|本期目录/Table of Contents|

[1]张佳慧,阎晗,胡德庆.利用CRISPR/Cas9技术构建Aff4基因敲除B16-F10细胞系及AFF4的多克隆抗体制备[J].天津医科大学学报,2023,29(04):372-378.
　ZHANG Jia-hui,YAN Han,HU De-qing.Aff4 gene knockout stable B16-F10 cell line generation with CRISPR/Cas9 system and anti-AFF4 polyclonal antibody preparation[J].Journal of Tianjin Medical University,2023,29(04):372-378.
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利用CRISPR/Cas9技术构建Aff4基因敲除B16-F10细胞系及AFF4的多克隆抗体制备(PDF)
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《天津医科大学学报》[ISSN:1006-8147/CN:12-1259/R]

卷:
29卷
期数:
2023年04期
页码:
372-378
栏目:
基础医学
出版日期:
2023-07-10

文章信息/Info

Title:
Aff4 gene knockout stable B16-F10 cell line generation with CRISPR/Cas9 system and anti-AFF4 polyclonal antibody preparation
文章编号:
1006-8147（2023）04-0372-07
作者:
张佳慧; 阎晗; 胡德庆
天津医科大学基础医学院细胞生物学系，天津300070
Author(s):
ZHANG Jia-hui; YAN Han; HU De-qing
Department of Cell Biology，School of Basic Medical Sciences，Tianjin Medical University，Tianjin 300070，China
关键词:
AFF4; CRISPR/Cas9; 表观遗传; 原核表达; 多克隆抗体
Keywords:
AFF4; CRISPR/Cas9; epigenetic; prokaryotic expression; polyclonal antibody
分类号:
R3
DOI:
-
文献标志码:
A
摘要:
目的：利用CRISPR/Cas9技术构建ALF转录伸长因子4（AFF4）基因敲除的小鼠黑色素瘤细胞（B16-F10），自主制备特异的AFF4多克隆抗体。方法：设计一对分别靶向小鼠Aff4基因第3个外显子和第4个内含子的向导RNA（sgRNA）。将构建成功的重组质粒转染至B16-F10细胞中，并利用抗性筛选出单克隆细胞，进行基因型鉴定和AFF4表达水平检测。验证自主制备的AFF4多克隆抗体的特异性。结果：通过基因组DNA鉴定获得两株正确的单克隆细胞系，其Aff4基因mRNA水平（t=53.08，P<0.001）和AFF4蛋白表达水平（t=15.17，P<0.001）显著降低。免疫共沉淀实验和蛋白免疫印迹实验证明自主制备的抗人/鼠AFF4多克隆抗体特异性良好（t=264.7，P<0.0001）。结论：成功构建了Aff4基因敲除的B16-F10稳定细胞系，并且成功制备抗AFF4蛋白的多克隆抗体。
Abstract:
Objective：ALF transcription elongation factor 4（ AFF4） gene knockout mouse melanoma cells（ B16-F10） were constructed using CRISPR/Cas9 technology，and the specific AFF4 polyclonal antibody was prepared independently. Methods：A pair of guide RNA （ sgRNA） targeting the third exon and the fourth intron of mouse Aff4 gene were designed. The successfully constructed recombinant plasmid was transfected into B16-F10 cells，and monoclonal cells were selected by resistance screening. The genotype was identified and the expression level of AFF4 was detected. The specificity of self-prepared AFF4 polyclonal antibody was verified. Results：Two correct monoclonal cell lines were obtained by genomic DNA identification，and their Aff4 mRNA level （ t=53.08，P<0.001）and AFF4 protein expression level（ t=15.17，P<0.001） were significantly decreased . Protein immunoprecipitation and Western blotting showed that the self-prepared anti-human/mouse AFF4 polyclonal antibody had good specificity（ t=264.7，P<0.000 1）. Conclusion：Two Aff4 knockout B16-F10 cell lines are successfully established. The polyclonal antibody of AFF4 protein is successfully prepared.

