CN108929863A - Mettl3基因敲除的细胞系及其构建方法与干扰载体 - Google Patents

Mettl3基因敲除的细胞系及其构建方法与干扰载体 Download PDF

Info

Publication number
CN108929863A
CN108929863A CN201810733097.5A CN201810733097A CN108929863A CN 108929863 A CN108929863 A CN 108929863A CN 201810733097 A CN201810733097 A CN 201810733097A CN 108929863 A CN108929863 A CN 108929863A
Authority
CN
China
Prior art keywords
mettl3
gene
shrna
fatty acid
acid absorption
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Granted
Application number
CN201810733097.5A
Other languages
English (en)
Other versions
CN108929863B (zh
Inventor
宗鑫
汪以真
赵婧
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Zhejiang University ZJU
Original Assignee
Zhejiang University ZJU
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Zhejiang University ZJU filed Critical Zhejiang University ZJU
Priority to CN201810733097.5A priority Critical patent/CN108929863B/zh
Priority to PCT/CN2018/096554 priority patent/WO2020006787A1/zh
Priority to US17/417,764 priority patent/US11339370B2/en
Publication of CN108929863A publication Critical patent/CN108929863A/zh
Application granted granted Critical
Publication of CN108929863B publication Critical patent/CN108929863B/zh
Active legal-status Critical Current
Anticipated expiration legal-status Critical

Links

Classifications

    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/113—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing
    • C12N15/1137—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing against enzymes
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/10—Transferases (2.)
    • C12N9/1003—Transferases (2.) transferring one-carbon groups (2.1)
    • C12N9/1007—Methyltransferases (general) (2.1.1.)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/85—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells
    • C12N15/86—Viral vectors
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/06—Animal cells or tissues; Human cells or tissues
    • C12N5/0602—Vertebrate cells
    • C12N5/0625—Epidermal cells, skin cells; Cells of the oral mucosa
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/06—Animal cells or tissues; Human cells or tissues
    • C12N5/0602—Vertebrate cells
    • C12N5/0679—Cells of the gastro-intestinal tract
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y201/00—Transferases transferring one-carbon groups (2.1)
    • C12Y201/01—Methyltransferases (2.1.1)
    • C12Y201/01062—Methyltransferases (2.1.1) mRNA (2'-O-methyladenosine-N6-)-methyltransferase (2.1.1.62)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2310/00—Structure or type of the nucleic acid
    • C12N2310/10—Type of nucleic acid
    • C12N2310/14—Type of nucleic acid interfering N.A.
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2310/00—Structure or type of the nucleic acid
    • C12N2310/50—Physical structure
    • C12N2310/53—Physical structure partially self-complementary or closed
    • C12N2310/531—Stem-loop; Hairpin
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2510/00—Genetically modified cells
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2740/00—Reverse transcribing RNA viruses
    • C12N2740/00011—Details
    • C12N2740/10011—Retroviridae
    • C12N2740/15011—Lentivirus, not HIV, e.g. FIV, SIV
    • C12N2740/15041—Use of virus, viral particle or viral elements as a vector
    • C12N2740/15043—Use of virus, viral particle or viral elements as a vector viral genome or elements thereof as genetic vector
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2740/00—Reverse transcribing RNA viruses
    • C12N2740/00011—Details
    • C12N2740/10011—Retroviridae
    • C12N2740/16011—Human Immunodeficiency Virus, HIV
    • C12N2740/16041—Use of virus, viral particle or viral elements as a vector
    • C12N2740/16043—Use of virus, viral particle or viral elements as a vector viral genome or elements thereof as genetic vector

Landscapes

  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Genetics & Genomics (AREA)
  • Biomedical Technology (AREA)
  • Chemical & Material Sciences (AREA)
  • Organic Chemistry (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Wood Science & Technology (AREA)
  • Zoology (AREA)
  • Biotechnology (AREA)
  • General Engineering & Computer Science (AREA)
  • Biochemistry (AREA)
  • General Health & Medical Sciences (AREA)
  • Microbiology (AREA)
  • Molecular Biology (AREA)
  • Physics & Mathematics (AREA)
  • Virology (AREA)
  • Biophysics (AREA)
  • Plant Pathology (AREA)
  • Cell Biology (AREA)
  • Dermatology (AREA)
  • Medicinal Chemistry (AREA)
  • Gastroenterology & Hepatology (AREA)
  • Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
  • Micro-Organisms Or Cultivation Processes Thereof (AREA)

