产品分类
CDK2 Knockout HEK293 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: CDK2 Knockout HEK293 Cell Pool
- 商品编号: LM01024123374
- Gene Symbol: CDK2 CDKN2
- Ensembl ID: ENSG00000123374
- Uniprot ID: P24941
- 宿主细胞 / 类型: HEK293/人胚肾细胞
- NCBI Gene ID: 1017
- 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: ~24-45 hours
- 生长培养基: DMEM＋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: Serine/threonine-protein kinase involved in the control of the cell cycle; essential for meiosis, but dispensable for mitosis. Phosphorylates CTNNB1, USP37, p53/TP53, NPM1, CDK7, RB1, BRCA2, MYC, NPAT, EZH2. Triggers duplication of centrosomes and DNA. Acts at the G1-S transition to promote the E2F transcriptional program and the initiation of DNA synthesis, and modulates G2 progression; controls the timing of entry into mitosis/meiosis by controlling the subsequent activation of cyclin B/CDK1 by phosphorylation, and coordinates the activation of cyclin B/CDK1 at the centrosome and in the nucleus. Crucial role in orchestrating a fine balance between cellular proliferation, cell death, and DNA repair in human embryonic stem cells (hESCs). Activity of CDK2 is maximal during S phase and G2; activated by interaction with cyclin E during the early stages of DNA synthesis to permit G1-S transition, and subsequently activated by cyclin A2 (cyclin A1 in germ cells) during the late stages of DNA replication to drive the transition from S phase to mitosis, the G2 phase. EZH2 phosphorylation promotes H3K27me3 maintenance and epigenetic gene silencing. Phosphorylates CABLES1 (By similarity). Cyclin E/CDK2 prevents oxidative stress-mediated Ras-induced senescence by phosphorylating MYC. Involved in G1-S phase DNA damage checkpoint that prevents cells with damaged DNA from initiating mitosis; regulates homologous recombination-dependent repair by phosphorylating BRCA2, this phosphorylation is low in S phase when recombination is active, but increases as cells progress towards mitosis. In response to DNA damage, double-strand break repair by homologous recombination a reduction of CDK2-mediated BRCA2 phosphorylation. Phosphorylation of RB1 disturbs its interaction with E2F1. NPM1 phosphorylation by cyclin E/CDK2 promotes its dissociates from unduplicated centrosomes, thus initiating centrosome duplication. Cyclin E/CDK2-mediated phosphorylation of NPAT at G1-S transition and until prophase stimulates the NPAT-mediated activation of histone gene transcription during S phase. Required for vitamin D-mediated growth inhibition by being itself inactivated. Involved in the nitric oxide- (NO) mediated signaling in a nitrosylation/activation-dependent manner. USP37 is activated by phosphorylation and thus triggers G1-S transition. CTNNB1 phosphorylation regulates insulin internalization. Phosphorylates FOXP3 and negatively regulates its transcriptional activity and protein stability (By similarity). Phosphorylates CDK2AP2 (PubMed:12944431). Phosphorylates ERCC6 which is essential for its chromatin remodeling activity at DNA double-strand breaks (PubMed:29203878).
- 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:- CDK2 CDKN2
-
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（蛋白降解药物相关靶点）
细胞系信息
Gene Symbol
CDK2 CDKN2
NCBI Gene ID
1017
Ensembl ID
ENSG00000123374
Uniprot ID
P24941
筛选标记
N/A
宿主细胞 / 类型
HEK293/人胚肾细胞
规格
1×10^6 cells/ 冻存管
生长培养基
DMEM＋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
~24-45 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
Serine/threonine-protein kinase involved in the control of the cell cycle; essential for meiosis, but dispensable for mitosis. Phosphorylates CTNNB1, USP37, p53/TP53, NPM1, CDK7, RB1, BRCA2, MYC, NPAT, EZH2. Triggers duplication of centrosomes and DNA. Acts at the G1-S transition to promote the E2F transcriptional program and the initiation of DNA synthesis, and modulates G2 progression; controls the timing of entry into mitosis/meiosis by controlling the subsequent activation of cyclin B/CDK1 by phosphorylation, and coordinates the activation of cyclin B/CDK1 at the centrosome and in the nucleus. Crucial role in orchestrating a fine balance between cellular proliferation, cell death, and DNA repair in human embryonic stem cells (hESCs). Activity of CDK2 is maximal during S phase and G2; activated by interaction with cyclin E during the early stages of DNA synthesis to permit G1-S transition, and subsequently activated by cyclin A2 (cyclin A1 in germ cells) during the late stages of DNA replication to drive the transition from S phase to mitosis, the G2 phase. EZH2 phosphorylation promotes H3K27me3 maintenance and epigenetic gene silencing. Phosphorylates CABLES1 (By similarity). Cyclin E/CDK2 prevents oxidative stress-mediated Ras-induced senescence by phosphorylating MYC. Involved in G1-S phase DNA damage checkpoint that prevents cells with damaged DNA from initiating mitosis; regulates homologous recombination-dependent repair by phosphorylating BRCA2, this phosphorylation is low in S phase when recombination is active, but increases as cells progress towards mitosis. In response to DNA damage, double-strand break repair by homologous recombination a reduction of CDK2-mediated BRCA2 phosphorylation. Phosphorylation of RB1 disturbs its interaction with E2F1. NPM1 phosphorylation by cyclin E/CDK2 promotes its dissociates from unduplicated centrosomes, thus initiating centrosome duplication. Cyclin E/CDK2-mediated phosphorylation of NPAT at G1-S transition and until prophase stimulates the NPAT-mediated activation of histone gene transcription during S phase. Required for vitamin D-mediated growth inhibition by being itself inactivated. Involved in the nitric oxide- (NO) mediated signaling in a nitrosylation/activation-dependent manner. USP37 is activated by phosphorylation and thus triggers G1-S transition. CTNNB1 phosphorylation regulates insulin internalization. Phosphorylates FOXP3 and negatively regulates its transcriptional activity and protein stability (By similarity). Phosphorylates CDK2AP2 (PubMed:12944431). Phosphorylates ERCC6 which is essential for its chromatin remodeling activity at DNA double-strand breaks (PubMed:29203878).
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。