CN113201515A - 一种与动物不孕相关的基因、sgRNA及应用和构建动物模型的方法 - Google Patents
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Abstract
本发明提供了一种与动物不孕相关的基因、sgRNA及应用和构建动物模型的方法,涉及动物模型技术领域。本发明提供了一种与动物不孕相关的基因,所述基因包括动物dguok基因。本发明基于所述基因设计sgRNA并构建雌性不孕动物模型,所述模型100%不孕,表现为:卵子无法成熟、不能成功受精、无法分裂、无法进入8细胞期和囊胚期;所述雌性不孕小鼠模型有助于深入研究分子水平上雌性不孕发生的具体分子机制,并有助于研发治疗不孕相关的药物。
Description
技术领域
本发明属于动物模型技术领域,具体涉及了一种与动物不孕相关的基因、sgRNA及应用和构建动物模型的方法。
背景技术
目前,不孕不育已成为常见多发病,在生殖医学领域,不孕症动物模型的制备是开展相关研究的基础。目前导致不孕症的因素有很多,如排卵障碍(中枢神经系统性无排卵、下丘脑性无排卵、垂体性无排卵、卵巢性无排卵、多囊卵巢综合征等)、输卵管因素(炎症或阻塞)、胚胎因素(受精异常、卵裂异常、早期胚胎发育阻滞等)、子宫因素(发育畸形、肌瘤及内膜病变等),此外还有盆腔因素、宫颈因素、免疫因素等。据中华中医药学会中药实验药理专业委员会2018年发布的《雌性不孕症动物模型制备规范(草案)》显示,不孕症小鼠模型的制备主要以排卵障碍和生殖道因素为主,鲜有胚胎因素导致的不孕症小鼠模型,而且得到的模型稳定性差,同批次间差异显著。
发明内容
有鉴于此,本发明的目的在于提供一种与动物不孕相关的基因、sgRNA及应用和构建动物模型的方法,可解决雌性不孕动物模型诱导成功率低及同批次诱导个体间差异大的技术问题。
为了实现上述发明目的,本发明提供以下技术方案:
本发明提供了一种与动物不孕相关的基因,所述基因包括动物dguok基因。
优选的,所述基因的敲除靶标片段包括动物dguok基因第二个外显子及两侧部分内含子区。
优选的,所述基因的敲除靶标片段核苷酸序列包括SEQ ID NO.1和SEQ ID NO.2所示的核苷酸序列。
优选的,所述动物包括哺乳动物。
本发明还提供了一组靶向上述基因的sgRNA,所述sgRNA包括M-DguoK-E2be-gRNAup、M-DguoK-E2be-gRNA down、M-DguoK-E2af-gRNA up和M-DguoK-E2af-gRNA down;
所述M-DguoK-E2be-gRNA up的核苷酸序列如SEQ ID NO.3所示;
所述M-DguoK-E2be-gRNA down的核苷酸序列如SEQ ID NO.4所示;
所述M-DguoK-E2af-gRNA up的核苷酸序列如SEQ ID NO.5所示;
所述M-DguoK-E2af-gRNA down的核苷酸序列如SEQ ID NO.6所示。
优选的,所述M-DguoK-E2be-gRNA up和M-DguoK-E2be-gRNA down靶向SEQ IDNO.1所示的核苷酸序列;
所述M-DguoK-E2af-gRNA up和M-DguoK-E2af-gRNA down靶向SEQ ID NO.2所示的核苷酸序列。
本发明还提供了上述基因或上述sgRNA在构建雌性不孕动物模型中的应用。
本发明还提供了一种构建雌性不孕动物模型的方法,包括以下步骤:将上述dguoksgRNA和CAS9 mRNA注射到动物的受精卵内,然后移植到假孕的动物输卵管,筛选雌性阳性F0动物;
将雌性阳性F0动物与野生型动物交配得到F1代,筛选阳性F1杂合动物并进行自交,F2纯合雌性动物为所述雌性不孕动物模型。
优选的,所述筛选包括PCR筛选,所述PCR筛选的引物对包括M-DguoK-F、M-DguoK-R和M-Dguok-deletion;
所述M-DguoK-F的核苷酸序列如SEQ ID NO.7所示;
所述M-DguoK-R的核苷酸序列如SEQ ID NO.8所示;
所述M-Dguok-deletion的核苷酸序列如SEQ ID NO.9所示。
本发明还提供了上述方法构建得到的雌性不孕动物模型在解析雌性不孕分子机制及筛选或制备治疗不孕药物中的应用。
