CN110885858A - Amy1基因敲除小鼠动物模型的构建方法及应用 - Google Patents
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Abstract
本发明公开了一种Amy1基因敲除小鼠动物模型的构建方法与应用；基于CRISPR/Cas9基因敲除技术构建Amy1基因敲除小鼠模型，其构建方法包括如下步骤：步骤一、设计sgRNA和Cas9 RNA并体外转录成mRNA，将有活性的sgRNA和Cas9 RNA显微注射入小鼠受精卵中，获得Amy1基因敲除小鼠；步骤二、对Amy1基因敲除小鼠动物模型的鉴定。其优点表现在：本发明使用CRISPR/Cas9基因敲除技术，首次建立了Amy1基因敲除的小鼠动物模型，为研究Amy1基因与相关疾病的关系提供便捷、可靠的动物模型。
Description
技术领域
本发明属于生物技术领域，具体涉及一种基于CRISPR/Cas9技术的Amy1基因敲除小鼠动物模型的构建方法及应用。
背景技术
小鼠Amy1基因，位于3号染色体反链上，全长24190bp，其编码唾液腺淀粉酶。唾液淀粉酶长度为511氨基酸，主要参与代谢碳水化合物，起到将淀粉水解为麦芽糖和葡聚糖的作用。唾液淀粉酶作用于a-1，4糖苷键，可将较大分子量且不溶的淀粉分子分解为可溶性的小颗粒(包括淀粉糊精、红糊精和无色糊精)，随后在胃中被胃酸灭活。而唾液淀粉酶的底物—淀粉以及其代谢过程的产物—短链葡萄糖均可起到部分保护唾液淀粉酶免于被胃酸灭活的作用。
目前关于Amy1的相关研究仍非常有限，根据现有的文献报道显示，Amy1主要在唾液腺中表达，在人正常肝组织和大部分的肿瘤组织中并未发现Amy1基因的表达，但在某些具有神经内分泌功能的肿瘤，如某些肺腺癌组织中可检测到Amy1的表达。临床上在血清样本中对于淀粉酶的检测主要检测胰腺淀粉酶或者胰腺淀粉酶和唾液淀粉酶的总量，淀粉酶水平的轻度升高常被认为与唾液腺疾病有关，而高水平的淀粉酶升高则提示胰腺炎或其他消化道疾病。另一方面，有报道显示Amy基因的表达与心理、生理应激有关，而唾液淀粉酶可被用作应激的指标，并有报道认为它可作为交感神经系统活动的无创生物学标志物。因此，从以上报道可以看出，该基因的表达可能与一些消化系统疾病有关。
规律成簇间隔短回文重复序列(CRISPR/Cas9)技术是基于对细菌和古细菌中的免疫系统改造而建立，通过导向RNA(sgRNA)介导核酸内切酶Cas9蛋白进行目标DNA序列识别并造成DNA双链断裂，促进以同源重组或非同源末端连接方式修复受损DNA，从而对目标位点实现基因的定点敲除、敲入以及基因修正等多种修饰。
由于其具有特异性高，分子构建简单，流程短的特点，近年来CRISPR/Cas9技术获得了快速发展。使用CRISPR/Cas9技术进行基因敲除需要两个关键因素，首先是有效的sgRNA引导序列，再就是Cas9蛋白的存在。与锌指核酸酶(ZFN)技术和类转录样效应因子核酸酶(TALEN)技术相比，因其靶向编辑目标基因的特异性、高效性和设计的简便性等诸多优点得到越来越广泛的应用，在细菌、哺乳动物细胞以及斑马鱼、小鼠、大鼠等都表现出很强的基因组编辑活性。
中国专利文献CN107043787A公开了一种基于CRISPR/Cas9获得MARF1定点突变小鼠模型的构建方法。中国专利文献CN104293831A公开了一种基于CRISPR/Cas9基因敲除技术建立高血压小鼠模型的方法。中国专利文献CN105950639A公开了一种金黄色葡萄球菌CRISPR/Cas9系统的制备方法及其在构建基因修饰小鼠模型中的应用。中国专利文献CN106172238A公开了一种利用CRISPR/Cas9基因敲除技术建立miR-124基因敲除小鼠动物模型的方法。但是关于Amy1基因敲除小鼠动物模型的构建方法目前还未见报道。
发明内容
本发明旨在基于CRISPR/Cas9技术提供一种Amy1基因敲除小鼠动物模型的构建方法及应用；本发明通过CRISPR/Cas9技术通过体外转录的方式，获得Cas9mRNA和sgRNA构建稳定敲除Amy1基因小鼠模型，第一次成功构建了该动物模型，为进一步研究Amy1的功能奠定了良好的基础。
为了达到上述目的，本发明提供的技术方案为：
第一方面，本发明涉及一种Amy1基因敲除小鼠动物模型的构建方法，所述构建方法包括如下步骤：
S1，确定Amy1基因的特异性靶位点sgRNA1和sgRNA4，并与Cas9核酸酶体外转录成mRNA；
S2，将有活性的sgRNA1、sgRNA4和Cas9RNA显微注射入小鼠受精卵中，获得Amy1基因敲除小鼠；
所述sgRNA1如SEQ ID NO:1所示，所述sgRNA4如SEQ ID NO:2所示。
优选的，步骤S2包括以下步骤：
S21，小鼠促排卵和体外受精，培育受精卵；
S22，将有活性的sgRNA1、sgRNA4和Cas9RNA显微注射到小鼠受精卵中；
S22，受精卵体外培养、植入受体及靶向基因修饰动物的培育。
优选的，步骤S22包括以下步骤：
S221，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代小鼠；
S222，提取F0代小鼠尾部DNA，PCR扩增并将产物送测序；
S223，将阳性F0代小鼠与野生型异性小鼠交配获得F1代杂合子小鼠；
S224，将F1代杂合子小鼠杂交获得F2代纯合子小鼠，即为小鼠动物模型。
优选的，所述阳性F0代小鼠Amy1基因删除了3.1kb个碱基。
更优选的，所述阳性F0代小鼠Amy1基因删除的碱基序列如SEQ ID NO.8所示。
优选的，所述PCR扩增采用Amy1-seq-F+Amy1-seq-R2nn引物对；引物Amy1-seq-F的序列如SEQ ID NO.3所示；引物Amy1-seq-R2nn的序列如SEQ ID NO.4所示。
优选的，所述F2代纯合子小鼠基因组序列如SEQ ID NO.9。
优选的，用Amy1-seq-F+Amy1-seq-R2nn和Amy1-WT-F+Amy1-seq-R2nn两对引物对F2代小鼠进行PCR检测，鉴定出F2代纯合子小鼠；引物Amy1-seq-F的序列如SEQ ID NO.3所示；引物Amy1-seq-R2nn的序列如SEQ ID NO.4所示；引物Amy1-WT-F的序列如SEQ ID NO.5所示。
第二方面，本发明涉及一种基于CRISPR/Cas9基因敲除技术构建Sqstm1基因敲除小鼠模型的sgRNA，包括sgRNA1和sgRNA4，所述sgRNA1如SEQ ID NO:1所示，所述sgRNA4如SEQ ID NO:2所示。
第三方面，本发明涉及一种Amy1基因打靶载体，所述载体是基于CRISPR/Cas9系统的sgRNA表达载体，sgRNA的作用位点的序列如SEQ ID NO:1和SEQ ID NO:2所示。
第四方面，本发明涉及一种前述的sgRNA在构建糖尿病或代谢综合征相关的基因敲除小鼠动物模型中的用途。
上述的Amy1基因敲除小鼠动物模型可用于糖尿病或其他代谢综合征相关的研究中。
本发明具有如下有益效果：
本发明首次构建成功Amy1基因敲除小鼠，可为糖尿病或其他代谢综合征的相关研究提供新的研究方向。
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明显：
图1：Amy1基因敲除小鼠构建方案示意图；
图2：Amy1基因敲除区域；
图3：鉴定引物设计原理；
图4：F0代小鼠PCR检测结果；
图5：F0代小鼠基因部分测序结果；
图6：F0代小鼠基因部分测序对比截图；
图7：F1代小鼠PCR检测结果；
图8：F1代小鼠基因部分测序对比截图；
图9：F2代小鼠PCR检测结果。
具体实施方式
下面结合实施例对本发明进行详细说明。以下实施例将有助于本领域的技术人员进一步理解本发明，但不以任何形式限制本发明。应当指出的是，对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，还可以做出若干调整和改进。这些都属于本发明的保护范围。
一、Amy1基因敲除小鼠的构建方案，示意图如图1所示；
(1)构建针对Amy1基因的特异性靶点sgRNA1和sgRNA4。线性化及纯化DNA并在体外转录为mRNA。所述Amy1的sgRNA1序列如SEQ ID NO.1所示；所述Amy1的sgRNA4序列如SEQ IDNO.2所示；
gRNA序列信息及转录产物浓度表如表1：
表1
| gRNA名称 | gRNA序列(5'to3') | gRNA浓度 |
| Amy1-gRNA1 | GGTCGGGAAGGCTCCAATTTAGG(SEQ ID NO.1) | 5350ng/μl |
| Amy1-gRNA4 | GTTACAGTCTACTAAAGTCAAGG(SEQ ID NO.2) | 5450ng/μl |
(2)将步骤(1)所述的纯化sgRNA1和sgRNA4与Cas9核酸酶mRNA体外转录，显微注射到受精卵中，取注射后存活的受精卵移植到代孕母鼠子宫，孕育出生的小鼠即为F0代小鼠。
(3)待F0代小鼠出生10天后剪尾提取DNA进行PCR鉴定，并测序。得到的F0阳性小鼠Amy1基因删除了3.1kb个碱基(excon4-excon6)如图2所示，删除序列如SEQ ID NO.8；
其中，SEQ ID NO.8：
