GeneCopeia提供病毒和非病毒载体的人、小鼠和大鼠全基因组OmicsLink™ shRNA表达克隆,shRNA序列经专利算法设计包括茎(19-29对核苷酸)和环,确保高效的抑制作用和最低的脱靶效应。
shRNA抑制效率保证
对于构建到慢病毒和哺乳动物载体的shRNA克隆,我们针对每个基因提供4个克隆,保证至少有1个克隆通过qRT-PCR检测对靶基因的表达抑制水平达到70%以上。
靶向任意细胞
使用慢病毒和非病毒载体系统,既可以转染或转导shRNA进入常规的分裂细胞,也可以进入难转染的非分裂细胞,如原代细胞、神经元细胞等。
灵活的载体选择
每个shRNA克隆提供5 µg纯化质粒,也提供客户定制的慢病毒载体的shRNA克隆。定制该克隆,请填写表格。
标签及报告基因
载体中带有的mCherry和eGFP报告基因可以用于监测转染和转导效率,嘌呤霉素标记可用于稳定细胞系的筛选。
全长测序的表达框架
载体的插入序列已经过测序,包括启动子、编码shRNA的正义和反义链、发夹结构、终止子和其他接头序列。
图1. shRNA克隆的病毒和非病毒表达载体
产品应用 |
基因表达下调 — 以包含无义序列的克隆为对照,研究shRNA克隆对单个基因表达的下调效果。在每个shRNA克隆订单中赠送对照克隆。
信号通路研究 — 我们将克隆按照不同的信号传导、代谢、疾病通路、基因家族等进行了分类,并将已知同一通路的shRNA表达克隆以96孔或384孔板的形式排列在一起,可一次研究一组基因在某一信号通路中的功能。目前我们提供针对人类全部激酶基因的shRNA表达克隆,并已按照按不同激酶的功能进行了分类。
靶点及功能验证 — OmicsLink™ ORF表达克隆和在shRNA靶序列带有沉默突变的ORF表达克隆一起使用可用于shRNA靶点验证和基因功能拯救研究。
图2. shRNA抑制基因表达的机制
shRNA表达克隆对基因表达抑制效应的检测方法 |
qRT-PCR — shRNA克隆的表达能导致靶标基因所转录的mRNA被切割和降解,从而降低蛋白的表达量。所以推荐检测表达抑制效率的第一步是qRT-PCR。GeneCopeia提供验证过的对应靶标基因和其他已报导的转录本的qRT-PCR引物。
Western Blot检测 — 大部分情况下,检测shRNA对基因表达的抑制效率需要用Western Blot方法来检测蛋白表达水平。
功能检测 — 检测表达抑制效率还可用生物和生化的终点分析方法,如细胞增殖、克隆形成、细胞周期、细胞迁移等。
报告基因检测表达水平 — 当研究的靶标基因内源转录水平很低,且不适宜使用qRT-PCR和WB检测时,表达抑制效率的检测也可通过共转导shRNA克隆和一个含靶标基因并带了报告基因的表达克隆,检测报告基因的表达水平即可。靶基因的mRNA遭shRNA的破坏,同时导致了报告基因的mRNA遭破坏,从而表现为报告蛋白的表达量下降。因而表达抑制效率可以用报告蛋白的酶活性分析结果来进行量化。
图3. shRNA(psi-sH1载体)对人激酶家族成员表达抑制效率的验证。psiCheck-AP载体以CMV为启动子,包括靶标激酶的ORF区和用于比色实验的碱性磷酸酶(AP)基因。A. 通过比色实验量化AP在没有shRNA的情况下的表达。B. 共转染shRNA和AP ORF表达克隆后,AP ORF的转录本被降解,AP基因翻译受阻导致荧光消失。
OmicsLink shRNA表达克隆载体 
载体 | 启动子 | 筛选标记 | 报告基因 | 病毒类型 |
psi-H1 | H1 | Puromycin | eGFP | N/A |
psi-U6 | U6 | Puromycin | eGFP | N/A |
psi-mH1 | H1 | Puromycin | mCherry | N/A |
psi-mU6 | U6 | Puromycin | mCherry | N/A |
psi-nH1 | H1 | Puromycin | N/A | N/A |
psi-nU6 | U6 | Puromycin | N/A | N/A |
psi-LVRH1GP | H1 | Puromycin | eGFP | HIV |
psi-LVRU6GP | U6 | Puromycin | eGFP | HIV |
psi-LVRH1MP | H1 | Puromycin | mCherry | HIV |
psi-LVRU6MP | U6 | Puromycin | mCherry | HIV |
psi-LVRH1P | H1 | Puromycin | N/A | HIV |
psi-LVRU6P | U6 | Puromycin | N/A | HIV |
psi-LVRU6GH | U6 | Hygromycin | EGFP | HIV |
psi-LVRH1GH | H1 | Hygromycin | EGFP | HIV |
psi-LVRU6MH | U6 | Hygromycin | mcherry | HIV |
psi-LVRH1MH | H1 | Hygromycin | mcherry | HIV |
psi-LVRU6H | U6 | Hygromycin | N/A | HIV |
psi-LVRH1H | H1 | Hygromycin | N/A | HIV |
shRNA表达克隆客户定制服务
预制的shRNA克隆套装
GeneCopeia提供载体可选的超过24,000条人、20,000条小鼠和10,000条大鼠预制的shRNA克隆套装。
慢病毒载体的shRNA表达克隆
GeneCopoeia 提供定制慢病毒载体的shRNA表达克隆的服务, 浏览价格信息,请填写表格。
shRNA表达克隆定制服务
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shRNA Clones Cancellation, QA, License, and Warranty |
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