CN114540310B - 一种小鼠胚胎干细胞及其制备方法和应用 - Google Patents
一种小鼠胚胎干细胞及其制备方法和应用 Download PDFInfo
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Abstract
本发明涉及一种小鼠胚胎干细胞株SXMUe002‑A‑1,其保藏编号为CGMCC No:23035,于2021年9月28日保藏于国家典型培养物保藏中心。本发明还提供了小鼠胚胎干细胞株SXMUe002‑A‑1在制备小鼠血友病模型中的应用。本发明获得的突变细胞株SXMUe002‑A‑1该突变株的生长繁殖能力及细胞形态与野生型高度相似,其在外显子上没有碱基的缺失及插入,仅仅突变了一个碱基,更接近临床病例。所述突变细胞株SXMUe002‑A‑1具有小鼠胚胎干细胞的特性,还可以进行小鼠胚胎干细胞嵌合注射得到血友病小鼠模型,对于进一步进行血友病无义突变的机制研究、血友病的药物筛选及基因治疗有重大意义。
Description
技术领域
本发明涉及生物技术领域,特别是涉及一种鼠胚胎干细胞及其制备方法和应用。
背景技术
血友病是一种X染色体连锁隐性遗传性出血性疾病。F8基因缺陷导致血友病A(hemophilia A,HA),F9基因缺陷导致血友病B(hemophilia B,HB),在男性人群中,发病率分别为1/5,000及1/25,000(杨仁池.血友病诊断与治疗中国专家共识(2017年版)[J].中华血液学杂志,2017,38(05):364-370)。中国是一个人口大国,预计有80,000–120,000血友病患者,其中约50%为重型。血友病现有的治疗手段仍以凝血因子制品输注的替代治疗为主,对家庭和社会造成极大负担[2]。此外,长期输注凝血因子导致抑制物的产生使得替代治疗疗效降低甚至无效,研究显示无义突变是凝血因子抑制物产生的高危因素[3]。故针对无义突变所致的血友病,探索新的廉价的个性化治疗方法并揭示其分子机制具有重要的意义。由于血友病是单基因遗传病,因此对于血友病,基因治疗应该是最理想的方式。而小鼠模型的我们研究各种疾病的良好模型,包括血友病。
CRISPR/Cas9基因编辑技术的发展为我们构建疾病模型提供了方便,它可以精准的实现目的基因的改造。以往的血友病小鼠模型是敲除F8/F9基因而导致凝血因子合成障碍,但血友病的基因突变种类繁多,这种敲除小鼠模型不能够很好反应血友病具体的基因突变情况。这就需要构建更符合病例条件下的基因突变类型。
山西医科大学第二医院筛查出一个带有无义突变的血友病B患者(F9.223C >T),Factor IX Gene(F9)Variant Database(http://www.factorix.org/)显示3713 个个案报告中有1094种F9变体,其中无义突变有87种,约占8%。F9 C.223C >T有73个患者报导,中国有7例,其中中国大陆5例,中国台湾2例。另一个数据库(https://omim.org/)显示有几千种无义突变与疾病有关。为了更深入研究血友病及无义突变的发病机制及治疗,我们在前期的研究基础中,进一步利用 CRISPR/Cas9技术在小鼠胚胎干细胞上构建到了一株无义突变的(F9 c.223C> T)小鼠胚胎干细胞模型。
发明内容
基于此,本发明的目的之一在于提供一种小鼠胚胎干细胞株SXMUe002-A-1。
包括如下技术方案:
一种小鼠胚胎干细胞株SXMUe002-A-1,其保藏编号为CGMCC No:23035,于2021年9月28日保藏于国家典型培养物保藏中心。
所述小鼠胚胎干细胞株具有F9基因发生c.223C>T无义突变。
本发明的第二目的是提供所述小鼠胚胎干细胞株SXMUe002-A-1在制备小鼠血友病细胞模型中的应用
本发明的第三目的是提供一种小鼠血友病模型,其通过嵌合注射上述的小鼠胚胎干细胞株SXMUe002-A-1而制备得到。
本发明的第四目的是提供上述的小鼠胚胎干细胞株SXMUe002-A-1或上述小鼠血友病模型的应用。
上述的小鼠胚胎干细胞株SXMUe002-A-1或上述小鼠血友病模型在制备、筛选血友病治疗药物中的应用。
上述的小鼠胚胎干细胞株SXMUe002-A-1或上述小鼠血友病模型在制备、筛选血友病治疗药物中的应用在制备血友病检测或分类、和/或治疗或预后监测的试剂中的应用。
上述的小鼠胚胎干细胞株SXMUe002-A-1或上述小鼠血友病模型在研究血友病无义突变发病的分子机制、无义突变的治疗策略中的应用。
本发明的第五目的是提供上述的小鼠胚胎干细胞株SXMUe002-A-1的制备方法
上述小鼠胚胎干细胞株SXMUe002-A-1的制备方法,包括如下步骤:
构建具有SEQ ID NO.43、SEQ ID NO.44、和SEQ ID NO.45所示序列的模板质粒;
将具有如SEQ ID NO.23和SEQ ID NO.24所示序列的sgRNA构建到px330 质粒中;
将构建后的px330质粒和所述模板质粒导入E14细胞中,筛选获得小鼠胚胎干细胞株SXMUe002-A-1。
本发明所述小鼠胚胎干细胞株SXMUe002-A-1,已于2021年9月28日在中国微生物菌种保藏管理委员会普通微生物中心保藏,保藏编号为CGMCC No: 23035。地址为北京市朝阳区北辰西路1号院3号,中国科学院微生物研究所,邮编:100101。
本发明的小鼠胚胎干细胞株SXMUe002-A-1具有以下优势:
1.本发明的发明人,通过筛选获得了一株人血友病B相关的无义突变的(F9c.223C>T)的小鼠胚胎干细胞株SXMUe002-A-1。这为血友病无义突变的机制研究、血友病的药物筛选及基因治疗提供了非常重要资源。
2.所获得的这株小鼠胚胎干细胞株SXMUe002-A-1生长繁殖能力及细胞形态与野生型高度相似,其在外显子上没有碱基的缺失及插入,仅仅是一个碱基发生了突变,更接近临床病例。
3.通过嵌合注射这株所述小鼠胚胎干细胞株SXMUe002-A-1,可获得小鼠胚胎干细胞模型,而这是人iPS伦理不允许的,这对于进一步进行血友病无义突变的机制研究、血友病的药物筛选及基因治疗有重大意义。