Abstract

本发明公开了一种METTL3基因敲除的细胞系及其构建方法与干扰载体。METTL3基因敲除的细胞系，利用m6A甲基转移酶基因Mettl3在通过基因编辑方法获得耐受LPS刺激导致脂肪酸吸收障碍的猪肠道上皮细胞系。一种耐受LPS刺激导致脂肪酸吸收障碍的猪肠道上皮细胞系。一种针对猪源m6A甲基转移酶基因Mettl3的基因干扰载体。本发明公开了Mettl3基因的一种新功能，参与调控肠道上皮细胞脂肪酸吸收转运，并提供了通过基因编辑手段改善LPS刺激导致的脂肪酸吸收障碍的方法。本发明不仅为研究Mettl3在脂肪酸吸收过程中的作用奠定基础，还利用基因编辑的方法改善了LPS刺激导致的脂肪酸吸收障碍。

Description

METTL3基因敲除的细胞系及其构建方法与干扰载体
技术领域
本发明属于基因工程领域，涉及一种METTL3基因敲除的细胞系及其构建方法与干扰载体。
背景技术
我国现在普遍采用集约型模式进行养猪生产，生猪在每个生长环节都会受到病原微生物的感染，容易造成仔猪肠道（屏障）功能损伤，导致养分消化吸收障碍，并往往伴随着抵抗力下降和较严重的腹泻。在主要养分中，又以作为主要能量物质的脂肪酸的吸收利用影响较大。针对这一现象，目前主要采取抗病育种和长期高剂量添加药物两方面的措施。由于抗病育种世代周期长和普及面小等问题难以大范围推广，因此目前普遍采用的措施是长期高剂量添加饲用抗生素和氧化锌，然而其存在的问题也是显而易见的：极易引发细菌耐药性、肉产品残留、扰乱动物肠道微生态区系平衡、环境污染。因此寻求解决这一问题的新方法迫在眉睫。
m6A甲基化的修饰主要位于mRNA的腺苷A第六号氮原子上，是一种动态可逆的RNA修饰，Mettl3作为其甲基转移酶，通常和Mettl14形成复合物，执行写入m6A的功能。有文章报道， Mettl3所调控的RNA甲基化水平变化参与到生物机体从基因的转录到蛋白质的翻译的整个过程，如敲除Mettl3能够显著提高神经胶质瘤干细胞或类干细胞的增殖和自我更新(Cui et al., 2017)；Mettl3能够介导m6A依赖的cir-RNA的翻译(Yang et al., 2017)；Mettl3能够通过与DNA启动子区域相结合，调控基因转录，发挥着基因转录开关的作用(Barbieri et al., 2017)；敲除Mettl3能够通过阻止炎症相关通路关键因子MyD88的剪切，进而阻止炎症反应的发生(Feng et al., 2018；江一舟等，2017)；同时有文章报道Mettl3在成脂分化过程中能够通过促进细胞周期进而促进脂肪生成(Batista et al.,2014)。但是其在脂肪酸吸收转运过程中的作用，尚未报道。
发明内容
为了克服现有技术的不足，本发明提供了一种METTL3基因敲除的细胞系及其构建方法与干扰载体。
一种METTL3基因敲除的细胞系的构建方法，利用m6A甲基转移酶基因Mettl3在通过基因编辑方法获得耐受LPS刺激导致脂肪酸吸收障碍的猪肠道上皮细胞系。
所述的建立方法，包括以下步骤：
1）根据所述的m6A甲基转移酶基因Mettl3设计shRNA，
所述的Mettl3序列如SEQ ID NO.1所示，所述的shRNA序列如SEQ ID NO.2、SEQ IDNO.3所示；
2）针对m6A甲基转移酶基因Mettl3 shRNA进行磷酸化；
3）将磷酸化的Mettl3 shRNA连接到慢病毒表达载体获得Mettl3 shRNA慢病毒表达载体；
4）以293A细胞作为受体，将步骤3）获得的Mettl3 shRNA慢病毒表达载体进行慢病毒包被；
5）将步骤4）获得的Mettl3 shRNA慢病毒侵染肠道上皮细胞，利用嘌呤霉素进行筛选，获得基因编辑敲除m6A甲基转移酶基因Mettl3的肠道上皮细胞。
所述的Mettl3 shRNA慢病毒表达载体的构建方法具体如下：对shRNA慢病毒表达载体利用BbsI进行酶切，并进行胶回收获得线性载体；以步骤2）获得磷酸化的Mettl3shRNA，采用T4连接酶，连接到pRSI9-U6慢病毒表达载体。
一种根据所述的建立方法得到的耐受LPS刺激导致脂肪酸吸收障碍的猪肠道上皮细胞系。
一种针对猪源m6A甲基转移酶基因Mettl3的基因干扰载体，
所述的干扰载体是将针对所述的m6A甲基转移酶基因Mettl3设计shRNA，插入到shRNA慢病毒表达载体所得，用于敲除肠道上皮细胞内Mettl3的表达。
本发明的有益效果为：
本发明公开了Mettl3基因的一种新功能，参与调控肠道上皮细胞脂肪酸吸收转运，并提供了通过基因编辑手段改善LPS刺激导致的脂肪酸吸收障碍的方法。Mettl3能够调控肠道上皮细胞脂肪酸吸收为首次报道。转录水平和蛋白水平分析表明进行基因编辑的肠道上皮细胞Mettl3表达量显著降低。脂肪酸吸收功能验证实验表明进行基因编辑的肠道上皮细胞能够耐受LPS刺激导致的脂肪酸吸收障碍。本发明不仅为研究Mettl3在脂肪酸吸收过程中的作用奠定基础，还利用基因编辑的方法改善了LPS刺激导致的脂肪酸吸收障碍。
