收藏本站
收藏 | 论文排版

Inhibiting alternative end-joining(Alt-EJ) pathway enhances CRISPR/Cas9 mediated site-specific knock-in efficiency in CHO-K1 cells

Chuanjie Wang  Ming Wang  Zhaolin Sun  Hongxu Cao  Kai Tu  Jingqing Liu  Zeliang Wei  Beifeng Shen  Jiannan Feng  Jing Wang  
【摘要】:Chinese hamster ovary(CHO) cells are the preferred host to produce a wide array of biopharmaceuticals,especially in antibody-drug production.However,development of recombinant CHO cell lines has been hampered by unstable and variable transgene expression caused by random integration.Although CRISPR/Cas9 with the homology-directed repair(HDR) pathway enables precise integration of transgenes into target genomic sites.However,inherent recalcitrance to HDR-mediated targeted integration of transgenes in CHO cells results in low targeting efficiency.In mammalian cells,DSBs are predominantly repaired by non-homologous endjoining(NHEJ) and HDR.Repair of DSBs via NHEJ encompasses two major sub-pathways:canonical/classical NHEJ(cNHEJ),and non-canonical,alternative end-joining(alt-EJ).Recent reports have shown that inactivation of a-family DNA polymerase θ(Polθ) encoded by the POLQ gene in mouse ES cells and Nalm6 cells could substantially improve the efficiency of gene targeting through blocking alternative end-joining(Alt-EJ) pathway.Whereas,whether this consequence suits for CHO cells has not been demonstrated.Based on above discovery,we establish deficiency of Polθ in CHO-K1 cells by CRISPR-Cas9 and test the efficiency of gene targeting in ROSA26 safe locus using different reporter vectors including fluorescence and puromycin-resistance system.In flow cytometry analysis,we found the in-targeting insertion efficiency was increased nearly 20-fold compared with wild type.Further experiments showed that about 100% integration efficiency of stable cells screened by puromycin had been identified by junction PCR.To our astonishment,there was no obvious impairment of growth to POLQ~(-/-) CHO-K1 cells in contrast to ES cells in previous studies.The establishment of POLQ~(-/-) cell line not only improve the exogenous gene targeting integration efficiency,which accelerating the selection of high-producing and stable cell clones,but also may diminish the side-effect to the host cell by the application of ROSA26 safe locus,which benefits for the continuous production.In short our work provides a simple and efficiency strategy for targeted generation of stable CHO-K1 production cell lines for industrial application in future.

知网文化
【相似文献】
中国期刊全文数据库 前20条
1 Somaya A.Abdelrahman;Moustafa T.Gabr;;Emerging small-molecule therapeutic approaches for Alzheimer's disease and Parkinson's disease based on targeting microRNAs[J];Neural Regeneration Research;2022年02期
2 Zhidi Pan;Jie Chen;Xiaodong Xiao;Yueqing Xie;Hua Jiang;Baohong Zhang;Huili Lu;Yunsheng Yuan;Lei Han;Yuexian Zhou;Huifang Zong;Lei Wang;Rui Sun;Jianwei Zhu;;Characterization of a novel bispecific antibody targeting tissue factor-positive tumors with T cell engagement[J];Acta Pharmaceutica Sinica B;2022年04期
3 Hongxia Duan;Yanhong Liu;Zhonggao Gao;Wei Huang;;Recent advances in drug delivery systems for targeting cancer stem cells[J];Acta Pharmaceutica Sinica B;2021年01期
4 Lei Wang;Mary C.Casey;Sanjeev Kumar V.Vernekar;Rajkumar Lalji Sahani;Karen A.Kirby;Haijuan Du;Huanchun Zhang;Philip R.Tedbury;Jiashu Xie;Stefan G.Sarafianos;Zhengqiang Wang;;Novel PF74-like small molecules targeting the HIV-1 capsid protein: Balance of potency and metabolic stability[J];Acta Pharmaceutica Sinica B;2021年03期