附图说明
图1.SXMUe002-A-1细胞系的基因模式图。
图2.SXMUe002-A-1细胞系的特征描述:其中A为细胞形态图,比例尺,25μm; B为细胞核型图。
图3为多能性标志物Nanog及Oct4的细胞免疫染色图,比例尺,50μm。
图4 SXMUe002-A-1细胞系表型分析实验结果示意图,其中,A为细胞株多能性标志物Naong,Klf4,Sox2,Oct4的Real-time PCR检测;B为EB分化形成的拟胚体,比例尺,100μm;C为EB分化后第6天Real-time PCR检测多能性退出,及向三胚层分化情况。
具体实施方式
下列实施例中未注明具体条件的实验方法,通常按照常规条件,例如 Sambrook等人,分子克隆:实验室手册(New York:Cold Spring Harbor Laboratory Press,1989)中所述的条件,或按照制造厂商所建议的条件。实施例中所用到的各种常用化学试剂,均为市售产品。
除非另有定义,本发明所使用的所有的技术和科学术语与属于本发明的技术领域的技术人员通常理解的含义相同。本发明的说明书中所使用的术语只是为了描述具体的实施例的目的,不用于限制本发明。本发明所使用的术语“和/ 或”包括一个或多个相关的所列项目的任意的和所有的组合。
此外,如本发明所使用的,术语“或”是包含性的“或”符号,并且等同于术语“和/或”,除非上下文另有明确规定。
为了便于理解本发明,下面将对本发明进行更全面的描述。本发明可以以许多不同的形式来实现,并不限于本文所描述的实施例。相反地,提供这些实施例的目的是使对本发明公开内容的理解更加透彻全面。
本发明的发明人课题组前期筛查了山西地区血友病患者基因突变类型,在血友病B中选择了无义突变F9 c.223C>T(p.R75X)位点作为研究对象。Factor IX Gene(F9)Variant Database(http://www.factorix.org/)显示3713个个案报告中有 1094种F9变体,其中无义突变有87种,约占8%。c.223C>T有73个患者报导,中国有7例,其中中国大陆5例,中国台湾2例。故构建血友病B无义突变(F9 c.223C>T)体对研究无义突变有重要意义。
通过NCBI比对,人和小鼠的F9 c.223C>T同源。以此为基础,我们设计了F9c.223C>T的模板质粒及sgRNA,电转后进行嘌呤霉素抗性及红色荧光,从众多突变株中,筛选得到一个碱基发生突变的单克隆细胞株SXMUe002-A-1,一代测序进一步证实了无义突变的发生。已于2021年9月28日在中国微生物菌种保藏管理委员会普通微生物中心保藏,保藏编号为CGMCC No:23035。
我们对细胞株SXMUe002-A-1进行表型分析,该突变细胞株与野生型细胞株的形态、多能性及三胚层分化能力没有检测出区别。利用该突变株,我们可以在细胞水平研究血友病无义突变发病的分子机制、无义突变的治疗。将该细胞株进行小鼠嵌合注射,还可以得到无义突变的小鼠模型进而进行更深入的研究。
实施例1
1、突变位点:从我院血友病B患者中检测到突变F9 c.223C>T,该突变为无义突变。
2、质粒构建:从E14细胞基因组中PCR扩增左臂(LA),利用定点突变将左臂上的目的位点突变及扩增右臂;筛选标记选择嘌呤霉素+mCherry红色荧光基因,嘌呤霉素及mCherry之间用P2A连接,两端各加上一个方向相同的Loxp 序列。用Goldgate克隆方法(E1601(BsaI-HFV2))将LA、RA及筛选标记(PGK-Puro,mCherry)组装到PGGA质粒中得到模板质粒即donor,如图1。在网站中(https://zlab.bio/guide-design-resources)设计sgRNA,通过各种方式筛选,最终获得一合适的sgRNA(Guide RNA-2(px330)),构建到px330质粒中。
Guide RNA-2(px330):在F9的第二个内含子设计sgRNA。
Donor上的左臂点突变,并带有puro抗性及mCherry红色荧光,在标记基因两端添加方向相同的loxp位点,筛选细胞株后在Cre酶会把loxp之间的序列删除。
(参见图1)
3、细胞培养:
E14细胞(129小鼠ES细胞)在MES培养基中培养,该培养基由DEME(Hyclone) +20%fetal bovine serum(FBS,Gibco),1mM非必须氨基酸(Gibco),1×谷氨酰胺(Gibco),1mM丙酮酸钠(Gibco),50units/ml青霉素和50μg/ml链霉素(Hyclone),0.1mMβ-巯基乙醇,1000U/ml白血病抑制因子和2i抑制剂(3μM CHIR99021和μM PD0325901),EB分化培养基不加2i及白血病抑制因子。5% CO2,温度37℃。细胞密度达到80%左右时,胰酶消化,1:10~1:50的比例传代。每天更换培养基。
EB分化:消化离心后,拿EB分化培养基重悬细胞并计数,20ul每滴的密度(0.5x105)点在15cm培养皿的盖子上,倒扣在培养皿上,皿中加一些PBS保持湿润,悬滴培养四天后,吸出EB球在6cm皿培养2-5天。
4、突变细胞株的筛选:
E14细胞在0.1%明胶铺盖的6cm培养皿中生长到80%左右密度时消化细胞,根据说明(Ltd.P3 Primary Cell 4D-NucleofectorTM X Kit L(V4XP-3012)取1x106的细胞数进行电转染,总质粒8ug(donor:sgRNA=3:1),电转后细胞在10cm皿中继续用MES培养,电转第二天培养基中添加嘌呤霉素(1μg/mL,Gibco),传两代后,待细胞长到60%左右,手工挑取有红色荧光的单克隆细胞到24孔中培养,细胞长起来后提取基因组(Tiangen Genomic DNAKit)进行PCR(Phanta Max Super-Fidelity DNA Polymerase,vazyme)检测及Sanger测序(生工生物工程股份有限公司,太原)。
