CN114410691B - Slc35e1基因敲除小鼠动物模型的构建方法和应用 - Google Patents
Slc35e1基因敲除小鼠动物模型的构建方法和应用 Download PDFInfo
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Abstract
本发明公开了SLC35E1基因敲除小鼠动物模型的构建方法和应用,属于生物技术领域。构建方法包括:根据SLC35E1基因序列确定SLC35E1小鼠待敲除基因的两对特异性靶点gRNA1和gRNA2、gRNA3和gRNA4,并与Cas9核酸酶体外转录成mRNA,显微注射入小鼠受精卵并移植至代孕母鼠输卵管内,待产出小鼠后传代培养,经基因型鉴定得到稳定遗传的SLC35E1基因敲除小鼠动物模型。通过构建的SLC35E1基因敲除小鼠动物模型研究银屑病或分枝杆菌感染性疾病,并验证SLC35E1基因在制备和筛选治疗银屑病或分枝杆菌感染性疾病药物中的应用,为人分枝杆菌感染性疾病的作用机制和治疗提供依据。
Description
技术领域
本发明涉及生物技术领域,具体涉及SLC35E1基因敲除小鼠动物模型的构建方法和应用。
背景技术
溶质转运载体35(Solute carrier 35,SLC35)家族,属于SLC家族中的第35个成员,分为7个亚型(A-G),是一种由300–400个氨基酸组成的疏水性蛋白质,属于Ⅲ型跨膜转运蛋白分子。SLC35多数成员定位在高尔基复合体膜上,另有一小部分定位在细胞膜上,主要转运细胞内相应单糖核苷酸并保证糖基化过程的顺利进行,SLC35被认为是糖基化途径中的关键调节因子。对SLC35缺陷型多细胞生物的生长发育研究及病理表型分析发现,SLC35转运蛋白缺乏可参与多种结缔组织病和遗传性疾病的发生,并且在肿瘤的发生及其转移等许多病理过程中发挥重要作用。
现有技术中,已知SLC35缺陷引起的疾病包括人类II型遗传性糖基化紊乱(CDGIIc)和II型遗传性包涵体肌病两种,近来还发现SLC35家族中某些成员与肿瘤的关系密切。此外,有研究显示在麻风患者中SLC35D1、SLC35D2的mRNA表达水平升高,提示SLC35D家族在麻风发病机制中可能具有重要作用,研究还发现SLC35E1在麻风病中发挥重要作用,通过对8个家系29个麻风患者及正常对照组进行全外显子测序,进一步扩大验证后发现麻风病患者研究中存在SLC35E1位点的突变;结合临床症状分析发现,SLC35E1是与皮损改变相关的易感基因。同时,SLC35E1的另一亚型SLC35E2B在斑块型银屑病患者的全外显子测序中被发现是突变基因,由此推断SLC35E1也可能参与银屑病的发病。
目前有关SLC35E1的研究甚少,其功能尚不清楚,SLC35E家族的成员SLC35E2B缺陷时可出现免疫缺陷及肌肉萎缩等症状,这与麻风病免疫功能紊乱及肌肉萎缩有相似临床症状,推测中国的麻风病患者发病可能与SLC35E2B基因突变有关。因此,建立敲除SLC35E1基因的C57小鼠模型,并在此基础上构建银屑病及分枝杆菌感染的模型对探究SLC35E1在银屑病及分枝杆菌感染中发挥的作用、相关机制及可能参与的分子通路具有重要意义,同时为指导银屑病和分枝杆菌感染性疾病的治疗提供新的理论依据。
发明内容
本发明的第一目的是提供SLC35E1基因敲除小鼠动物模型的构建方法。
本发明的第二个目的是提供SLC35E1基因在制备和筛选治疗银屑病或分枝杆菌感染性疾病药物中的应用。
为实现上述目的,本发明采用如下技术方案:
本发明提供的SLC35E1基因敲除小鼠动物模型的构建方法,基于CRISPR/Cas9基因敲除技术构建得到,包括以下步骤:
(1)根据SLC35E1基因序列确定SLC35E1小鼠待敲除基因的两对特异性靶点gRNA1和gRNA2、gRNA3和gRNA4,并与Cas9核酸酶体外转录成mRNA;
(2)将两对特异性靶点gRNA1和gRNA2、gRNA3和gRNA4和mRNA一起显微注射入小鼠受精卵内,显微注射后的受精卵移植至代孕母鼠输卵管内;
(3)待产出小鼠后继续传代培养,经基因型鉴定,得到稳定遗传的SLC35E1基因敲除小鼠动物模型;
