CN114958852A - 一种Ifnar1基因敲除小鼠动物模型的构建方法及其应用 - Google Patents
一种Ifnar1基因敲除小鼠动物模型的构建方法及其应用 Download PDFInfo
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Abstract
本发明属于转基因技术领域,具体地涉及一种Ifnar1基因敲除小鼠动物模型的构建方法和应用,所述方法包括使用CRISPR‑Cas9靶向敲除小鼠Ifnar1基因,包括以下步骤:选择小鼠Ifnar1基因的外显子2作为敲除区域,crRNA设计在外显子2两边的内含子中;合成针对Ifnar1基因的crRNA1,序列如SEQ ID N0.1所示;合成针对Ifnar1基因的crRNA2,序列如SEQ ID N0.2所示。本发明利用CRISPR/Cas9系统能够快速高效的将目的Ifnar1基因大片段敲除,并且不留下外源基因片段。
Description
技术领域
本发明属于转基因技术领域,具体地涉及一种Ifnar1基因敲除小鼠动物模型的构建方法和应用。
背景技术
Ifnar1基因编码的蛋白质是一种I型膜蛋白,它形成干扰素α和β受体的两条链之一。受体的结合和激活会刺激Janus蛋白激酶,进而磷酸化几种蛋白质,包括STAT1和STAT2。该蛋白质属于II型细胞因子受体家族,具有抗病毒因子的作用。
在人和小鼠中,Ifnar1蛋白在氨基酸组成上同源性只有48%。在氨基酸数量上也不太一致,小鼠中该基因编码590个氨基酸,人类中该基因编码557个氨基酸。
通常建立基因敲除动物模型,是通过基于同源重组的敲除载体来实现的。这种打靶载体构建繁琐,周期长,费用高,而且效率极低。而且抗性筛选基因通常残留在基因组上,对小鼠表型可能产生影响,即使删除后也会留下Ioxp或者FRT 的重组酶位点序列片段。
基于CRISPR(Clustered regularly interspaced short palindromicrepeats)/Cas9 系统介导的基因组编辑技术,是继锌指核酸酶(Zinc-finger nucleases,ZFNs)和类转录激活因子效应物核酸酶(Transcription activa-tor-1ike effectornuclease, TALEN)后的第三代基因组编辑技术,是基于细菌的获得性免疫系统改造而成。CRISPR/Cas9是基于细菌或古细菌规律成簇的间隔短回文重复 CRISPR(clusteredregularly interspaced short palindromic repeats)介导的获得性免疫系统衍生而来的基因编辑技术。该技术通过RNA碱基互补配对识别DNA,指导Cas9核酸酶切割识别的双链DNA,诱发同源重组(HDR,homologous directed repair)或非同源末端链接(NHEJ,non-homologous end-joining),进而实现目的DNA编辑。该技术的基本要求之一就是设计识别受体细胞内基因组序列的sgRNA(sgRNA,single guiding RNA)位点,该分子负责识别特异性的基因编辑位点。然后介导结合Cas9蛋白行使DNA酶切活性,在设计的位点引入DNA双链断裂损伤,通过胞内的NHEJ或HDR修复途径引入突变。因此,sgRNA是该技术的重要组成部分。目前还未有通过CRISPR/Cas9技术制备Ifnar1基因敲除小鼠的报道。
发明内容
为了解决上述技术问题,发明人公开了一种Ifnar1基因敲除小鼠动物模型的构建方法及其应用。
本发明的技术方案如下:
一种Ifnar1基因敲除小鼠动物模型的构建方法,所述方法包括使用 CRISPR-Cas9靶向敲除小鼠Ifnar1基因。发明人通过对小鼠Ifnar1基因进行分析,进行同源比对,选择同源性最低的基因序列做为靶序列,并设计了crRNA序列,与Cas9蛋白一起对对小鼠Ifnar1基因进行了敲除,有效的提高了靶序列的特异性,使利用该靶序列获得的小鼠模型特异性敲除了Ifnar1基因,减少脱靶的可能性。
