CN110257426A - 一种研究鸡ESCs向PGCs分化过程中lin28调节Blimp1表达的方法 - Google Patents

一种研究鸡ESCs向PGCs分化过程中lin28调节Blimp1表达的方法 Download PDF

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CN110257426A
CN110257426A CN201910547131.4A CN201910547131A CN110257426A CN 110257426 A CN110257426 A CN 110257426A CN 201910547131 A CN201910547131 A CN 201910547131A CN 110257426 A CN110257426 A CN 110257426A
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pgcs
micrna
let7
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李碧春
姜景译
左其生
王曼
李建成
张明
石祥
袁霞
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Abstract

本发明涉及一种研究鸡ESCs向PGCs分化过程中lin28调节Blimp1表达的方法，属于生物技术领域。本发明将Lin28通过micRNA‑let7间接调控PGCs标记基因Blimp1的研究扩展到家禽领域，并准确定位到gga‑let‑7a‑2‑3p上，刷新了不同物种的Lin28‑let7‑Blimp1调节链的功能范畴。本方法简单易行，思路清晰，操作可行性高，在普通研究室就能完成。

Description

一种研究鸡ESCs向PGCs分化过程中lin28调节Blimp1表达的 方法
技术领域
本发明涉及一种研究鸡ESCs向PGCs分化过程中lin28调节Blimp1表达的方法,具体是一种研究鸡ESCs向PGCs分化过程中lin28靶向gga-let7a-2-3p调节Blimp1表达的方法，属于生物技术领域。
背景技术
自West等在2009年发现Lin28是一个潜在的PGCs发育的关键调控因子以来，研究者们便开始对其具体的调控机制进行研究。已有研究发现Lin28A/B作为RNA结合蛋白，可以通过调控let-7 microRNA的成熟从而调节其他基因的表达参与多种生物过程。
目前Lin28s在肿瘤发生和细胞多潜能性方面的研究较多，在生殖细胞发育中的作用机制研究较少，哺乳动物中已有少量研究报道Lin28可以通过抑制let-7microRNA成熟促进其靶基因Blimp1的表达，调控PGCs的形成。然而到目前为止，在家禽上未有报道，且仍未找到参与家禽PGCs生成的关键let-7 microRNA。
发明内容
本发明的目的是针对上述现有技术的不足，提供一种研究鸡ESCs向PGCs分化过程中lin28调节Blimp1表达的方法,具体是一种研究鸡ESCs向PGCs分化过程中lin28靶向gga-let7a-2-3p调节Blimp1表达的方法。
本发明的技术方案如下：
一种研究鸡ESCs向PGCs分化过程中lin28调节Blimp1表达的方法,其特征是,包括以下步骤:
通过在线软件筛选鸡的micRNA-let7s成熟体，并在ESCs、PGCs和DF1三种细胞中共同干扰或过表达Lin28A/B，通过qRT-PCR筛选出关键候选的micRNA-let7；
进一步通过在线软件预测候选micRNA-let7的靶基因，并筛选其中参与PGCs形成的关键靶基因；
构建micRNA-let7模拟物和抑制物，在PGCs和DF1细胞中通过qRT-PCR检测micRNA-let7对其靶基因的调控作用；
对靶基因3’UTR区域的micRNA-let7结合位点进行缺失，构建了靶基因3’UTR野生型和突变型载体，通过双荧光素酶报告系统验证micRNA-let7对靶基因的靶向作用。
本发明的有益效果如下：
本方法简单易行，思路清晰，操作可行性高，在普通研究室就能完成。实验者两个月就能完成。本发明将Lin28通过micRNA-let7间接调控PGCs标记基因Blimp1的研究扩展到家禽领域，并准确定位到gga-let-7a-2-3p上，刷新了不同物种的Lin28-let7-Blimp1调节链的功能范畴。
具体实施方式
1.Lin28A/B靶向的gga-let7s筛选
利用在线软件http://mirdb.org/cgi-bin/search.cgi预测鸡micRNA Let7s，共筛选到17个gga-let7s。
分别选取生长状态良好的DF1细胞、ESCs和PGCs，各分3组，一组转染siLin28A/B，一组转染oeLin28A/B，一组不做处理。其中DF1细胞在完全培养基中进行转染，ESCs和PGCs在因子培养基中进行转染。于37℃，5％CO2饱和湿度下培养48h后，取样提总RNA。
按照miRNA提取分离试剂盒操作程序提取总RNA，按照反转录试剂盒操作程序反转录合成cDNA，设计micRNA-let7s定量引物并以U6为内参检测检测micRNA-let7s相对表达量变化。qRT-PCR反应参考miRNA荧光定量检测试剂盒说明书，具体体系为：cDNA 50ng；2×miRucte Plus miRNA PreMix 10ul；上游引物0.4ul；Reverse Primer 0.4ul；ddH2O补足至总体积为20ul。PCR反应程序为：95℃15min预变性；94℃20s，63℃30s，72℃34s，5循环；94℃20s，60℃34s退火/延伸，40循环；标准溶解曲线分析。
2.gga-let-7a-2-3p靶基因筛选
利用在线软件http://mirdb.org/cgi-bin/search.cgi预测gga-let-7a-2-3p的靶基因，如表1所示，接着根据Target Score由高到低进行热谱分析，对target score在95分以上的基因进行筛选，筛选出PGCs标记基因Blimp1(PRDM1)。
表1miRDB预测的gga-let-7a-2-3p的靶基因(部分)
表1续
3.检测gga-let-7a-2-3p对靶基因的靶向作用
a)gga-let-7a-2-3p模拟物、抑制物合成及效率验证
设计并合成gga-let-7a-2-3p模拟物、抑制物，并验证其效率。
b)Blimp1 3’UTR野生型和突变型载体构建
通过生物信息学对gga-let-7a-2-3p和Blimp1-3’UTR区域的分析，预测gga-let-7a-2-3p在Blimp1 3’UTR区域的结合位点，结合序列为“UUGUACA”，将这些结合位点进行全部缺失，构建Blimp1 3’UTR野生型和突变型载体，基因序列合成由武汉金开瑞生物工程有限公司完成。之后，将Blimp1 3’UTR野生型和突变型基因序列连接至pMIR-reportLuciferase载体上，经HindIII和Sac I双酶切和测序鉴定载体构建成功。构建成功的野生型命名为Blimp1-3'UTR-WT，突变型命名为Blimp1-3'UTR-Mut。
c)双荧光素报告基因检测gga-let-7a-2-3p对Blimp1的靶向作用
以双荧光素酶报告基因检测系统检测gga-let-7a-2-3p对Blimp1的靶向作用，即将Blimp1-3'UTR-WT或Blimp1-3'UTR-Mut和PRL-TK以质量体积比10:1的比例共转染DF1细胞，并在此基础上添加gga-let-7a-2-3p模拟物或抑制物，同时设置阴性对照。
具体实验步骤：
提前一天以2×105/孔不含抗生素的DF1细胞接种24孔板，当DF1细胞覆盖率达到50％～60％时，用50ul Opti-MEM稀释模拟物(或抑制物，转染细胞的终浓度为50uM)、Blimp1-3'UTR-WT(或Blimp1-3'UTR-Mut，转染细胞的总质量为1ug)和PRL-TK，轻轻混匀作为A液；
用50ul Opti-MEM稀释4ul FuGENE HD转染试剂，轻轻混匀，室温静置5min作为B液。将A液和B液混合后轻轻吹3-5次混匀，室温下静置20min，37℃孵育10～15min。将混合液缓缓加入细胞培养孔内混匀，加入400μL完全培养基，于37℃、5％CO2恒温培养箱中培养。
每次转染3个孔，重复3次，转染48h后收集细胞，每管加70ul细胞裂解液，轻轻混匀，每孔细胞中加入等体积即70μL荧光液，轻轻混匀，放入酶标仪中读取萤火虫荧光值，然后加入70μL STOP终止液，轻轻吹打混匀，再放入酶标仪中读取海肾荧光值，记录3次读取结果。具体操作参照Promega公司双荧光素酶报告基因检测试剂盒说明书。相对荧光活性的数值为3次重复试验结果的“平均值±标准差”。