其中,所述gRNA1的靶点序列如SEQ ID NO:1所示,所述gRNA2的靶点序列如SEQ IDNO:2所示,所述gRNA3的靶点序列如SEQ ID NO:3所示,所述gRNA4的靶点序列如SEQ ID NO:4所示。
优选地,步骤(3)中,显微注射后的受精卵移植至代孕母鼠输卵管内,产出小鼠,即为F0代小鼠;提取F0代小鼠尾部DNA,PCR扩增并将产物测序,将阳性小鼠与野生型异性小鼠交配获得F1代杂合子小鼠,将F1代杂合子小鼠杂交获得F2代纯合子小鼠,直至得到稳定遗传的SLC35E1基因敲除小鼠动物模型;其中,若为一个680bp的条带,为阳性纯合子;若为两个990bp和680bp的条带,为阳性杂合子;若为一个990bp的条带,为野生型对照。
本发明还提供一种SLC35E1基因敲除小鼠动物模型,通过上述SLC35E1基因敲除小鼠动物模型的构建方法得到。
本发明还提供SLC35E1基因敲除小鼠动物模型在银屑病或分枝杆菌感染性疾病研究中的应用。
本发明还提供SLC35E1基因在制备和筛选治疗银屑病药物中的应用。
本发明还提供SLC35E1基因在制备和筛选治疗分枝杆菌感染性疾病药物中的应用。
与现有技术相比,本发明的有益效果在于:
(1)本发明基于CRISPR/Cas9基因敲除技术设计SLC35E1小鼠待敲除基因的特异性靶点gRNA1、gRNA2、gRNA3和gRNA4,并成功构建SLC35E1基因敲除小鼠动物模型,经鉴定SLC35E1基因的exon1-4、exon2-5被敲除,为银屑病或分枝杆菌感染性疾病研究提供主要动物实验材料。
(2)本发明通过咪喹莫特乳膏构建SLC35E1基因缺陷小鼠银屑病模型、和用分枝杆菌感染构建SLC35E1基因缺陷小鼠感染模型,利用分子生物学、免疫组化染色、单细胞测序等手段研究SLC35E1基因在分枝杆菌感染过程中对银屑病、分枝杆菌感染性疾病的临床表现、病理特征、疾病状态、发病机制及可能的分子通路之间的关系,为研究SLC35E1家族在人分枝杆菌感染性疾病中的作用机制及相关治疗研究提供理论依据。
我们将参考以下详细说明更易于理解本发明的上述以及其他特征、方面和优点。
附图说明
通过阅读参照以下附图对非限制性实施例所作的详细描述,本发明的其它特征、目的和优点将会变得更显著:
图1是实施例中构建SLC35E1基因敲除小鼠动物模型的方法流程图。
图2是非目标gRNA分析引物;其中:(a)gRNA1的非目标分析序列;(b)gRNA2的非目标分析序列;(c)gRNA3的非目标分析序列;(d)gRNA4的非目标分析序列。
图3是咪喹莫特乳膏构建SLC35E1基因敲除小鼠银屑病模型的方法流程图。
图4是海鱼分枝杆菌构建SLC35E1基因敲除小鼠分枝杆菌感染模型的方法流程图。
图5是海鱼分枝杆菌构建的SLC35E1基因敲除小鼠分枝杆菌感染模型。
图6是实施例3中小鼠足垫脓液涂片的Ziehl-Neelsen抗酸染色。
图7是实施例3中小鼠足垫组织涂片的Ziehl-Neelsen抗酸染色。
具体实施方式
为使本发明实施例的目的、技术方案和优点更加清楚,下面将结合本发明实施例的附图,对本发明实施例的技术方案进行清楚、完整地描述。显然,所描述的实施例是本发明的一部分实施例,而不是全部的实施例。基于所描述的本发明的实施例,本领域普通技术人员在无需创造性劳动的前提下所获得的所有其它实施例,都属于本发明保护的范围。
以下实施例中敲除SLC35E1基因的基本信息为:敲除基因名称(Ensembl):ENSMUSG00000019731,敲除基因GenBank编码:NM_177766.3;敲除基因的exon数量:6,敲除针对的exon:exon1-4和exon2-5;构建的gRNA数目:2对;显微共注射:将体外转录生成的Cas9 mRNA和gRNA显微共注射到小鼠受精卵内。
实施例1
如图1和2所示,基于CRISPR/Cas9基因敲除技术构建SLC35E1基因敲除小鼠动物模型,步骤如下:
(1)根据SLC35E1基因序列(GenBank编码:NM_177766.3)确定SLC35E1小鼠待敲除基因的两对特异性靶点:
