产品分类
METTL3 Knockout HT29 Cell Pool
- 产品描述
- 细胞复苏
- 细胞传代
- 细胞冻存
- 抗体验证结果
-
- 品牌: ELEMok138cn太阳集团529
- 商品名称: METTL3 Knockout HT29 Cell Pool
- 商品编号: LM01022165819
- Gene Symbol: METTL3 MTA70
- Ensembl ID: ENSG00000165819
- Uniprot ID: Q86U44
- 宿主细胞 / 类型: HT29/人结肠癌细胞
- NCBI Gene ID: 56339
- 规格: 1×10^6 cells/ 冻存管
- 筛选标记: N/A
- 生长特性: 贴壁细胞，上皮细胞样
- 培养条件: 37℃,5% CO2 的培养箱，1/3 到 1/4传代
- 倍增时间: ~36-60 hours
- 生长培养基: McCoy’s 5A＋10% FBS＋1% P/S
- 参考换液频率: 2~3次/周
- 支原体检测结果: 阴性
- 敲除效率（Sanger测序）: >70%
- 蛋白质组验证结果: N/A
- 抗体货号: 添加中
- 目标基因介绍: The METTL3-METTL14 heterodimer forms a N6-methyltransferase complex that methylates adenosine residues at the N(6) position of some RNAs and regulates various processes such as the circadian clock, differentiation of embryonic and hematopoietic stem cells, cortical neurogenesis, response to DNA damage, differentiation of T-cells and primary miRNA processing (PubMed:22575960, PubMed:24284625, PubMed:25719671, PubMed:25799998, PubMed:26321680, PubMed:26593424, PubMed:27627798, PubMed:27373337, PubMed:27281194, PubMed:28297716, PubMed:30428350, PubMed:29506078, PubMed:29348140, PubMed:9409616). In the heterodimer formed with METTL14, METTL3 constitutes the catalytic core (PubMed:27627798, PubMed:27373337, PubMed:27281194). N6-methyladenosine (m6A), which takes place at the 5'-[AG]GAC-3' consensus sites of some mRNAs, plays a role in mRNA stability, processing, translation efficiency and editing (PubMed:22575960, PubMed:24284625, PubMed:25719671, PubMed:25799998, PubMed:26321680, PubMed:26593424, PubMed:28297716, PubMed:9409616). M6A acts as a key regulator of mRNA stability: methylation is completed upon the release of mRNA into the nucleoplasm and promotes mRNA destabilization and degradation (PubMed:28637692). In embryonic stem cells (ESCs), m6A methylation of mRNAs encoding key naive pluripotency-promoting transcripts results in transcript destabilization, promoting differentiation of ESCs (By similarity). M6A regulates the length of the circadian clock: acts as an early pace-setter in the circadian loop by putting mRNA production on a fast-track for facilitating nuclear processing, thereby providing an early point of control in setting the dynamics of the feedback loop (By similarity). M6A also regulates circadian regulation of hepatic lipid metabolism (PubMed:30428350). M6A regulates spermatogonial differentiation and meiosis and is essential for male fertility and spermatogenesis (By similarity). Involved in the response to DNA damage: in response to ultraviolet irradiation, METTL3 rapidly catalyzes the formation of m6A on poly(A) transcripts at DNA damage sites, leading to the recruitment of POLK to DNA damage sites (PubMed:28297716). M6A is also required for T-cell homeostasis and differentiation: m6A methylation of transcripts of SOCS family members (SOCS1, SOCS3 and CISH) in naive T-cells promotes mRNA destabilization and degradation, promoting T-cell differentiation (By similarity). Inhibits the type I interferon response by mediating m6A methylation of IFNB (PubMed:30559377). M6A also takes place in other RNA molecules, such as primary miRNA (pri-miRNAs) (PubMed:25799998). Mediates m6A methylation of Xist RNA, thereby participating in random X inactivation: m6A methylation of Xist leads to target YTHDC1 reader on Xist and promote transcription repression activity of Xist (PubMed:27602518). M6A also regulates cortical neurogenesis: m6A methylation of transcripts related to transcription factors, neural stem cells, the cell cycle and neuronal differentiation during brain development promotes their destabilization and decay, promoting differentiation of radial glial cells (By similarity). METTL3 mediates methylation of pri-miRNAs, marking them for recognition and processing by DGCR8 (PubMed:25799998). Acts as a positive regulator of mRNA translation independently of the methyltransferase activity: promotes translation by interacting with the translation initiation machinery in the cytoplasm (PubMed:27117702). Its overexpression in a number of cancer cells suggests that it may participate in cancer cell proliferation by promoting mRNA translation (PubMed:27117702).
