p53凋亡刺激蛋白2基因肝脏条件性敲除小鼠的构建和鉴定*

doi:10.3969/j.issn.1672-5069.2022.02.005

实用肝脏病杂志 ›› 2022, Vol. 25 ›› Issue (2): 170-173.doi: 10.3969/j.issn.1672-5069.2022.02.005

p53凋亡刺激蛋白2基因肝脏条件性敲除小鼠的构建和鉴定^*

寇卜心, 柴梦音, 豆双双, 庞丽君, 陈德喜, 刘晓霓

100069 北京市首都医科大学附属北京佑安医院/北京市肝病研究所/北京市肝炎与肝癌精准医疗及转化工程技术研究中心

收稿日期:2021-04-28 出版日期:2022-03-10 发布日期:2022-03-15
通讯作者: 刘晓霓,E-mail: liuxiaoni888@ccmu.edu.cn
作者简介:寇卜心,女,32岁,大学本科,实验员。主要从事肝病实验动物模型研究。 E-mail: koubuxin@sina.com
基金资助:
*北京市自然科学基金资助项目(编号:7192084);首都卫生发展科研专项基金资助项目(编号:2020-2-1152);北京市属医学科研院所公益发展改革试点项目(编号:京医研2019-6)

Construction and identification of liver conditional knockout mice with p53 apoptosis stimulating protein 2 gene

Kou Buxin, Chai Mengyin, Dou Shuangshuang, et al

Beijing Institute of Hepatology, Beijing Precision Medicine and Transformation Engineering, Technology Research Center of Hepatitis and Liver Cancer, You'an Hospital Affiliated to Capital Medical University, Beijing 100069, China

Received:2021-04-28 Online:2022-03-10 Published:2022-03-15

摘要/Abstract

摘要： 目的采用Cre-LoxP系统构建p53凋亡刺激蛋白2(ASPP2)基因肝脏条件性敲除小鼠。方法利用自行设计的sgRNA 序列,在ASPP2基因两端插入LoxP序列,构建ASPP2-flox 小鼠。采用PCR法和测序法对F0代和F1代小鼠进行基因型鉴定。将ASPP2-flox F1小鼠与特异性表达Alb-Cre的工具鼠交配,获得肝脏敲除ASPP2基因小鼠。采用PCR法进行基因型鉴定。采用Westernblot和免疫组化法检测ASPP2基因肝脏敲除小鼠肝脏ASPP2蛋白表达。结果对F0代和F1代动物基因行PCR和测序结果表明,构建ASPP2-flox 小鼠成功;F1代杂合子的基因型为ASPP2 fl/-;将F1代杂合子与Alb-Cre小鼠进行杂交后,回交ASPP2 fl/-小鼠,其基因型为ASPP2fl/fl CreT, 即为ASPP2肝脏条件性敲除小鼠;经Western blot和免疫组化法检测显示ASPP2fl/fl CreT小鼠肝脏ASPP2 蛋白表达显著减少。结论基于Cre-LoxP系统成功构建ASPP2基因肝脏条件性敲除小鼠,为后续实验作了良好的基础。

关键词: Cre-LoxP系统, p53凋亡刺激蛋白2, 条件性敲除, 小鼠

Abstract: Objective The aim of this experiment was to construct p53 apoptosis stimulating protein 2 (ASPP2) gene liver conditional knockout mice by Cre-loxP system. Methods The ASPP2-flox mice were constructed by inserting loxP sequence at both ends of ASPP2 gene with designed sgRNA sequence. The F0 and F1 mice were genotyped by PCR amplification and gene sequencing. The ASPP2-flox F1 mice were mated with Alb-Cre expressing tool mice to obtain liver ASPP2 liver conditional knockout mice, and the genotypes were identified by PCR. The expression of ASPP2 protein in liver tissues of ASPP2 gene knockout mice was detected by Western blot and immunohistochemistry. Results The PCR and sequencing results of F0 and F1 generation mice showed that the ASPP2-flox mice were successfully constructed, and the genotype of F1 generation heterozygotes was ASPP2fl/-; after hybridization with Alb-Cre mice, the F1 generation heterozygotes were backcrossed with ASPP2fl/- mice, and the genotype was ASPP2fL/fLCreT, namely ASPP2 liver conditional knockout mice; the ASPP2 protein expression in liver tissues of ASPP2fL/fLCreT mice by Western blot and immunohistochemistry detection was significantly decreased. Conclusion The ASPP2 gene liver conditional knockout mice is successfully constructed based on Cre-loxP system, which might be beneficial to further experimental study.

Key words: Cre-loxP system, p53 apoptosis stimulating protein 2, Conditional knockout, Mice

寇卜心, 柴梦音, 豆双双, 庞丽君, 陈德喜, 刘晓霓. p53凋亡刺激蛋白2基因肝脏条件性敲除小鼠的构建和鉴定^*[J]. 实用肝脏病杂志, 2022, 25(2): 170-173.

Kou Buxin, Chai Mengyin, Dou Shuangshuang, et al. Construction and identification of liver conditional knockout mice with p53 apoptosis stimulating protein 2 gene[J]. Journal of Practical Hepatology, 2022, 25(2): 170-173.

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p53凋亡刺激蛋白2基因肝脏条件性敲除小鼠的构建和鉴定^*

Construction and identification of liver conditional knockout mice with p53 apoptosis stimulating protein 2 gene

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