本发明提供了一种与动物不孕相关的基因,所述基因为基因组编码的线粒体定位的脱氧鸟苷酸激酶。本发明发现所述基因与不孕相关,实施例中采用小鼠全身dguok基因敲除的方式构建自发性的雌性小鼠不孕模型,相比于药物诱导的雌鼠不孕动物模型,所述雌性不孕小鼠模型具有更好的稳定性以及更低的同批次诱导个体间差异,所述构建方法产生的dguok基因缺失雌鼠100%不孕,表现为:卵子无法成熟、不能成功受精、无法分裂、无法进入8细胞期和囊胚期;所述雌性不孕小鼠模型有助于深入研究分子水平上雌性不孕发生的具体分子机制,并有助于研发治疗不孕相关的药物。
附图说明
图1为本发明dguok(-/-)缺失小鼠构建方法的基本流程图;
图2为本发明构建得到的dguok(-/-)敲除小鼠的敲除位点图;
图3为dguok基因缺失小鼠和对照小鼠的基因型鉴定图;
图4为dguok(-/-)基因缺失小鼠雌性不孕鉴定流程图;
图5为对照小鼠的卵子体外受精后发育图;
图6为dguok(-/-)基因缺失雌性小鼠的卵子受精后发育图;
图7为对照和dguok(-/-)基因缺失雌性小鼠的卵子发育及受精比率的统计图。
具体实施方式
本发明提供了一种与动物不孕相关的基因,所述基因包括动物dguok基因。
本发明所述动物优选包括哺乳动物,更优选包括啮齿类动物。在本发明实施例中,优选对小鼠基因组内所述dguok基因的编码区进行功能验证,并构建雌性不孕动物模型。在本发明中,在小鼠中dguok基因的基因ID:27369,基因定位于Chromosome 6。
本发明所述基因的敲除靶标片段优选包括动物dguok基因第二个外显子及两侧部分内含子区。在本发明实施例中,优选以SEQ ID NO.1(GGCCCTGGCTCCCATGAGATGGG)和SEQID NO.2(GGCAGGAGCAATAGTCAACGAGG)所示的序列为靶向序列设计sgRNA,从而实现dguok基因的敲除。
本发明还提供了一组靶向上述基因的sgRNA,所述sgRNA包括M-DguoK-E2be-gRNAup、M-DguoK-E2be-gRNA down、M-DguoK-E2af-gRNA up和M-DguoK-E2af-gRNA down;所述M-DguoK-E2be-gRNA up的核苷酸序列如SEQ ID NO.3所示:TAGGCCCTGGCTCCCATGAGAT;所述M-DguoK-E2be-gRNA down的核苷酸序列如SEQ ID NO.4所示:AAACATCTCATGGGAGCCAGGG;所述M-DguoK-E2af-gRNA up的核苷酸序列如SEQ ID NO.5所示:TAGGCAGGAGCAATAGTCAACG;所述M-DguoK-E2af-gRNA down的核苷酸序列如SEQ ID NO.6所示:AAACCGTTGACTATTGCTCCTG。
本发明所述M-DguoK-E2be-gRNA up和M-DguoK-E2be-gRNA down优选靶向SEQ IDNO.1所示的核苷酸序列;所述M-DguoK-E2af-gRNA up和M-DguoK-E2af-gRNA down优选靶向SEQ ID NO.2所示的核苷酸序列。
本发明对所述sgRNA的合成方法并没有特殊限定,利用本领域的常规合成方法即可,本发明实施例中优选委托擎科昆明合成部进行合成。
本发明还提供了上述基因或上述sgRNA在构建雌性不孕动物模型中的应用。
利用上述sgRNA,可靶向SEQ ID NO.1和SEQ ID NO.2所示的核苷酸序列,并实现dguok基因的靶向敲除,从而获得雌鼠不孕率为100%,避免了药物诱导的不确定性,且不孕表现为:卵子无法成熟、不能成功受精、无法分裂、无法进入8细胞期和囊胚期,可得到一种胚胎因素导致的不孕症小鼠模型。
本发明还提供了一种构建雌性不孕动物模型的方法,包括以下步骤:将上述dguoksgRNA和CAS9 mRNA注射到动物的受精卵内,然后移植到假孕的动物输卵管,筛选雌性阳性F0动物;
将雌性阳性F0动物与野生型动物交配得到F1代,筛选阳性F1杂合动物并进行自交,F2纯合雌性动物为所述雌性不孕动物模型。
