利用CRISPR/Cas9技术构建脂多糖结合蛋白基因敲除小鼠

doi:10.12300/j.issn.1674-5817.2022.002

实验动物与比较医学 ›› 2022, Vol. 42 ›› Issue (4): 294-300.DOI: 10.12300/j.issn.1674-5817.2022.002

• 动物实验技术与方法专题 • 上一篇下一篇

利用CRISPR/Cas9技术构建脂多糖结合蛋白基因敲除小鼠

李思迪¹(), 付彬¹, 郭中坤¹, 林颖杰¹^,², 张振宇¹, 米传靓¹, 王可洲¹()()

^1.山东第一医科大学(山东省医学科学院)实验动物学院(省实验动物中心), 济南 250002
^2.山东第一医科大学附属皮肤病医院(山东省皮肤病医院), 山东省皮肤病性病防治研究所, 济南 250022

收稿日期:2022-01-10 修回日期:2022-04-27 出版日期:2022-08-25 发布日期:2022-09-01
通讯作者: 王可洲（1974—），男，研究员，硕士研究生导师，主要从事药理毒理学研究。E-mail： wangkezhou_cn@163.com。ORCID： 0000-0003-1919-272X
作者简介:李思迪（1996—），女，硕士研究生，药理学专业。E-mail：1322701578@qq.com

Construction of Lipopolysaccaride Binding Protein Knockout Mice Using CRISPR/Cas9 Technology

Sidi LI¹(), Bin FU¹, Zhongkun GUO¹, Yingjie LIN¹^,², Zhenyu ZHANG¹, Chuanliang MI¹, Kezhou WANG¹()()

^1.School of Laboratory Animal & Shandong Laboratory Animal Center, Shandong First Medical University & Shandong Academy of Medical Sciences ), Jinan 250002, China
^2.Shandong Provincial Hospital for Skin Diseases & Shandong Provincial Institute of Dermatology and Veneorology, Shandong First Medical University & Shandong Academy of Medical Sciences, Jinan 250022, China

Received:2022-01-10 Revised:2022-04-27 Published:2022-08-25 Online:2022-09-01
Contact: WANG Kezhou (ORCID: 0000-0003-1919-272X), E-mail: wangkezhou_cn@163.com

摘要/Abstract

摘要：

目的利用成簇的规律间隔的短回文重复序列（clustered regularly interspaced short palindromic repeats，CRISPR）/CRISPR相关蛋白核酸酶9（CRISPR-associated nuclease 9，Cas9）基因编辑技术构建稳定遗传的脂多糖结合蛋白（lipopolysaccaride binding protein，Lbp）基因敲除小鼠。方法根据C57BL/6J小鼠的Lbp基因序列特征，设计sgRNA靶点，删除Lbp基因5’-端蛋白编码保守序列并引入移码突变，使编码LBP失活。提取F0、F1、F2、F3代小鼠基因组，PCR鉴定并测序验证Lbp基因敲除效果，RT-PCR测序验证Lbp基因转录水平，蛋白质印迹法验证F2代LBP蛋白表达水平。结果 F0代获得5只杂合子敲除小鼠（Lbp^+/-）、F1代获得3只Lbp^+/-小鼠，F2代获得4只Lbp纯合子敲除小鼠（Lbp^-/-），F3代获得30只Lbp^-/-小鼠。RT-PCR测序表明，Lbp^-/-小鼠mRNA缺失244 bp且发生移码突变，导致翻译提前终止。蛋白质印迹证明Lbp^-/-小鼠肝脏组织中无LBP蛋白表达。结论通过CRISPR/Cas9技术成功构建可以稳定遗传的Lbp基因敲除小鼠，为后续进一步研究LBP的免疫及其生理作用提供基础。

关键词: 脂多糖结合蛋白, CRISPR/Cas9技术, 稳定遗传, 基因敲除小鼠

Abstract:

Objective To construct a stable hereditary lipopolysaccaride binding protein (Lbp) gene knockout mice by using clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated nuclease 9 (Cas9) gene editing technology. Methods According to the sequence characteristics of Lbp gene in C57BL/6J mice, the target of sgRNA was designed, the 5'-end protein coding conserved sequence of Lbp gene was deleted and the shift mutation was introduced to inactivate LBP. The genome of F0, F1, F2, F3 generation mice was extracted; PCR was used to identify and sequence Lbp knockout; RT-PCR was used to verify Lbp gene transcription, and Western blotting was used to verify LBP protein expression in F2 generation. Results Five Lbp^+/- mice from F0 generation, three Lbp^+/- mice from F1 generation, four Lbp^-/- mice from F2 generation and thirty Lbp^-/- mice from F3 generation were obtained. RT-PCR showed that Lbp^-/- mice mRNA was 244 bp and the translation was stopped early by code-shifting mutation. Western blotting showed that LBP protein was not expressed in the liver of Lbp^-/- mice. Conclusion The Lbp gene knockout mice were successfully constructed by CRISPR/Cas9 technique, which will provide a basis for further study of the immune and physiological effects of LBP.

