CN112410333B - 一种naaa基因敲除鼠模型的构建方法与应用 - Google Patents
一种naaa基因敲除鼠模型的构建方法与应用 Download PDFInfo
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Abstract
本发明涉及一种NAAA基因敲除的鼠模型的构建方法,本发明首先基于NAAA基因序列设计得到活性好的sgRNA,然后将其与Cas9蛋白配合,经Crispr‑Cas9重组系统,得到了NAAA基因敲除的小鼠模型。本发明所构建的NAAA基因敲除的小鼠具有天然的抗炎和疼痛失敏行为,且血糖含量、胰岛素浓度低,构建方法简单,构建过程时间短,对于炎症、疼痛和糖代谢疾病研究具有重大意义。
Description
技术领域
本发明属于基因工程领域,具体涉及NAAA基因敲除鼠模型的构建方法与应用。
技术背景
内源性大麻素N-花生四烯酰乙醇胺(ananadamide,AEA)和棕榈酰乙醇胺(N-Palmitoylethanolamide,PEA)是内源性的具有保护性的调节物质。AEA和PEA在体内有着广泛的分布,在中枢神经系统、肝脏、肺、胃肠道等脏器中均可检测到AEA和PEA的存在。AEA可以通过直接激活内源性大麻素受体(CB),产生抗炎、镇痛等多种药理作用。PEA可通过激动细胞核受体过氧化物酶体增殖物激活受体-α(PPAR-α),同样产生抗炎、镇痛等多种药理作用。AEA和PEA都可以被N-酰基乙醇胺水解酶(N-acylethanolamide hydrolyzing acidamidase,NAAA)水解。NAAA的编码基因(NAAA基因)包含362个氨基酸(小鼠)或359个氨基酸(人)序列。小鼠和人的NAAA基因同源性达76.7%,是研究NAAA在人体中功能的较好模型。研究显示,NAAA与多种疾病密切相关,例如,在风湿性关节炎和多发性硬化的动物模型中,NAAA的含量明显升高;在前列腺癌患者的病灶中,也发现了NAAA的表达出现增高。而抑制NAAA的活性,可提高体内的AEA和PEA含量,有效治疗炎症,疼痛等多种疾病。NAAA可能在多种疾病的发生发展过程中扮演重要角色,因此NAAA的基因敲除鼠在研究NAAA的功能及成药安全性方面有着不可替代的作用。然而,迄今为止,NAAA的基因敲除鼠研究进展十分缓慢。仅有的一种NAAA敲除鼠(EMMA编号:07382)为欧洲Infrafrontier科研协作组采用Cre-loxP和Flp-FRT系统构建,但该构建方法构建时间长。因此,研究NAAA的基因敲除鼠构建方法仍有非常重大的意义。
发明内容
本发明提供一种sgRNA,所述sgRNA的核酸序列如SEQ ID NO.1或SEQ ID NO.2所示。
本发明还提供所述的sgRNA应用于NAAA基因敲除动物模型建立的用途。
本发明提供一种NAAA基因敲除鼠模型的构建方法,包括以下步骤:如SEQ ID NO.1所示的sgRNA与Cas9 RNA或如SEQ ID NO.2所示的sgRNA与Cas9蛋白转入到鼠受精卵中,经过基因型鉴定,得到NAAA基因敲除的模型鼠。
根据本发明的一个实施方案,所述NAAA基因敲除鼠模型的构建方法包括如下步骤:
(1)针对NAAA基因构建sgRNA;
(2)将步骤(1)中所述的sgRNA与Cas9核酸酶mRNA体外转录之后,得到用于的sgRNA及Cas9 RNA;将所述用于注射的sgRNA及Cas9 RNA按照1:2比例混合后注射到小鼠的受精卵中,移植到假孕母鼠体内,所生产的小鼠为F0代小鼠;所述注射优选显微注射;
(3)提取F0代小鼠尾部DNA,鉴定筛选出成功敲除NAAA基因的纯合子小鼠;
(4)将成功敲除NAAA基因的F0代与野生型小鼠交配,获得F1代杂交鼠;
(5)将F1代小鼠杂交,获得F2代小鼠,从中筛选出NAAA基因敲除的纯合子小鼠;
其中,步骤(1)所述sgRNA的核酸序列如SEQ ID NO.1或SEQ ID NO.2所示。