附图说明
图1 为PCR鉴定Mettl3 shRNA慢病毒表达载体琼脂糖凝胶电泳图（Agarose gelelectrophoresis）；
图2为pRSI9-U6慢病毒载体的示意图；
图3A为Mettl3 shRNA慢病毒侵染上皮细胞之后Mettl3的蛋白表达水平Western-blot图；
图3B为基因编辑后的肠道上皮细胞Mettl3的转录水平；数据平均值代表3个生物学重复，误差线表示标准误差(SE)。显著性分析*表示p < 0.05，**表示p< 0.01。
图4为激光共聚焦检测基因编辑肠道上皮细胞的脂肪酸吸收情况图。
图5为流式细胞法检测基因编辑肠道上皮细胞的脂肪酸吸收情况，左部分是不同处理组脂肪酸的吸收情况图，右部分是对左图的量化统计；数据平均值代表3个生物学重复。误差线表示标准误差(SE)。显著性分析*表示p < 0.05，**表示p< 0.01。
图6 为紫外分光法检测基因编辑肠道上皮细胞的脂肪酸吸收情况；数据平均值代表3个生物学重复。误差线表示标准误差(SE)。显著性分析*表示p < 0.05，**表示p< 0.01。
图7 为转入Mettl3质粒对基因编辑敲除Mettl3基因的肠道上皮细胞脂肪酸吸收的影响。
具体实施方式
以下结合附图和实施例对本发明做进一步的阐述。
实施例1 Mettl3 shRNA慢病毒表达载体的获得
本发明中的Mettl3 shRNA是利用在线的shRNA设计工具：http://www.broadinstitute.org/rnai/public/seq/search，根据所述的m6A甲基转移酶基因Mettl3设计；其中，Mettl3基因序列如如SEQ ID NO.１所示，shRNA序列包括上游序列P1，下游序列P2，如下所示：
P1（上游序列）：ACCGG GAGATCCTAGAACTATTAAAT CTCGAG ATTTAATAGTTCTAG GATCTCTTTTTTG （SEQ ID NO.2）；
P2（下游序列）: CGAACAAAAAA GAGATCCTAGAACTATTAAAT CTCGAG ATTTAATAGTTCTAGGATCTC C （SEQ ID NO.3）。
并利用T4 Polynucleotide Kinase，37 °C反应30分钟后，95 °C 反应2分钟，然后自然冷却至室温，对Mettl3 shRNA进行磷酸化。
对shRNA慢病毒表达载体利用BbsI进行酶切，并进行胶回收获得线性载体；以获得磷酸化的Mettl3 shRNA，采用T4连接酶，室温孵育2小时，将其连接到pRSI9-U6慢病毒表达载体上，通过热激法转化大肠杆菌DH5α菌株，提取质粒进行PCR鉴定，引物序列如下所示，
Forward: 5`- GAGGGCCTATTTCCCATGATTCC-3`（SEQ ID NO.4），
Reverse: 5`-ACAGTCCGAAACCCCAAACGCACGAA-3`（SEQ ID NO.5）。
反应条件为反应条件94℃预 变性3 min，94℃变性15s，56℃退火30s，72℃延伸2min，25个反应循环，获得阳性克隆，如图1所示，所挑克隆PCR产物均大于阴性对照组，表明Mettl3的shRNA确实插入到如图2所示的pRSI9-U6慢病毒表达载体上。
实施例2 Mettl3 shRNA慢病毒的包被
将2μg实施例1中的方法获得的Mettl3 shRNA慢病毒表达质粒，利用脂质体Lipo2000，同时转入慢病毒元件0.25μg VsVg和1μg PsPax；24小时后换成1.5ml完全培养基；48小时后收集病毒，获得Mettl3 shRNA的慢病毒。
实施例3 基因编辑肠道上皮细胞系的获得
将500μl实施例2中的方法获得的Mettl3 shRNA慢病毒加入到肠道上皮细胞IPEC-J2中，24小时后换成完全培养基，48小时后加入4μg/ml嘌呤霉素进行筛选，至通过荧光定量PCR及Western-blot法验证获得基因编辑敲除Mettl3基因的肠道上皮细胞。如图3A-3B所示，与空载体组相比，Mettl3 shRNA慢病毒侵染组的肠道上皮细胞Mettl3的表达水平显著降低。
实施例4　激光共聚焦法分析基因编辑肠道上皮细胞的脂肪酸吸收情况
用实施例3中的方法获得的基因编辑敲除Mettl3基因的肠道上皮细胞，以正常细胞（Scramble）作为对照，分别用100μg/ml脂多糖LPS刺激0小时，3小时，6小时后；加入Bodipy标记的脂肪酸，37℃孵育5min，激光共聚焦检测脂肪酸的吸收情况。结果表明基因编辑敲除Mettl3基因的肠道上皮细胞对LPS刺激导致的脂肪酸吸收障碍的耐受显著高于正常细胞，如图4所示，分别针对对照组和Mettl3基因编辑后的肠道上皮细胞进行LPS刺激处理，结果表明，对照组在LPS刺激条件下，脂肪酸的吸收明显受阻，但是Mettl3基因编辑后的肠道上皮细胞则表面出明显的耐受。
实施例5 流式细胞分析法基因编辑肠道上皮细胞的脂肪酸吸收情况