进一步的,上述Ifnar1基因敲除小鼠动物模型的构建方法,包括以下步骤:
合成针对Ifnar1基因的crRNA1,序列如SEQ ID N0.1所示;
合成针对Ifnar1基因的crRNA2,序列如SEQ ID N0.2所示。
使用上述crRNA,减少脱靶的可能性。
进一步的,上述Ifnar1基因敲除小鼠动物模型的构建方法,选择小鼠Ifnar1 基因的外显子2作为敲除区域,该基因的外显子2靠近基因的N端,且敲除后会形成移码从而不产生目标蛋白,因此将crRNA设计在外显子2两边的内含子中。
进一步的,上述Ifnar1基因敲除小鼠动物模型的构建方法,包括以下步骤:
从NCBI数据库中下载小鼠Ifnar1基因的核苷酸序列,并进行同源比对。根据crRNA的PAM设计原则及同源比对的结果,合成了crRNA1和crRNA2,序列分别为:
crRNA1序列=SEQ ID NO.1=
5’-GUAGGUGAAUCAGUAGGGACGUUUUAGAGCUAUGCUGUUUUG-3,
crRNA2序列=SEQ ID NO.2=
5’-UACAAGUUUAGCUCAUGGACGUUUUAGAGCUAUGCUGUUUUG-3'。
本发明利用CRISPR/Cas9系统能够快速高效的将目的Ifnar1基因大片段敲除,并且不留下外源基因片段。
进一步的,上述Ifnar1基因敲除小鼠动物模型的构建方法,包括以下步骤:
1)合成针对Ifnar1基因的crRNA1,序列如SEQ ID NO.1所示;合成针对 Ifnar1基因的crRNA2,序列如SEQ ID NO.2所示;
2)PMSG处理C57BL/6雌性小鼠,再注射hCG,与雄性小鼠合笼交配,次日取受精卵进行显微注射,将步骤1)所述的crRNA,tracrRNA与Cas9蛋白共注射到受精卵中,取注射后存活的受精卵移植到假孕母鼠体内,产出小鼠,即为 F0小鼠;
3)提取F0代小鼠尾部DNA,PCR扩增并将产物送测序,鉴定是否为嵌合体;
4)待雄性Founder小鼠到8周龄,雌性小鼠到6周龄,可分别与野生型异性小鼠交配获得F1代杂合子小鼠,小鼠出生14天后PCR鉴定,若有阳性小鼠出生,则表示转基因已经整合到生殖细胞。
本发明所述的方法仅需28天就可以生产F0小鼠,相比现有方法大大节省时间,并提高了成功率。
进一步的,上述Ifnar1基因敲除小鼠动物模型的构建方法,所述步骤2)包括:PMSG处理C57BL/6雌性小鼠,再注射hCG,与雄性小鼠合笼交配,次日取受精卵进行显微注射,将步骤1)所述的crRNA,tracrRNA与Cas9蛋白共注射到受精卵中,取注射后存活的受精卵移植到假孕母鼠体内,胚胎移植的小鼠将会在手术后19-21天出生,即为F0代小鼠,待小鼠出生7天后剪尾提取DNA并进行PCR鉴定,DNA抽提及PCR检测时间为2-3天,整个步骤2)实验周期28-31 天。
上述鉴定方法简单,整个试验周期短。
进一步的,上述Ifnar1基因敲除小鼠动物模型的构建方法,所述步骤3)包括:待雄性Founder小鼠到8周龄,雌性小鼠到6周龄,可分别与野生型异性小鼠交配获得F1代杂合子小鼠,小鼠出生14天后PCR鉴定,若有阳性小鼠出生,则表示转基因已经整合到生殖细胞,这个过程耗时小于120天。上述鉴定方法简单,成功率高。
进一步的,上述Ifnar1基因敲除小鼠动物模型的构建方法,所述步骤4) PCR鉴定中,鉴定引物为:
Forward primer(F1)=SEQ ID NO.3=5’-ACCCACCTGCCAAGGATTGAG-3’
Reverse primer(R1)=SEQ ID NO.4= 5’-AGGGTCAACAATACTGATGTGGC-3’