5 Liang Han;Chen Jiang;;Evolution of blood-brain barrier in brain diseases and related systemic nanoscale brain-targeting drug delivery strategies[J];Acta Pharmaceutica Sinica B;2021年08期
6 Qi Qiao;Xiong Liu;Ting Yang;Kexin Cui;Li Kong;Conglian Yang;Zhiping Zhang;;Nanomedicine for acute respiratory distress syndrome: The latest application, targeting strategy, and rational design[J];Acta Pharmaceutica Sinica B;2021年10期
7 Zhijian Luo;Yan Dai;Huile Gao;;Development and application of hyaluronic acid in tumor targeting drug delivery[J];Acta Pharmaceutica Sinica B;2019年06期
8 Jianwei Zhang;Ruilan Dong;Lin Shen;;Evaluation and reflection on claudin 18.2 targeting therapy in advanced gastric cancer[J];Chinese Journal of Cancer Research;2020年02期
9 Qing Yu;Yihan Jiang;Yi Sun;;Anticancer drug discovery by targeting cullin neddylation[J];Acta Pharmaceutica Sinica B;2020年05期
10 Alexandra M.Iavorovschi;Aijun Wang;;Engineering mesenchymal stromal/stem cell-derived extracellular vesicles with improved targeting and therapeutic efficiency for the treatment of central nervous system disorders[J];Neural Regeneration Research;2020年12期
11 Jing Bai;Jialun Duan;Rui Liu;Yafei Du;Qian Luo;Yinuo Cui;Zhanbo Su;Jiarui Xu;Ying Xie;Wanliang Lu;;Engineered targeting tLyp-1 exosomes as gene therapy vectors for efficient delivery of siRNA into lung cancer cells[J];Asian Journal of Pharmaceutical Sciences;2020年04期
12 Lei Wang;Xiaoli Xu;Zhengyu Jiang;Qidong You;;Modulation of protein fate decision by small molecules:targeting molecular chaperone machinery[J];Acta Pharmaceutica Sinica B;2020年10期
13 Lu Zhong;Yanying Liu;Lu Xu;Qingsong Li;Dongyang Zhao;Zhenbao Li;Huicong Zhang;Haotian Zhangb;Qiming Kan;Jin Sun;Zhonggui He;;Exploring the relationship of hyaluronic acid molecular weight and active targeting efficiency for designing hyaluronic acid-modified nanoparticles[J];Asian Journal of Pharmaceutical Sciences;2019年05期
14 ;In vivo epidermal migration requires focal adhesion targeting of ACF7[J];Science Foundation in China;2016年04期
15 Qiang-Nan Feng;Yan Zhang;Sha Li;;Tonoplast targeting of VHA-a3 relies on a Rab5-mediated but Rab7-independent vacuolar trafficking route[J];Journal of Integrative Plant Biology;2017年04期
16 Keishiro Aoyagi;Kikuo Kouhuji;Junya Kizaki;Taro Isobe;Kousuke Hashimoto;Kazuo Shirouzu;;Molecular targeting to treat gastric cancer[J];World Journal of Gastroenterology;2014年38期
17 Li-Sha Chen;An-Xin Wang;Bing Dong;Ke-Feng Pu;Li-Hua Yuan;Yi-Min Zhu;;A new prospect in cancer therapy: targeting cancer stem cells to eradicate cancer[J];癌症;2012年12期
18 Jill Wykosky;Tim Fenton;Frank Furnari;Webster K. Cavenee;;Therapeutic targeting of epidermal growth factor receptor in human cancer: successes and limitations[J];癌症;2011年01期
19 刘菡萏;徐威;王石刚;柯遵纪;;Hydrodynamic modeling of ferrofluid flow in magnetic targeting drug delivery[J];Applied Mathematics and Mechanics(English Edition);2008年10期
20 ;New Anti-cancer Progress Scored in Vascular-targeting Treatment[J];Bulletin of the Chinese Academy of Sciences;2006年03期
中国重要会议论文全文数据库 前20条
1 肖俊;;Targeting 7-Dehydrocholesterol Reductase Integrates Cholesterol Metabolism and IRF3 Activation to Eliminate Infection[A];第十四届全国免疫学学术大会壁报交流集[C];2021年
2 Xiaobao Yang;Yu Liu;Dakang Xu;;Targeting CXCR3 chemokines as a novel therapeutic strategy to suppress the Pancreatic tumor progression through subset T cells[A];第十四届全国免疫学学术大会学术报告[C];2021年
3 Defeng Jiao;Xiaohu Zheng;Xianghui Du;Dong Wang;Ziming Hu;Rui Sun;Zhigang Tian;Binqing Fu;Haiming Wei;;Immunogenic Senescence Sensitizes Lung Cancer to LUNX-Targeting Therapy[A];第十四届全国免疫学学术大会学术报告[C];2021年
4 Jinghao Wang;Xu Chen;Wei Huang;;MicroRNA-369 attenuates hypoxia-induced cardiomyocytes apoptosis and inflammation via targeting TRPV3[A];2022第27届广东省药师周大会论文集[C];2021年