5、Real time:用EZ-press RNA纯化试剂盒提取总RNA(EZBioscience)。然后移除基因组DNA,使用RT SuperMix for qPCR(Vazyme)将RNA反转录成cDNA。在CFX Connect(Bio-Rad)上使用2x RealStar Green Power Mixture (GenStar),在以下条件下通过实时qPCR对cDNA进行3个重复的定量:在95℃初始变性。95℃10s,60℃10s,72℃20s,循环40次。
表1基因扩增引物序列
Donor质粒序列:
LA:
caaacaggcttctgtccttcgcgggatatttgttgcaaagtcattgggagactacacactggaaacagcccagccagaggaatgatagatcacct tggaacgatcctgtactgagctattttcaagagatgacaaagtgggaacttaactgcttagttgagaactttctttttcatccaaagttaaacacaaatacaactgaaatgtaaccttttattgctggcactctaccaattaaaaataaaattgaattttaattcctaaatttccatgtgtatactgtgaaaaaaaaatcatagtttttggctctatgccccaaagagaaattagctatggaattacatgaacccaaaacaaaccttttctttaaaaacatatttagttttctgaggtttttttttttcctaatactaaagaactatacttttaaatttcagttttccttgatcgtgaaaatgccaccaaaattcttacccgtccaaagagatataattcagga aaactagaagagtttgttcgaggaaaccttgaaagagagtgtatagaagaaagatgtagttttgaagaagcatgagaagtttttgaaaacactgaaaaaactgtgagtatacccacatcatacctgaataatatgtcccagagcaaaagatagaaaatctctattttcaaaaggagtgtggaactagagagg tgactccatatttaggagcactctgctattccagagcacacagattcaaattcaagcatctaggaaacccaacactctcttctgacctttttgagtagtacatacacagg(SEQ ID NO.43)
Loxp-PGK-puro-P2A-mCherry-Loxp:
ataacttcgtataatgtatgctatacgaagttatgggtaggggaggcgcttttcccaaggcagtctggagcatgcgctttagcagccccgctgg gcacttggcgctacacaagtggcctctggcctcgcacacattccacatcccccggtaggcgccaaccggctccgttctttggtggccccttcgcgccaccttctactcctcccctagtcaggaagttcccccccgccccgcagctcgcgtcgtgcaggacgtgacaaatggaagtagcacgtctcact agtctcgtgcagatggacagcaccgctgagcaatggaagcgggtaggcctttggggcagcggccaatagcagctttgctccttcgctttctgggctcagaggctgggaaggggtgggtccgggggcgggctcaggggcgggctcaggggcggggcgggcgcccgaaggtcctccggaggcc cggcattctgcacgcttcaaaagcgcacgtctgccgcgctgttctcctcttcctcatctccgggcctttcgacctgcagcccaagctagcttaccatgaccgagtacaagcccacggtgcgcctcgccacccgcgacgacgtccccagggccgtacgcaccctcgccgccgcgttcgccgactaccc cgccacgcgccacaccgtcgatccggaccgccacatcgagcgggtcaccgagctgcaagaactcttcctcacgcgcgtcgggctcgacatcggcaaggtgtgggtcgcggacgacggcgccgcggtggcggtctggaccacgccggagagcgtcgaagcgggggcggtgttcgccgagat cggcccgcgcatggccgagttgagcggttcccggctggccgcgcagcaacagatggaaggcctcctggcgccgcaccggcccaaggagcccgcgtggttcctggccaccgtcggcgtctcgcccgaccaccagggcaagggtctgggcagcgccgtcgtgctccccggagtggaggcgg ccgagcgcgccggggtgcccgccttcctggagacctccgcgccccgcaacctccccttctacgagcggctcggcttcaccgtcaccgccgacgtcgaggtgcccgaaggaccgcgcacctggtgcatgacccgcaagcccggtgccgccactaacttcagcttgttgaagcaggccggagacg tcgaagagaacccgggtccaatggtgagcaagggcgaggaggataacatggccatcatcaaggagttcatgcgcttcaaggtgcacatggagggctccgtgaacggccacgagttcgagatcgagggcgagggcgagggccgcccctacgagggcacccagaccgccaagctgaaggtga