Reverse primer(R2)=SEQ ID NO.4 =5’-CTGCCCACAAGTAAGTATAGTTCTC-3’。
上述鉴定引物设计巧妙,鉴定成功率高。
进一步的,上述Ifnar1基因敲除小鼠动物模型的构建方法在免疫药物研究中的应用。与IFNAR1相关的疾病包括丙型肝炎、黄热病、麻疹、乳头状瘤、病毒感染性疾病等。在其相关的途径中有免疫反应IFNα/β信号途径和DAP12受体在 NK细胞中的免疫反应作用。
本发明具有以下有益效果:
1、本技术方案发明人通过对小鼠Ifnar1基因进行分析,进行同源比对,选择同源性最低的基因序列做为靶序列,并设计了crRNA序列,与tracrRNA和 Cas9蛋白一起对对小鼠Ifnar1基因进行了敲除,有效的提高了靶序列的特异性,使利用该靶序列获得的小鼠模型特异性敲除了Ifnar1基因,减少脱靶的可能性。
2、本技术方案选用的CRISPR/Cas9系统,CRISPR-Cas9里面需要两个组件,一个是crRNA+tracrRNA,另外一个是内切酶也就是Cas9。gRNA是由crRNA (CRISPRRNA)与tracrRNA(trans_activating CRISPR RNA)嵌合构成的一个约100 个核苷酸大小的RNA复合物,能够与靶向的DNA形成RNA-DNA复合物,这段靶向DNA序列叫做前间隔序列。crRNA与trancrRNA-起构成的gRNA与Cas9 相互作用形成核糖核蛋白。gRNA的5'端的约20bp(对应的是crRNA)通过 RNA-DNA互补配对指引Cas9与靶序列结合进而对靶位点进行切割,造成DNA 双链断裂(double strand break,DSB)。在细胞中,核酸酶造成的DSB在没有修复模板的情况下,以非同源末端连接(nonhomologous end_joining,NHEJ)的方式进行修复。NHEJ能够引起随机长度的碱基插入或缺失,可以破坏编码基因的翻译阅读框架,因此,本发明能更简单、高效的对基因进行修饰,实现了仅合成一对crRNA就能对小鼠Ifnar1进行基因编辑的效果,同时方法简单、高效。
附图说明
附图1为敲除策略;
附图2为F0小鼠PCR鉴定结果:5,6,15和17号小鼠鉴定为阳性F0小鼠;
附图3为为阳性小鼠测序分析,基因组间删除了1561bp;
附图4为为F1小鼠鉴定情况,1和3号小鼠鉴定为阳性F1;
附图5为PCR引物在基因组上相对位置。
具体实施方式
下面结合实例对本发明的方法做进一步说明,实施例中未注明具体条件的实验方法,通常可按常规条件,如J.萨姆布鲁克(Sambrook)等编写的《分子克隆实验指南》中所述的条件,或按照制造厂商所建议的条件进行。本领域相关的技术人员可以借助实施例更好地理解和掌握本发明。但是,实现本发明的方法不应限于本发明实施例所记载的具体方法步骤。
实施例1
靶向敲除基因crRNA的合成:
从NCBI数据库中下载小鼠Ifnar1基因的核苷酸序列,并进行同源比对。根据crRNA的PAM设计原则及同源比对的结果,合成了crRNA1和crRNA2,敲除策略见附图1,序列分别为:
1、合成crRNA1序列=SEQ ID NO.1:
5’-GUAGGUGAAUCAGUAGGGACGUUUUAGAGCUAUGCUGUUUUG-3',
2、合成crRNA2序列=SEQ ID NO.2:
5’-UACAAGUUUAGCUCAUGGACGUUUUAGAGCUAUGCUGUUUUG-3'。
实施例2
F0小鼠的生产和鉴定
PMSG处理C57BL/6雌性小鼠,再注射hCG,与雄性小鼠合笼交配,次日取受精卵进行显微注射,将步骤(1)所述的crRNA,tracrRNA和Cas9蛋白(NEB 公司产品(货号M0646))共注射到受精卵中,取注射后存活的受精卵移植到假孕母鼠体内,胚胎移植的小鼠将会在手术后21天左右出生,即为F0代小鼠,待小鼠出生7天后剪尾提取DNA 并进行PCR鉴定,结果见附图2所示,测序结果分析见附图3。DNA抽提及PCR检测时间为2-3天。因此这个周期所需时间约为31天。