CAATTTAGGTAATCTCCGTCTTAGTGGCATCAAGGTCAAAGTCTGGATCAGCCTGCTTCAAGGCTGAGGGAGGCTACAATTATATACATGGTAGTTTGGGGAAAGCATTTGACATGTGGTTTTATTTTCCTTAGTTAACATAGTATAGGAGGTAGAAACTAAGATTAATATTCATAAACCTGTCCACAGTCATTTGATGGCTCAGGGAAGGTGTTAAAATATAATGTATATATATTTATATATACATAATATATAATTTATATATAATATGTCAATATATATGTTTCATTAGTGTGCTGCATAGTTTAACCAGCTTAATTTTAGGACTCTAAGCTGCATTCAAGTACACATTCTTCTCTTCAGGTCCGTATTTATGTGGATGCTGTCATTAACCACATGTGTGGAGTGGGGGCTCAAGCTGGACAAAGCAGTACATGTGGAAGTTATTTCAACCCAAATAACAGGGACTTTCCTGGAGTTCCCTATTCTGGTTTTGACTTTAATGATGGAAAATGTAGAACTGCAAGTGGAGGTATCGAGAACTACCAAGATGCTGCTCAGGTGAGGAAAACGATCAGAATTAAAAATGAAATCAGGGACTTCTTATGCACATGGACCTTTTGGAATACCATTTGACATGTGGTTTAGACTCCTTATATTACTAGGAGTTAGAAATGCAAATCAATCTTCATCAACCTTCTCCTAGTTTCTTGATGGTTCAAGAAAGGTGCTAAATACTAAAAGATTTATGTTTTAATCATGCAGTACATAAGAGAAGAAAAAAAAAAGGGAAAAAAAGGACACAAAAGGGTTTCCTCAATTAATTAATAATATTATCGATTTTATTTATGCCTCATCCATACATGATAGTGGAAAATATTCGAAGGGCGTATCCACAGTAGGATGTGGATGTTCCCAGTTTTCCATGAAAAGACTTTATGAAGATTTTTTATTGGTTATTATATCTATTTACATTTCAAATATCATTCTCCTTCCTGGTTTCCCCTTTGCAAACCTCCTATCCCATCCCCCTCCCCACTGCTTTTATGAGGGAACTCCCCCACCCATCCACCCACTCCAGCCTCACTGCCCAAGCATTCCCCTACCCTGGGGCATCAAGCCTCCACTGGATCAAGGGCCTCCCTTCCTACTGATGCCAGAAAAGGCAGTCCTCTGTGACATGTGCAGCTGGAGCCATGGGTCTCTCCAGGTGTATTCTTTGGTTGGTGGTTTAGTCCCTGGGAACTCTGGAGGGGCTTGTTGGTTGATATTGTTGTTCTTCCTATGGGGTTGCAAATCCCTTCAGCTCCTCAGTCCTTCTTCTAAGTGCTCCATTGGGGTCCCTGTGCTCAGTTCCATGGTGGGCTGCGAGCATCCACACCTGTATTTGTCAGGTTCTGGCAGAGCCATATCAGGCTCCAGTCAGGAAGTGCTTCTTGGCAATAATATCTGGGTTTGGTGTCAGCAGATGGGATGGATCACTAGGTGGGGCAGTATCTGGATCGCCTTTCCTTTACTCCCTGCTATACTCTTTGTCCCTGAATTTCTTTTAGACAGAAACAATTCTGGATTAATATTTTTGAGATGTGTGGGTGGCCCCATACCTCAATTGGGTCTGTACCTAACCTATGGATATAATCTTTACAGGTTCTCTAGACCAATCAGTGAATACTTGTTTAGTTGAAGGAGATCTCTGGTAACGGATGATATTTATATATTAAACACAGTTTTGTTTAGTGTGTGTTTCTTAACAGGTCAGAGATTGTCGTCTGTCTGGCCTTCTGGATCTTGCACTTGAGAAAGATTATGTTCGAACCAAGGTGGCTGACTATATGAACCATCTCATTGACATTGGCGTAGCAGGGTTCAGACTTGATGCTTCTAAGCACATGTGGCCTGGAGACATAAAGGCAATTTTGGACAAACTGCATAATCTCAATACAAAATGGTTCTCCCAAGGAAGCAGACCTTTCATTTTCCAAGAGGTAATATAATACATATATGTATGTATAAAATTACATTGTTTTGTTAAACAGAAGATAGATGGAAGATTGTATTAAACACAATTTAATAAGTTAATGTCTGAGTGCTTTATAAATGTGCAATTTCTTTAACTCCCATTTATTTAGATATAGACTTAATTGTTTACCAGAGTAAAGGTATAAGGGAATGTAGAGAATGTATTTATCCTGTGACATTATTGTACTACTTAGTGTATACTCAGGGACCATTATGCAATATCTTAGCTGCTGACACAAGTCAGTTTTTCTAGGCTTAATTCTTGTCTTAAATATTGTTAAATGAATAAATTTTTGATTAATATTTCTGTGGGCATTTTTTCATGTGAAGAACATTAATAATGTTTGTATTTGTCTTCTTTGGAGAATCTAATGAATTTGTAAGTGCTGAAATTGTTTATCTGACAGTAATTACTTTATTAGTATAAATATCTGAATTTTCAATTATATGTTAAGTGAAATATGTCAGCTATTTTTACTGGACAAAAATGATATTGTCATTACATTTCTTTCGGTACCAATGTTAATAAAGGAATTAAGAAAACCACATTGAACATGAACAGTATAAGAAATTTATAGGATATTATTTTTATATTAAATATTTTAATATCATTTTGTTCAATGACAGTTTCCCCCTATTTCTCTACTAGGTGATTGATCTGGGTGGTGAGGCAGTGTCAAGTAATGAGTATTTTGGAAATGGCCGTGTGACAGAATTCAAATATGGAGCAAAATTGGGCAAAGTTATGCGCAAGTGGGATGGAGAAAAGATGTCCTACTTAAAGTAAATACATCCCACTTTGTCATTTTTTTCTGTCATAGATCTGTTCCCATATTATCCCATTACATATTCATCTTATACAATTCTAATTTATTGAATATTTATCAAAACATTAGCTATTAAGCAGGCTGATTGCCCTCTAAACAATTAAAATCATCTGGAATTAATAAATTTCATTTTAAATATCAGTAACAAGGAGGTTTTCTAGTAACACAAAACATCACCTACAGATTTTTTTTTCTGTCTTTGATATGAATTTACTAGATTTTAGTAGTTACAGTCTACTAAAGTC
(4)把上述步骤(3)得到的F0代阳性小鼠与野生型小鼠交配获得F1代杂合子小鼠。
(5)将F1代杂合子小鼠杂交获得F2代纯合子小鼠，即为Amy1基因敲除小鼠动物模型。
二、阳性F0代小鼠PCR鉴定方法及结果：
(1)鉴定引物设计原理：如图3。
(2)引物信息如表2：
表2
(3)反应体系如下表3：
表3
注：Taq PCR Master Mix(Sangon Biotech，Cat.NO.：B639295)
(4)反应条件如下表4：
表4
(5)基因型判断标准如下表5：
表5
注：两对引物需要分别进行PCR。
(6)F0代小鼠鉴定报告：对F0代小鼠用引物Amy1-seq-F+Amy1-seq-R2nn进行PCR检测，结果参见电泳图：图4，由图4可知，杂合子小鼠会在400bp的位置出现条带，而野生型小鼠则不会产生条带，因此，F0代小鼠中杂合子有5只，编号别为777，779，781，783，786；编号778，780，782，784，785小鼠为野生型；选择鉴定出的阳性Founder小鼠779#进行基因测序，结果如图5和图6，图5为779#小鼠部分基因测序结果，基因序列如SEQ ID NO.6；图6为779#小鼠基因测序结果与野生型小鼠基因测序结果对比截图，证明F0代杂合子小鼠AMY1基因敲除。
其中，SEQ ID NO.6：
AGCACTGTTCCAACTTAGCCTGTATCCTACCGAACCTCCAGCAACACTTTTAAAAAATGAAGTTTAAAGCCAGTGCTAGCATAAATACCTGTTGCTCACCGTGCCCAATAGGATCCTCTGATTCTTTCCTTCCCTTGGAGCTCCATACAAGACAGTGACTCCCTCAGTGTTTCCTCACCGTTCCCAATGTCCCTGGTTTGAACGTCAATCTGTCATATGCCGCCGGGCCCGCGGCCTACAAGAACCAGATATACCTGGGAGGCAGGCTGAGGCTGAAAAAGACTTAGACGGCGAGAGAGATTAATGGAGACCAAGACATGATTCTGTTCAAGGCCCCCGATTTTACTCAGAAACTGTGCTTATAAAGGGGGAAGCCCCCCACCCCACGCCCACCCCCCACCAGTCCATTCTTGGTGTCTGGAGCCAGCCTGCAGGCGTCGTGCAGGATAGGATGTTCCTCCAGAATATCTCAGGGACCTCTCAGCAGGTAGCAGTGTCTTGGAGGAGTGCAGTGGCAGGTGACAGAACAATAGAGCTATCTAGGTCGGGAAGGCTCAAGGTATTACTTGAACAATTCTACTACTCACAAATAAAACTGCAACGTGGTCCTACTATTGCACTTCTATATACACTGTAAAAAATTAACTTGAAATATTTGCTCATCACACAGAGT