ccaagggtggccccctgcccttcgcctgggacatcctgtcccctcagttcatgtacggctccaaggcctacgtgaagcaccccgccgacatccccgactacttgaagctgtccttccccgagggcttcaagtgggagcgcgtgatgaacttcgaggacggcggcgtggtgaccgtgacccaggactc ctccctgcaggacggcgagttcatctacaaggtgaagctgcgcggcaccaacttcccctccgacggccccgtaatgcagaagaagacgatgg gctgggaggcctcctccgagcggatgtaccccgaggacggcgccctgaagggcgagatcaagcagaggctgaagctgaaggacggcggccactacgacgctgaggtcaagaccacctacaaggccaagaagcccgtgcagctgcccggcgcctacaacgtcaacatcaagttggacatcac ctcccacaacgaggactacaccatcgtggaacagtacgaacgcgccgagggccgccactccaccggcggcatggacgagctgtacaagtaaataacttcgtataatgtatgctatacgaagttat(SEQ ID NO.44)
RA:
Gtgcatgtatgcacacagatagaacactcatacacataaaacaaaatacaaagtattatagatcaattattaaaggaaaattgtatttcaaatcttaa aaatatcagttcatatcatatcatctattacaaatgttctaaaaggaaaggaatccatatcaaaatactttaaacactatcattaagctgtcctcctttttccttacagactgaattttggaagcagtatgttggtaagcaattcattttattttattttatttcctacctgctatatgaaacacttgagaattgtgccttttttct atatagagaggttgtacagtctcagtaaaaaaaaaaaaaatcaggaaaaaaaccaaacaactgcatcttagagctaaatgtacatttactgtaactagtagattcagagatgattgggatggtttccaatgccctccgtgtcttgacctaccatcccttcttgttgccctctgaccacctctactactactgccct cattccacaaaggggctccttgcatgccctagcactgttcccatattagaggaacaatcttactatccctgttagctgagtcttcccagcttcttctaagctaacttccttaccttgctcagggacttgtccaaaattcctccaactcagtaagtcctcccttgataaggtattaatattccaacctgctacctggtcg ctctgtcattttagacacatgtgttctggtc(SEQ ID NO.45)
6、免疫荧光:免疫荧光染色在室温下进行,将细胞固定在4%多聚甲醛中15分钟。用0.5%Triton X-100的PBS溶液渗透5分钟后,将细胞用0.5%Triton X-100和10%山羊血清封闭30分钟。接下来,将细胞与在10%山羊血清中稀释的一抗在4℃下孵育过夜,然后与在10%山羊血清中稀释的二抗在室温下孵育 1小时。细胞核用DAPI复染5分钟。使用共焦系统(Zeiss LSM700,德国)获取图像。
表2细胞免疫染色的抗体
7、核型分析:当细胞达到对数期时,将Colcemid(秋水仙碱)加入终浓度为 20μg/ml的溶液中2小时。除去上清液,并将沉淀重悬于8ml的0.075M KCl 中,并在37℃下孵育20分钟。细胞用新鲜的Carnoy固定剂(甲醇:冰醋酸的比例为3:1)固定。使用Ikaros(MetaStstems,德国)在Olympus BX51显微镜上以450-500谱带分辨率分析了20个中期细胞。
8、STR分析:送检单位为上海启达生物科技有限公司,未见其他细胞系污染。
结果:
取1x106经电穿孔转染细胞,Cas9蛋白在sgRNA的引导下切割F9基因造成双键断裂,细胞发生同源重组修复,donor上的LA+PGK-puro-mCherry-RA代替基因组上的LA和RA,细胞基因组完成突变。我们通过puro及荧光筛选挑出了单克隆细胞株,并通过测序证实了目的位点的突变。
我们设计了若干条sgRNA,其中,sgRNA1 (F:caccgAAACTAGAAGAGTTTGTTCG,
R:aaacCGAACAAACTCTTCTAGTTTc,SEQ ID NO.46和SEQ ID NO.47) 和sgRNA2(Guide RNA-2)效率较高,sgRNA1在突变位点附近,即外显子上,挑取的45个克隆中有3个克隆发生靶点的编辑,并且实现了点突变,但是在cas9 切割处发生额外的碱基缺失,故并不可取。SgRNA2在内含子上,挑取72个克隆中有11个发生了靶点的编辑,即donor上的LA+PGK-puro-mCherry-RA代替基因组上的LA和RA,但是只有一个克隆发生了点突变。而且发现得到的这个点突变细胞株存在PGK-puro-mCherry,为了去掉这个外来插入的基因,我们再次电转了Cre-RNA,它能够在细胞内翻译出Cre蛋白,该蛋白能够识别Loxp 序列,把方向相同的Loxp序列之间的序列切掉。电转后我们再手工挑取单克隆到24孔中培养,扩增之后每个克隆分成两组,一组用嘌呤霉素筛选,一组不用嘌呤霉素,被切掉PGK-puro-mCherry在嘌呤霉素环境下不能存活,而且显微镜下没有观察到红色荧光。Cre-RNA效率较高,电转后挑取24个克隆,有22个能够被嘌呤霉素杀死。选状态最好的单克隆细胞株进行后续的实验。最终得到本发明的所述无义突变的小鼠胚胎干细胞株。