Pair1:gRNA1:aacgggtgagtagctacgcatgg(SEQ ID NO:1);
gRNA2:ggggatccacacataagtcctgg(SEQ ID NO:2);
Pair2:gRNA3:cgtgagaatccgacgcaccttgg(SEQ ID NO:3);
gRNA4:gtctacaagatatatgctcgagg(SEQ ID NO:4);
根据两对特异性靶点设计扩增引物序列如下:
SLC35E1-pair1-F:cattacagggccatttgaggagg(SEQ ID NO:5)
SLC35E1-pair1-R:gtgctgagtgaagcctggacctt(SEQ ID NO:6);
SLC35E1-pair2-F:ctgtgtctactatggtacgcgctgag(SEQ ID NO:7);
SLC35E1-pair2-R:gattcgcatcatacttggtctgtgtg(SEQ ID NO:8);
按照如表1所示的PCR反应体系及反应条件进行PCR扩增,与Cas9核酸酶体外转录成mRNA;
(2)将两对特异性靶点gRNA1和gRNA2、gRNA3和gRNA4和mRNA一起显微注射入小鼠受精卵内,显微注射后的受精卵移植至代孕母鼠输卵管内;
(3)显微注射后的受精卵移植至代孕母鼠输卵管内,产出小鼠,即为F0代小鼠;提取F0代小鼠尾部DNA,PCR扩增并将产物测序,将阳性小鼠与野生型异性小鼠交配获得F1代杂合子小鼠,将F1代杂合子小鼠杂交获得F2代纯合子小鼠,直至得到稳定遗传的SLC35E1基因敲除小鼠动物模型,其中按照如下基因鉴定引物进行基因型鉴定:
Mouse SLC35E1-F:ctgtgtctactatggtacgcgctgag(SEQ ID NO:9);
Mouse SLC35E1-R:gattcgcatcatacttggtctgtgtg(SEQ ID NO:10);
Mouse SLC35E1-Wt/He-F:atctgcccgagtatagatgtgccc(SEQ ID NO:11);
若为一个680bp的条带,为阳性纯合子;若为两个990bp和680bp的条带,为阳性杂合子;若为一个990bp的条带,为野生型对照,统计结果如表2所示。
表1
表2
实施例2
通过实施例1构建的SLC35E1基因敲除小鼠动物模型进一步构建银屑病动物模型验证SLC35E1基因在制备和筛选治疗银屑病药物中的应用。
按照如图3所示的方法,采用分子生物学、免疫组化染色和单细胞测序等手段研究SLC35E1基因与分枝杆菌感染过程中银屑病之间的关系。选取经基因型鉴定后的SLC35E1基因敲除小鼠,在SPF级环境中饲养至8周时,用咪喹莫特乳膏构建银屑病动物模型。
实施例3
通过实施例1构建的SLC35E1基因敲除小鼠动物模型进一步构建海鱼分枝杆菌感染动物模型验证SLC35E1基因在制备和筛选治疗分枝杆菌感染性疾病药物中的应用。
按照如图4所示的方法,采用分子生物学、免疫组化染色和单细胞测序等手段研究SLC35E1基因与分枝杆菌感染性疾病之间的关系。选取经基因型鉴定后的SLC35E1基因敲除小鼠,在SPF级环境中饲养至8周时,用海鱼分枝杆菌进行足垫的皮下注射构建分枝杆菌感染动物模型,如图5所示。
取上述小鼠足垫脓液进行涂片Ziehl-Neelsen抗酸染色,可见大量抗酸杆菌,如图6所示;取上述小鼠足垫组织及进行涂片Ziehl-Neelsen抗酸染色,可见抗酸杆菌如图7所示。
序列表
<110> 南方医科大学皮肤病医院(广东省皮肤病医院、广东省皮肤性病防治中心、中国麻风防治研究中心)
<120> SLC35E1基因敲除小鼠动物模型的构建方法和应用
<141> 2021-08-02
<160> 11
<170> SIPOSequenceListing 1.0
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aacgggtgag tagctacgca tgg 23