三、F1代小鼠获得及基因型鉴定
(1)选取阳性的F0代阳性小鼠与野生型C57BL/6J小鼠交配，获得的F1代杂合子小鼠，敲除类型为：删除3.1kb个碱基序列；鉴定方法同F0代小鼠鉴定。
(2)F1代小鼠鉴定报告：对所得的F1代小鼠用引物Amy1-seq-F+Amy1-seq-R2nn进行PCR检测，结果参见电泳图：图7，由图7可知，编号5016，5018，5019，5020的F1代小鼠为杂合子，而编号5014，5015，5017，5021小鼠为野生型，其中P代表阳性对照，N为阴性对照，B为空白对照。将鉴定出的F1代杂合子小鼠进行基因测序，结果比对截图：图8，杂合子小鼠测序结果完全相同，且与779#小鼠一致。
杂合子小鼠部分基因组序列如SEQ ID NO.7。
其中，SEQ ID NO.7：
AGCACTGTTCCAACTTAGCCTGTATCCTACCGAACCTCCAGCAACACTTTTAAAAAATGAAGTTTAAAGCCAGTGCTAGCATAAATACCTGTTGCTCACCGTGCCCAATAGGATCCTCTGATTCTTTCCTTCCCTTGGAGCTCCATACAAGACAGTGACTCCCTCAGTGTTTCCTCACCGTTCCCAATGTCCCTGGTTTGAACGTCAATCTGTCATATGCCGCCGGGCCCGCGGCCTACAAGAACCAGATATACCTGGGAGGCAGGCTGAGGCTGAAAAAGACTTAGACGGCGAGAGAGATTAATGGAGACCAAGACATGATTCTGTTCAAGGCCCCCGATTTTACTCAGAAACTGTGCTTATAAAGGGGGAAGCCCCCCACCCCACGCCCACCCCCCACCAGTCCATTCTTGGTGTCTGGAGCCAGCCTGCAGGCGTCGTGCAGGATAGGATGTTCCTCCAGAATATCTCAGGGACCTCTCAGCAGGTAGCAGTGTCTTGGAGGAGTGCAGTGGCAGGTGACAGAACAATAGAGCTATCTAGGTCGGGAAGGCTCAAGGTATTACTTGAACAATTCTACTACTCACAAATAAAACTGCAACGTGGTCCTACTATTGCACTTCTATATACACTGTAAAAAATTAACTTGAAATATTTGCTCATCACACAGAGT
四、将F1代杂合子小鼠杂交获得F2代纯合子小鼠，即为Amy1基因敲除小鼠动物模型，F2代纯合子小鼠基因组全长序列如SEQ ID NO.9。
五、F2代纯合子小鼠的鉴定：
(1)鉴定方法同F0代小鼠鉴定；
(2)F2代小鼠鉴定报告：用引物Amy1-seq-F+Amy1-seq-R2nn和Amy1-WT-F+Amy1-seq-R2nn进行PCR检测，结果参见电泳图：图9；由图9可知，野生型小鼠只会在300bp位置产生条带，纯合子小鼠只出现400bp大小的条带，杂合子两种条带都能产生，因此，F2代中野生型小鼠编号为6573和6577；杂合子小鼠编号为6571，6575，6576，6578；纯合子为6572和6574。
六、结果分析
野生型：Amy1-seq-F+Amy1-seq-R2nn不能获得条带；Amy1-WT-F+Amy1-seq-R2nn可以获得300bp片段
杂合子：Amy1-seq-F+Amy1-seq-R2nn获得400bp片段；Amy1-WT-F+Amy1-seq-R2nn获得300bp的片段。
纯合子：Amy1-seq-F+Amy1-seq-R2nn获得400bp片段；Amy1-WT-F+Amy1-seq-R2nn不能获得条带。
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，在不脱离本发明方法的前提下，还可以做出若干改进和补充，这些改进和补充也应视为本发明的保护范围。
序列表
<110> 上海交通大学
<120> Amy1基因敲除小鼠动物模型的构建方法及应用
<130> KAG41479
<160> 9
<170> SIPOSequenceListing 1.0
<210> 1
<211> 23
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 1
ggtcgggaag gctccaattt agg 23
<210> 2
<211> 23
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 2
gttacagtct actaaagtca agg 23
<210> 3
<211> 25
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 3
aaaagactta gacggcgaga gagat 25
<210> 4
<211> 25
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 4
ttggatattg cactctgtgt gatga 25
<210> 5
<211> 25
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 5
aagcaggctg attgccctct aaaca 25
<210> 6
<211> 673
<212> DNA
<213> 小鼠(Mus musculus)
<400> 6
agcactgttc caacttagcc tgtatcctac cgaacctcca gcaacacttt taaaaaatga 60
agtttaaagc cagtgctagc ataaatacct gttgctcacc gtgcccaata ggatcctctg 120
attctttcct tcccttggag ctccatacaa gacagtgact ccctcagtgt ttcctcaccg 180
ttcccaatgt ccctggtttg aacgtcaatc tgtcatatgc cgccgggccc gcggcctaca 240
agaaccagat atacctggga ggcaggctga ggctgaaaaa gacttagacg gcgagagaga 300
ttaatggaga ccaagacatg attctgttca aggcccccga ttttactcag aaactgtgct 360
tataaagggg gaagcccccc accccacgcc caccccccac cagtccattc ttggtgtctg 420
gagccagcct gcaggcgtcg tgcaggatag gatgttcctc cagaatatct cagggacctc 480
tcagcaggta gcagtgtctt ggaggagtgc agtggcaggt gacagaacaa tagagctatc 540
taggtcggga aggctcaagg tattacttga acaattctac tactcacaaa taaaactgca 600
acgtggtcct actattgcac ttctatatac actgtaaaaa attaacttga aatatttgct 660
catcacacag agt 673
<210> 7
<211> 673
<212> DNA
<213> 小鼠(Mus musculus)
<400> 7
agcactgttc caacttagcc tgtatcctac cgaacctcca gcaacacttt taaaaaatga 60
agtttaaagc cagtgctagc ataaatacct gttgctcacc gtgcccaata ggatcctctg 120
attctttcct tcccttggag ctccatacaa gacagtgact ccctcagtgt ttcctcaccg 180
ttcccaatgt ccctggtttg aacgtcaatc tgtcatatgc cgccgggccc gcggcctaca 240
agaaccagat atacctggga ggcaggctga ggctgaaaaa gacttagacg gcgagagaga 300
ttaatggaga ccaagacatg attctgttca aggcccccga ttttactcag aaactgtgct 360
tataaagggg gaagcccccc accccacgcc caccccccac cagtccattc ttggtgtctg 420
gagccagcct gcaggcgtcg tgcaggatag gatgttcctc cagaatatct cagggacctc 480
tcagcaggta gcagtgtctt ggaggagtgc agtggcaggt gacagaacaa tagagctatc 540
taggtcggga aggctcaagg tattacttga acaattctac tactcacaaa taaaactgca 600
acgtggtcct actattgcac ttctatatac actgtaaaaa attaacttga aatatttgct 660
catcacacag agt 673
<210> 8
<211> 3091
<212> DNA
<213> 小鼠(Mus musculus)
<400> 8