我们的实验结果发现,该突变株呈现出典型的mESC形态(图2A,比例尺, 25μm):Donor整合后细胞能在嘌呤霉素环境中存活并且在荧光显微镜下能看到红色荧光。G带分析进行了核型分析,结果显示二倍体40,XY(图2B)。 Sanger测序证实了目的位点突变(图1)。
免疫荧光(IF)染色证实了多能性标记OCT4和Nanog的表达(图3,比例尺,100μm)。实时聚合酶链反应(RT-qPCR)结果显示,突变株的多能性与三胚层分化能力与野生型相似(图4)。
STR分析:该株细胞鉴定结果为小鼠细胞系,DNA分型在细胞系检索中未找到匹配的细胞系。本次检测在该细胞系中,没有发现多等位基因。本次检测细胞分型结果良好。
上述实验中,细胞免疫荧光染色及RT-qPCR两方面证明了突变株在蛋白水平和RNA水平都能表达多能性标志物。体外自由分化显示其能够向中内外三个胚层分化。本发明获得的突变细胞株SXMUe002-A-1该突变株的生长繁殖能力及细胞形态与野生型高度相似,其在外显子上没有碱基的缺失及插入,仅仅突变了一个碱基,更接近临床病例。综上所述,突变株SXMUe002-A-1具有小鼠胚胎干细胞的特性,可以做为干细胞水平研究血友病发病机制和治疗的疾病模型,还可以进行小鼠胚胎干细胞嵌合注射得到血友病小鼠模型,为我们研究血友病奠定了基础。
以上所述实施例仅表达了本发明的几种实施方式,其描述较为具体和详细,但并不能因此而理解为对发明专利范围的限制。应当指出的是,对于本领域的普通技术人员来说,在不脱离本发明构思的前提下,还可以做出若干变形和改进,这些都属于本发明的保护范围。因此,本发明专利的保护范围应以所附权利要求为准。
序列表
<110> 山西医科大学
山西大学
<120> 一种小鼠胚胎干细胞及其制备方法和应用
<150> 2022101387887
<151> 2022-02-15
<160> 47
<170> SIPOSequenceListing 1.0
<210> 1
<211> 25
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 1
aactttggca ttgtggaagg gctca 25
<210> 2
<211> 25
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 2
ttggcagcac cagtggatgc aggga 25
<210> 3
<211> 20
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 3
ctcaagtcct gaggctgaca 20
<210> 4
<211> 20
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 4
tgaaacctgt ccttgagtgc 20
<210> 5
<211> 20
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 5
taggtgagcc gtctttccac 20
<210> 6
<211> 20
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 6
gcttagccag gttcgaggat 20
<210> 7
<211> 23
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 7
ctgcagtaca actccatgac cag 23
<210> 8
<211> 21
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 8
ggacttgacc acagagccca t 21
<210> 9
<211> 20
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 9
aacatgcccg gacttacaaa 20
<210> 10
<211> 21
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 10
ttcaagggaa tcctggtctt c 21
<210> 11
<211> 19
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 11
cgagccaaag cggagtctc 19
<210> 12
<211> 19
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 12
tgccaaggtc aacgccttc 19
<210> 13
<211> 19
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 13
ccctacccag cctacatgg 19
<210> 14
<211> 22
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 14
acatatcgag attggggtgt ct 22
<210> 15
<211> 24
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 15
catcggaaca gctctccaac ctat 24
<210> 16
<211> 21
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 16
gtgggctggc gttatgactc a 21
<210> 17
<211> 19
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 17
acgcagtgct ttccaaacc 19
<210> 18
<211> 19
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 18
cccgcaagtg gatgtctgg 19