根据本发明,所述步骤(3)、(5)包括进一步对所得到的小鼠利用特异性引物通过PCR方法进行基因型鉴定。
其中,步骤(3)中进行基因型鉴定的引物为:
NAAA(AJ6+7)-F:5’-tgcccttacaatcctcctgc-3’(SEQ ID NO.11)
NAAA(AJ6+7)-R:5’-tgccatctagatccctgaca-3’(SEQ ID NO.12);
其中,步骤(5)中进行纯合子筛选所用的引物为:
F:tgtccatgctgagacgag(SEQ ID NO.13)
R:cttcgacagttcctaggccttaac(SEQ ID NO.14)。
本发明还提供上述方法构建的NAAA基因敲除鼠模型。
本发明还提供所述的NAAA基因敲除鼠模型在疾病研究或药物安全性评价中的应用。
具体地,所述疾病选自炎症、疼痛、糖代谢相关疾病的研究。
本发明所述的炎症相关疾病包括由脂多糖诱发的肺炎和脓毒血症。
本发明的发明人发现,采用本发明技术制备的NAAA基因敲除鼠,具有天然的抗炎行为,可以有效拮抗由脂多糖(LPS)诱发的肺炎和脓毒血症;并且本发明制备的NAAA基因敲除鼠,具有天然的对疼痛失敏行为,对机械痛阈值较高。
本发明的发明人进一步发现,采用本发明制备的NAAA基因敲除鼠,血浆中血糖含量与胰岛素浓度均较低,可以用于研究NAAA在糖代谢相关疾病中的作用。
有益效果:
(1)本发明经过对待敲除序列的分析和sgRNA活性检测得到了活性好的sgRNA,其与Cas9蛋白配合,经Crispr-Cas9重组系统实现基因敲除,得到了NAAA基因敲除的小鼠。
(2)本发明所构建的NAAA基因敲除的小鼠具有天然的抗炎和疼痛失敏行为,且血糖含量、胰岛素浓度低,对于炎症、疼痛和糖代谢疾病研究具有重大意义。
(3)本发明提供的构建NAAA基因敲除鼠模型的构建方法,操作简便,构建过程时间短,能有效得到NAAA基因敲除的小鼠。
附图说明
图1:针对NAAA基因的外显子区域进行的sgRNA筛选结果。
图2:胞浆注射前后RNA变性琼脂糖凝胶电泳结果。
图3:F0代小鼠基因型检测PCR扩增条带;WT:野生型C57BL/6小鼠;ZP-N16与ZP-18:NAAA敲除鼠纯合子;其中箭头所示的为NAAA基因缺失的小鼠样本。
图4:野生型C57BL/6小鼠(WT)、ZP-N18型(A)和ZP-N16型(B)NAAA基因敲除鼠的基因序列对比图。
图5:F2代小鼠基因型检测PCR扩增条带;ZP-N16:NAAA敲除鼠纯合子;F2代:F2代杂交小鼠;WT:野生型C57BL/6小鼠。
图6:野生型C57BL/6小鼠(WT)、ZP-N16型NAAA基因敲除鼠在接受LPS(5mg/kg)滴鼻3天之后,肺组织切片的HE染色图。
图7:野生型C57BL/6小鼠(WT)和ZP-N16型NAAA基因敲除鼠疼痛耐受测试结果。
图8:野生型C57BL/6小鼠(WT),ZP-N16型,ZP-N18型NAAA基因敲除鼠空腹血糖含量。
具体实施方式
下文将结合具体实施例对本发明的制备方法做更进一步的详细说明。应当理解,下列实施例仅为示例性地说明和解释本发明,而不应被解释为对本发明保护范围的限制。凡基于本发明上述内容所实现的技术均涵盖在本发明旨在保护的范围内。
下述实施例中所使用的实验方法如无特殊说明,均为常规方法;下述实施例中所用的试剂、材料等,如无特殊说明,均可从商业途径得到。
下述实施例中所用小鼠及小鼠受精卵来源于厦门大学实验动物中心。
实施例1:sgRNA的设计
如图1所示,NAAA有4个同分异构体,我们挑选其共有的功能性区域所在并比较靠前的外显子(要求基因靠后部分无ATG序列),通过网站http://crispr.mit.edu/进行sgRNA设计,选取评分最高的sgRNA。本次实验我们共设计了针对两个外显子区域的6条sgRNA。
实施例2:sgRNA效率的测试
