CN111206054B - 一种利用CRISPR-Cas9条件性敲除肝脏HO-1基因动物模型的构建方法 - Google Patents
一种利用CRISPR-Cas9条件性敲除肝脏HO-1基因动物模型的构建方法 Download PDFInfo
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Abstract
本发明提供了一种利用CRISPR‑Cas9条件性敲除肝脏HO‑1基因动物模型的构建方法,属于基因工程技术领域,包括以下步骤:1)将sgRNA序列、Cas9基因序列和donor基因序列导入小鼠的受精卵中,得到含有打靶序列的F0代小鼠;2)将所述步骤1)得到的含有打靶序列的F0代小鼠与野生型小鼠杂交,得到含有打靶序列的杂合性小鼠;3)将所述步骤2)得到的含有打靶序列的杂合性小鼠与Alb‑Cre阳性小鼠杂交,得到敲除肝脏HO‑1基因动物模型。
Description
技术领域
本发明属于基因工程技术领域,尤其涉及一种利用CRISPR-Cas9条件性敲除HO-1基因动物模型的构建方法。
背景技术
血红素氧合酶-1(heme oxygenase-1,HO-1,Hmox1)是血红素代谢的起始酶和限速酶,主要功能是催化血红素产生胆红素、一氧化碳和游离铁,减轻血红素对细胞膜的直接损伤及其产生的氧自由基导致的细胞损伤。目前研究表明,HO-1表达增加为机体对抗氧化应激和脂质过氧化反应的一种保护机制,在氧中毒、脂多糖、缺血再灌注等多种肝损伤中发挥保护肝细胞、抗炎和抗纤维化作用。HO-1缺陷小鼠应激能力下降,可导致肝细胞发生炎症、坏死。采用胆碱蛋氨酸缺乏(methionine-choline deficient,MCD)饮食分别喂养C57BL/6J小鼠4周、8周诱导非酒精性脂肪性肝炎/肝纤维化模型,结果发现模型组小鼠肝组织中HO-1表达明显上调,表明在非酒精性脂肪性肝炎/肝纤维化中存在HO-1的激活,其表达和肝细胞的炎性坏死、纤维增生及细胞凋亡等密切相关。进一步应用HO-1化学诱导剂血晶素、腺病毒Ad-HO-1及HO-1抑制剂锌原卟啉调节HO-1表达,结果表明内源性升高的HO-1和外源性诱导HO-1可抑制氧化应激反应,下调促炎、促纤维化相关基因表达,减缓肝脏炎症及纤维化进展。但是现有技术中还未有关于肝脏条件性敲除肝脏HO-1基因动物模型的报道。
发明内容
有鉴于此,本发明的目的在于提供一种利用CRISPR-Cas9条件性敲除肝脏HO-1基因动物模型的构建方法,采用本发明提供的方法能够得到敲除HO-1基因动物模型。
为了实现上述发明目的,本发明提供了以下技术方案和构建模型的特点:
本发明提供了一种利用CRISPR-Cas条件性敲除肝脏HO-1基因动物模型的构建方法,包括以下步骤:
1)将sgRNA序列、Cas9基因序列和donor基因序列导入小鼠的受精卵中,得到含有打靶序列的F0代小鼠;
2)将所述步骤1)得到的含有打靶序列的F0代小鼠与野生型小鼠杂交,得到含有打靶序列的杂合性小鼠;
3)将所述步骤2)得到的含有打靶序列的杂合性小鼠与Alb-Cre阳性小鼠杂交,得到敲除HO-1基因动物模型。
优选的,所述小鼠包括C57BL/6J小鼠。
本发明提供了一种利用CRISPR-Cas9条件性敲除肝脏HO-1基因动物模型的构建方法,包括以下步骤:1)将sgRNA序列、Cas9基因序列和donor基因序列导入小鼠的受精卵中,得到含有打靶序列的F0代小鼠;2)将所述步骤1)得到的含有打靶序列的F0代小鼠与野生型小鼠杂交,得到含有打靶序列的杂合性小鼠;3)将所述步骤2)得到的含有打靶序列的杂合性小鼠与Alb-Cre阳性小鼠杂交,得到敲除肝脏HO-1基因动物模型。
附图说明
图1为设计Hmox1条件性基因敲除小鼠策略图;
图2为中靶序列:绿色为外显子,红色为LoxP位点;
图3为引物设计策略;
图4为F1代小鼠鉴定电泳图。
具体实施方式
本发明提供了一种利用CRISPR-Cas9条件性敲除肝脏HO-1基因动物模型的构建方法,包括以下步骤:
1)将sgRNA序列、Cas9基因序列和donor基因序列导入小鼠的受精卵中,得到含有打靶序列的F0代小鼠;
2)将所述步骤1)得到的含有打靶序列的F0代小鼠与野生型小鼠杂交,得到含有打靶序列的杂合性小鼠;
3)将所述步骤2)得到的含有打靶序列的杂合性小鼠与Alb-Cre阳性小鼠杂交,得到敲除肝脏HO-1基因动物模型。
在本发明中,所述敲除肝脏HO-1基因动物模型委托南京大学南京生物医药研究院构建得到,用到的sgRNA、donor基因序列和Cas9基因序列以及具体的操作步骤由南京大学南京生物医药研究院提供。
在本发明中,所述小鼠优选包括C57BL/6J小鼠,本发明对所述C57BL/6J小鼠的来源没有特殊限定,采用常规市售即可。
下面结合实施例对本发明提供的技术方案进行详细的说明,但是不能把它们理解为对本发明保护范围的限定。
实施例1
敲除肝脏HO-1基因动物模型委托南京大学南京生物医药研究院构建得到:
1、小鼠Hmox1(HO-1)基因的sgRNA设计、构建和纯化