<210> 19
<211> 21
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 19
gcagatgcaa aagtccaggt g 21
<210> 20
<211> 22
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 20
caggttgcga agaactctgt tt 22
<210> 21
<211> 19
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 21
ccctgaagtc gaggagctg 19
<210> 22
<211> 19
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 22
ctgctgcacc tctaagcga 19
<210> 23
<211> 25
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 23
caccgaaagg agtgtggaac tagag 25
<210> 24
<211> 25
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 24
aaacctctag ttccacactc ctttc 25
<210> 25
<211> 33
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 25
catgaatttc agtgaaatgc ttccatcact ggc 33
<210> 26
<211> 38
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 26
cctgtgtatg tactactcaa aaaggtcaga agagagtg 38
<210> 27
<211> 34
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 27
gaagaaagat gtagttttga agaagcatga gaag 34
<210> 28
<211> 34
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 28
cttctcatgc ttcttcaaaa ctacatcttt cttc 34
<210> 29
<211> 45
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 29
cccaagtaat aggggtaggg gaggcgcttt tcccaaggca gtctg 45
<210> 30
<211> 34
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 30
gaagttagtg gcggcaccgg gcttgcgggt catg 34
<210> 31
<211> 40
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 31
caagcccggt gccgccacta acttcagctt gttgaagcag 40
<210> 32
<211> 48
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 32
ggtcagggtg caggcagtga aaaaaatgct ttatttgtga aatttgtg 48
<210> 33
<211> 37
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 33
ggctacggtc tctggagcaa acaggcttct gtccttc 37
<210> 34
<211> 48
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 34
ggctacggtc tcatacgaag ttatcctgtg tatgtactac tcaaaaag 48
<210> 35
<211> 48
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 35
ggctacggtc tcacgtataa tgtatgctat acgaagttat gggtaggg 48
<210> 36
<211> 35
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 36
ggctacggtc tcctttccag gaaggcgggc acccc 35
<210> 37
<211> 34
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 37
ggctacggtc tccgaaacct ccgcgccccg caac 34
<210> 38
<211> 50
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 38
ggctacggtc tcgaacttcg tatagcatac attatacgaa gttatttact 50
<210> 39
<211> 43
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 39
ggctacggtc tcgagttatg tgcatgtatg cacacagata gaa 43
<210> 40
<211> 46
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 40
ggctacggtc tcgatgggac cagaacacat gtgtctaaaa tgacag 46
