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产品目录号
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GKC-LMP010237
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产品名称
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Human TRIM21-A549 KO Cell Pool
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基因编号
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TRIM21
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Uniprot_id
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P19474
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宿主细胞
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A549
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组织来源
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人非小细胞肺癌细胞
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规格
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1×106cells/T25培养瓶或1×106cells/冻存管
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培养基
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MEM+10%FBS+1%P/S
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筛选标记
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N/A
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生长特性
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贴壁细胞,上皮细胞样
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培养条件
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37℃,5% CO2的培养箱
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传代比例
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1/2到1/3传代,2-3天长满
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筛选标记
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N/A
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换液频率
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2-3天换液
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支原体检测结果
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阴性
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蛋白组验证结果
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N/A
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抗体验证结果
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N/A
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目标基因介绍
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E3 ubiquitin-protein ligase whose activity is dependent on E2 enzymes, UBE2D1, UBE2D2, UBE2E1 and UBE2E2. Forms a ubiquitin ligase complex in cooperation with the E2 UBE2D2 that is used not only for the ubiquitination of USP4 and IKBKB but also for its self-ubiquitination. Component of cullin-RING-based SCF (SKP1-CUL1-F-box protein) E3 ubiquitin-protein ligase complexes such as SCF(SKP2)-like complexes. A TRIM21-containing SCF(SKP2)-like complex is shown to mediate ubiquitination of CDKN1B ('Thr-187' phosphorylated-form), thereby promoting its degradation by the proteasome. Monoubiquitinates IKBKB that will negatively regulates Tax-induced NF-kappa-B signaling. Negatively regulates IFN-beta production post-pathogen recognition by polyubiquitin-mediated degradation of IRF3. Mediates the ubiquitin-mediated proteasomal degradation of IgG1 heavy chain, which is linked to the VCP-mediated ER-associated degradation (ERAD) pathway. Promotes IRF8 ubiquitination, which enhanced the ability of IRF8 to stimulate cytokine genes transcription in macrophages. Plays a role in the regulation of the cell cycle progression. Enhances the decapping activity of DCP2. Exists as a ribonucleoprotein particle present in all mammalian cells studied and composed of a single polypeptide and one of four small RNA molecules. At least two isoforms are present in nucleated and red blood cells, and tissue specific differences in RO/SSA proteins have been identified. The common feature of these proteins is their ability to bind HY RNAs.2. Involved in the regulation of innate immunity and the inflammatory response in response to IFNG/IFN-gamma. Organizes autophagic machinery by serving as a platform for the assembly of ULK1, Beclin 1/BECN1 and ATG8 family members and recognizes specific autophagy targets, thus coordinating target recognition with assembly of the autophagic apparatus and initiation of autophagy. Regulates also autophagy through FIP200/RB1CC1 ubiquitination and subsequent decreased protein stability (PubMed:36359729).
Acts as an autophagy receptor for the degradation of IRF3, hence attenuating type I interferon (IFN)-dependent immune responses (PubMed:26347139, PubMed:16297862, PubMed:16316627, PubMed:16472766, PubMed:16880511, PubMed:18022694, PubMed:18361920, PubMed:18641315, PubMed:18845142, PubMed:19675099).
Represses the innate antiviral response by facilitating the formation of the NMI-IFI35 complex through 'Lys-63'-linked ubiquitination of NMI (PubMed:26342464).
During viral infection, promotes cell pyroptosis by mediating 'Lys-6'-linked ubiquitination of ISG12a/IFI27, facilitating its translocation into the mitochondria and subsequent CASP3 activation (PubMed:36426955).
When up-regulated through the IFN/JAK/STAT signaling pathway, promotes 'Lys-27'-linked ubiquitination of MAVS, leading to the recruitment of TBK1 and up-regulation of innate immunity (PubMed:29743353).
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细胞系生成
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采用CRISPR方法生成Human TRIM21-A549 KO Cell Pool
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数据说明
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Sanger 测序结果显示Human TRIM21-A549 KO Cell Pool敲除效率为100%
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应用
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体内和体外测定
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复苏
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1)在37℃水浴中预热完全培养基。
2)将冻存管在 37℃水浴中解冻1-2分钟。
3)将冻存管转移到生物安全柜中,并用70%乙醇擦拭表面。
4)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。
5)在室温下以125g离心5-7分钟,弃上清。
6)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。
7)将细胞转移到37℃,5% CO2的培养箱中培养。
8)参考传代比例:1/2到1/3传代,2-5天长满。
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传代
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1)待培养瓶中细胞汇合度至80%-90%以上,可进行细胞传代。
2)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。
3)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。
4)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。
5)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。
6)将细胞悬液转移至15mL离心管,悬液300g离心5min,弃上清。
7)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。
8)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% CO4培养箱中。
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细胞冻存
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1)准备冻存液,并提前预冷。
2)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。
3)对细胞进行消化及离心处理(具体步骤参考传代培养流程)
4)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。
5)将细胞放在程序降温盒中,在-80℃冰箱中冷冻。
6)后续将细胞转移到液氮罐中,以便长期储存。
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