中国组织工程研究

中国组织工程研究 ›› 2022, Vol. 26 ›› Issue (32): 5179-5184.doi: 10.12307/2022.892

• 组织构建实验造模 experimental modeling in tissue construction • 上一篇    下一篇

CRISPR/Cas9结合Cre-loxP技术制备溴结构域蛋白4基因敲除小鼠

王向宇1，朱睿智1，赵志平1，2，张聿达1，张永涛3，王昌耀3   

  1. 1青岛大学医学部，山东省青岛市  266071；2黄冈市中心医院，湖北省黄冈市  438000；3青岛大学附属医院，山东省青岛市  266071
  • 收稿日期:2021-12-05 接受日期:2022-01-05 出版日期:2022-11-18 发布日期:2022-05-14
  • 通讯作者: 王昌耀，博士，副主任医师，青岛大学附属医院，山东省青岛市 266071
  • 作者简介:王向宇，男，1995年生，山东省龙口市人，汉族，青岛大学在读硕士，主要从事骨科方面的研究。
  • 基金资助:
    国家自然科学基金项目(81772329)，负责人：王昌耀

Construction of bromodomain-containing protein 4 gene knockout mice by CRISPR/Cas9 combined with Cre-loxP technology

Wang Xiangyu1, Zhu Ruizhi1, Zhao Zhiping1, 2, Zhang Yuda1, Zhang Yongtao3, Wang Changyao3   

  1. 1Medical Department of Qingdao University, Qingdao 266071, Shandong Province, China; 2Huanggang Central Hospital, Huanggang 438000, Hubei Province, China; 3Affiliated Hospital of Qingdao University, Qingdao 266071, Shandong Province, China
  • Received:2021-12-05 Accepted:2022-01-05 Online:2022-11-18 Published:2022-05-14
  • Contact: Wang Changyao, MD, Associate chief physician, Affiliated Hospital of Qingdao University, Qingdao 266071, Shandong Province, China
  • About author:Wang Xiangyu, Master candidate, Medical Department of Qingdao University, Qingdao 266071, Shandong Province, China
  • Supported by:
    the National Natural Science Foundation of China, No. 81772329 (to WCY)

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摘要：

文题释义：
CRISPR/Cas9：是一种由小导向RNA介导的对靶基因定向编辑技术，可以快速、有效地修饰各种物种和细胞类型的内源基因。
Cre-loxP：Cre-loxP系统具有很高的组织特异性或空间特异性，在该系统的衍生系统中，Cre ERT-它莫昔芬系统在翻译后水平对Cre入核时间进行调控，具备优良的时间特异性，可以做到控制目的基因在何时、何处精确表达。

背景：目前CRISPR/Cas9结合Cre-loxP技术制备溴结构域蛋白4(bromodomain-containing protein 4，BRD4)基因敲除小鼠的实验方法非常少见。
目的：运用CRISPR/Cas9技术敲除小鼠基因组中BRD4基因片段，构建BRD4基因敲除小鼠。
方法：根据BRD4基因的外显子序列，设计一段gRNA并合成。gRNA体外转录后和Cas9 mRNA以及含loxp位点的质粒共同注射入受精卵细胞中，Cas9通过识别gRNA先导链切割目的片段，然后loxp插入到切割位点，在与Cre配种后，Cre酶会切割loxp位点实现最终的特异性删除效果。注射后的受精卵细胞移植至C57BL/6N雌性小鼠获得子代小鼠，对子代小鼠进行测序鉴定其基因型。将成功导入loxp位点的小鼠BRD4-loxP+/-(F0代)与野生型C57BL/6N小鼠配繁后筛选可稳定遗传的小鼠BRD4-loxP+/-(F1代)，BRD4-loxP+/-小鼠一部分互相交配，一部分与CAGGCre-ERTM鼠交配，获得BRD4-loxP+/+小鼠和BRD4-loxP+/-、CAGGCre-ERTM共表达的小鼠(F2代)；F2代小鼠互相交配可以获得纯合子BRD4-loxP+/+、CAGGCre-ERTM小鼠。将6-8周纯合子小鼠腹腔注射它莫昔芬75 mg/kg(溶于玉米油中)，连续7 d即可完成BRD4基因的诱导敲除。取基因敲除后的小鼠尾部片段，吸附柱法提取DNA，通过琼脂糖凝胶电泳检测BRD4基因片段在小鼠尾部组织中的表达。
结果与结论：①通过PCR筛选鉴定出F1代小鼠7，8，9，10，12，14，16，19，20，21，25，42，43和47号小鼠为成功插入2条loxP片段等位基因的杂合型小鼠；②F2代交互繁育出的F3代纯合子用它莫昔芬诱导后，经PCR凝胶电泳验证表明纯合子小鼠中BRD4基因片段被成功敲除；③提示利用CRISPR/Cas9技术结合Cre-loxP技术成功构建出了BRD4基因敲除小鼠。
缩略语：溴结构域蛋白4：bromodomain-containing protein 4，BRD4