构建合适抗性的Cas9 sgRNA质粒,转染细胞,采用抗性药杀转染的细胞和未转染的细胞,未转染的细胞全部死亡后取转染Cas9/sgRNA的细胞,提取基因组。设计在sgRNA切割位点前后各300bp的引物,PCR,回收PCR产物,连接到PBKS质粒载体上,涂板,挑菌16个,进行测序,统计16个菌中有敲除的比例,选取敲除效率高于50%的进行下步实验。
如图1所示,根据NCBI网站给出的NAAA基因序列(NCBI网站Gene ID:67111,更新时间2019年05月12日),NAAA基因序列共有9个外显子,导入到snapgene软件给出的sgRNA位点以及后期对sgRNA活性的检测,选取作用在第二个外显子上的两个sgRNA,即sgRNA-1-1和sgRNA-1-2。在本实验中,我们优选出作用在第二个外显子上的一对sgRNA,序列如下:
sgRNA-1-1:5’-ggtcggagaaattgtgtcga-3’(SEQ ID NO.1)
sgRNA-1-2:5’-gctgcggatctcgtcggtga-3’(SEQ ID NO.2)
实施例3:sgRNA和Cas9的体外转录
3.1sgRNA体外转录
3.11sgRNA体外转录模板的准备
3.11a设计sgRNA与Cas9的引物
具体引物序列如下:
表1.sgRNA与Cas9的PCR引物序列
3.11b PCR反应体系及条件
按表2所示的条件,采用PCR扩增方法,应用表1中的引物序列,进行体外扩增,得到体外转录所需的sgRNA模板。
表2.sgRNA的PCR扩增体系及条件
3.11c PCR产物回收
(参考Tiangen普通DNA产物纯化试剂盒DP214说明书).
1)柱平衡步骤:向吸附柱CB2中(吸附柱放入收集管中)加入500μL的平衡液BL,12000rpm离心1min,倒掉收集管中的废液,将吸附柱CB2重新放入收集管中。
2)向PCR反应体系(200μL)中加入3倍体积(600μL)的结合液PB,充分混匀。
3)将上一步所得溶液加入一个吸附柱CB2(吸附柱放入收集管)中,室温放置2min,12000rpm离心1min,倒掉收集管中的废液,将吸附柱CB2放入收集管中。
4)向吸附柱CB2中加入600μL漂洗液PW(使用前先检查是否加入无水乙醇),先室温放置5min,然后12000rpm离心1min,倒掉收集管中的废液,将吸附柱CB2放入收集管中。
5)重复步骤4)。
6)将吸附柱CB2放回收集管中,12000rpm离心2min,尽量去除漂洗液。
7)将吸附柱CB2打开盖子放置10分钟,以彻底晾干吸附柱内的乙醇残留。
8)将吸附柱CB2放入一个1.5mL离心管中,向吸附膜中间位置悬空滴加——(sgRNA每50μLPCR体系产物溶10μL,Cas9每50μLPCR体系产物溶15μL)无RNA酶的水,室温放置2min,12000rpm离心2min收集DNA溶液。
9)重复步骤8),移除吸附柱,将DNA收集液移入另一个新的离心管中,并在离心管盖上标注好DNA名称,此DNA为储存模板DNA,用于体外转录。
10)分光光度计测量DNA浓度,并在离心管侧壁做好标注,标注内容为:DNA回收日期,DNA浓度(单位为ng/μL),260/280,260/230。
11)在2%的琼脂糖胶中进行凝胶电泳2μL,以检测回收结果。提取后的DNA保存于-20℃。
质控要求:凝胶电泳电泳时显示为单一清晰的条带,无非特异条带。PCR产物浓度应高于50ng/μL;260/280在1.7-2.0之间;260/230>1.8。若纯度达不到要求,可重新进行PCR产物纯化。
结果:DNA浓度为90ng/μL,260/280=1.83,260/230=2.12。以上数据表明纯化后的DNA浓度和纯度均可满足下游实验。
3.12sgRNA的体外转录
3.12a体外转录(Ambion体外转录试剂盒MEGAshortscriptTM Kit Cat#AM1354)
按照顺序加入以下成分(20μL体系):
