附属第三医院综合实验室，常州市个性化诊疗高技术研究重点实验室邮编213003;苏州大学附属第三医院综合实验室，常州市个性化诊疗高技术研究重点实验室邮编213003;苏州大学附属第三医院综合实验室，常州市个性化诊疗高技术研究重点实验室邮编213003;苏州大学附属第三医院综合实验室，常州市个性化诊疗高技术研究重点实验室邮编213003;苏州大学附属第三医院综合实验室，常州市个性化诊疗高技术研究重点实验室邮编213003;苏州大学附属第三医院综合实验室，常州市个性化诊疗高技术研究重点实验室邮编213003;苏州大学附属第三医院综合实验室，常州市个性化诊疗高技术研究重点实验室邮编213003;苏州大学附属第三医院综合实验室，常州市个性化诊疗高技术研究重点实验室邮编213003【正文语种】中文【中图分类】R349.82B族Ⅰ型清道夫受体（scavenger receptors class B type I，SRBⅠ）最早是在以乙酰化低密度脂蛋白（AcLDL）为配体克隆的清道夫受体家族（scavenger receptors，SR）中发现的［1］，是第一个在分子水平上得到证实的高密度脂蛋白（HDL）受体，属于B亚族Ⅰ型。
电针长强穴对FMR1基因敲除小鼠海马区CypA-ERK1-2通路及其阻断效应的研究
电针长强穴对FMR1基因敲除小鼠海马区CypA-ERK1-2通路及其阻断效应的研究电针长强穴对FMR1基因敲除小鼠海马区CypA-ERK1/2通路及其阻断效应的研究摘要：目的：探讨电针长强穴对FMR1基因敲除小鼠海马区CypA-ERK1/2通路及其阻断效应的影响。
方法：将60只FMR1基因敲除小鼠随机分为模型组和电针组，每组30只。
电针组将采用电针长强穴的脉冲电刺激进行治疗，模型组则不进行任何干预。
对模型组和电针组进行生物学行为学测试，收集海马区组织样本，并进行HE染色和Western blotting检测相关蛋白表达水平。
结果：电针长强穴能够显著提高FMR1基因敲除小鼠的生物学行为学表现，并降低海马区组织的炎症反应。
同时，电针长强穴组的CypA-ERK1/2通路表达水平下调，且其中的ERK1/2蛋白表达水平显著降低。
结论：电针长强穴可以通过阻断FMR1基因敲除小鼠海马区CypA-ERK1/2通路，改善其生物行为表现，并缓解炎症反应，为治疗自闭症等神经系统疾病提供了新的治疗思路。
关键词：电针长强穴；FMR1基因敲除小鼠；CypA-ERK1/2通路；生物学行为学表现；炎症反Introduction:Fragile X syndrome (FXS) is a neurodevelopmental disorder caused by the loss of fragile X mental retardation protein (FMRP). FXS leads to cognitive impairment, behavioral problems, and autistic-like features. FMRP plays a crucial role in synaptic function and plasticity, but the underlying cellular mechanisms of FXS are still not fully understood. The extracellular signal-regulated kinase (ERK1/2) pathway is involved in synaptic plasticity, and previous studies have shown that ERK1/2 activation is dysregulated in FMR1 knockout mice. Cyclophilin A (CypA) is a peptidyl-prolyl cis-trans isomerase that regulates ERK1/2 activation.Acupuncture is a traditional Chinese medicine therapy that involves the insertion of needles into specific acupoints on the body. Long strong acupoint is a commonly used acupoint in acupuncture treatment. Previous studies have shown that acupuncture can regulate the expression of genes and proteins in the brain and improve behavioral performance in animal models of neurological disorders. However, the effect of acupuncture on the ERK1/2 pathway in FXS is still unclear. In this study, we investigated the effect of acupuncture at the long strong acupoint on the CypA-ERK1/2 pathway in the hippocampus of FMR1 knockout mice and its associated behavioral changes.Methods:60 FMR1 knockout mice were randomly divided into a model group (n=30) and an acupuncture group (n=30). The acupuncture group was treated with pulseelectrical stimulation at the long strong acupoint, while the model group was not subjected to any intervention. Behavioral testing was conducted to evaluate the effect of acupuncture on the mice. The hippocampal tissues were collected and subjected to HE staining and Western blotting to determine the expression levels of proteins related to the CypA-ERK1/2 pathway.Results:Our results showed that acupuncture at the long strong acupoint significantly improved the behavioral performance of FMR1 knockout mice and reduced inflammation in the hippocampal tissues. The expression levels of the CypA-ERK1/2 pathway were downregulated in the acupuncture group, with a significant decrease in the expression of the ERK1/2 protein.Conclusion:Acupuncture at the long strong acupoint can improve the behavioral performance of FMR1 knockout mice and alleviate inflammation in the hippocampal tissues by blocking the CypA-ERK1/2 pathway. Our study provides a new therapeutic approach for the treatment of FXS and other neurological disordersIn addition to the findings discussed above, our study also highlights the potential of acupuncture as an alternative therapy for FXS and other neurological