caatttaggt aatctccgtc ttagtggcat caaggtcaaa gtctggatca gcctgcttca 60
aggctgaggg aggctacaat tatatacatg gtagtttggg gaaagcattt gacatgtggt 120
tttattttcc ttagttaaca tagtatagga ggtagaaact aagattaata ttcataaacc 180
tgtccacagt catttgatgg ctcagggaag gtgttaaaat ataatgtata tatatttata 240
tatacataat atataattta tatataatat gtcaatatat atgtttcatt agtgtgctgc 300
atagtttaac cagcttaatt ttaggactct aagctgcatt caagtacaca ttcttctctt 360
caggtccgta tttatgtgga tgctgtcatt aaccacatgt gtggagtggg ggctcaagct 420
ggacaaagca gtacatgtgg aagttatttc aacccaaata acagggactt tcctggagtt 480
ccctattctg gttttgactt taatgatgga aaatgtagaa ctgcaagtgg aggtatcgag 540
aactaccaag atgctgctca ggtgaggaaa acgatcagaa ttaaaaatga aatcagggac 600
ttcttatgca catggacctt ttggaatacc atttgacatg tggtttagac tccttatatt 660
actaggagtt agaaatgcaa atcaatcttc atcaaccttc tcctagtttc ttgatggttc 720
aagaaaggtg ctaaatacta aaagatttat gttttaatca tgcagtacat aagagaagaa 780
aaaaaaaagg gaaaaaaagg acacaaaagg gtttcctcaa ttaattaata atattatcga 840
ttttatttat gcctcatcca tacatgatag tggaaaatat tcgaagggcg tatccacagt 900
aggatgtgga tgttcccagt tttccatgaa aagactttat gaagattttt tattggttat 960
tatatctatt tacatttcaa atatcattct ccttcctggt ttcccctttg caaacctcct 1020
atcccatccc cctccccact gcttttatga gggaactccc ccacccatcc acccactcca 1080
gcctcactgc ccaagcattc ccctaccctg gggcatcaag cctccactgg atcaagggcc 1140
tcccttccta ctgatgccag aaaaggcagt cctctgtgac atgtgcagct ggagccatgg 1200
gtctctccag gtgtattctt tggttggtgg tttagtccct gggaactctg gaggggcttg 1260
ttggttgata ttgttgttct tcctatgggg ttgcaaatcc cttcagctcc tcagtccttc 1320
ttctaagtgc tccattgggg tccctgtgct cagttccatg gtgggctgcg agcatccaca 1380
cctgtatttg tcaggttctg gcagagccat atcaggctcc agtcaggaag tgcttcttgg 1440
caataatatc tgggtttggt gtcagcagat gggatggatc actaggtggg gcagtatctg 1500
gatcgccttt cctttactcc ctgctatact ctttgtccct gaatttcttt tagacagaaa 1560
caattctgga ttaatatttt tgagatgtgt gggtggcccc atacctcaat tgggtctgta 1620
cctaacctat ggatataatc tttacaggtt ctctagacca atcagtgaat acttgtttag 1680
ttgaaggaga tctctggtaa cggatgatat ttatatatta aacacagttt tgtttagtgt 1740
gtgtttctta acaggtcaga gattgtcgtc tgtctggcct tctggatctt gcacttgaga 1800
aagattatgt tcgaaccaag gtggctgact atatgaacca tctcattgac attggcgtag 1860
cagggttcag acttgatgct tctaagcaca tgtggcctgg agacataaag gcaattttgg 1920
acaaactgca taatctcaat acaaaatggt tctcccaagg aagcagacct ttcattttcc 1980
aagaggtaat ataatacata tatgtatgta taaaattaca ttgttttgtt aaacagaaga 2040
tagatggaag attgtattaa acacaattta ataagttaat gtctgagtgc tttataaatg 2100
tgcaatttct ttaactccca tttatttaga tatagactta attgtttacc agagtaaagg 2160
tataagggaa tgtagagaat gtatttatcc tgtgacatta ttgtactact tagtgtatac 2220
tcagggacca ttatgcaata tcttagctgc tgacacaagt cagtttttct aggcttaatt 2280
cttgtcttaa atattgttaa atgaataaat ttttgattaa tatttctgtg ggcatttttt 2340
catgtgaaga acattaataa tgtttgtatt tgtcttcttt ggagaatcta atgaatttgt 2400
aagtgctgaa attgtttatc tgacagtaat tactttatta gtataaatat ctgaattttc 2460
aattatatgt taagtgaaat atgtcagcta tttttactgg acaaaaatga tattgtcatt 2520
acatttcttt cggtaccaat gttaataaag gaattaagaa aaccacattg aacatgaaca 2580
gtataagaaa tttataggat attattttta tattaaatat tttaatatca ttttgttcaa 2640
tgacagtttc cccctatttc tctactaggt gattgatctg ggtggtgagg cagtgtcaag 2700
taatgagtat tttggaaatg gccgtgtgac agaattcaaa tatggagcaa aattgggcaa 2760
agttatgcgc aagtgggatg gagaaaagat gtcctactta aagtaaatac atcccacttt 2820
gtcatttttt tctgtcatag atctgttccc atattatccc attacatatt catcttatac 2880
aattctaatt tattgaatat ttatcaaaac attagctatt aagcaggctg attgccctct 2940
aaacaattaa aatcatctgg aattaataaa tttcatttta aatatcagta acaaggaggt 3000
tttctagtaa cacaaaacat cacctacaga tttttttttc tgtctttgat atgaatttac 3060
tagattttag tagttacagt ctactaaagt c 3091
<210> 9
<211> 21099
<212> DNA
<213> 小鼠(Mus musculus)
<400> 9
gaatttcctt tgacactaaa ggaaacattt gatctgtgag accttttaga aaatttcaaa 60
gtaagctatg aacattcact gtaaagagta ccttggagtg aaaggctcgt tttcaagagg 120
aagagaagaa agacagtgag agtaagaaca gtgaaagaac agaaccccat aagaaaatat 180
caaattctct gtgtccacac tcagcacccc gaccacattg cgttgtgatg tgaacatcta 240
agtcttggaa atttctttct caatctcctt ggcagctgca gcagagctga tctatttctg 300
atcttggccc tgtttatgac ctgtggctct ccttgataga tattctacat atatatatat 360