用实施例3中的方法获得的基因编辑敲除Mettl3基因的肠道上皮细胞，以正常细胞（Scramble）作为对照，分别用100μg/ml脂多糖LPS刺激0小时，3小时，6小时后；加入Bodipy标记的脂肪酸，37℃孵育5min，流式细胞仪（波长488nm）检测脂肪酸的吸收情况。结果表明基因编辑敲除Mettl3基因的肠道上皮细胞对LPS刺激导致的脂肪酸吸收障碍的耐受显著高于正常细胞。如图5所示，分别针对对照组（Scramble）和Mettl3基因编辑后的肠道上皮细胞（shMettl3）进行LPS刺激处理0小时，3小时，6小时。流式细胞法检测细胞荧光标记脂肪酸的吸收情况。
实施例6紫外分光法检测基因编辑肠道上皮细胞的脂肪酸吸收情况
分别将正常的和基因编辑的IPCE-J2细胞种植明胶包被、96孔、黑色、透明底板，过夜培养后，用无FBS培养基对血清进行饥饿培养至少8小时，用1mg/ml LPS处理0小时、3小时、6小时，然后用磷酸盐缓冲盐水（PBS）进行短暂洗涤。同时在PBS中加入BodiPy FA（分子探针γ-D3823）和无脂肪酸牛血清白蛋白（BSA）（2:1摩尔比）在37℃水浴中预孵育10分钟；加入细胞后在37℃下孵育5分钟，用加入0.5%BSA的PBS洗涤2次，每次2min ，为了抑制细胞外荧光，加入0.4%台盼蓝（每孔50uL），并用紫外分光光度计立即测量细胞内荧光（激发488 nm，发射515 nm，cut-off 495 nm）。如图6所示，分别针对对照组（Scramble）和Mettl3基因编辑后的肠道上皮细胞（shMettl3）进行LPS刺激处理0小时，3小时，6小时。紫外分光法检测细胞荧光标记脂肪酸的吸收情况。
实施例7 转入Mettl3质粒对基因编辑敲除Mettl3基因的肠道上皮细胞脂肪酸吸收的影响
用实施例3中的方法获得的基因编辑敲除Mettl3基因的肠道上皮细胞，用Lipo2000分别转入pcDNA3.1空质粒和Mettl3质粒，24小时后，分别用100μg/ml脂多糖LPS刺激0小时，3小时，6小时后；加入Bodipy标记的脂肪酸，37℃孵育5min，流式细胞仪（波长488nm）检测脂肪酸的吸收情况。结果表明相比于空载体组，转入Mettl3质粒组在LPS刺激下脂肪酸吸收水平显著降低，表明确实是Mettl3在耐受脂肪酸吸收障碍中发挥着重要作用。如图7所示，对基因编辑敲除Mettl3基因的肠道上皮细胞转入pcDNA3.1空质粒和Mettl3质粒后，进行LPS刺激处理0小时，3小时，6小时。激光共聚焦法检测细胞荧光标记脂肪酸的吸收情况。
序列表
<110> 浙江大学
<120> METTL3基因敲除的细胞系及其构建方法与干扰载体
<141> 2018-07-05
<160> 5
<170> SIPOSequenceListing 1.0
<210> 1
<211> 2088
<212> DNA
<213> 猪(Sus scrofa)
<400> 1
cactgaaaaa catatccgcc actccgctaa catttaaggt ggctcctcat ttcagttcca 60
aagcgagagc gaaacgggaa atgactttct atctggcgca tctctcagga cctccttccg 120
gttagccttg gggagtacgc gtgagagttg gaaatttcgt ggagccagtg ctgggaggtg 180
ctagtcggct accccttgtt cgagacgtgt cccggctgtg ggactaaaat gtcggacacg 240
tggagctcta tccaggccca caaaaagcag ctggactcgc tgagggaaag gctgcggcgg 300
aggcggaagc aggactcagg gcacttggat cttcggaatc cagaggcagc actgtctcca 360
accttccgta gtgacagccc agtgcctact gtacccactt ctggtggccc taagcccagc 420
acagcttcag cagttcctga gctagctaca gaccctgaat tagagaagaa gttgctacac 480
cacctttctg atctggcgct aacattgccc actgatgctg tctccatccg tcttgccatc 540
tccacgccag atgcccctgc cactcaggat ggagtggaaa gcctcttaca gaagtttgca 600
gctcaggagt tgattgaagt aaagcgaagt ctcctacaag atgatgcaca ccctactctt 660
gtgacctatg ctgatcattc caagctctct gccatgatgg gtgctgtggc agaaaagaag 720
