产品分类
SYK Knockout HAP1 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: SYK Knockout HAP1 Cell Pool
- 商品编号: LM01010165025
- Gene Symbol: SYK
- Ensembl ID: ENSG00000165025
- Uniprot ID: P43405
- 宿主细胞 / 类型: HAP1/慢性粒细胞白血病
- NCBI Gene ID: 6850
- 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 1/5 传代
- 倍增时间: ~16 hours
- 生长培养基: IMDM＋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: LMAb01165025
- 目标基因介绍: Non-receptor tyrosine kinase which mediates signal transduction downstream of a variety of transmembrane receptors including classical immunoreceptors like the B-cell receptor (BCR). Regulates several biological processes including innate and adaptive immunity, cell adhesion, osteoclast maturation, platelet activation and vascular development. Assembles into signaling complexes with activated receptors at the plasma membrane via interaction between its SH2 domains and the receptor tyrosine-phosphorylated ITAM domains. The association with the receptor can also be indirect and mediated by adapter proteins containing ITAM or partial hemITAM domains. The phosphorylation of the ITAM domains is generally mediated by SRC subfamily kinases upon engagement of the receptor. More rarely signal transduction via SYK could be ITAM-independent. Direct downstream effectors phosphorylated by SYK include VAV1, PLCG1, PI-3-kinase, LCP2 and BLNK. Initially identified as essential in B-cell receptor (BCR) signaling, it is necessary for the maturation of B-cells most probably at the pro-B to pre-B transition. Activated upon BCR engagement, it phosphorylates and activates BLNK an adapter linking the activated BCR to downstream signaling adapters and effectors. It also phosphorylates and activates PLCG1 and the PKC signaling pathway. It also phosphorylates BTK and regulates its activity in B-cell antigen receptor (BCR)-coupled signaling. In addition to its function downstream of BCR plays also a role in T-cell receptor signaling. Plays also a crucial role in the innate immune response to fungal, bacterial and viral pathogens. It is for instance activated by the membrane lectin CLEC7A. Upon stimulation by fungal proteins, CLEC7A together with SYK activates immune cells inducing the production of ROS. Also activates the inflammasome and NF-kappa-B-mediated transcription of chemokines and cytokines in presence of pathogens. Regulates neutrophil degranulation and phagocytosis through activation of the MAPK signaling cascade (By similarity). Required for the stimulation of neutrophil phagocytosis by IL15 (PubMed:15123770). Also mediates the activation of dendritic cells by cell necrosis stimuli. Also involved in mast cells activation. Involved in interleukin-3/IL3-mediated signaling pathway in basophils (By similarity). Also functions downstream of receptors mediating cell adhesion. Relays for instance, integrin-mediated neutrophils and macrophages activation and P-selectin receptor/SELPG-mediated recruitment of leukocytes to inflammatory loci. Plays also a role in non-immune processes. It is for instance involved in vascular development where it may regulate blood and lymphatic vascular separation. It is also required for osteoclast development and function. Functions in the activation of platelets by collagen, mediating PLCG2 phosphorylation and activation. May be coupled to the collagen receptor by the ITAM domain-containing FCER1G. Also activated by the membrane lectin CLEC1B that is required for activation of platelets by PDPN/podoplanin. Involved in platelet adhesion being activated by ITGB3 engaged by fibrinogen. Together with CEACAM20, enhances production of the cytokine CXCL8/IL-8 via the NFKB pathway and may thus have a role in the intestinal immune response (By similarity).
- 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:- SYK
-
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 1/5 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。 - 采用特异性单克隆抗体对野生型细胞和KO细胞的裂解液同时进行Western Blot(WB)检测，目标靶蛋白条带在WT细胞中显示，在KO细胞中消失或减弱。
产品类型： 基因敲除细胞池（蛋白水平已验证）
细胞系信息
Gene Symbol
SYK
NCBI Gene ID
6850
Ensembl ID
ENSG00000165025
Uniprot ID
P43405
筛选标记
N/A
宿主细胞 / 类型
HAP1/慢性粒细胞白血病
规格
1×10^6 cells/ 冻存管
生长培养基
IMDM＋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 1/5 传代
倍增时间
~16 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
LMAb01165025
抗体验证结果
细胞系说明
目标基因介绍
Non-receptor tyrosine kinase which mediates signal transduction downstream of a variety of transmembrane receptors including classical immunoreceptors like the B-cell receptor (BCR). Regulates several biological processes including innate and adaptive immunity, cell adhesion, osteoclast maturation, platelet activation and vascular development. Assembles into signaling complexes with activated receptors at the plasma membrane via interaction between its SH2 domains and the receptor tyrosine-phosphorylated ITAM domains. The association with the receptor can also be indirect and mediated by adapter proteins containing ITAM or partial hemITAM domains. The phosphorylation of the ITAM domains is generally mediated by SRC subfamily kinases upon engagement of the receptor. More rarely signal transduction via SYK could be ITAM-independent. Direct downstream effectors phosphorylated by SYK include VAV1, PLCG1, PI-3-kinase, LCP2 and BLNK. Initially identified as essential in B-cell receptor (BCR) signaling, it is necessary for the maturation of B-cells most probably at the pro-B to pre-B transition. Activated upon BCR engagement, it phosphorylates and activates BLNK an adapter linking the activated BCR to downstream signaling adapters and effectors. It also phosphorylates and activates PLCG1 and the PKC signaling pathway. It also phosphorylates BTK and regulates its activity in B-cell antigen receptor (BCR)-coupled signaling. In addition to its function downstream of BCR plays also a role in T-cell receptor signaling. Plays also a crucial role in the innate immune response to fungal, bacterial and viral pathogens. It is for instance activated by the membrane lectin CLEC7A. Upon stimulation by fungal proteins, CLEC7A together with SYK activates immune cells inducing the production of ROS. Also activates the inflammasome and NF-kappa-B-mediated transcription of chemokines and cytokines in presence of pathogens. Regulates neutrophil degranulation and phagocytosis through activation of the MAPK signaling cascade (By similarity). Required for the stimulation of neutrophil phagocytosis by IL15 (PubMed:15123770). Also mediates the activation of dendritic cells by cell necrosis stimuli. Also involved in mast cells activation. Involved in interleukin-3/IL3-mediated signaling pathway in basophils (By similarity). Also functions downstream of receptors mediating cell adhesion. Relays for instance, integrin-mediated neutrophils and macrophages activation and P-selectin receptor/SELPG-mediated recruitment of leukocytes to inflammatory loci. Plays also a role in non-immune processes. It is for instance involved in vascular development where it may regulate blood and lymphatic vascular separation. It is also required for osteoclast development and function. Functions in the activation of platelets by collagen, mediating PLCG2 phosphorylation and activation. May be coupled to the collagen receptor by the ITAM domain-containing FCER1G. Also activated by the membrane lectin CLEC1B that is required for activation of platelets by PDPN/podoplanin. Involved in platelet adhesion being activated by ITGB3 engaged by fibrinogen. Together with CEACAM20, enhances production of the cytokine CXCL8/IL-8 via the NFKB pathway and may thus have a role in the intestinal immune response (By similarity).
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 1/5 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。