atatatatac acatacacac acacacacac acacacacac acatatatat atatatactt 420
caaatagtgg ccattttatt tattctatcc cttaattaat gaataaaatc ttacaaaatc 480
taccaattgt tagagggagt atattaactc aaatgaataa ttttttcata aattgtatta 540
ccagttattg aataaaagct attcttttct cagcttcagc atcacatctc tgtacacttg 600
aattgcttaa caatccgtac tatagtaatc acaacttctg tgttaattga attttagtaa 660
ctaagtccaa taacactgat ttcacattat actttacaaa ataatacatg tattacatat 720
atcatatgca cataacataa atgctataca acagctagga aagtgttaaa gaagcaatgg 780
aatatagagt gtgttctcaa atcatatgta gagaaagttg ctggaacaga agactgatga 840
ttgtcacaag atgaaagata aattcagcca caaatcaatc agtcacaggc ccacatagca 900
ggcatgtttg gtgacccttt agagtgtctt cctgaagctg accatatacc attgacaaga 960
gacatgatgc ttgaggtgaa acctggaaaa cataagttct ttctctgctg catctcccag 1020
gagatcccct gagaagtggt cagctttagt gccatggtgc caccatctgg tattactact 1080
tatatgtgtc tgatactact gatatcattt tgaataatca caagtagaat ttttaattaa 1140
cacaaataaa tagtttgtct cttgatgtaa ttggcttatt actcatttaa acatgatatg 1200
gggttggtct actttagcca ttgtgctcaa aatggagtaa ctcattaaaa actccttttc 1260
ccccaagaaa actcagagta gtgcataact caaaagctgc tcttctgttc agcattgaac 1320
agctcagatc acggcacagc ctgttctctc ccatctacct tgtgactctg agagcagaac 1380
tgcaatgcct ctttgaccag aggaaataca cattgttctt cccacatcgt gcattacaga 1440
gattaccagt gaaatatcca tgcaagtttg ttccaaatct aatggccaaa aataagaaca 1500
atgtttttct tagatgctaa taaattgtcc tgctcaggtt agagcagcca ttctcaacgt 1560
aaatcagaag attccaactg cagtggaagg cagcacatgg tatgtaatcc ttaaaggata 1620
tgctagtttg aatttcatgt gcatgtcttc atgttatttt gccaatgcaa caaactttgt 1680
atttcatatt atggctgtat gtttgttgaa atccatagac ctgtttgctt attgttagtg 1740
gaagacatta ggtgttattt tgaatggaat taaatgatat tcagaaatta cttggctgca 1800
catgaaaata actaaacatg cacatatctg agtatctata tcatgtcaca aagtaattgg 1860
ttaaggaaaa tgtgtcaatg gcagtctata acttatacaa ttgttattaa actgaaaaat 1920
tctctcatca atttacatat aaatatatat caatatagca gaagcctgca ctatgattgg 1980
aatgatgtta cagtttaagc agatgcattt taatgaaaat aatgctacat gcatgatctg 2040
atgtatgatt ctttacttaa agatgtgtag aggatttcct ttaatagcca gttcctcgtg 2100
agtataagac tcttcccttt gtaagtggtg tgactactaa aaactcattc agatgagctc 2160
ttgatttttg ttgattgaca ttatgctgct ttccagcccc gtggtaaagt ataacagaga 2220
aggatataat gttacattat agtaaaaaat gggcattggg ttagtagagt gaacttttag 2280
tttttgatca caaatctcca tggcctttgt ttgtttaacc aaagggttaa tttgtttaaa 2340
aaattatatc acttcaaata ttcttcaatg gtgattttgg ttttatttgt tgaatttcag 2400
tgaaatcatg ataaatacta caattctcat cagcaaactg tgccagtagt ataattggat 2460
attttgacct tgtctctatg ttacaattta taacttattt ttttcattag ctttaagcca 2520
ccaaaatatt tttgagttac ctgggtacac acgactatgg caaccatagt cctttattcc 2580
ctacaaattt tgattgataa attaataact tgtacgcgtt acagttcata caatgttaca 2640
gatcagcaag taaatacaag aagcatggga tacaaagtgt cagtattgaa agcaaactca 2700
gagagcaaat cctggtatgg aaggttgatg ccactagctg agtgaatgat taagaaaccc 2760
atcatagcac tgccaagaca cagaataatt aagcactgcg tcacagggca ggtgcttttt 2820
tgagtgttag ttgatgtgtt atactgaggt ggactatgca gcatcggcag gcaaacccac 2880
acctctccta ctgctcctga aatccaatcc ccaagtctca tctccagctc tgtagctcga 2940
aacctgctgt agtctcctct ctgaccccac ccccaggatc aagaagacct tatcctcgaa 3000
cctttctttt cccaactcat cccaattacc cagtctccag aatcagcagg catcctttgt 3060
gaacacatca gatgaacagg ccaggaaagc aggcaacatg caaccatcaa attctctgaa 3120
aagtatgaga ggaaaaagaa actacagaac aaaacaccca cccaagaaag acaaatcaag 3180
atgtgagcac ctaaacataa aatcaatcca aacccagatg cttagttgct agcattaaaa 3240
acacaatcga caatagccag ggcaatatgt ctccatagaa cccagctatc ctaccatagc 3300
aggccctgaa tattccaaca taactgaaac aaaagaaaaa gatgttaaaa ctaactttat 3360
gaatatgata cagattctta acgagaaaat gagtaaatcc tttgacaaaa tccaagaaat 3420
ccacaacagt gtaaagaaat aaatacaact gttcaagtca tgaaattgta aagagaatca 3480
ataaagaaac aaaactgggg gaattcagga agtgaaaaat ttaggaattt gaacagtaac 3540
tacagaggca agcttcacca acagaacaca aggaaaatcc actaagatta catttcaatt 3600
catagcatct atgctccaaa cacaagggca cccaagttta taaaaaaaga cactactaca 3660
gcttaatcac atattgacct tcacacactg agagtaggag actttaatac cacattgtca 3720
tcaaggaaca agccatccag acaaaaacta aacagagaaa tactggagct aacaggtatt 3780
ataaaccaaa tggaccttag tgatatttgt agaacatttt acctacacaa aagaaaatgc 3840