<210> 41
<211> 33
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 41
catgaatttc agtgaaatgc ttccatcact ggc 33
<210> 42
<211> 26
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 42
tgctacttcc atttgtcacg tcctgc 26
<210> 43
<211> 801
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 43
caaacaggct tctgtccttc gcgggatatt tgttgcaaag tcattgggag actacacact 60
ggaaacagcc cagccagagg aatgatagat caccttggaa cgatcctgta ctgagctatt 120
ttcaagagat gacaaagtgg gaacttaact gcttagttga gaactttctt tttcatccaa 180
agttaaacac aaatacaact gaaatgtaac cttttattgc tggcactcta ccaattaaaa 240
ataaaattga attttaattc ctaaatttcc atgtgtatac tgtgaaaaaa aaatcatagt 300
ttttggctct atgccccaaa gagaaattag ctatggaatt acatgaaccc aaaacaaacc 360
ttttctttaa aaacatattt agttttctga ggtttttttt tttcctaata ctaaagaact 420
atacttttaa atttcagttt tccttgatcg tgaaaatgcc accaaaattc ttacccgtcc 480
aaagagatat aattcaggaa aactagaaga gtttgttcga ggaaaccttg aaagagagtg 540
tatagaagaa agatgtagtt ttgaagaagc atgagaagtt tttgaaaaca ctgaaaaaac 600
tgtgagtata cccacatcat acctgaataa tatgtcccag agcaaaagat agaaaatctc 660
tattttcaaa aggagtgtgg aactagagag gtgactccat atttaggagc actctgctat 720
tccagagcac acagattcaa attcaagcat ctaggaaacc caacactctc ttctgacctt 780
tttgagtagt acatacacag g 801
<210> 44
<211> 1957
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 44
ataacttcgt ataatgtatg ctatacgaag ttatgggtag gggaggcgct tttcccaagg 60
cagtctggag catgcgcttt agcagccccg ctgggcactt ggcgctacac aagtggcctc 120
tggcctcgca cacattccac atcccccggt aggcgccaac cggctccgtt ctttggtggc 180
cccttcgcgc caccttctac tcctccccta gtcaggaagt tcccccccgc cccgcagctc 240
gcgtcgtgca ggacgtgaca aatggaagta gcacgtctca ctagtctcgt gcagatggac 300
agcaccgctg agcaatggaa gcgggtaggc ctttggggca gcggccaata gcagctttgc 360
tccttcgctt tctgggctca gaggctggga aggggtgggt ccgggggcgg gctcaggggc 420
gggctcaggg gcggggcggg cgcccgaagg tcctccggag gcccggcatt ctgcacgctt 480
caaaagcgca cgtctgccgc gctgttctcc tcttcctcat ctccgggcct ttcgacctgc 540
agcccaagct agcttaccat gaccgagtac aagcccacgg tgcgcctcgc cacccgcgac 600
gacgtcccca gggccgtacg caccctcgcc gccgcgttcg ccgactaccc cgccacgcgc 660
cacaccgtcg atccggaccg ccacatcgag cgggtcaccg agctgcaaga actcttcctc 720
acgcgcgtcg ggctcgacat cggcaaggtg tgggtcgcgg acgacggcgc cgcggtggcg 780
gtctggacca cgccggagag cgtcgaagcg ggggcggtgt tcgccgagat cggcccgcgc 840
atggccgagt tgagcggttc ccggctggcc gcgcagcaac agatggaagg cctcctggcg 900
ccgcaccggc ccaaggagcc cgcgtggttc ctggccaccg tcggcgtctc gcccgaccac 960
cagggcaagg gtctgggcag cgccgtcgtg ctccccggag tggaggcggc cgagcgcgcc 1020
ggggtgcccg ccttcctgga gacctccgcg ccccgcaacc tccccttcta cgagcggctc 1080
