CN114150023B - 内皮细胞特异性pfn1基因敲除小鼠模型的构建方法 - Google Patents

内皮细胞特异性pfn1基因敲除小鼠模型的构建方法 Download PDF

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CN114150023B
CN114150023B CN202111078460.2A CN202111078460A CN114150023B CN 114150023 B CN114150023 B CN 114150023B CN 202111078460 A CN202111078460 A CN 202111078460A CN 114150023 B CN114150023 B CN 114150023B
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陈美芳
杨达峰
杨梅
肖智林
李振宇
丁志勇
蹇静
喻南
张丽辉
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Xiangya Hospital of Central South University
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Abstract

本发明属于动物模型及其应用领域，尤其涉及一种内皮细胞特异性pfn1基因敲除小鼠模型的构建方法。其首先将构建好的以C57BL/6基因背景的pfn1‑flox+/+小鼠与C57BL/6基因背景的VE‑Cdherin‑Cre小鼠杂交，得到第1代杂合后代小鼠；选取双阳性的小鼠继续相互杂交得到第2代杂合后代小鼠至第5代杂合后代小鼠；选取4周大小的第5代杂合后代小鼠雄性小鼠；通过连续腹腔注射5天他莫昔芬，诱导小鼠特异性敲除内皮细胞pfn1。