3.12b消化模板DNA
将擦干的体外转录反应体系拿入超净工作台后去除封口膜,每20μL反应体系内再加入1μL的TURBO DNase,封口后轻敲离心管底部4-5次,使体系混匀。用封口膜封口后,放入37℃水浴锅消化15min。
3.12c琼脂糖凝胶电泳初步验证
取1μL反应产物进行2%的非变性琼脂糖凝胶电泳(DNA胶),200V,10min,有明显条带,即体外转录成功,可进行下一步纯化实验(质控步骤)。
3.12d sgRNA的纯化
(参考Ambion MEGAclearTM Kit试剂盒说明书cat#AM1908)
1)用小离心机将体外转录体系离心5s后,向体系中加入稀释液(ElutionSolution)80μL,以使总体系达到100μL,用枪头轻柔吹吸混匀。
2)向每100μL的反应体系中加入浓缩液350μL,用枪头轻柔混匀。
3)向每个体系中再加入250μL无水乙醇,用枪头轻柔吹吸混匀,得到700μL的总样品。
4)将收集柱放到收集管上,再将700μL的总样品加入收集柱中,盖上收集管的帽子,10000rpm离心30s,倒掉收集管中的液体。
5)向吸附柱中加入500μL洗涤液,盖上收集管的帽子,10000rpm离心30s。
6)重复上述洗脱步骤一次。
7)倒掉收集管中的液体,盖上收集管的帽子,10000rpm离心1min,以去除残余的洗涤液。
8)将收集柱拿回超净工作台后倒掉收集管中可能存在的残余液体,打开收集管的帽子,室温放置2min。
9)将95度预热的稀释液50μL加入到收集柱的正中央,盖上收集管的帽子,10000rpm离心60s。
10)将离心管内的RNA收集液在超净工作台内转到另一个新的1.5mL离心管内,盖上盖子并标记上RNA名称,此RNA即为实验用贮存RNA。
11)取4μL回收后的RNA到另一个新的离心管中,并标注好RNA名称,用于测量RNA浓度和电泳。
12)用分光光度计测量RNA浓度。在离心管侧壁做好标注,标注内容为:RNA回收日期,RNA浓度(单位为ng/μL),260/280,260/230。在实验用贮存RNA离心管上也作上同样的标注。
13)在2%的变性琼脂糖凝胶中电泳2μL,以检测回收结果。
14)纯化后的RNA如果分光光度计测量结果可以达到要求,且变性琼脂糖凝胶电泳均达到要求立即进行分装:RNA稀释成500ng/μL左右浓度(稀释后也要进行分光光度计浓度检测),每管5μL分装,并且每管都要在管帽及管壁上标注RNA名称,纯化时间,浓度及260/280,260/230的值。
质控要求:电泳时显示为单一清晰的条带,在琼脂糖胶前方无明显脱尾,且与marker相比,有与分光光度计大致相同的定量结果。RNA原始浓度应高于1μg/μL;260/280大于2.4,260/230大于2.4。
结果:纯化后sgRNA的浓度为1494.9ng/μL,达到了可用于下游实验的浓度要求;同时,sgRNA的260/280=2.35,说明纯化所得sgRNA为无蛋白质及DNA污染的高纯度单链RNA。260/230=2.57,说明该sgRNA无有机试剂污染。以上指标说明纯化所得sgRNA产物的质量可满足下游实验的需求。
3.12e sgRNA的变性琼脂糖凝胶电泳
1)DEPC(焦炭酸二乙酯)水
加入转子,在生物安全柜内室温避光搅拌过夜,即为生DEPC水。
121℃,0.11MPa高压40min,此时大部分DEPC会分解为乙醇,即为熟DEPC水。
注意:DEPC有香味,有挥发性,但是有毒性,因此搅拌在生物安全柜内进行。
2)10×3-(N-吗啉基)丙磺酸(MOPS)电泳缓冲液的配制:
搅拌均匀,室温避光保存。
3)1×MOPS变性电泳缓冲液的配制
搅拌均匀,室温避光保存。
4)变性琼脂糖凝胶电泳的配制
充分混匀,倒入模具中,等至琼脂糖全部凝固(约30min)之后将琼脂糖放入电泳槽内,浸泡在1×MOPS变性电泳缓冲液中30min后使用。
5)如果电泳看到干净的RNA条带及与分光光度计相同的定量结果则保存在-80℃,准备进行注射实验。