- 细胞开发路径: 采用CRISPR-RNP方法生成稳定KO Cell Pool；Sanger 测序结果显示KO Cell Pool敲除效率>70%。
- 应用: 高敲除效率的基因敲除细胞池（KO Cell Pool），特别适用于初步功能分析、复杂疾病模型的开发、精准药物筛选以及广泛的基因发现研究。KO pool能够无需繁琐的单克隆挑选过程，直接应用于多种类型的测定和分析，大幅提升实验效率。
关键词:- METTL3 MTA70
-
01. 在 37℃水浴中预热完全培养基。
02. 将冻存管在 37℃水浴中解冻 1-2 分钟。
03. 将冻存管转移到生物安全柜中，并用 70% 乙醇擦拭表面。
04. 拧开冻存管管盖，将细胞悬液轻轻转移到含有 9mL 完全培养基的无菌离心管中。
05. 在室温下以 125g 离心 5-7 分钟，弃上清。
06. 用 5mL 的完整培养基重悬细胞沉淀，将细胞悬液转移到 T25 培养瓶中。
07. 将细胞转移到 37℃,5% CO2 的培养箱中培养。
08. 参考传代比例：1/3 到 1/4 传代，2-3 天长满。 -
01. 待培养瓶中细胞汇合度至 80%-90% 以上，可进行细胞传代。
02. 将培养基、PBS、胰酶（0.25%Trypsin_EDTA Gibco 25200-056） 等从 4℃冰箱中拿出， 置于 37℃水浴中温度接近 37℃时取出并在瓶子表面喷洒 75% 酒精后置于生物安全柜中。03. 从培养箱中取出待传代的培养瓶，瓶身喷洒 75% 酒精后置于生物安全柜中。
04. 为避免冲散细胞，沿培养瓶上壁 PBS 润洗细胞，清洗细胞后弃去，T25 加 2mL。
05. 加入对应体积的胰酶（T75 加 1.5mL， T25 加 0.5mL） ，并轻轻晃动瓶身使胰酶平铺满细胞 底部。可根据实际情况适当增加或减少用量。约 1-2min 后大部分细胞脱落时，加入对应体积的完全培养基终止消化，并用 5mL 移液管轻轻吹打至细胞全部脱落。
06. 将细胞悬液转移至 15mL 离心管，悬液 300g 离心 5min，弃上清。
07. 移取 5mL 完全培养基重悬细胞，按需求调整接种比例，并补充培养瓶中完全培养基，T75 加至 13-15mL，T25 加至 5mL，加 1% 双抗。
08. 盖上瓶盖拧紧后轻轻晃动瓶身，使细胞混合均匀后置于 37℃,5% CO2 培养箱中。 -
01. 准备冻存液，并提前预冷。
02. 确保待冻存的细胞满足冻存要求，用显微镜检查以下状态：健康的外观及形态特征、所处生 长周期（对数晚期）、无污染或衰退迹象。
03. 对细胞进行消化及离心处理（具体步骤参考传代培养流程）
04. 按照每管 1mL 的量添加冻存液重悬细胞，吹打均匀后分装至冻存管。
05. 将细胞放在程序降温盒中，在 -80℃冰箱中冷冻。
06. 后续将细胞转移到液氮罐中，以便长期储存。 - 抗体验证中
产品类型： 基因敲除细胞池
细胞系信息
Gene Symbol
METTL3 MTA70
NCBI Gene ID
56339
Ensembl ID
ENSG00000165819
Uniprot ID
Q86U44
筛选标记
N/A
宿主细胞 / 类型
HT29/人结肠癌细胞
规格
1×10^6 cells/ 冻存管
生长培养基
McCoy’s 5A＋10% FBS＋1% P/S
生长特性
贴壁细胞，上皮细胞样
培养条件
37℃,5% CO2 的培养箱，1/3 到 1/4传代
倍增时间
~36-60 hours
参考换液频率
2~3次/周
支原体检测结果
阴性
敲除验证
敲除效率（Sanger测序）
>70%
蛋白质组验证结果
N/A
抗体货号
添加中
抗体验证结果
细胞系说明
目标基因介绍
The METTL3-METTL14 heterodimer forms a N6-methyltransferase complex that methylates adenosine residues at the N(6) position of some RNAs and regulates various processes such as the circadian clock, differentiation of embryonic and hematopoietic stem cells, cortical neurogenesis, response to DNA damage, differentiation of T-cells and primary miRNA processing (PubMed:22575960, PubMed:24284625, PubMed:25719671, PubMed:25799998, PubMed:26321680, PubMed:26593424, PubMed:27627798, PubMed:27373337, PubMed:27281194, PubMed:28297716, PubMed:30428350, PubMed:29506078, PubMed:29348140, PubMed:9409616). In the heterodimer formed with METTL14, METTL3 constitutes the catalytic core (PubMed:27627798, PubMed:27373337, PubMed:27281194). N6-methyladenosine (m6A), which takes place at the 5'-[AG]GAC-3' consensus sites of some mRNAs, plays a role in mRNA stability, processing, translation efficiency and editing (PubMed:22575960, PubMed:24284625, PubMed:25719671, PubMed:25799998, PubMed:26321680, PubMed:26593424, PubMed:28297716, PubMed:9409616). M6A