ggccctgggg aggtagccgg gaccatcaca gggcagaaga ggcgtgcaga acaggactcg 780
accacagtag ctgcctttgc aagctctctg acctctggtc tggcctcttc agcatcagaa 840
gtagccaagg agccaaccaa gaaatcaagg aaacatgctg cctcagatgt tgacctggag 900
atagagagcc ttctgaacca acaatctact aaggaacaac agagcaagaa ggttagtcaa 960
gagatcctag aactattaaa tactacaaca gccaaggaac aatccattgt tgaaaagttt 1020
cgttcacgag gtcgggctca agtgcaagaa ttctgtgact atggaaccaa ggaggagtgc 1080
atgaaagcca gtgatgctga ccggccttgt cgcaagctgc acttcagacg gatcatcaat 1140
aaacacacgg atgagtcatt aggtgactgc tctttcctta acacatgttt ccacatggat 1200
acctgcaaat atgttcacta tgaaattgat gcttgcatgg attctgaggc tcctggaagc 1260
aaagaccata caccaagcca ggagcttgcc cttacacaga gcgttggagg ggactccaat 1320
gcagatcgac tcttcccacc tcagtggatc tgttgtgata tccgctacct ggacgtcagt 1380
atcttgggca agtttgcagt tgtgatggct gacccaccct gggatattca catggagctg 1440
ccctatggga ccctgacaga tgatgagatg cgcaggctca acataccagt actgcaggat 1500
gatggctttc tcttcctctg ggtcacaggc agggccatgg agttgggcag agaatgtctg 1560
aacctctggg gttacgaacg ggtagatgaa attatctggg tgaagacaaa tcaactgcag 1620
cgcatcattc ggacaggccg tacaggtcac tggttgaacc atgggaagga acactgcttg 1680
gttggtgtca aaggaaatcc ccaaggattc aaccagggtc tggattgtga tgtgatcgta 1740
gccgaggttc gttccaccag tcataaacca gatgaaatct atggcatgat tgagagactg 1800
tcccctggca ctcgaaagat tgaattgttt ggacgaccac acaatgtgca acctaattgg 1860
atcacccttg gaaatcaact ggatgggatc catctactag acccagatgt ggttgcccgg 1920
ttcaagcaaa ggtatccaga tggtatcatc tctaaaccta agaatttata gaagtacttt 1980
gaaacctaag catccatggc catggctctt caggctgtac ctgaagagta atatttgtac 2040
aatagctttt attccttatt taaataaaag tttgtattgt agttggga 2088
<210> 2
<211> 60
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 2
accgggagat cctagaacta ttaaatctcg agatttaata gttctaggat ctcttttttg 60
<210> 3
<211> 60
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 3
cgaacaaaaa agagatccta gaactattaa atctcgagat ttaatagttc taggatctcc 60
<210> 4
<211> 23
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 4
gagggcctat ttcccatgat tcc 23
<210> 5
<211> 26
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 5
acagtccgaa accccaaacg cacgaa 26