注意:甲醛及DEPC都有毒性及挥发性,要避免与皮肤直接接触及吸入,所有操作尽量在通风橱内进行。
3.2Cas9的PCR扩增体系及条件
3.21Cas9模板PCR Phusion酶扩增体系及条件
3.22Cas9 RNA的体外转录
按照顺序加入以下成分(20μL体系):
离心管封口,水浴反应。条件:37℃,2.0hrs3.22b消化模板DNA
将擦干的体外转录反应体系拿入超净工作台后去除封口膜,每20μL反应体系内再加入1μL的TURBO DNase,封口后轻敲离心管底部4-5次,使体系混匀。同样用封口膜封口后,用同样的方式放入37℃水浴锅消化20min。
3.22c琼脂糖凝胶电泳初步验证
取1μL反应产物进行1%的非变性琼脂糖凝胶电泳(DNA胶),200V,10min,有明显条带,即体外转录成功,可进行下一步加尾实验。
3.23Cas9 RNA的加尾
RNA加尾(Ambion加尾试剂盒Poly(A)Tailing Kit cat#AM1350)
按照顺序加入以下成分:
离心管封口,水浴反应。条件:37℃,45min3.24Cas9 RNA纯化
(参考Qiagen RNeasy Mini Kit试剂盒说明书Cat#74104)
1)向每100μL的加尾反应体系中加入缓冲液RLT 350μL,用移液器轻柔混匀。
2)向每个体系中再加入250μL无水乙醇,用移液器轻柔混匀。
3)将700μL的总样品加入RNeasy收集柱(RNeasy mini spin column)中,收集柱自带2mL的收集管,12000rpm离心15s,倒掉收集柱中的液体。
4)向收集管中加入500μL的缓冲液RPE(第一次使用前要向瓶中加入4倍体积,即44mL的无水乙醇,并在瓶上标注),12000rpm离心15s,倒掉收集柱中的液体。
5)向收集管中加入500μL的缓冲液RPE,12000rpm离心15s,倒掉收集柱中的液体。
6)将收集柱转到一个新的2mL收集管上,12000rpm离心2min。
7)将收集柱放到超净工作台上室温放置5min,然后将收集柱放到1.5mL离心管上,悬空滴加室温35μL无RNA酶的H2O到收集柱的正中央,盖上收集柱的盖子,室温放置1分钟。
8)12000rpm离心1min,之后将离心管内的RNA收集液在超净工作台内转到另一个新的1.5mL离心管内,盖上盖子并标记上RNA名称,此RNA即为实验用贮存RNA。
9)取4μL回收后的RNA到另一个新的离心管中,并标注好RNA名称,用于测量RNA浓度和电泳。
10)用分光光度计测量RNA浓度,在离心管侧壁做好标注,标注内容为:RNA回收日期,RNA浓度(单位为ng/μL),260/280,260/230。在实验用贮存RNA离心管上也作上同样的标注。
11)取2μL在1%的变性琼脂糖凝胶电泳,以检测回收结果。
12)纯化后的RNA如果分光光度计测量结果可以达到要求,且变性琼脂糖凝胶电泳均达到要求立即进行分装:RNA稀释成1000ng/μL左右浓度,每管5μL分装,并且每管都要在管帽及管壁上标注RNA名称,纯化时间,浓度及260/280,260/230的值。
质控要求:电泳时显示为单一清晰的条带,在琼脂糖胶前方无明显脱尾,且与marker相比,有与分光光度计相同的定量结果。加尾后条带有100-200bp shift,纯化后条带有特异性。RNA原始浓度要高于500ng/μL;260/280以及260/230必须均大于2.4。
结果:纯化后的RNA浓度为860ng/μL达到了可用于下游实验的浓度要求同时,RNA的260/280=2.52,说明纯化所得RNA为无蛋白质及DNA污染的高纯度单链RNA。260/230=2.63,说明该RNA无有机试剂污染。以上指标说明纯化所得RNA产物的质量可满足下游实验的需求。
3.25RNA变性琼脂糖凝胶电泳
具体步骤同sgRNA变性凝胶电泳。
实施例4:胞浆注射