实施例3
F1小鼠的生产与鉴定
待雄性F0小鼠到8周龄,雌性小鼠到6周龄,可分别与野生型异性小鼠交配获得F1代杂合子小鼠,小鼠出生14天后PCR鉴定,若有阳性小鼠出生,则表示转基因已经整合到生殖细胞,结果见附图4所示,这个过程需要120天左右。
实施例4
PCR鉴定引物的设计
鉴定的PCR引物F1设计在敲除区域上游外侧,R1设计在敲除区域下游外侧,R2设计在敲除区域内;如附图5为PCR鉴定引物在基因组上相对位置:
下述为鉴定所用的引物序列:
Forward primer(F1)=SEQ ID NO.3=5’-ACCCACCTGCCAAGGATTGAG-3’
Reverse primer(R1)=SEQ ID NO.4= 5’-AGGGTCAACAATACTGATGTGGC-3’
Reverse primer(R2)=SEQ ID NO.4 =5’-CTGCCCACAAGTAAGTATAGTTCTC-3’。
PCR混合物体系见表1所示,反应条件见表2。
表1 PCR混合物体系
表2 PCR反应条件
由以上实施例可知:本技术方案发明人通过对小鼠Ifnar1基因进行分析,进行同源比对,选择同源性最低的基因序列做为靶序列,并设计了crRNA序列,与tracrRNA和Cas9蛋白一起对对小鼠Ifnar1基因进行了敲除,有效的提高了靶序列的特异性,使利用该靶序列获得的小鼠模型特异性敲除了Ifnar1基因,减少脱靶的可能性。本发明能更简单、高效的对基因进行修饰,实现了仅合成一对 crRNA就能对小鼠Ifnar1基因进行基因编辑的效果,同时方法简单、高效。
以上仅为本发明的较佳实施例而已,不能以此限定本发明的保护范围,即大凡依本发明权利要求书及发明内容所做的简单的等效变化与修改,皆仍属于本发明专利申请的保护范围。
SEQUENCE LISTING
<110> 赛业(苏州)生物科技有限公司
<120> 一种Ifnar1基因敲除小鼠动物模型的构建方法及其应用
<130> 2022
<160> 5
<170> PatentIn version 3.5
<210> 1
<211> 42
<212> RNA
<213> artificial
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guaggugaau caguagggac guuuuagagc uaugcuguuu ug 42
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<212> RNA
<213> artificial
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uacaaguuua gcucauggac guuuuagagc uaugcuguuu ug 42
<210> 3
<211> 21
<212> DNA
<213> artificial
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acccacctgc caaggattga g 21
<210> 4
<211> 23
<212> DNA
<213> artificial
<400> 4
agggtcaaca atactgatgt ggc 23
<210> 5
<211> 25
<212> DNA
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ctgcccacaa gtaagtatag ttctc 25
Claims (9)
1.一种Ifnar1基因敲除小鼠动物模型的构建方法,其特征在于,所述方法包括使用CRISPR-Cas9靶向敲除小鼠Ifnar1基因。