Claims (5)

1.一种METTL3基因敲除的细胞系的构建方法，其特征在于，利用m6A甲基转移酶基因Mettl3在通过基因敲除方法获得耐受LPS刺激导致脂肪酸吸收障碍的猪肠道上皮细胞系。
2.根据权利要求1所述的建立方法，其特征在于，包括以下步骤：
1）根据所述的m6A甲基转移酶基因Mettl3设计shRNA，
所述的Mettl3序列如SEQ ID NO.1所示，所述的shRNA序列如SEQ ID NO.2、SEQ IDNO.3所示；
2）针对m6A甲基转移酶基因Mettl3 shRNA进行磷酸化；
3）将磷酸化的Mettl3 shRNA连接到慢病毒表达载体获得Mettl3 shRNA慢病毒表达载体；
4）以293A细胞作为受体，将步骤3）获得的Mettl3 shRNA慢病毒表达载体进行慢病毒包被；
5）将步骤4）获得的Mettl3 shRNA慢病毒侵染肠道上皮细胞，利用嘌呤霉素进行筛选，获得基因编辑敲除m6A甲基转移酶基因Mettl3的肠道上皮细胞。
3.根据权利要求1所述的建立方法，其特征在于，所述的Mettl3 shRNA慢病毒表达载体的构建方法具体如下：对shRNA慢病毒表达载体利用BbsI进行酶切，并进行胶回收获得线性载体；以步骤2）获得磷酸化的Mettl3 shRNA，采用T4连接酶，连接到pRSI9-U6慢病毒表达载体。
4.一种根据权利要求1所述的建立方法得到的耐受LPS刺激导致脂肪酸吸收障碍的猪肠道上皮细胞系。
5.一种针对猪源m6A甲基转移酶基因Mettl3的基因干扰载体，其特征在于，
所述的干扰载体是将针对所述的m6A甲基转移酶基因Mettl3设计shRNA，插入到shRNA慢病毒表达载体所得，用于敲除肠道上皮细胞内Mettl3的表达。
CN201810733097.5A 2018-07-05 2018-07-05 Mettl3基因敲除的细胞系及其构建方法与干扰载体 Active CN108929863B (zh)