按Cas9 RNA100 ng/μL和sgRNA 50ng/μL比例混合后进行0.5天受精卵的显微注射。共注射180个受精卵,移植了6只ICR假孕母鼠,19天后获得24只仔鼠,断尾取样进行鉴定。
为检测注射后的sgRNA是否被降解,影响敲除效率,我们通过RNA变性琼脂糖凝胶电泳对比了注射前后的RNA浓度变化。如图2所示,泳道3为注射前的RNA,泳道4为注射后的RNA,二者并无显著的浓度差异。所以,我们的sgRNA经显微注射后不会因降解而降低敲除效率。
实施例5:F0小鼠鉴定:
F0小鼠断尾巴取样,并配制适量的组织消化液,鼠尾PCR试剂盒(来源于Bimake)中Protease Plus和Buffer L的体积比为1:50,每只鼠尾加约100μL消化液,55℃水浴15min,再置于95℃水浴中5min,以灭活消化液中的蛋白酶,12000rpm离心5min,取上清进行后续实验。根据sgRNA的位置设计PCR引物,并按表3条件进行PCR扩增。配制100mL 2%琼脂糖凝胶,微波炉加热,溶解后加入7μL核酸染料,混合均匀后倒入胶槽凝胶30min。将胶块转移至电泳槽内,调节功率150W,电压150V,电流500mA,35min后取出胶块,显影。
表3.F0小鼠NAAA基因PCR检测条件
结果显示,所得野生型PCR产物应为734bp。如图3所示,多份样本与野生型基因扩增产物不同。对图3中编号为ZP-N16和ZP-N18的小鼠进行基因测序后发现:ZP-N16雄性小鼠是缺失256bp(缺失258bp,插入2bp)基因的纯合子,其第187-265bp的NAAA基因序列如SEQID NO.9所示;ZP-N18雄性小鼠缺失47bp基因的纯合子,其第192-248bp的NAAA基因序列如SEQ ID NO.10所示。缺失的基因如图4所示。
实施例6:NAAA基因敲除鼠的传代建系
将ZP-N16或者ZP-N18突变小鼠与野生C57BL/6小鼠配对,获得F1代杂合小鼠作为亲本繁育F2代小鼠,从F2代小鼠中筛选出NAAA基因敲除的ZP-N16或者ZP-N18纯合子小鼠。
表4.F2小鼠NAAA基因PCR检测条件
采用H16-2引物序列:
F:tgtccatgctgagacgag(SEQ ID NO.13)
R:cttcgacagttcctaggccttaac(SEQ ID NO.14)
PCR程序如下:
PCR产物经纯化后琼脂糖凝胶电泳显示片段大小为500bp,如图5所示。
实施例7:NAAA基因敲除鼠对炎症的反应
野生型C57BL/6小鼠与ZP-N16型NAAA敲除鼠滴鼻灌注LPS(5mg/kg)溶液,3天后获得小鼠急性肺炎模型(ALI)。如图6所示,HE染色结果显示,野生型C57BL/6小鼠在LPS刺激下,出现了免疫细胞浸润,出血,水肿等现象;而ZP-N16型NAAA敲除鼠则具有明显的抗炎现象,免疫细胞浸润,出血,水肿等现象较少,各项指标接近正常小鼠。
实施例8:NAAA基因敲除鼠对疼痛的反应
我们采用针刺小鼠脚底,考察小鼠发生抬脚时的机械力阈值来评价野生型C57BL/6小鼠与ZP-N16型NAAA敲除鼠对疼痛的反应程度。NAAA基因敲除鼠在足底皮下注射LPS(1mg)1天后,采用电子式机械测痛仪检测小鼠对机械刺激的抬脚阈值,结果如图7所示,ZP-N16型NAAA敲除鼠对机械疼痛不敏感,表明本发明的NAAA基因敲除鼠可以作为研究痛觉传导的相关模型。
实施例9:NAAA基因敲除鼠具有自发低血糖的属性
我们对野生型C57BL/6小鼠与ZP-N16及ZP-N18型NAAA敲除鼠的空腹10h的血糖进行检测。如图8所示,发现ZP-N16与ZP-N18型NAAA敲除鼠具有显著的自发性低血糖,表明本发明的NAAA基因敲除鼠可应用于糖尿病,低血糖等糖代谢相关疾病的研究。
以上,对本发明的实施方式进行了说明。但是,本发明不限定于上述实施方式。凡在本发明的精神和原则之内,所做的任何修改、等同替换、改进等,均应包含在本发明的保护范围之内。