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<213> Artificial Sequence
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ggggatccac acataagtcc tgg 23
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<213> Artificial Sequence
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cgtgagaatc cgacgcacct tgg 23
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gtctacaaga tatatgctcg agg 23
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<212> DNA
<213> Artificial Sequence
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cattacaggg ccatttgagg agg 23
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<212> DNA
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gtgctgagtg aagcctggac ctt 23
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ctgtgtctac tatggtacgc gctgag 26
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gattcgcatc atacttggtc tgtgtg 26
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ctgtgtctac tatggtacgc gctgag 26
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gattcgcatc atacttggtc tgtgtg 26
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atctgcccga gtatagatgt gccc 24
Claims (1)
1.SLC35E1基因敲除小鼠动物模型的构建方法,其特征在于,基于CRISPR/Cas9基因敲除技术构建,包括以下步骤:
(1)根据SLC35E1基因序列确定SLC35E1小鼠待敲除基因的两对特异性靶点gRNA1和gRNA2、gRNA3和gRNA4,并与Cas9核酸酶体外转录成mRNA,其中:
SLC35E1基因的GenBank编码为NM_177766.3;
所述gRNA1的靶点序列如SEQ ID NO:1所示,所述gRNA2的靶点序列如SEQ ID NO:2所示,所述gRNA3的靶点序列如SEQ ID NO:3所示,所述gRNA4的靶点序列如SEQ ID NO:4所示;
(2)将两对特异性靶点gRNA1和gRNA2、gRNA3和gRNA4和mRNA一起显微注射入小鼠受精卵内,显微注射后的受精卵移植至代孕母鼠输卵管内;
(3)显微注射后的受精卵移植至代孕母鼠输卵管内产出小鼠,为F0代小鼠;提取F0代小鼠尾部DNA,PCR扩增并将产物测序,阳性小鼠与野生型异性小鼠交配获得F1代阳性杂合子小鼠,F1代阳性杂合子小鼠杂交获得F2代阳性纯合子小鼠,经基因型鉴定:
若为一个680bp的条带,为阳性纯合子;若为两个990bp和680bp的条带,为阳性杂合子;若为一个990bp的条带,为野生型对照,得到稳定遗传的SLC35E1基因敲除小鼠动物模型。
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