本发明优选将dguok sgRNA和CAS9 mRNA注射到动物的受精卵的核区,所述dguoksgRNA和CAS9 mRNA的浓度优选分别为0.1g/L和0.05g/L。本发明所述筛选优选包括PCR筛选,所述PCR筛选的引物对优选包括M-DguoK-F、M-DguoK-R和M-Dguok-deletion;所述M-DguoK-F的核苷酸序列如SEQ ID NO.7所示:5’-CTCCCGCACTCAGTACTACAGCT-3’;所述M-DguoK-R的核苷酸序列如SEQ ID NO.8所示:5’-AGTCCAAGTCACAGGGTCCAATA-3’;所述M-Dguok-deletion的核苷酸序列如SEQ ID NO.9所示:5’-gcgacagaacctatagcagagtg-3’,(跟M-DguoK-R在野生型中产生800bp的条带)。本发明对所述PCR筛选的体系和程序并没有特殊限定,实施例中优选采用25μl体系:模板DNA 5μl,M-DguoK-F引物0.5μl和M-DguoK-R引物0.5μl;或M-DguoK-R引物0.5μl和M-Dguok-deletion引物0.5μl(引物的浓度各为10μM),vazyme 2xRapid taq MasterMix 12.5μl,ddH2O 6.5μl;并选择以下程序:95℃预变性5min;95℃变性30s,56℃退火30s,72℃延伸90s,35个循环;72℃再延伸5min。
本发明所述阳性F1杂合动物的筛选方法,优选与上述相同,在此不再赘述。本发明实施例中,在得到所述阳性F0小鼠后,与野生型C57BL/6品系小鼠交配得到F1代小鼠,并进行基因型鉴定阳性F1小鼠,阳性F1杂合小鼠自交产生野生、杂合与纯合F2代小鼠。
本发明所述鉴定阳性F1小鼠的方法优选与上述相同,在此不再赘述。在本发明中,无论是F0代还是F1代或F2代,杂合小鼠会产生一条1.7kb和一条1kb的条带;野生小鼠会产生一条1.7kb的条带。同时,对F2代进行鉴定时,优选还包括利用引物M-DguoK-deletion(SEQID NO.9)和M-DguoK-R进行PCR鉴定,杂合和野生小鼠都会产生一条800bp的条带,纯合小鼠不能PCR出条带。
本发明还提供了利用上述方法构建得到的雌性不孕动物模型,在本发明实施例中,优选构建了雌性不孕小鼠模型,但是不能仅将其认定为本发明的保护范围。本发明所述所述雌性不孕动物模型为100%不孕,可用来体内体外研究,尤其是研究雌性不孕发生的分子机制。
本发明还提供了上述方法构建得到的雌性不孕动物模型在解析雌性不孕分子机制及筛选或制备治疗不孕药物中的应用。
下面结合实施例对本发明提供的一种与动物不孕相关的基因、sgRNA及应用和构建动物模型的方法进行详细的说明,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
根据图1所示流程构建dguok缺失(Dguok(-/-))小鼠:
委托擎科昆明合成部设计dguok的sgRNA(SEQ ID NO.3~6)。
将体外合成dguok sgRNA和CAS9 mRNA显微注射到C57BL/6品系小鼠(南京模式动物中心)的受精卵的核区;再将小鼠受精卵细胞移植到假孕C57BL/6品系小鼠的输卵管中,获得F0代小鼠并进行基因型鉴定(利用引物M-DguoK-F、M-DguoK-R和M-Dguok-deletion);
将阳性F0小鼠(M-DguoK-R和M-Dguok-deletion只产生一条800bp的条带)与野生型C57BL/6品系小鼠交配得到F1代小鼠,利用上述方法进行基因型鉴定,将所述阳性F1杂合小鼠(M-DguoK-F和M-DguoK-R产生1.7kb条带,M-DguoK-R和M-Dguok-deletion产生800bp左右条带)自交产生野生(只M-DguoK-F和M-DguoK-R产生一条1.7kb的条带)、杂合与纯合F2代小鼠(只M-DguoK-R和M-Dguok-deletion产生一条800bp的条带)。同时利用引物M-DguoK-deletion和M-DguoK-R对F2代小鼠进行再次进行验证,其中杂合和野生小鼠都会产生一条800bp的条带;纯合小鼠不能PCR出条带。
通过基因敲除技术得到的小鼠模型的雌鼠不孕率为100%,避免了药物诱导的不确定性;并且同批次野生和dguok基因缺失的小鼠个体由dguok杂合雌鼠与雄鼠杂交得到,所有dguok基因缺失纯合小鼠和野生小鼠的培育条件保持一致,有效降低小鼠个体间差异(图2显示敲除位点)。
实施例2