Description

内皮细胞特异性pfn1基因敲除小鼠模型的构建方法
技术领域
本发明属于动物模型及其应用领域，尤其涉及一种内皮细胞特异性pfn1基因敲除小鼠模型的构建方法。
背景技术
基因敲除是自80年代末以来发展起来的一种新型分子生物学技术，是通过一定的途径使机体特定的基因失活或缺失的技术。通常意义上的基因敲除主要是应用DNA同源重组原理，用设计的同源片段替代靶基因片段，从而达到基因敲除的目的。随着基因敲除技术的发展，除了同源重组外，新的原理和技术也逐渐被应用，比较成功的有基因的插入突变和iRNA，它们同样可以达到基因敲除的目的。
诱导性基因敲除是以Cre/loxp系统为基础，利用控制Cre表达的启动子的活性或所表达的Cre酶活性具有可诱导的特点，通过对诱导剂给予时间的控制或利用Cre基因定位表达系统中载体的宿主细胞特异性和将该表达系统转移到动物体内的过程在时间上的可控性，从而在1oxP动物的一定发育阶段和一定组织细胞中实现对特定基因进行遗传修饰之目的的基因敲除技术。
发明内容
针对上述现有技术，本发明的目的是要提供一种内皮细胞特异性pfn1基因敲除小鼠模型的构建方法。
为了解决上述技术问题，本发明采取的技术方案如下：
内皮细胞特异性pfn1基因敲除小鼠模型的构建方法，其具体包括以下步骤：
(1)基于sgRNA的设计原则，在鼠尾5’端和3’端靶位点构建Cas9/sgRNA 质粒，构建打靶载体；
(2)Cas9/sgRNA、打靶载体显微注射到小鼠受精卵中，将构建好的以 C57BL/6基因背景的pfn1-flox+/+小鼠与C57BL/6基因背景的VE-Cdherin-Cre小鼠杂交，得到第1代杂合后代小鼠；
(3)对第1代杂合后代小鼠经过基因鉴定，选取pfn1-flox+/+和 VE-Cadherin-Cre+/+双阳性的小鼠继续相互杂交得到第2代杂合后代小鼠；
(4)重复步骤(2)至第5代杂合后代小鼠；
(5)选取4周大小的第5代杂合后代小鼠雄性
pfn1-flox+/+-VE-Cdherin-Cre+/+的小鼠；
(6)为步骤(4)选取的雄性pfn1-flox+/+-VE-Cdherin-Cre+/+的小鼠连续腹腔注射5天他莫昔芬，注射剂量为10mg/kg，诱导pfn1-flox+/+-Ed-Cre+/+ 小鼠特异性敲除内皮细胞pfn1。
上述的内皮细胞特异性pfn1基因敲除小鼠模型的构建方法，其所述 VE-Cdherin为内皮细胞特异性标记物。
上述的一种内皮细胞特异性pfn1基因敲除小鼠模型的构建方法，其所述 Cas9/sgRNA质粒构建包括：
(1)基于sgRNA的设计原则，在鼠尾5’端和3’端靶位点区域共设计14条 sgRNA：
5’Guide
Guide#1：CGGGAGGGTACCGGATATAG GGG
Guide#2：TTCTTAGGAGCCCGGACGCC TGG
Guide#3：GGGGGAGCGGAAGTTCGAAT GGG
Guide#4：ATATCCGGTACCCTCCCGCC CGG
Guide#5：AAGTTCCCCTACGCCGGGCC CGG
Guide#6：AAGTTCCCCTACGCCGGGCC CGG
Guide#7：ACTTAATTAGAGAGCCCCCG GGG
3’Guide
Guide#8：ATACCACTATAGAGGGGCCT AGG
Guide#9：CCATTACATACTAATTCAAC TGG
Guide#10：CCAGTTGAATTAGTATGTAA TGG
Guide#11：GCAGGCTTGGACCCATTCTC TGG
Guide#12：GAGAATGGGTCCAAGCCTGC GGG
Guide#13：CCAAGCCTGCGGGTGAGGCA GGG
Guide#14：CCCTGCCTCACCCGCAGGCT TGG
(2)按照已设计sgRNA序列合成oligos，通过退火聚合的方式连入pCS载体，连接产物转化后送样测序验证正确。
有益效果：
本发明的内皮细胞条件性敲除pfn1基因小鼠模型的构建方法，是在小鼠体内以一种细胞特异性方式引入突变,从而使pfn1靶基因的缺失发生在试验动物的某一特定的细胞，最大限度达到机理研究的可控性。
附图说明
图1为特异性内皮细胞pfn1基因敲除小鼠模型构建技术路线图；
图2为Flox+/+：pfn1-flox+/+阳性小鼠基因鉴定图；
图3为Cre+/+：VE-Cdherin-Cre+/+阳性小鼠基因鉴定图；
图4为鼠尾5’端靶位点区域的sgRNA；
图5为鼠尾3’端靶位点区域的sgRNA；
图6为PCR引物；
图7为检测结果。
具体实施方式
下面结合实施例对本发明作进一步的说明。然而，本发明的范围并不限于下述实施例。本领域的专业人员能够理解，在不背离本发明的精神和范围的前提下，可以对本发明进行各种变化和修饰。
实施例
参照图1、图2和图3，本发明的构建方法包括以下步骤：
1.靶序列的测序确认
不同品系，基因序列可能有差异。为了保证所设计CRISPR/sgRNA的效率，首先需要对B6鼠尾靶位点序列进行PCR扩增并测序验证，以保证sgRNA识别序列与构建品系小鼠DNA序列完全一致。参照图6，PCR引物如图所示。
对B6鼠尾DNA进行PCR扩增并测序，结果表明：B6鼠尾靶序列与Genebank和Ensembl所给序列一致。
2.CRISPR/sgRNA设计及构建
2.1sgRNA的设计
参照图4、图5，基于sgRNA的设计原则，在靶位点区域共设计14条sgRNA；
2.2Cas9/sgRNA质粒的构建
按照已设计sgRNA序列合成oligos，通过退火聚合的方式连入pCS载体，连接产物转化后送样测序验证正确。
sgRNA的活性检测采用一种自主研发的CRISPR/Cas9活性检测方法-UCATM方式进行。其具有无物种限制、高通量、适应性广、灵敏度高、简便性等优点(具体实验方法详见公司网站)。综合选择EGE-HJL-007-sgRNA7和 EGE-HJL-007-sgRNA11进行下一步实验。
参照图7，检测结果如图所示。
将EGE-HJL-007-sgRNA7和EGE-HJL-007-sgRNA11连入带T7启动子质粒载体上并进行体外转录，得到用于显微注射的RNA。根据打靶方案，设计引物构建打靶载体，并通过酶切鉴定和测序，确认打靶载体构建完成。
3.Cas9/sgRNA、打靶载体显微注射到小鼠受精卵中，以C57BL/6基因背景的 pfn1-flox+/+小鼠与C57BL/6基因背景的VE-Cdherin-Cre小鼠杂交，VE-Cdherin 为内皮细胞特异性标记物，得到第1代杂合后代小鼠；对第1代杂合后代小鼠经过基因鉴定，选取pfn1-flox+/+和VE-Cadherin-Cre+/+双阳性的小鼠继续相互杂交得到第2代杂合后代小鼠；对第2代杂合后代小鼠经过基因鉴定，选取 pfn1-flox+/+和VE-Cadherin-Cre+/+双阳性的小鼠继续相互杂交得到第3代杂合后代小鼠，以此类推，直至得到第5代杂合后代小鼠；选取4周大小的第5代杂合后代小鼠雄性pfn1-flox+/+-VE-Cdherin-Cre+/+的小鼠；为被选取的雄性 pfn1-flox+/+-VE-Cdherin-Cre+/+的小鼠连续腹腔注射5天他莫昔芬，注射剂量为10mg/kg，诱导pfn1-flox+/+-Ed-Cre+/+小鼠特异性敲除内皮细胞pfn1。
以上所述仅为本发明的实施例，并非因此限制本发明的专利范围，凡是利用本发明说明书及附图内容所作的等效结构或等效流程变换，或直接或间接运用在其他相关的技术领域，均同理包括在本发明的专利保护范围内。
SEQUENCE LISTING
<110> 中南大学湘雅医院
<120> 内皮细胞特异性pfn1基因敲除小鼠模型的构建方法
<130> 2021.12.24
<160> 14
<170> PatentIn version 3.5
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Claims (3)

