The Construction of Ring Finger Protein 126 Gene Knockout Mouse by Using CRISPR/Cas9 Technique

doi:10.3969/j.issn.1674-5817.2019.01.005

Abstract

Abstract: Objective To establish ring finger protein 126 (RNF126) gene knockout mouse model by CRISPR/Cas9 technique and predict the biological function of RNF126 gene. Methods The knockout primers were designed for exon 4 of Rnf126 gene of C57BL/6 mice to construct sgRNA recombinant expression vector. The mixture of sgRNA and Cas9 mRNA , obtained through transcription in vitro, was injected into the fertilized eggs of C57BL/6 mice by microinjection and the fertilized eggs were transferred into surrogate mice. The DNA of offsprings were screened by PCR identification. Results SgRNA for Rnf126 gene and Cas9 mRNA were successfully obtained. A total of 82 fertilized and microinjected zygotes were successfully transferred into 3 surrogate mice. 11 offsprings were obtained in F₀ generation. One of the positive mouse with 62 bases missing as the founder was selected for propagation and the mutation was also detected in F₁ and F₂ generations. Conclusion Rnf126 gene knockout C57BL/6 mouse model was successfully derived, it may be used as a tool for studying the biological function of RNF126 gene.

Key words: CRISPR/Cas9, Ring finger protein 126 (RNF126), Gene knockout, Animal model

CLC Number:

Q95-33

GAO Meng-qiao, AI Dong-xu, LI Yu, SUN Fei, WANG Jin, FAN Jun-wen, YUAN Zheng, LIU Yuan, SUN Zhao-zeng. The Construction of Ring Finger Protein 126 Gene Knockout Mouse by Using CRISPR/Cas9 Technique[J]. Laboratory Animal and Comparative Medicine, 2019, 39(1): 21-25.

References

[1] Zeng T, Wang Q, Fu J, et al.Impeded Nedd4-1-mediated Ras degradation underlies Ras-driven tumorigenesis[J]. Cell Rep, 2014, 7(3):871-882.
[2] Mei Z, Zhang D, Hu B, et al.FBXO32 targets c-Myc for proteasomal degradation and inhibits c-Myc activity[J]. J Biol Chem, 2015, 290(26):16202-16214.
[3] Lina W, Xin W, Yuehan Z, et al.E3 Ubiquitin ligase RNF126 regulates the progression of tongue cancer[J]. Cancer Med, 2016, 5(8):2043-2047.
[4] Yang X, Pan Y, Qiu Z, et al.RNF126 as a biomarker of a poor prognosis in invasive breast cancer and CHEK1 inhibitor efficacy in breast cancer cells[J]. Clin Cancer Res, 2018, 24(7):1629-1643.
[5] Yangang M, Peng L, Mengyou L, et al.A novel Zn(II) coordination polymer with a honeycomb network: inhibiting human glioma cell strains[J]. Lat Am J Pharm, 2017, 36(8):1575-1579.
[6] Wang Y, Deng O, Feng Z, et al.RNF126 promotes homologous recombination via regulation of E2F1-mediated BRCA1 expression[J]. Oncogene, 2016, 35(11):1363-1372.
[7] Carroll D.Genome engineering with targetable nucleases[J]. Annu Rev Biochem, 2014, 83:409-439.
[8] Deveau H, Barrangou R, Garneau JE, et al.Phage response to CRISPR-encoded resistance in Streptococcus thermophilus[J]. J Bacteriol, 2008, 190:1390-1400.
[9] Mussolino C, Cathomen T.RNA guides genome engineering[J]. Nat Biotechnol, 2013, 31:208-209.
[10] Lieber, MR.The mechanism of double-strand DNA break repair by the nonhomologous DNA end-joining pathway[J]. Annu Rev Biochem, 2010, 79:181-211.
[11] Zhi X, Zhao D, Wang Z, et al.E3 ubiquitin ligase RNF126 promotes cancer cell proliferation by targeting the tumor suppressor p21 for ubiquitinmediated degradation[J]. Cancer Res, 2013, 73:385-394.
[12] Zheng M, Morgan-Lappe SE, Yang J, et al.Growth inhibition and radiosensitization of glioblastoma and lung cancer cells by small interfering RNA silencing of tumor necrosis factor receptor-associated factor 2[J]. Cancer Res, 2008, 68:7570-7578.