Priority Applications (3)

Application Number Priority Date Filing Date Title
CN201810733097.5A CN108929863B (zh) 2018-07-05 2018-07-05 Mettl3基因敲除的细胞系及其构建方法与干扰载体
PCT/CN2018/096554 WO2020006787A1 (zh) 2018-07-05 2018-07-21 Mettl3基因敲除的细胞系及其构建方法与干扰载体
US17/417,764 US11339370B2 (en) 2018-07-05 2018-07-21 Cell line with METTL3 gene knocked out, its construction method and interference vector

Applications Claiming Priority (1)

Application Number Priority Date Filing Date Title
CN201810733097.5A CN108929863B (zh) 2018-07-05 2018-07-05 Mettl3基因敲除的细胞系及其构建方法与干扰载体

Publications (2)

Publication Number Publication Date
CN108929863A true CN108929863A (zh) 2018-12-04
CN108929863B CN108929863B (zh) 2019-11-22

Family

ID=64446862

Family Applications (1)

Application Number Title Priority Date Filing Date
CN201810733097.5A Active CN108929863B (zh) 2018-07-05 2018-07-05 Mettl3基因敲除的细胞系及其构建方法与干扰载体

Country Status (3)

Country Link
US (1) US11339370B2 (zh)
CN (1) CN108929863B (zh)
WO (1) WO2020006787A1 (zh)

Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN111206034A (zh) * 2020-01-10 2020-05-29 浙江大学 猪GADD45a基因的新用途及高表达细胞系的构建和应用
CN111676222A (zh) * 2020-06-18 2020-09-18 暨南大学 抑制Mettl3基因表达的shRNA及其重组腺相关病毒与应用
CN113813403A (zh) * 2021-07-20 2021-12-21 南方医科大学口腔医院 Mettl3在制备修复牙髓损伤的药物中的用途

Families Citing this family (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN111440826A (zh) * 2020-03-16 2020-07-24 中山大学附属第一医院 靶向RNA的6-甲基腺嘌呤修饰CasRx载体系统及制备方法和应用
CN115176760B (zh) * 2022-07-07 2023-11-17 电子科技大学 一种构建视网膜色素变性疾病模型的方法、应用及繁育方法

Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN109457029A (zh) * 2018-12-30 2019-03-12 王增艳 Mettl3基因的应用及其检测方法

Family Cites Families (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN107349217B (zh) 2017-07-21 2020-06-09 深圳大学 一种基于mettl3的小干扰rna及其药物和应用

Patent Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN109457029A (zh) * 2018-12-30 2019-03-12 王增艳 Mettl3基因的应用及其检测方法

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
XINXIA WANG ET AL: "mRNA M6A methylation downregulates adipogenesis in porcine adipocyte", 《BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATION》 *

Cited By (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN111206034A (zh) * 2020-01-10 2020-05-29 浙江大学 猪GADD45a基因的新用途及高表达细胞系的构建和应用
CN111206034B (zh) * 2020-01-10 2021-09-28 浙江大学 猪GADD45a基因的新用途及高表达细胞系的构建和应用
CN111676222A (zh) * 2020-06-18 2020-09-18 暨南大学 抑制Mettl3基因表达的shRNA及其重组腺相关病毒与应用
CN113813403A (zh) * 2021-07-20 2021-12-21 南方医科大学口腔医院 Mettl3在制备修复牙髓损伤的药物中的用途
CN113813403B (zh) * 2021-07-20 2024-04-02 南方医科大学口腔医院 Mettl3在制备修复牙髓损伤的药物中的用途