5 Mengtao Cao;Hanchao Gao;;Salt-inducible Kinases inhibitor HG-9-91-01 targets RIPK3 kinase activity to alleviate necroptosis-mediated inflammatory injury[A];第十四届全国免疫学学术大会论文摘要汇编[C];2021年
6 Gui Qian;Yu Wang;Liuyan Li;Yihua Zhang;Manman Li;Sirui Li;Dapeng Yan;;An HIV protein suppresses immunity by targeting STING[A];第十四届全国免疫学学术大会论文摘要汇编[C];2021年
7 Yiqing Shen;;Targeting CD24 anti-hepatocellular carcinoma treatment[A];第十四届全国免疫学学术大会论文摘要汇编[C];2021年
8 Chuanjie Wang;Ming Wang;Zhaolin Sun;Hongxu Cao;Kai Tu;Jingqing Liu;Zeliang Wei;Beifeng Shen;Jiannan Feng;Jing Wang;;Inhibiting alternative end-joining(Alt-EJ) pathway enhances CRISPR/Cas9 mediated site-specific knock-in efficiency in CHO-K1 cells[A];第十四届全国免疫学学术大会论文摘要汇编[C];2021年
9 Xiaoxiao Yu;Jiayao Yan;Xiaotong Chen;Baorui Liu;;Identification of a specific peptide binding to cancer-testis antigen KK-LC-1 from a phage-displayed peptide library[A];2021年中国肿瘤标志物学术大会暨第十五届肿瘤标志物青年科学家论坛论文集[C];2021年
10 lin Cheng;wen Di;guanglei zhuang;;Therapeutic Targeting of CDK12/CDK13 in Epithelial Ovarian Cancer[A];2021年中国肿瘤标志物学术大会暨第十五届肿瘤标志物青年科学家论坛论文集[C];2021年
11 Guoping Wang;Yan Zhang;Yan Guifang;;miR-525-5p regulates radio sensitivity of gastric cancer by targeting RECQL5[A];2021年中国肿瘤标志物学术大会暨第十五届肿瘤标志物青年科学家论坛论文集[C];2021年
12 Yiwen Ma;Xiaorui Li;Xun Qiu;Mingyang Li;Fang Cheng;Tao Sun;;Nanoparticles targeting HER2(TAA0287) are effective in overcoming drug resistance in HER2-positive breast cancer[A];2021年中国肿瘤标志物学术大会暨第十五届肿瘤标志物青年科学家论坛论文集[C];2021年
13 Xuebing Zhou;Xue Wang;Ludan Zhang;Renbo An;Xiangshan Ren;;Crowberry(Empetrum nigrum var. japonicum) inhibits cell proliferation and migration by targeting DEK via the Akt signaling pathway in cholangiocarcinoma cells[A];2021年中国肿瘤标志物学术大会暨第十五届肿瘤标志物青年科学家论坛论文集[C];2021年
14 Xiaohui Zhang;Tingyu Li;Yongzhan Nie;Daiming Fan;Kaichun Wu;Yuanyuan Lu;Xiaodi Zhao;;miR-125b promotes colorectal cancer migration and invasion by dual-targeting CFTR and CGN[A];2021年中国肿瘤标志物学术大会暨第十五届肿瘤标志物青年科学家论坛论文集[C];2021年
15 来鲁华;;Ligand discovery targeting intrinsically disordered proteins[A];2021年全国生物相分离和相变学术研讨会论文集[C];2021年
16 Ning Wang;Yuhao Zhao;Weiwei Sheng;Shuyan Chen;Zhen Li;Fei Wang;;miR-132-3p aggravates lipopolysaccharide-induced injury in endothelial cells by targeting Nrf2[A];2020长三角老年医学高峰论坛、浙江省老年医学学术大会、华东老年医学中心联盟会议、浙江省老年医学联盟会议、浙江省老年病专科联盟会议论文汇编[C];2020年
17 陈学栋;张会江;李朋;李恭会;;Targeting TR4 nuclear receptor alters prostate cancer cell MDV3100-sensitivity[A];2018年浙江省男科学、泌尿外科学学术年会论文汇编[C];2018年
18 Xuedong Chen;;Targeting TR4 nuclear receptor-mediated p21 downregulation to increase apoptosis for reverse of the Enzalutamide-resistance in prostate cancer[A];2019年浙江省医学会泌尿外科学、男科学学术大会论文汇编[C];2019年
19 Jian Wu;;MicroRNA-335-5p inactivates AKT/FOXO1 pathway activity in renal cell carcinoma proliferation inhibition along with directly targeting CCNE2 and CDK2[A];2019年浙江省医学会泌尿外科学、男科学学术大会论文汇编[C];2019年
20 Zerui Wu;Lin Cai;Jianglong Lu;Zhipeng Su;;MicroRNA-93 mediates cabergoline-resistance by targeting ATG7 in prolactinoma[A];2018年浙江省神经外科学学术年会论文汇编[C];2018年
中国博士学位论文全文数据库 前2条
1 朴哲男(Pak Chol Nam);使用转录激活子样因子核酸酶对(TALENs)山羊iPS细胞中PRNP基因的靶向敲除[D];浙江大学;2014年
2 Le Van Minh;[D];华东理工大学;2015年
中国硕士学位论文全文数据库 前8条
1 赵盈盈;结肠癌细胞中CacyBP/SIP核转位后其下游信号分子的筛选[D];宁夏医科大学;2012年
2 Abdul Malik;[D];江苏大学;2017年
3 尹田乐;[D];陕西师范大学;2011年
4 董睿;广告语言的性别差异研究[D];广西师范大学;2011年
5 李蕊;NRDR及其选择性剪接亚型的表达、抗体制备和亚细胞定位[D];汕头大学;2007年
6 王立成;信号肽介导的量子点定位标记细胞的研究[D];吉林大学;2007年
7 Divine Setsofia;通货膨胀目标制对撒哈拉以南非洲货币政策的影响:综合控制方法[D];浙江科技学院;2020年
8 Lamin Rahman Mansaray;[D];长安大学;2014年
 快捷付款方式  订购知网充值卡  订购热线  帮助中心
  • 400-819-9993
  • 010-62982499
  • 010-62783978