2.根据权利要求1所述的一种Ifnar1基因敲除小鼠动物模型的构建方法,其特征在于,包括以下步骤:
合成针对Ifnar1基因的 crRNA1,序列如SEQ ID N0.1所示;
合成针对Ifnar1基因的crRNA2,序列如SEQ ID N0.2所示。
3.根据权利要求2所述的一种Ifnar1基因敲除小鼠动物模型的构建方法,其特征在于,选择小鼠Ifnar1基因的外显子2作为敲除区域,crRNA设计在外显子2两边的内含子中。
4.根据权利要求3所述的一种Ifnar1基因敲除小鼠动物模型的构建方法,其特征在于,包括以下步骤:
从NCBI数据库中下载小鼠Ifnar1基因的核苷酸序列,并进行同源比对。
5.根据crRNA的PAM设计原则及同源比对的结果,合成了crRNA1和crRNA2,序列分别为:
crRNA1=SEQ ID NO.1 =
5’-GUAGGUGAAUCAGUAGGGACGUUUUAGAGCUAUGCUGUUUUG-3 ,
crRNA2=SEQ ID NO.2 =
5’-UACAAGUUUAGCUCAUGGACGUUUUAGAGCUAUGCUGUUUUG-3 '。
6.根据权利要求4所述的一种Ifnar1基因敲除小鼠动物模型的构建方法,其特征在于,包括以下步骤:
1) 合成针对Ifnar1基因的 crRNA,包括crRNA1和crRNA2,crRNA1序列如SEQ ID N0.1所示;crRNA2序列如SEQ ID N0.2所示;
2)PMSG处理C57BL/6雌性小鼠,再注射hCG,与雄性小鼠合笼交配,次日取受精卵进行显微注射,将步骤1)所述的crRNA与 tracrRNA和Cas9蛋白共注射到受精卵中,取注射后存活的受精卵移植到假孕母鼠体内,产出小鼠,即为F0小鼠;
3)提取F0代小鼠尾部DNA,PCR扩增并将产物送测序,鉴定是否为嵌合体;
4)待雄性F0小鼠到8周龄,雌性小鼠到6周龄,可分别与野生型异性小鼠交配获得F1代杂合子小鼠,小鼠出生14天后PCR鉴定,若有阳性小鼠出生,则表示转基因已经整合到生殖细胞。
7.根据权利要求4所述的一种Ifnar1基因敲除小鼠动物模型的构建方法,其特征在于,所述步骤2)包括:PMSG处理C57BL/6雌性小鼠,再注射hCG,与雄性小鼠合笼交配,次日取受精卵进行显微注射,将步骤1)所述的crRNA和tracrRNA与Cas9蛋白共注射到受精卵中,取注射后存活的受精卵移植到假孕母鼠体内,胚胎移植的小鼠将会在手术后19-21天出生,即为F0代小鼠,待小鼠出生7天后剪尾提取DNA 并进行PCR鉴定,DNA抽提及PCR检测时间为2-3天,整个步骤2)实验周期28-31天。
8.根据权利要求4所述的一种Ifnar1基因敲除小鼠动物模型的构建方法,其特征在于,所述步骤3)包括:待雄性Founder小鼠到8周龄,雌性小鼠到6周龄,可分别与野生型异性小鼠交配获得F1代杂合子小鼠,小鼠出生14天后PCR鉴定,若有阳性小鼠出生,则表示转基因已经整合到生殖细胞,这个过程耗时小于120天;
根据权利要求4所述的一种Ifnar1基因敲除小鼠动物模型的构建方法,其特征在于,所述步骤4)PCR鉴定中,鉴定引物为:
Forward primer (F1)=SEQ ID NO.3= 5’-ACCCACCTGCCAAGGATTGAG-3’
Reverse primer (R1) =SEQ ID NO.4= 5’-AGGGTCAACAATACTGATGTGGC-3’
Reverse primer (R2)= SEQ ID NO.4 =5’-CTGCCCACAAGTAAGTATAGTTCTC-3’。
9.如权利要求1-8任一项所述的Ifnar1基因敲除小鼠动物模型的构建方法在免疫药物研究中的应用。
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