[1]	Xin LIU, Shaobo SHI, Cui ZHANG, Bo YANG, Chuan QU. Construction and Evaluation of End-to-side Anastomosis Model of Autologous Arteriovenous Fistula in Mice [J]. Laboratory Animal and Comparative Medicine, 2023, 43(6): 595-603.
[2]	Shuwu XIE, Ruling SHEN, Jinxing LIN, Chun FAN. Progress in Establishment and Application of Laboratory Animal Models Related to Development of Male Infertility Drugs [J]. Laboratory Animal and Comparative Medicine, 2023, 43(5): 504-511.
[3]	Yanjuan CHEN, Ruling SHEN. Progress in the Application of Animal Disease Models in the Medical Research on Colorectal Cancer [J]. Laboratory Animal and Comparative Medicine, 2023, 43(5): 512-523.
[4]	Rui ZHANG, Meiyu LÜ, Jianjun ZHANG, Jinlian LIU, Yan CHEN, Zhiqiang HUANG, Yao LIU, Lanhua ZHOU. Research Progress on Establishing and Evaluation of Acne Animal Models [J]. Laboratory Animal and Comparative Medicine, 2023, 43(4): 398-405.
[5]	Jin LU, Jian WANG, Lian ZHU, Guofeng YAN, Zhengwen MA, Yao LI, Jianjun DAI, Yinqiu ZHU, Jing ZHOU. Establishment of Preeclampsia Model in Goat and Evaluation on Maternal Biological Characteristics [J]. Laboratory Animal and Comparative Medicine, 2023, 43(4): 371-380.
[6]	Jiahui YU, Qian GONG, Lenan ZHUANG. Animal Models of Pulmonary Arterial Hypertension and Their Application in Drug Research [J]. Laboratory Animal and Comparative Medicine, 2023, 43(4): 381-397.
[7]	Yasheng DENG, Jiang LIN, Chiling GAN, Guanfeng ZENG, Jiayin HUANG, Huifang DENG, Yingxian MA, Siyin HAN. Literature Analysis of the Preparation Elements of Animal Models of Skin Photoaging and the Data of Subjects [J]. Laboratory Animal and Comparative Medicine, 2023, 43(4): 406-414.
[8]	Xue WANG, Yonghe HU. Analysis of Common Types and Construction Elements of Diabetic Mouse Models [J]. Laboratory Animal and Comparative Medicine, 2023, 43(4): 415-421.
[9]	Hui HUANG, Yasheng DENG, Tianwei LIANG, Yiqing ZHENG, Yanping FAN, Na RONG, Jiang LIN. Evaluation and Analysis of Modeling Methods for Animal Models with Diminished Ovarian Reserve [J]. Laboratory Animal and Comparative Medicine, 2023, 43(4): 422-428.
[10]	Lei XIANG, Jinzhu JING, Zhen LIANG, Guoqiang YAN, Wenfeng GUO, Meng ZHANG, Wei ZHANG, Yajun LIU. A Visual Analysis on Animal Model of Sarcopenia Based on VOSviewer [J]. Laboratory Animal and Comparative Medicine, 2023, 43(4): 429-439.
[11]	Zhigang TAN, Jinxin LIU, Chuya ZHENG, Wenfeng LIAO, Luping FENG, Hongli PENG, Xiu YAN, Zhenjian ZHUO. Advances and Applications in Animal Models of Neuroblastoma [J]. Laboratory Animal and Comparative Medicine, 2023, 43(3): 288-296.
[12]	Can LAI, Lele LI, Tala HU, Yan MENG. Recent Advances of Animal Models of Renal Interstitial Fibrosis [J]. Laboratory Animal and Comparative Medicine, 2023, 43(2): 163-172.
[13]	Ling HU, Zhibin HU, Yunqing HU, Yuqiang DING. Overview of Studies in Animal Models of Schizophrenia [J]. Laboratory Animal and Comparative Medicine, 2023, 43(2): 145-155.
[14]	Danyang YIN, Yi HU, Rengfei SHI. Advances in Animal Aging Models [J]. Laboratory Animal and Comparative Medicine, 2023, 43(2): 156-162.
[15]	Haosheng WU, Hang SU, Chao ZHU, Wenhui WANG, Shengbing WU, Shuai CUI, Meiqi ZHOU. Research Progress of Animal Models of Stress Cardiomyopathy [J]. Laboratory Animal and Comparative Medicine, 2023, 43(2): 173-179.