剪取小鼠脚趾并对其编号,上肢脚趾从左到右,代表十位数。下肢脚趾从左到右,代表个位数。将剪下的脚趾放入裂解液中,提取DNA。利用M-DguoK-F和M-DguoK-R以及M-DguoK-R和M-Dguok-deletion的引物组合进行PCR,结果如图3中A和图3中B所示,dguok(-/+)杂合小鼠(产生1.7kb和1kb左右的两条带),wt野生小鼠(只产生一条1.7kb的条带)、dguok-/-纯合小鼠(只产生一条1kb的条带)。
实施例3
将基因型鉴定后的野生小鼠和纯和小鼠处死解剖,取其肝脏和肺组织,组织破碎后提取蛋白,用WesternBlot检测蛋白水平的Dguok表达,在纯合小鼠组织中Dguok无表达。结果如图3中C所示。
实施例4
用Dguok(-/-)雄性小鼠和Dguok(-/-)雌性小鼠交配繁殖后代,发现其无法产生后代,为了检测是Dguok(-/-)雄性小鼠还是Dguok(-/-)雌性小鼠导致受孕失败,选用Dguok(-/-)雄性小鼠和WT雌性小鼠交配,结果如图4所示,发现能够产生后代,说明Dguok(-/-)雄性小鼠功能正常;选用Dguok(-/-)雌性小鼠和WT雄性小鼠交配,发现不能产生后代,说明Dguok(-/-)雌性小鼠生殖功能异常,不能受孕,参考图4。由于植入前、后胚胎发育在体内进行,无法观察和记录,随后设计了体外受精和体外培养实验进行探究。
实施例5
体外受精实验:野生型对照,六周周龄的WT雌性小鼠-2day下午17:30注射10IUPMSG,在0day下午17:30注射10IU HCG,在1day上午9:00取卵;并取一只已验证生育能力正常的雄鼠取其附睾尾,缓慢推出精子,将其转移到精子获能液中。将获能后的精子转移到卵母细胞培养皿中进行体外受精。随后在8h,30h,56h,96h观察卵母细胞受精情况与发育情况。
结果如图5所示,体外受精8小时观察,可见雌雄原核的形成,第二极体排出;体外受精30小时观察,可见2细胞期胚胎形成;体外受精56小时观察,可见8细胞期胚胎形成;体外受精96小时观察,可见扩张期囊胚形成。说明WT对照雌性小鼠的体外受精及发育潜能正常。
实施例6
六周周龄的Dguok(-/-)雌性小鼠-2day下午17:30注射10IU PMSG,在0day下午17:30注射10IU HCG,在1day上午9:00取卵;并取一只已验证生育能力正常的雄鼠(与WT雌鼠体外受精实验是同一雄鼠)取其附睾尾,缓慢推出精子,将其转移到精子获能液中。将获能后的精子转移到卵母细胞培养皿中进行体外受精。随后在8h,30h,56h,96h观察卵母细胞受精情况与发育情况。
结果如图6所示,体外受精8小时观察,可见多数卵母细胞仍处于GV期,未见明显卵周隙,未见第一极体排出;体外受精30小时观察,未见明显卵周隙,未见第一极体排出,且少数卵母细胞凋亡;体外受精56小时观察,未见明显卵周隙,未见第一极体排出,且多数卵母细胞凋亡;体外受精96小时观察,未见明显卵周隙,未见第一极体排出,且卵母细胞全部凋亡。说明Dguok(-/-)雌性小鼠卵母细胞成熟阻滞,且多数卵母细胞未能恢复减数分裂进程,少部分生发泡破裂的卵母细胞也未能完成第一次减数分裂并阻滞。
实施例7
分别统计6周周龄的WT雌性小鼠、Dguok(+/-)雌性小鼠、Dguok(-/-)雌性小鼠在获卵后3小时的卵子退化率、成熟率,在体外受精8小时后的受精率,在体外受精30小时后的卵裂率(≥2cells stage),在体外受精56小时后的≥8cells stage胚胎形成率,在体外受精96小时后的囊胚形成率(含早期囊胚、扩张囊胚和孵出囊胚),实验重复3次,原始数据如表1:其中,2细胞率=2细胞数/排极体的卵数;8细胞率=8细胞数/排极体的卵数;囊胚率=囊胚数/2细胞数。使用Graphpad Prism 8.0进行统计分析,结果如图7所示野生型小鼠和dguok(-/+)杂合小鼠的卵细胞在体外成熟率,受精率,卵裂率,8细胞率及囊胚率。
表1体外受精情况原始数据统计图
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> 云南大学
<120> 一种与动物不孕相关的基因、sgRNA及应用和构建动物模型的方法
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<170> SIPOSequenceListing 1.0