https://orcid.org/0000-0002-0792-4025 (王向宇)

中国组织工程研究杂志出版内容重点：组织构建；骨细胞；软骨细胞；细胞培养；成纤维细胞；血管内皮细胞；骨质疏松；组织工程

关键词: CRISR/Cas9, Cre-loxP, BRD4, 基因敲除, 小鼠, 它莫昔芬

Abstract: BACKGROUND: Currently, the combined use of CRISPR/Cas9 and CRE-LOXP to prepare bromodomain-containing protein 4 (BRD4) gene knockout mice is very rare. 
OBJECTIVE: To construct a BRD4 gene knockout mouse model by using CRISPR/Cas9 technology combined with Cre-loxP technology that can be used to knock out the BRD4 gene fragment in the mouse genome. 
METHODS: According to the exon sequence of BRD4 gene, a gRNA was designed and synthesized. After gRNA was transcribed in vitro, Cas9 mRNA and plasmid containing loxp site were injected into fertilized oocytes. Cas9 cut the target fragment by recognizing the leading strand of the gRNA, and then loxp was inserted into the cleavage site. After breeding with Cre, the Cre enzyme cut the loxp site to achieve the final specific deletion effect. The fertilized oocytes were transplanted into C57BL/6N recipient female mice to obtain progeny mice. The offspring mice were sequenced to identify their genotypes. The BRD4-loxP +/- (F0 generation) mice with loxp loci being successfully introduced was bred with wild-type C57BL/6N mice, and the stably inherited mice BRD4-loxP +/- (F1 generation) were selected. Some of the BRD4-loxP +/- mice interbred with each other and some interbred with CAGGcre-ERTM mice. BRD4-loxP +/+ mice were co-expressed with BRD4-loxP+/- and CAGGcre-ERTM mice (F2 generation). The F2 generation mice were bred to obtain homozygous BRD4-loxP +/+ and CAGGcre-ERTM mice. BRD4 gene knockout was induced by intraperitoneal injection of tamoxifen (75 mg/kg, dissolved in corn oil) in 6-8 weeks homozygous mice for 7 consecutive days. DNA was extracted from the tail fragment of the knockout mice using adsorption column method. The expression of BRD4 gene fragment in mouse tail tissue was detected by agarose gel electrophoresis.
RESULTS AND CONCLUSION: Screening using PCR revealed that mice 7, 8, 9, 10, 12, 14, 16, 19, 20, 21, 25, 42, 43, and 47 of F1 generation were identified as heterozygous mice that were successfully inserted with two alleles of loxP fragment. The F3 homozygotes bred from F2 generation were induced with tamoxifen, and PCR gel electrophoresis verified that the BRD4 gene fragment was successfully knocked out in the homozygous mice. We therefore successfully constructed BRD4 gene knockout mice using CRISPR/Cas9 technology combined with CRE-LOXP technology in this study. 

Key words: CRISR/Cas9, Cre-loxP, BRD4, gene knockout, mouse, tamoxifen

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