设计Hmox1条件性基因敲除小鼠策略,如图1所示。根据策略,确定flox区域为Hmox1基因的exon2,使用麻省理工学院的CRISPR Design工具(http://crispr.mit.edu/),依据Score的高低设计一对长度为20bp的针对Hmox1基因DNA的寡聚核苷酸链序列用于制备sgRNA,将设计的序列合成PAGE的产物。根据sgRNA序列设计一个携带靶位点同源区域及loxp位点的Donor vector,并在该靶区域设计引物用于后续小鼠的基因鉴定。订购相应Oligo sgRNA序列如下:
表1 Oligo sgRNA序列
sgRNA名称 | sgRNA序列(5'-3‘) | 序号 | PAM |
5'end | tgtgtaacatcacactccgc | SEQIDNo:1 | TGG |
3'end | aagagctcgagctcggattg | SEQIDNo.2 | TGG |
中靶载体序列如图2所示;引物设计策略如图3;
将合成后的2条单链寡聚核苷酸sgRNA序列退火(95℃5min后自然降至室温)形成双链DNA,与sgRNA的骨架质粒进行连接,16度连接1h,将连接产物转化到DH5a感受态细胞中,涂布到卡那霉素抗性的LB平板,挑取单克隆进行培养及鉴定,得到sgRNA序列正确***的克隆,用试剂盒提取质粒。连接体系:
表2连接体系
囊胚注射筛选高效的上述两个sgRNA,根据筛选获得的高效sgRNA制作携带靶位点同源区域以及loxP位点的片段的重组质粒(Donor vector),将重组质粒转化到DH5a感受态细胞中,通过氨苄抗性以及***片段的测序,对阳性克隆质粒进行筛选及鉴定,挑选正确的菌落克隆,扩大培养后提取质粒并纯化,获得的donor片段产物用于注射使用。
2、体外转录:将sgRNA的表达载体线性化,经酚氯仿抽提纯化后,作为模板用于体外转录,并依照MEGAshortscript Kit试剂盒体外合成sgRNA(表1)。以上述质粒为模板,以高保值酶对sgRNA进行PCR扩增,循环数设为30个,25μl反应体系。然后将产物纯化后用作体外转录模板,体外转录参照MEGAshortscript Kit说明进行。转录后获得的单链序列用于后续的受精卵注射。
3、Cas9/sgRNA的显微注射:将上述转录产物sgRNA和cas9以及纯化后的donor片段混合并调整浓度,使用显微注射仪将混合物显微注射到C57BL/6J小鼠受精卵中,再将受精卵移植到假孕的C57BL/6J母鼠子宫中,等待F0代小鼠出生。
4、F0小鼠的鉴定:对出生的99只F0代小鼠进行基因型鉴定。小鼠出生后5-7天剪尾,采用剪脚趾法标记小鼠,并将剪取鼠尾组织经酚氯仿法提取DNA进行PCR鉴定,鉴定使用的引物和程序见表3和4。回收PCR产物送商业公司进行测序,确认小鼠的基因型,含有打靶序列的即为阳性。结果见表5。
表3引物信息
表4反应程序
结果显示获得3只阳性F0代鼠,如下:
表5阳性结果
ID | Gender | Color | Gty | Gen |
98 | ♀ | B | 阳性 | F0 |
129 | ♂ | B | 阳性 | F0 |
265 | ♀ | B | 阳性 | F0 |
选择以上F0代小鼠分别配繁C57BL/6J,以获得阳性F1代小鼠。
5、F0代小鼠的可遗传性检测:将PCR以及测序正确的F0代小鼠与野生型C57BL/6小鼠进行交配,产生F1代小鼠,依据F0代小鼠的鉴定方法对F1代小鼠进行鉴定,获得的阳性F1代杂合子小鼠即可稳定遗传。
表6阳性F1代小鼠
ID | Gender | Color | Gty | Gen | F/M |
266 | ♂ | B | F1/wt | F1 | 265#/C57BL/6J |
268 | ♀ | B | F1/wt | F1 | 265#/C57BL/6J |
270 | ♀ | B | F1/wt | F1 | 265#/C57BL/6J |
F1代阳性鼠鉴定报告如图4。
结论:266#、268#、270#:Fl/wt,其余为野生型。
阳性F1代小鼠进一步与Alb-Cre阳性小鼠配繁,即获得HO-1肝脏特异性基因敲除小鼠模型。
以上所述仅是本发明的优选实施方式,应当指出,对于本技术领域的普通技术人员来说,在不脱离本发明原理的前提下,还可以做出若干改进和润饰,这些改进和润饰也应视为本发明的保护范围。
序列表
<110> 河北医科大学第三医院
<120> 一种利用CRISPR-Cas9条件性敲除肝脏HO-1基因动物模型的构建方法
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Claims (1)
1.一种利用CRISPR-Cas9条件性敲除肝脏HO-1基因动物模型的构建方法,其特征在于,包括以下步骤:
1)将sgRNA序列、Cas9基因序列和donor基因序列导入小鼠的受精卵中,得到含有打靶序列的F0代小鼠;
所述小鼠为C57BL/6J小鼠;
所述sgRNA序列如SEQ ID No:1和SEQ ID No:2所示;
2)将所述步骤1)得到的含有打靶序列的F0代小鼠与野生型小鼠杂交,得到含有打靶序列的杂合性小鼠;
3)将所述步骤2)得到的含有打靶序列的杂合性小鼠与Alb-Cre阳性小鼠杂交,得到敲除肝脏HO-1基因动物模型。
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