acts as a key regulator of mRNA stability: methylation is completed upon the release of mRNA into the nucleoplasm and promotes mRNA destabilization and degradation (PubMed:28637692). In embryonic stem cells (ESCs), m6A methylation of mRNAs encoding key naive pluripotency-promoting transcripts results in transcript destabilization, promoting differentiation of ESCs (By similarity). M6A regulates the length of the circadian clock: acts as an early pace-setter in the circadian loop by putting mRNA production on a fast-track for facilitating nuclear processing, thereby providing an early point of control in setting the dynamics of the feedback loop (By similarity). M6A also regulates circadian regulation of hepatic lipid metabolism (PubMed:30428350). M6A regulates spermatogonial differentiation and meiosis and is essential for male fertility and spermatogenesis (By similarity). Involved in the response to DNA damage: in response to ultraviolet irradiation, METTL3 rapidly catalyzes the formation of m6A on poly(A) transcripts at DNA damage sites, leading to the recruitment of POLK to DNA damage sites (PubMed:28297716). M6A is also required for T-cell homeostasis and differentiation: m6A methylation of transcripts of SOCS family members (SOCS1, SOCS3 and CISH) in naive T-cells promotes mRNA destabilization and degradation, promoting T-cell differentiation (By similarity). Inhibits the type I interferon response by mediating m6A methylation of IFNB (PubMed:30559377). M6A also takes place in other RNA molecules, such as primary miRNA (pri-miRNAs) (PubMed:25799998). Mediates m6A methylation of Xist RNA, thereby participating in random X inactivation: m6A methylation of Xist leads to target YTHDC1 reader on Xist and promote transcription repression activity of Xist (PubMed:27602518). M6A also regulates cortical neurogenesis: m6A methylation of transcripts related to transcription factors, neural stem cells, the cell cycle and neuronal differentiation during brain development promotes their destabilization and decay, promoting differentiation of radial glial cells (By similarity). METTL3 mediates methylation of pri-miRNAs, marking them for recognition and processing by DGCR8 (PubMed:25799998). Acts as a positive regulator of mRNA translation independently of the methyltransferase activity: promotes translation by interacting with the translation initiation machinery in the cytoplasm (PubMed:27117702). Its overexpression in a number of cancer cells suggests that it may participate in cancer cell proliferation by promoting mRNA translation (PubMed:27117702).