Key words: Lipopolysaccaride binding protein, CRISPR / cas9 technology, Stable inheritance, Gene knockout mice

中图分类号:

R-332

李思迪, 付彬, 郭中坤, 林颖杰, 张振宇, 米传靓, 王可洲. 利用CRISPR/Cas9技术构建脂多糖结合蛋白基因敲除小鼠[J]. 实验动物与比较医学, 2022, 42(4): 294-300.

Sidi LI, Bin FU, Zhongkun GUO, Yingjie LIN, Zhenyu ZHANG, Chuanliang MI, Kezhou WANG. Construction of Lipopolysaccaride Binding Protein Knockout Mice Using CRISPR/Cas9 Technology[J]. Laboratory Animal and Comparative Medicine, 2022, 42(4): 294-300.

图/表 4

图1 Lbp基因敲除示意图注：Lbp指脂多糖结合蛋白基因。下方为靶点Lbp-L4和Lbp-R1的序列，序列标灰为原间隔序列临近基序（PAM）

Figure 1 Lbp gene knockout diagramNote：Lbp， lipopolysaccaride binding protein gene. Below is the sequences of Lbp-L4 and Lbp-R1， and the sequence gray is protospacer adjacent motif （PAM）.

表1 针对Lbp基因的sgRNA靶点名称及序列

Table 1 Names and sequences of lipopolysaccaride binding protein (Lbp) gene-small guide RNA (gRNA) target

靶点名称

Target name

靶点序列（5’→3’）

Target sequence (5’→3’)

Lbp-L1

Lbp-L2

Lbp-L3

Lbp-L4

Lbp-L5

Lbp-R1

Lbp-R2

Lbp-R3

Lbp-R4

Lbp-R5

AGTTGCTCCCATCATCCCTGG

AGCCTGTGCTGTGACTGCCGG

ACCCCCTGACAACCCTGCAGG

TACACAGACCTAGTACTAAGG

TGGTTAGAAAACTGCCACAGG

GAACAAAGGCCAAAGTCATGG

GCCTTTGTTCCTTCTTCCTGG

GACAGACAGGTGTAACCCAGG

TGTAACCCAGGCCCCAGTGGG

AGAAAGAAACTCGGGACTCGG

图2 F0代及F2代Lbp基因敲除小鼠的基因型鉴定结果注：A为F0代鼠基因型鉴定结果（引物为F/R， M即DL2000 Marker，图中箭头所指条带即为突变条带）；B为F0代3号雌鼠测序峰图（碱基缺失1 457 bp）；C为F2代鼠基因型鉴定结果（左部分引物为F/R，右部分引物为F/WT-R，M即DL2000 Marker，图中箭头所指条带即为纯合子小鼠的突变条带）。KO为敲除，WT为野生型。

Figure 2 Genotyping results of F0 and F2 lipopolysaccaride binding protein （Lbp） gene knockout miceNote： A， the results of genotype identification of F0 generation mice （using F and R primers， M is DL2000 marker， the arrow in the figure points to the mutation strip）； B， the sequencing peak map of F0 offspring No.3 female mice （base deletion 1 457 bp）； C， F2 mice genotype identification results （using F and R primers in left， using F and WT-R primers in right， M is DL2000 marker， the arrow in the figure points to the mutant band of homozygous mice）. KO： knockout； WT： wild type.

图3 RT-PCR测序和蛋白质印迹法分别检测Lbp基因敲除鼠的mRNA转录及蛋白表达情况注：A为Lbp基因敲除鼠mRNA测序峰图；B为Lbp敲除鼠的mRNA序列与WT鼠mRNA序列对比图（最右边数字为每行最后一个碱基在整个Lbp mRNA序列对应的碱基数，第二行划框位置为Lbp-/-翻译终止位置）；C为蛋白质印迹鉴定F2代小鼠肝脏组织中LBP表达结果。KO为敲除，WT为野生型。HE为杂合子，HO为纯合子。

Figure 3 The expression of mRNA and protein in liver tissues of Lbp gene knockout mice detected by RT-PCR sequencing and Western blottingNote： A， mRNA sequencing peak mapping in Lbp knockout mice； B， the mRNA sequence of the Lbp gene knockout mice was aligned against the WT mice （the rightmost number is the base number corresponding to the last base in each line of Lbp mRNA sequence， and the second line is Lbp-/- termination）； C， western blotting of LBP expression in the liver of F2 generation mice. KO， knockout； WT， wild type.HE， heterozygote； HO， homozygote.

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利用CRISPR/Cas9技术构建脂多糖结合蛋白基因敲除小鼠

Construction of Lipopolysaccaride Binding Protein Knockout Mice Using CRISPR/Cas9 Technology

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