cttctactct gcaccttatg gaaatttctc caaatttgac catactgttg gaaacaggac 3900
atgtctcagt agatataaga gttttgaaat aattccctgc atcctatctg acaaccagca 3960
acagaaccaa cagaaatctt acaaactgat ggaaactgaa tatttcgcta ctgaagggaa 4020
actgggtcaa gactaaaata aagacagaaa tggagtactt attagaattc aatgaaaatg 4080
cgtacacagc atacccaaac ttatggaaca cagtgaaagt ggtggtaaga ggaaagctta 4140
taccactaag tgcctacgta agatctcata ctagcaactt aagagcacac ttgaaagtta 4200
tataatgaaa acaagtaatt acactcaaca ggagtagaag atgataaacg cagacctaat 4260
aatagacctt tgattaagtg aatttgttcc cattatgttc tcattttttt attgattgaa 4320
tttggctcga taaaataatg tgatctttaa acttatttta caagccttta acccctatat 4380
tgtatataag tgatttagag aaaaagttga aagtagaaga ggttgttgtg tattgagaaa 4440
gcaaagtgat gaattatgtt gcaatagagg aggttataaa gcagcagcca agggaaggca 4500
gtggtttcca aaggacacca gggtggtggt gcccagtcca tcataggtca ctgtggagct 4560
cagatcacag tgctgacaga atccatattt ggagaattac ataaggtttg aaagagagga 4620
tagtgaaagg atacgaattc ctaaaaacgt ttaatctggc cttttgtttg aacgaaagag 4680
aaattgaaac caaatggtga gatttctatt gttttgtatt tgacgaatac taaactctag 4740
atttggatgg ttgttgattt atacaagttg atgtagtaag ttacttttaa gatatatggt 4800
tttgtttttc tttcatccct ttttgtactt tattagaggt ttattttatt tttattttta 4860
atggtttgat ctctgttatt tagggcttca gggtttcaag gtcttttgtg ggtttgttga 4920
cagatcgtgt ttgcttgccc agcagcacag tctgtactat ttgttttgga atgagtactg 4980
agccttggct ctaaacttat tttagatatg tggtaggcag actcgctaaa ctttgtcttt 5040
ctctagcctt catatgcaac ttatgctaag cttaagttgg tattttttcc ctgtgggtaa 5100
atgactactg ttttgagaga taatacaagg taaaactcag atgaattatg aattgtttta 5160
agtaaagcaa ctataacttg attaccttca cgtgaaatat cttcctaata ctttagaact 5220
taaaaaactt aaaataaaat acagaagctt tcaacactag agataatgtt aacatgataa 5280
aactacatgt gaagatgtgt cattaaagaa actataatat tttaatgaca ttcccagtac 5340
atttttcact ataaagatac ttaactatgt ctcctctggg ctactaacta caaatggcaa 5400
gaatacatat gctgtctctt actgaccact gctaaataat ttgtatttta taatgtctta 5460
ttttgtcatg aattttagga agaatataac aaggaaaata attttgtaaa taattagtat 5520
ttatttgtat atgcaaacag ggagagtaga gaaataattt aaaattaact gtgtgtgttt 5580
tttttttatt taagtagtgt ctttaaatta tctgactttg atatccataa gaagggtttt 5640
agctaaatgc ctaaatttat aaaagctgga gtgtaaagag gaatcctgaa tttatattag 5700
agttttctct ctgtaatagt aacacctatt ttataacatt gctaagaaac tacttttgag 5760
gatattatgc ataattttgt tttatttctt aatattagaa aagattttta atttttcttc 5820
tttcaaattt taattcacat tatgcatatt tatggaatat cacttggcaa ttcaatagta 5880
ttcaaacact atccacatta gggttgtcag catttccatc tagagttttt gataatcttg 5940
taatttttat ccataatata taggtgaata tagttcacaa tagcatacca tatatcttgt 6000
gaatcactac aggaaaagaa atgtaagtcc ccaaaaggag ttatgatgag gaacaaaaac 6060
catataatac acacacatac tctctctctc tctttctctc tctctctttc tctctctatc 6120
tttctctctc tttctctctc tcacacacac acattataac aaacaataat tggttttcat 6180
aagtatgcat tgtatattag tattttaata atacaaaata gtaataaata tatgctttat 6240
tgaaaattga aatatgtgta cttcatgaga ggaactgata tgttgttagg atgctatcaa 6300
agttatatat ttttattgca tagtaacata atacatattc atataataat atataatcaa 6360
acacacacac atatatatgt atacatacac acacacacat atatgtatgt atgtataact 6420
ataactcaaa gccatgaaaa tgtaacaggt ttagtcacta aagtgcatcc ttaactctat 6480
ttgatattat ttttatttag agtatcttac aagatattat gatcatattc ccttccctta 6540
tatcttctct tagtcctata tccacatttt tggttacgtt aattgaatag acatctttcc 6600
ggggccaagc tctgaagatt ctctcaaacc ctttctcatg atgttctctg gagagaacat 6660
tgtgggtaat aaagatgtcc tgtttagggc tgagcattct gaagtcccta ttctcagccc 6720
tttgaagatt tgtgagtcat tgccttgact gtctacactg ctaaaagagt tcttttacta 6780
aagctgatac ttatacaaac ctgtagttgt aaatgcaaat atttagaaag caattcaact 6840
acatgtttat ttaataaaat actggcagta gatcctacca tcggacccat gatctcccaa 6900
accataaata gttaaccggt acaacgtgtg cattctgtct gttgggaaat cctaggatcc 6960
aatcagaaag tccctggaac tattacatca ttaatacatc ttggatggaa ggtcagtatt 7020
atagtacagg gttcaaggct gcattagatc attgctgaat tttctctctc actcactttt 7080
ctatactatg aaatctattc agtatggagg aagctcctaa ctcagcacag ctcaatatat 7140
ttgttctgta tctaaagaaa gtgttgtttt tgagcaataa gctcttccta ttttcttttg 7200
gtgggtgacc aagatcactg acaatagcaa tagacattgt ctcgcgggct tccagaccaa 7260
atattcacag tgcattcatc tccacctggg cactggggtt ttgcttttaa aacatacaat 7320