序列表
<110> 厦门稀土材料研究所
<120> 一种NAAA基因敲除鼠模型的构建方法与应用
<130> CPCN19410596
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ggtcggagaa attgtgtcga 20
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gctgcggatc tcgtcggtga 20
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gaaattaata cgactcacta taggggtcgg agaaattgtg tcgagtttta gagctagaaa 60
tagc 64
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aaaagcaccg actcggtgcc 20
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gaaattaata cgactcacta tagggctgcg gatctcgtcg gtgagtttta gagctagaaa 60
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aaaagcaccg actcggtgcc 20
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Claims (6)
1.一种NAAA基因敲除鼠模型的构建方法,其特征在于,将如SEQ ID NO.1和SEQ IDNO.2所示的核酸序列的一对sgRNA与Cas9 RNA转入到鼠受精卵中,经过基因型鉴定,得到NAAA基因敲除的模型鼠。
2.根据权利要求1所述的NAAA基因敲除鼠模型的构建方法,其特征在于,包括如下步骤:
(1)针对NAAA基因构建sgRNA,获得SEQ ID NO.1和SEQ ID NO.2所示的核酸序列的一对sgRNA;
(2)将步骤(1)中所述的sgRNA与Cas9核酸酶mRNA体外转录之后,得到用于注射的sgRNA及Cas9 RNA;将所述用于注射的sgRNA及Cas9 RNA按照1:2比例混合后注射到鼠的受精卵中,移植到假孕母鼠体内,所生产的鼠为F0代鼠;
(3)提取F0代鼠尾部DNA,鉴定筛选出成功敲除NAAA基因的纯合子鼠;
(4)将成功敲除NAAA基因的F0代与野生型鼠交配,获得F1代杂交鼠;
(5)将F1代鼠杂交,获得F2代鼠,从中筛选出NAAA基因敲除的纯合子鼠;
所述注射为显微注射。
3.根据权利要求1或2所述的NAAA基因敲除鼠模型的构建方法,其特征在于,所述鼠为小鼠或大鼠。
4.根据权利要求2所述的NAAA基因敲除鼠模型的构建方法,其特征在于,所述步骤(3)和/或步骤(5)包括进一步对所得到的鼠利用特异性引物通过PCR方法进行基因型鉴定;
所述步骤(3)中进行基因型鉴定的引物为:
F: 5’- tgcccttacaatcctcctgc-3’ (SEQ ID NO.11)
R: 5’- tgccatctagatccctgaca-3’ (SEQ ID NO.12);
步骤(5)中进行纯合子筛选所用的引物为:
F:tgtccatgctgagacgag(SEQ ID NO.13)
R:cttcgacagttcctaggccttaac(SEQ ID NO.14)。
5.权利要求1-2任一项所述的NAAA基因敲除鼠模型的构建方法所构建的鼠模型在疾病研究或药物安全性评价中的应用,所述应用为非疾病诊断和治疗目的的应用。
6.根据权利要求5所述的应用,其中,所述疾病选自炎症、疼痛、糖代谢相关疾病。
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