Claims (1)

1.一种研究鸡ESCs向PGCs分化过程中lin28靶向gga-let7a-2-3p调节Blimp1表达的方法,其特征是,包括以下步骤:
通过在线软件筛选鸡的micRNA-let7s成熟体，并在ESCs、PGCs和DF1三种细胞中共同干扰或过表达Lin28A/B，通过qRT-PCR筛选出关键候选的micRNA-let7；
进一步通过在线软件预测候选micRNA-let7的靶基因，并筛选其中参与PGCs形成的关键靶基因；
构建micRNA-let7模拟物和抑制物，在PGCs和DF1细胞中通过qRT-PCR检测micRNA-let7对其靶基因的调控作用；
对靶基因 3’UTR区域的micRNA-let7结合位点进行缺失，构建了靶基因3’UTR野生型和突变型载体，通过双荧光素酶报告系统验证micRNA-let7对靶基因的靶向作用。
CN201910547131.4A 2019-06-24 2019-06-24 一种研究鸡ESCs向PGCs分化过程中lin28调节Blimp1表达的方法 Pending CN110257426A (zh)

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Citations (2)

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US20140328858A1 (en) * 2011-03-25 2014-11-06 Children's Medical Center Corporation Lin28-mediated control of let-7 biogenesis
US20140363467A1 (en) * 2011-06-10 2014-12-11 University Of Georgia Research Foundation, Inc. Avian induced pluripotent stem cells and their use

Patent Citations (2)

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US20140328858A1 (en) * 2011-03-25 2014-11-06 Children's Medical Center Corporation Lin28-mediated control of let-7 biogenesis
US20140363467A1 (en) * 2011-06-10 2014-12-11 University Of Georgia Research Foundation, Inc. Avian induced pluripotent stem cells and their use

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JASON A. WEST等: "A role for Lin28 in primordial germ-cell development and germ-cell malignancy", 《NATURE》 *
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Application publication date: 20190920