参考文献/References:

[1] TAKI T，KANO H，TANIWAKI M，et al. AF5q31，a newly identified AF4-related gene，is fused to MLL in infant acute lymphoblastic leukemia with ins （ 5;11）（ q31;q13q23）[J]. Proc Natl Acad Sci U S A，1999，96（ 25）：14535-14540.
[2] GUO C，CHE Z，YUE J，et al. ENL initiates multivalent phase sepa－ration of the super elongation complex（ SEC） in controlling rapid transcriptional activation[J]. Sci Adv，2020，6（ 14）：eaay4858.
[3] CHEN Y，VOS S M，DIENEMANN C，et al. Allosteric transcription stimulation by RNA polymeraseⅡsuper elongation complex [J]. Mol Cell，2021，81（ 16）：3386-3399.
[4] YU Z，JIANG S，WANG Y，et al. CRISPR-Cas adaptive immune sys－tems in Sulfolobales：genetic studies and molecular mechanisms [J]. Sci China Life Sci，2021，64（ 5）：678-696.
[5] HILLE F，RICHTER H，WONG S P，et al. The biology of CRISPR-Cas：backward and forward [J]. Cell，2018，172（ 6）：1239-1259.
[6] CHANG H H Y，PANNUNZIO N R，ADACHI N，et al. Non-homolo－gous DNA end joining and alternative pathways to double-strand break repair [J]. Nat Rev Mol Cell Biol，2017，18（ 8）：495-506.
[7] MACLEOD G，BOZEK D A，RAJAKULENDRAN N，et al. Genome-wide CRISPR-Cas9 screens expose genetic vulnerabilities and mech－anisms of temozolomide sensitivity in glioblastoma stem cells[J]. Cell Rep，2019，27（ 3）：971-986.
[8] ZHAN T，RINDTORFF N，BETGE J，et al. CRISPR/Cas9 for cancer research and therapy [J]. Semin Cancer Biol，2019，55：106-119.
[9] SHIFRUT E，CARNEVALE J，TOBIN V，et al. Genome-wide CRISPR screens in primary human T cells reveal key regulators of immune function[J]. Cell，2018，175（ 7）：1958-1971.
[10] CYRANOSKI D. CRISPR gene-editing tested in a person for the first time [J]. Nature，2016，539（ 7630）：479.
[11] ISNARD P，COR魪N，NAQUET P，et al. Altered lymphoid develop－ment in mice deficient for the mAF4 proto-oncogene[J]. Blood，2000， 96（ 2）：705-710.
[12] IZUMI K，NAKATO R，ZHANG Z，et al. Germline gain-of-function mutations in AFF4 cause a developmental syndrome functionally linking the super elongation complex and cohesin[J]. Nat Genet，2015， 47（ 4）：338-344.
[13] URANO A，ENDOH M，WADA T，et al. Infertility with defective spermiogenesis in mice lacking AF5q31，the target of chromosomal translocation in human infant leukemia[J]. Mol Cell Biol，2005，25（15）：6834-6845.
[14] ETCHEGARAY J P，ZHONG L，LI C，et al. The histone deacetylase SIRT6 restrains transcription elongation via promoter -proximal pausing[J]. Mol Cell，2019，75（ 4）：683-699.
[15] BACON C W，D'ORSO I. CDK9：a signaling hub for transcriptional control [J]. Transcription，2019，10（ 2）：57-75.
[16] LLOYD S M，LEON D B，BRADY M O，et al. CDK9 activity switch associated with AFF1 and HEXIM1 controls differentiation initiation from epidermal progenitors [J]. Nat Commun，2022，13（ 1）：4408.
[17] LIANG K，VOLK A G，HAUG J S，et al. Therapeutic targeting of mll degradation pathways in mll-rearranged leukemia[J]. Cell，2017， 168（ 1-2）：59-72.e13.
[18] LIANG K，SMITH E R，AOI Y，et al. Targeting processive transcription elongation via SEC disruption for MYC-induced cancer therapy[J]. Cell，2018，175（ 3）：766-779.
[19] HSU T Y，SIMON L M，NEILL N J，et al. The spliceosome is a ther－apeutic vulnerability in MYC-driven cancer[J]. Nature，2015，525 （ 7569）：384-388.
[20] CHENG M，SHENG L，GAO Q，et al. The m（ 6）A methyltransferase METTL3 promotes bladder cancer progression via AFF4/NF-kap－paB/MYC signaling network[J]. Oncogene，2019，38（19）：3667-3680.

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备注/Memo

备注/Memo:
基金项目:国家自然科学基金资助项目（3187282
作者简介:张佳慧（1998-），女，硕士在读，研究方向：医学细胞生物学；
通信作者：胡德庆，E-mail：hudq@tmu.edu.cn。
更新日期/Last Update: 2023-07-10