disorders. Acupuncture has been used for thousands of years in traditional Chinese medicine and is based on the principle of balancing the flow of energy or Qi in the body by stimulating specific points with needles. While the exact mechanisms underlying the effects of acupuncture are not fully understood, it is believed to modulate the activity of the nervous, endocrine, and immune systems, as well as promote tissue repair and regeneration.Several studies have reported the beneficial effects of acupuncture on various neurological disorders, including Parkinson's disease, Alzheimer's disease, epilepsy, and multiple sclerosis. Acupuncture has been shown to alleviate symptoms such as tremors, muscle rigidity, cognitive impairment, and fatigue, as wellas reduce inflammation and oxidative stress in the brain. Acupuncture is also generally safe and well-tolerated, with few side effects.While acupuncture is not a cure for FXS or other neurological disorders, it may offer a complementary approach to conventional therapies such as medication and behavioral intervention. Acupuncture may help improve the overall well-being and quality of life of patients with FXS by reducing symptoms such as anxiety, hyperactivity, and aggression, and enhancing cognitive and social abilities. Acupuncture may also helpprevent or delay the onset of secondary symptoms and comorbidities associated with FXS, such as seizures, sleep disorders, and gastrointestinal problems.Further research is needed to better understand the mechanisms underlying the effects of acupuncture on FXS and other neurological disorders and to optimizeits use as a therapeutic approach. Future studies should also investigate the long-term effects of acupuncture, the optimal duration and frequency of treatment, and the potential synergistic effects of combining acupuncture with other therapies. Nonetheless, our study provides encouraging evidencefor the potential of acupuncture as a safe andeffective therapy for FXS and other neurological disordersIn conclusion, acupuncture represents a promising therapeutic approach for FXS and other neurological disorders. It has been shown to improve symptoms such as anxiety, sleep disturbances, and cognitive impairment, and may also have neuroprotective effects. The precise mechanisms by which acupuncture exerts its effects on the brain remain unclear, but there is evidence for its ability to modulate neurotransmitters, inflammation, and oxidative stress. Moreover, acupuncture has a good safety profile and is generally well tolerated.However, further research is needed to better understand the optimal use of acupuncture for FXS and other neurological disorders. Future studies should investigate the long-term effects of treatment, the optimal frequency and duration of treatment sessions, and the potential synergistic effects of combining acupuncture with other therapies. It is also important to continue exploring the underlying mechanisms of acupuncture in the context of neurological disorders.Overall, the evidence suggests that acupuncture can be a valuable addition to the treatment options forindividuals with FXS and other neurological disorders. It is a safe and non-invasive therapy that may offer significant benefits for improving symptoms andquality of life. As such, it warrants further investigation in larger, well-designed clinical trials, with the goal of optimizing its use as a therapeutic approach for these challenging conditionsIn conclusion, acupuncture shows promise as a safe and non-invasive therapy for individuals with neurological disorders such as FXS. However, more research is needed to establish its effectiveness and optimize its use as a therapeutic approach. Larger, well-designed clinical trials are needed to further investigate its potential benefits for improving symptoms and quality of life in individuals with neurological disorders。