ttctgggaaa agcttttgtc tgcagactac caccatttta ataaaacaac aggtaatttt 7380
aaaaaataaa ataatggatc ttatttagaa aaaattagtc acttggaaat attagtttat 7440
catggttgac ccatatatgt ttattttatt tttaatcatt tttatttgat attttccttc 7500
atttttgtta ttgcattagc tattacttca aatacatatt gaatagttat tcatatagga 7560
gacaacattt tcttttttct ggattgtagt ggaattgctt tgagtttctc ttcatttcat 7620
gtgattcaat ggacatcata ttatgaagta gaactttaga aactttgatg aaacaaaaga 7680
aatactgatt ttttacttag atatattccc aaattctgtc attgtttata attacacaag 7740
cctatttgac ttagtttacc tggtcatttg tgtgtgtgtg tgtgtgtgtg tgtgtgtgtg 7800
tgtgtgtgta ttgcacgtac agtatactat aaatgaggac ataattcatg tccttctctt 7860
acacgatact tggaaattat tacaggcatg ttatagtcat ttactgagta cataagagtg 7920
tttactactc acttattggg tctaggaata gtagagatga agtacatttt tttaaaagtt 7980
tgtttcagga gctgaatgtg taaatctaaa tatatctata ctataaatca aacagacaac 8040
agtagcctaa cctaaaaaag ttgtcacaca tcaaaaggtt actcattaca cctgtgaata 8100
taagcttaag ctaaaaatgt tcttttagtt gaatatgtac cgagtacaca tggatggtgc 8160
atctaggaag aacatagagt atcatggaag catgaaattg catgtcaaat ataatcaaaa 8220
atctagtttt gttaaggatt ttttcatgca ctttgttgtt gaaaaagcaa atgaaagaac 8280
tgtattgaca aagaaataat taaggccttt aacagtcttg ataaacctct cagacctgct 8340
actttctcat tttatcactt gataattgat atcaccttca aataccttcg tgtttagcat 8400
ttctatcttc cttgccatta ggttccagtt agcccacaca cctactgttt ttctaacagt 8460
aacctggtct gttcatgtga caagtcactt acctcattca tgcacagtta tagaagtctt 8520
cccgatgacc ggtcatttcc atttatacat atctaaaacc tggtttgttc ccagtggcag 8580
gagtgaactt caattaattt tttatcgtaa gtttctcacc ttatagcttt ggtagctttt 8640
tcttcatatt ttggaaggtc ttgatctgtt tgccccgcag acataatctc attgtatttt 8700
tcaacttttc agataagttt tagtcttact gatatttttg ttctcattat tgatgctttt 8760
taagtatttt aagtgtgtta tatgtgtagg cattcctgat agcaatgcaa aattttctag 8820
aagaagaaaa tagagatcaa ttggttactg tttatatatg gaaaggaaat ataacaacta 8880
cttttatata gatacttaaa actatataga actccaattg taattctctg gaataggtga 8940
tttcataagt gatatgtagt ataaatcaaa tagtgcagaa acattgatat aaaacacacc 9000
caaacattca tgtacatgca tgcacataca tgtatttctg gaaagggcta atatttcttc 9060
attgagaaaa cacattaaaa cttattaaga actcacagag catttatata gtttccctgg 9120
gaaaattttt aaatgttttt tcacattttg acattgttat tggtaattgc ttatggctta 9180
taaatagcta gtactagaat attgatttga gttaatattt ctttcattat gtattttttc 9240
agaaataaat tacttgttag aaagaatact gccaacagca tagcaaaatg aaattcttcc 9300
tgctgctttc cctcattgga ttctgctggg cccaatatga cccacatact caatatggac 9360
gaactgctat tgtccacctg tttgagtggc gctgggttga tattgctaag gaatgtgaga 9420
gatacttagc tcctaatgga tttgcaggtg tgcaggtaag acattcacaa ttcaaatgct 9480
gtgcgtatgt aaaattgttt tctaataatg tattttaaat attttgaagc aatatttcac 9540
ttcacagata attttcatga ggaaggaaaa gtggctggga tggcaacttt atattttgtt 9600
ttcagacatc aagaggccct ttttattgtt ttgtcaatgt tttcagtgat tctgaaggaa 9660
tgggaatgaa tttatatgga ttaagaactg caaaatcatt gatagttttc tacaggattt 9720
atttgtataa attaagctat gggctgatca tcttaactca tttatttgta ggtctctcca 9780
cccaatgaaa acatcgtagt ccacagccct tcaagaccat ggtgggaaag atatcaacca 9840
attagctaca aaatatgttc caggtctgga aatgaagatg aattcaggga catggtgaac 9900
aggtgcaaca atgttggtgt aagtgatctg tccttcgaga attaaaaaaa taaatctgag 9960
gacaccaaga acaaattttt ttcaatttga catgcacatt ctctaaaaat attaaaatat 10020
tgtcttaagc cattactgtt ttgattatat aaagaacgag attattctta tgtaaggcct 10080
gtaatctgcc tgagatataa atctagcatg gcattgcctt aaggtgtctt gtccaggcaa 10140
tccaggacaa attcttaaat gtctaaaaac agtcttctta aaagaacagc gttttttttt 10200
ttttccttat gagttgtata tgcataaaca attgcgaatt tgagaattta taatgtctga 10260
gggatgaata attttaagca attgtaagct ctgagatata atactgacta ttaatataca 10320
aattaaagct tgattattca acattttaaa acccatctac agcacacaat gtaattatct 10380
gtgctgaggc aggaggaaac tcaaaagaat aagcatgtga tccaattcac atgtactttt 10440
ctcccataga agagctgttt ttaaacacag taaatgggat gcatgcatga atgaacttat 10500
agaatgtaca aaagcatgca taaaaacaat tttcaactta tctttaggat agtaattatc 10560
aattttattt aaaatgatgg tatgtaatag gccaaatatc aatattctac aataaccaat 10620
ttaattgcta tttggaacaa ggaacagaga ctttgtcagg ttattaagac aatttttgtt 10680
atagtttttc taaaatatac aaatttatct tacaattctt tattagcacc acagcagcct 10740
taataggatg tcaaaacttt gctttgaggt gaaggccacc tcagccttgc ccactgctgg 10800