细胞开发路径
采用CRISPR-RNP方法生成稳定KO Cell Pool；Sanger 测序结果显示KO Cell Pool敲除效率>70%。
应用
高敲除效率的基因敲除细胞池（KO Cell Pool），特别适用于初步功能分析、复杂疾病模型的开发、精准药物筛选以及广泛的基因发现研究。KO pool能够无需繁琐的单克隆挑选过程，直接应用于多种类型的测定和分析，大幅提升实验效率。
细胞培养说明
细胞复苏
01. 在 37℃水浴中预热完全培养基。
02. 将冻存管在 37℃水浴中解冻 1-2 分钟。
03. 将冻存管转移到生物安全柜中，并用 70% 乙醇擦拭表面。
04. 拧开冻存管管盖，将细胞悬液轻轻转移到含有 9mL 完全培养基的无菌离心管中。
05. 在室温下以 125g 离心 5-7 分钟，弃上清。
06. 用 5mL 的完整培养基重悬细胞沉淀，将细胞悬液转移到 T25 培养瓶中。
07. 将细胞转移到 37℃,5% CO2 的培养箱中培养。
08. 参考传代比例：1/3 到 1/4 传代，2-3 天长满。
细胞传代
01. 待培养瓶中细胞汇合度至 80%-90% 以上，可进行细胞传代。
02. 将培养基、PBS、胰酶（0.25%Trypsin_EDTA Gibco 25200-056） 等从 4℃冰箱中拿出， 置于 37℃水浴中温度接近 37℃时取出并在瓶子表面喷洒 75% 酒精后置于生物安全柜中。
03. 从培养箱中取出待传代的培养瓶，瓶身喷洒 75% 酒精后置于生物安全柜中。
04. 为避免冲散细胞，沿培养瓶上壁 PBS 润洗细胞，清洗细胞后弃去，T25 加 2mL。
05. 加入对应体积的胰酶（T75 加 1.5mL， T25 加 0.5mL） ，并轻轻晃动瓶身使胰酶平铺满细胞 底部。可根据实际情况适当增加或减少用量。约 1-2min 后大部分细胞脱落时，加入对应体积的完全培养基终止消化，并用 5mL 移液管轻轻吹打至细胞全部脱落。
06. 将细胞悬液转移至 15mL 离心管，悬液 300g 离心 5min，弃上清。
07. 移取 5mL 完全培养基重悬细胞，按需求调整接种比例，并补充培养瓶中完全培养基，T75 加至 13-15mL，T25 加至 5mL，加 1% 双抗。
08. 盖上瓶盖拧紧后轻轻晃动瓶身，使细胞混合均匀后置于 37℃,5% CO2 培养箱中。
细胞冻存
01. 准备冻存液，并提前预冷。
02. 确保待冻存的细胞满足冻存要求，用显微镜检查以下状态：健康的外观及形态特征、所处生 长周期（对数晚期）、无污染或衰退迹象。
03. 对细胞进行消化及离心处理（具体步骤参考传代培养流程）
04. 按照每管 1mL 的量添加冻存液重悬细胞，吹打均匀后分装至冻存管。
05. 将细胞放在程序降温盒中，在 -80℃冰箱中冷冻。
06. 后续将细胞转移到液氮罐中，以便长期储存。