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ggccctggct cccatgagat ggg 23
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<213> Mus musculus
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ggcaggagca atagtcaacg agg 23
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<213> 人工序列(artificial sequence)
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taggccctgg ctcccatgag at 22
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taggcaggag caatagtcaa cg 22
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aaaccgttga ctattgctcc tg 22
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ctcccgcact cagtactaca gct 23
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Claims (10)
1.一种与动物不孕相关的基因,其特征在于,所述基因包括动物dguok基因。
2.根据权利要求1所述基因,其特征在于,所述基因的敲除靶标片段包括动物dguok基因第二个外显子及两侧部分内含子区。
3.根据权利要求1或2所述基因,其特征在于,所述基因的敲除靶标片段的核苷酸序列包括SEQ ID NO.1和SEQ ID NO.2所示的核苷酸序列。
4.根据权利要求1或2所述基因,其特征在于,所述动物包括哺乳动物。
5.一组靶向权利要求1~4任一项所述基因的sgRNA,其特征在于,所述sgRNA包括M-DguoK-E2be-gRNA up、M-DguoK-E2be-gRNA down、M-DguoK-E2af-gRNAup和M-DguoK-E2af-gRNAdown;
所述M-DguoK-E2be-gRNAup的核苷酸序列如SEQ ID NO.3所示;
所述M-DguoK-E2be-gRNAdown的核苷酸序列如SEQ ID NO.4所示;
所述M-DguoK-E2af-gRNAup的核苷酸序列如SEQ ID NO.5所示;
所述M-DguoK-E2af-gRNAdown的核苷酸序列如SEQ ID NO.6所示。
6.根据权利要求4所述sgRNA,其特征在于,所述M-DguoK-E2be-gRNA up和M-DguoK-E2be-gRNAdown靶向SEQ ID NO.1所示的核苷酸序列;
所述M-DguoK-E2af-gRNAup和M-DguoK-E2af-gRNAdown靶向SEQ ID NO.2所示的核苷酸序列。
7.权利要求1~4任一项所述基因或权利要求5或6所述sgRNA在构建雌性不孕动物模型中的应用。
8.一种构建雌性不孕动物模型的方法,其特征在于,包括以下步骤:将权利要求5或6所述dguok sgRNA和CAS9 mRNA注射到动物的受精卵内,然后移植到假孕的动物输卵管,筛选雌性阳性F0动物;
将雌性阳性F0动物与野生型动物交配得到F1代,筛选阳性F1杂合动物并进行自交,F2纯合雌性动物为所述雌性不孕动物模型。
9.根据权利要求8所述方法,其特征在于,所述筛选包括PCR筛选,所述PCR筛选的引物对包括M-DguoK-F、M-DguoK-R和M-Dguok-deletion;
所述M-DguoK-F的核苷酸序列如SEQ ID NO.7所示;
所述M-DguoK-R的核苷酸序列如SEQ ID NO.8所示;
所述M-Dguok-deletion的核苷酸序列如SEQ ID NO.9所示。
10.权利要求8或9所述方法构建得到的雌性不孕动物模型在解析雌性不孕分子机制及筛选或制备治疗不孕药物中的应用。
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