ggcttcaccg tcaccgccga cgtcgaggtg cccgaaggac cgcgcacctg gtgcatgacc 1140
cgcaagcccg gtgccgccac taacttcagc ttgttgaagc aggccggaga cgtcgaagag 1200
aacccgggtc caatggtgag caagggcgag gaggataaca tggccatcat caaggagttc 1260
atgcgcttca aggtgcacat ggagggctcc gtgaacggcc acgagttcga gatcgagggc 1320
gagggcgagg gccgccccta cgagggcacc cagaccgcca agctgaaggt gaccaagggt 1380
ggccccctgc ccttcgcctg ggacatcctg tcccctcagt tcatgtacgg ctccaaggcc 1440
tacgtgaagc accccgccga catccccgac tacttgaagc tgtccttccc cgagggcttc 1500
aagtgggagc gcgtgatgaa cttcgaggac ggcggcgtgg tgaccgtgac ccaggactcc 1560
tccctgcagg acggcgagtt catctacaag gtgaagctgc gcggcaccaa cttcccctcc 1620
gacggccccg taatgcagaa gaagacgatg ggctgggagg cctcctccga gcggatgtac 1680
cccgaggacg gcgccctgaa gggcgagatc aagcagaggc tgaagctgaa ggacggcggc 1740
cactacgacg ctgaggtcaa gaccacctac aaggccaaga agcccgtgca gctgcccggc 1800
gcctacaacg tcaacatcaa gttggacatc acctcccaca acgaggacta caccatcgtg 1860
gaacagtacg aacgcgccga gggccgccac tccaccggcg gcatggacga gctgtacaag 1920
taaataactt cgtataatgt atgctatacg aagttat 1957
<210> 45
<211> 731
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 45
gtgcatgtat gcacacagat agaacactca tacacataaa acaaaataca aagtattata 60
gatcaattat taaaggaaaa ttgtatttca aatcttaaaa atatcagttc atatcatatc 120
atctattaca aatgttctaa aaggaaagga atccatatca aaatacttta aacactatca 180
ttaagctgtc ctcctttttc cttacagact gaattttgga agcagtatgt tggtaagcaa 240
ttcattttat tttattttat ttcctacctg ctatatgaaa cacttgagaa ttgtgccttt 300
tttctatata gagaggttgt acagtctcag taaaaaaaaa aaaaatcagg aaaaaaacca 360
aacaactgca tcttagagct aaatgtacat ttactgtaac tagtagattc agagatgatt 420
gggatggttt ccaatgccct ccgtgtcttg acctaccatc ccttcttgtt gccctctgac 480
cacctctact actactgccc tcattccaca aaggggctcc ttgcatgccc tagcactgtt 540
cccatattag aggaacaatc ttactatccc tgttagctga gtcttcccag cttcttctaa 600
gctaacttcc ttaccttgct cagggacttg tccaaaattc ctccaactca gtaagtcctc 660
ccttgataag gtattaatat tccaacctgc tacctggtcg ctctgtcatt ttagacacat 720
gtgttctggt c 731
<210> 46
<211> 25
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 46
caccgaaact agaagagttt gttcg 25
<210> 47
<211> 25
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 47
aaaccgaaca aactcttcta gtttc 25
Claims (4)
1.一种小鼠胚胎干细胞株SXMUe002-A-1,其保藏编号为CGMCC No:23035,于2021年9月28日保藏于国家典型培养物保藏中心, 所述小鼠胚胎干细胞株具有F9基因发生c.223C>T无义突变,且在外显子上没有碱基的缺失及插入。
2.权利要求1所述小鼠胚胎干细胞株SXMUe002-A-1在制备小鼠血友病细胞模型中的应用。
3.一种小鼠血友病模型,其特征在于,通过嵌合注射权利要求1所述的小鼠胚胎干细胞株SXMUe002-A-1而制备得到。
4.权利要求1所述的小鼠胚胎干细胞株SXMUe002-A-1或权利要求3所述小鼠血友病模型在制备、筛选血友病治疗药物中的应用。
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