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产品目录号
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GKC-LMP01276
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产品名称
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Human NONO-A549 KO Cell Pool
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基因编号
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NONO
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Uniprot_id
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Q15233
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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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DNA- and RNA binding protein, involved in several nuclear processes (PubMed:11525732, PubMed:12403470, PubMed:26571461).
Binds the conventional octamer sequence in double-stranded DNA (PubMed:11525732, PubMed:12403470, PubMed:26571461).
Also binds single-stranded DNA and RNA at a site independent of the duplex site (PubMed:11525732, PubMed:12403470, PubMed:26571461).
Involved in pre-mRNA splicing, probably as a heterodimer with SFPQ (PubMed:11525732, PubMed:12403470, PubMed:26571461).
Interacts with U5 snRNA, probably by binding to a purine-rich sequence located on the 3' side of U5 snRNA stem 1b (PubMed:12403470).
Together with PSPC1, required for the formation of nuclear paraspeckles (PubMed:22416126).
The SFPQ-NONO heteromer associated with MATR3 may play a role in nuclear retention of defective RNAs (PubMed:11525732).
The SFPQ-NONO heteromer may be involved in DNA unwinding by modulating the function of topoisomerase I/TOP1 (PubMed:10858305).
The SFPQ-NONO heteromer may be involved in DNA non-homologous end joining (NHEJ) required for double-strand break repair and V(D)J recombination and may stabilize paired DNA ends (PubMed:15590677).
In vitro, the complex strongly stimulates DNA end joining, binds directly to the DNA substrates and cooperates with the Ku70/G22P1-Ku80/XRCC5 (Ku) dimer to establish a functional preligation complex (PubMed:15590677).
NONO is involved in transcriptional regulation. The SFPQ-NONO-NR5A1 complex binds to the CYP17 promoter and regulates basal and cAMP-dependent transcriptional activity (PubMed:11897684).
NONO binds to an enhancer element in long terminal repeats of endogenous intracisternal A particles (IAPs) and activates transcription (By similarity).
Regulates the circadian clock by repressing the transcriptional activator activity of the CLOCK-BMAL1 heterodimer (By similarity).
Important for the functional organization of GABAergic synapses (By similarity).
Plays a specific and important role in the regulation of synaptic RNAs and GPHN/gephyrin scaffold structure, through the regulation of GABRA2 transcript (By similarity).
Plays a key role during neuronal differentiation by recruiting TET1 to genomic loci and thereby regulating 5-hydroxymethylcytosine levels (By similarity).
Plays a role in the regulation of DNA virus-mediated innate immune response by assembling into the HDP-RNP complex, a complex that serves as a platform for IRF3 phosphorylation and subsequent innate immune response activation through the cGAS-STING pathway (PubMed:28712728, PubMed:30270045).
Promotes activation of the cGAS-STING pathway in response to HIV-2 infection: acts by interacting with HIV-2 Capsid protein p24, thereby promoting detection of viral DNA by CGAS, leading to CGAS-mediated inmmune activation (PubMed:30270045).
In contrast, the weak interaction with HIV-1 Capsid protein p24 does not allow activation of the cGAS-STING pathway (PubMed:30270045).
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细胞系生成
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采用CRISPR方法生成Human NONO-A549 KO Cell Pool
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数据说明
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Sanger 测序结果显示Human NONO-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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