1.一种内皮细胞特异性pfn1基因敲除小鼠模型的构建方法，其特征在于：具体包括以下步骤：
（1）基于sgRNA的设计原则，在鼠尾5’端和3’端靶位点构建Cas9/sgRNA质粒，构建打靶载体；
（2）Cas9/sgRNA、打靶载体显微注射到小鼠受精卵中，将构建好的以C57BL/6基因背景的pfn1-flox+/+小鼠与C57BL/6基因背景的VE-Cdherin-Cre小鼠杂交，得到第1代杂合后代小鼠；
（3）对第1代杂合后代小鼠经过基因鉴定，选取pfn1-flox+/+和VE-Cadherin-Cre+/+双阳性的小鼠继续相互杂交得到第2代杂合后代小鼠；
（4）重复步骤（2）至第5代杂合后代小鼠；
（5）选取4周大小的第5代杂合后代小鼠雄性pfn1-flox+/+-VE-Cdherin-Cre+/+的小鼠；
（6）为步骤（5）选取的雄性pfn1-flox+/+-VE-Cdherin-Cre+/+的小鼠连续腹腔注射5天他莫昔芬，注射剂量为10mg/kg，诱导pfn1-flox+/+-Ed-Cre+/+小鼠特异性敲除内皮细胞pfn1。
2.根据权利要求1所述的一种内皮细胞特异性pfn1基因敲除小鼠模型的构建方法，其特征在于：所述VE-Cdherin为内皮细胞特异性标记物。
3.根据权利要求1所述的一种内皮细胞特异性pfn1基因敲除小鼠模型的构建方法，其特征在于：所述Cas9/sgRNA质粒构建包括：
基于sgRNA的设计原则，在鼠尾5’端和3’端靶位点区域共设计14条sgRNA：
5’Guide
Guide#1：CGGGAGGGTACCGGATATAG GGG
Guide#2： TTCTTAGGAGCCCGGACGCC TGG
Guide#3： GGGGGAGCGGAAGTTCGAAT GGG
Guide#4： ATATCCGGTACCCTCCCGCC CGG
Guide#5： AAGTTCCCCTACGCCGGGCC CGG
Guide#6： AAGTTCCCCTACGCCGGGCC CGG
Guide#7： ACTTAATTAGAGAGCCCCCG GGG
3’Guide
Guide#8： ATACCACTATAGAGGGGCCT AGG
Guide#9： CCATTACATACTAATTCAAC TGG
Guide#10： CCAGTTGAATTAGTATGTAA TGG
Guide#11： GCAGGCTTGGACCCATTCTC TGG
Guide#12： GAGAATGGGTCCAAGCCTGC GGG
Guide#13： CCAAGCCTGCGGGTGAGGCA GGG
Guide#14： CCCTGCCTCACCCGCAGGCT TGG
（2）按照已设计sgRNA序列合成oligos，通过退火聚合的方式连入pCS载体，连接产物转化后送样测序验证正确。
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