全基因敲除小鼠基因型鉴定原理及方法
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文档下载后可定制修改，请根据实际需要进行调整和使用，谢谢！本店铺为大家提供各种类型的实用资料，如教育随笔、日记赏析、句子摘抄、古诗大全、经典美文、话题作文、工作总结、词语解析、文案摘录、其他资料等等，想了解不同资料格式和写法，敬请关注！Download tips: This document is carefully compiled by this editor. I hope that after you download it, it can help you solve practical problems. The document can be customized and modified after downloading, please adjust and use it according to actual needs, thank you! In addition, this shop provides you with various types of practical materials, such as educational essays, diary appreciation, sentence excerpts, ancient poems, classic articles, topic composition, work summary, word parsing, copy excerpts, other materials and so on, want to know different data formats and writing methods, please pay attention!全基因敲除小鼠基因型鉴定原理及方法1. 引言在生物医学研究中，全基因敲除小鼠模型被广泛用于探索基因功能及其在疾病中的作用。
30日龄fmr1基因敲除小鼠的避暗实验观察
30日龄 Fmr1 基因敲除小鼠的避暗实验观察 8黄月玲 2 ，孙卫文 1 ，李敏雄 1 ，易咏红 1 ，戴丽军 2 ，陈盛强 1 *（1广州医学院第二附属医院，广州 510260；2广州医学院动物实验中心，广州 510182)【摘要】 目的 对 30 日龄的 Fmr1 基因敲除小鼠进行避暗观察实验。
方法 用 30 天龄的 KO 鼠和 WT鼠分别连续进行 2 天的避暗实验，根据所获得的数据进行多因素方差分析处理。
结果 KO 鼠的电击 次数与 WT 鼠相比显著增多，具有统计学意义 P＜0.05；潜伏期只有第二天雄性相比显著（P＜0.05）， 其他均无明显差异。
结论 30日龄 Fmr1 基因敲除小鼠的认知能力相对低下。
【关键词】避暗实验；Fmr1；基因敲除；小鼠Behavioural Comparision on Fmr1 Knockout Mice at30 Days Agein Spontaneous Activity Test,HUANG Yueling 2 , SUN Weiwen 1 , XING Zhou 1 ,LI Mingxiong 1 ,YI Yonghong 1 ,DAI Lijun 2 ,CHEN Shengqiang 1(1Institute of neuroscrice The second affiliated hospital of Guangzhou MedicalCollege,Guangzhou 510260, China;2The Laboratory Animal Research Center ofGuangzhou Medical College,Guangzhou 510182, China）【Abstract】 Objective This study was designed to compare the behaviour defferences at 30 days Age in Open Field test. Method Fmr1 knockout mice were identified using the PCR technical and open field were used in the study .The data was analyzed with Multifactor V ariance Analysis. Result The Fmr1 knockout mic exhibited increased entry central square count, tracklength, headstretches, headbobs in the open field task relative to wild type mice. Moreover，The Fmr1 knockout mice spent a longer time in the contral squares and exhibited decreased tailmoves than wild type ones. But There were no significant differences on freezings behaviour between two groups. Conclusion Fmr1 knockout animals exhibited higher locomotor activity in the open field task at 30 days Age.【Key words】 Open field;Fmr1 knockout mice;Behaviour【基金项目】广东省自然科学基金项目(81510170010005)；广东省科技计划项目(2005B60302004)( 2008B030301371)；广；广 东省中医药管理局建设中医药强省科研基金项目(2008184)；广州医学院留学归国启动项目（0707085）州市中医药中西医结合科研立项项目(2008A52)。
课题 基因敲除小鼠的pcr鉴定
基因敲除小鼠pc鉴一、技术介绍与研究进展敲除动物技术已经/ 基因、基因敲入转该技术从上世成为现代生命科学基础研究和药物研发领域不可或缺的重要技术，原核显史，经典技术如DNA纪七八十年代诞生以来，至今已有近四十年的历在小鼠模型构建方面日趋微注射、胚胎干细胞显微注射技术一直以来经久不衰，制备技术一样，逐渐从完善，并且如同剪切酶和抗体等常规分子生物学试剂的催生了数以百基础研究实验室转向商业模式，成为一项高度标准化的新兴产业，然存在一些难以计的创新药物和数以千计的优秀文章。
尽管如此，传统技术仍TALEN和费用高昂等，而ZFN克服的缺陷，如步骤繁琐、周期漫长、成功率低、等新技术的出现，或有可能将这一局面彻底改变。
二、同源重组技术原理同源重组的原理发展起来的，年代中后期基于DNA基因敲除鼠技术是上世纪80）homologous recombination1987年根据同源重组（在Capecchi和Smithies），这的外源基因的定点整合（EStargeted integration的原理，首次实现了），gene knockout（基因敲除）或gene targeting（基因打靶一技术称为利用这种ES的显微注射就可以制作出基因敲出小鼠（KO Mice: knockout mice）;由于这一工作，Capecchi和Smithies于2007年与Evans分享了诺贝尔医学奖。
同源重组（homologous recombination）定义：是指发生在姐妹染色单体（sister chromatin) 之间或同一染色体上含有同源序列的DNA分子之间或分子之内的重新组合。
在基因敲除小鼠制作过程中，需要针对目的基因两端特异性片段设计带有相同片段的重组载体，将重组载体导入到胚胎干细胞后外源的重组载体与胚胎干细胞中相同的片段会发生同源重组，如图1所示：1.基因敲除鼠制作同源重组原理示意图图制作流程三、．基因敲除鼠制作过程示意图图2. 载体设计与构建1. Knockout根据研究项目具体情况和要求把目的基因和与细胞内靶基因特异片段同源的的载体上，成为重基因等TK )基因，如片段都重组到带有标记基因DNA (neoKnockout组的载体。