Also Published As

Publication number Publication date
CN108929863B (zh) 2019-11-22
WO2020006787A1 (zh) 2020-01-09
US11339370B2 (en) 2022-05-24
US20220010272A1 (en) 2022-01-13

Similar Documents

Publication Publication Date Title
CN108929863B (zh) Mettl3基因敲除的细胞系及其构建方法与干扰载体
Chuhma et al. Dopamine neurons control striatal cholinergic neurons via regionally heterogeneous dopamine and glutamate signaling
Bruno et al. Salmonella Typhimurium type III secretion effectors stimulate innate immune responses in cultured epithelial cells
Knittler et al. Chlamydia psittaci: New insights into genomic diversity, clinical pathology, host–pathogen interaction and anti-bacterial immunity
Jing et al. Environmental contaminant ammonia triggers epithelial-to-mesenchymal transition-mediated jejunal fibrosis with the disassembly of epithelial cell-cell contacts in chicken
Lund et al. Recognition of single-stranded RNA viruses by Toll-like receptor 7
Chen et al. Innate sensing of viruses by pattern recognition receptors in birds
Caron et al. Osmolarity determines the in vitro chondrogenic differentiation capacity of progenitor cells via nuclear factor of activated T-cells 5
CN106148286A (zh) 一种用于检测热原的细胞模型的构建方法和细胞模型及热原检测试剂盒
Edlin Gene regulation during bacteriophage T4 development: I. Phenotypic reversion of T4 amber mutants by 5-Fluorouracil
Wong et al. Evidence implicating the 5′ untranslated region of Listeria monocytogenes actA in the regulation of bacterial actin‐based motility
Connor et al. Yersinia pestis targets the host endosome recycling pathway during the biogenesis of the Yersinia-containing vacuole to avoid killing by macrophages
Kelly et al. Effect of cortisol on the physiology of cultured pavement cell epithelia from freshwater trout gills
Johnson et al. Alterations in Helicobacter pylori triggered by contact with gastric epithelial cells
Tahoun et al. Comparative analysis of EspF variants in inhibition of Escherichia coli phagocytosis by macrophages and inhibition of E. coli translocation through human-and bovine-derived M cells
Braithwaite et al. The immunomodulating agent gliotoxin causes genomic DNA fragmentation
Schwend et al. Zebrafish con/disp1 reveals multiple spatiotemporal requirements for Hedgehog-signaling in craniofacial development
Roe et al. Co‐ordinate single‐cell expression of LEE4‐and LEE5‐encoded proteins of Escherichia coli O157: H7
Campellone et al. Enterohaemorrhagic Escherichia coli Tir requires a C‐terminal 12‐residue peptide to initiate EspFU‐mediated actin assembly and harbours N‐terminal sequences that influence pedestal length
Zhao et al. gga-miR-99a targets SMARCA5 to regulate Mycoplasma gallisepticum (HS strain) infection by depressing cell proliferation in chicken
Guevara et al. Enterotoxigenic Escherichia coli CS21 pilus contributes to adhesion to intestinal cells and to pathogenesis under in vivo conditions
Eroglu et al. Epicardium-derived cells organize through tight junctions to replenish cardiac muscle in salamanders
Deng et al. AI-2/LuxS quorum sensing system promotes biofilm formation of lactobacillus rhamnosus GG and enhances the resistance to enterotoxigenic escherichia coli in germ-free zebrafish
Hrstka et al. Francisella tularensis strain LVS resides in MHC II-positive autophagic vacuoles in macrophages
Stamm et al. Epithelial and mesenchymal cells in the bovine colonic mucosa differ in their responsiveness to Escherichia coli Shiga toxin 1

Legal Events

Date Code Title Description
PB01 Publication
PB01 Publication
SE01 Entry into force of request for substantive examination
SE01 Entry into force of request for substantive examination
GR01 Patent grant
GR01 Patent grant