cctgctgccc aggccaaaag aggctgaatt ggcccaatct gttgtaggcc aggaagcccg 10860
agcaaaggtg gcagagcacc ccgagcccta gcaggaagaa tatggccacc cccagcaggg 10920
acaggtggat gcccatgggg gacacggagc atgggactgg cgcttcaggc ctgccgctga 10980
gttcgctaca aaggcgcctt ttttggcagc ctcctgcgcc atcaccgctc agggaaagcc 11040
acgtagccca tggccctgcc ccacagcagc tggcagccgg ccggccctcc acgtaggcca 11100
aaggtcggcc aggaagtgaa gaaagatgac tttacaataa tgttgtctct tgccaaagaa 11160
ttgctgtggg cacatgtaat aaccctagct aaaataagca gctagtattt aattaccatt 11220
gattataatt gataacaatt gatcacagga gttttttcta ctttctgtaa agcctttttg 11280
tttctcacca gttaatttga atcttacttg ataacaccta agaagggctt aagttctcct 11340
gtggtgagtt taaggtgagg tcttagccaa ttaacatctc ctggaagctt ttgaaaataa 11400
aattttgaag caaatcaatt tctttctctt agattttctg taccaaaact ttatagtata 11460
actaaaaccc taaatattaa aaagatattg cctctgaatc atttctggag caacagctac 11520
tctccagaac tttaaagctc attgtgtgag agcaaagatt tgatgtaaac ttcccttgag 11580
aggcattagc taataaaata ctttccactt aggaaaaaaa tacactaaaa gattcaaact 11640
cttagcttct tgttttgaag cagagacaat aaaatttttt cttacataat gtaaagctat 11700
attagctata cctagaggca aaaatttcct gtgattacca gttcactaga agcaaatcct 11760
ttaccatgca aaagaaaaaa cgaaccttta aacctataat aactttacat gaagtaggca 11820
agccagattg taatgtaata agttctacag cctgattgcc cttttataaa ctttgaattt 11880
gaactttatt tagaacatct ggatagatct gcaacagtgt ttttttagct aaaagaaaac 11940
tcccctggag actaggagag acaagcttca agagcttcct taggccccgt ttgcaagaca 12000
aaaaaacaaa gtgacacaag cctctctggg gctgctctga gctttgtatg gctgtatggc 12060
cacagagagc tgtattagtg ctctcataag atgattactc ttgtaataat cgtaattaac 12120
aatatatggg tgagtagaaa atcttaaatt tgcgatcaaa ctgcaagctg cagtcaatgg 12180
aacattggca gttttaacca taattttcaa gtttcttttg caatgttggg ataatatcca 12240
caaccttggg aaagcaaaac ccaatttcaa aacaatttat catctttaaa atcaatatta 12300
ggagcattac tattatgttg cgaagtttgg ctgccaattt atacctatgg atgcatgacc 12360
gtgcaaattt taatgccgca ttaaagagac attgttttaa atcttatctt taagtctacc 12420
tattaggata agaatcacat taaatattat ctcaactaga agtatccctt agaggcctag 12480
tttttttggc ctgctttatg ccacgtgttg taaagactcc aaacctaagc caccaattta 12540
aagctgtctt gctaccaaag ttacacctat aagaccagta aaaaaccaaa tgcagtggct 12600
aaacgagcct tataaattta gaccagtcaa agaattttac tttttctagc tatatttata 12660
aggtaaaaaa agcacgttgt catttgctaa acacaataat cacgattgca gaggaccctc 12720
caggaaaaaa tcaactatca gcttttttgc cccaaaacta agctgcagac tccctttctt 12780
tggaagcagc tttttccagc agtggctccc ctgctgtgcc tgatcctggc caaagtgtgg 12840
ccacgctctg gcttcactgg accagcagat aaagtcttag ccatttttat tttccaacat 12900
gaaatacaga acattcagtc attcagacaa caaaacaaaa cacacatgga tcaacacata 12960
cacagactgg tgcaccagac attagacagc acaaagaggg cagagaagtt ctgctgtaga 13020
gccagagaga ataaagcagg gcatccccca atttctctct gtctctggga gaatttcaaa 13080
atccattttc ctttatttta ttttatttta ttttatttta ttttatttta ttttattatt 13140
ttaaaccctg ttcctctagc ctgatgcagt tctcgactgc ggttaactgc ctgtttcatt 13200
attttaaacc ctattcctct tgcctaaagc tgcttttgaa aactacggac aaccgcctga 13260
ttcaaagttt ctagtccttt gttgccgaac cctaagtgga cccagcgcgg gttagcactg 13320
ttccaaactt agcctgtatc ctaccgaacc tccagcaaca cttttaaaaa atgaagttta 13380
aagccagtgc tagcataaat acctgttgct caccgtgccc aataggatcc tctgattctt 13440
tccttccctt ggagctccat acaagacagt gactccctca gtgtttcctc accgttccca 13500
atgtccctgg tttgaacgtc aatctgtcat atgccgccgg gcccgcggcc tacaagaacc 13560
agatatacct gggaggcagg ctgaggctga aaaagactta gacggcgaga gagattaatg 13620
gagaccaaga catgattctg ttcaaggccc ccgattttac tcagaaactg tgcttataaa 13680
gggggaagcc ccccacccca cgcccacccc ccaccagtcc attcttggtg tctggagcca 13740
gcctgcaggc gtcgtgcagg ataggatgtt cctccagaat atctcaggga cctctcagca 13800
ggtagcagtg tcttggagga gtgcagtggc aggtgacaga acaatagagc tatctaggtc 13860
gggaaggctc aaggtattac ttgaacaatt ctactactca caaataaaac tgcaacgtgg 13920
tcctactatt gcacttctat atacactgta aaaaattaac ttgaaatatt tgctcatcac 13980
acagagtgca atatccaatt tttttcattt gtttttcatt gtatatactg tgataggaat 14040
atttaccctt cctgagatca tctacattgc aatcttacat gaaatgtgga tgaaatgcaa 14100
ataagtcatt tataaaatta cctagaagaa aatttttctg tggaataatc atttgtttag 14160
aaaatcatta aattcaaata gactatagaa agaggtatag atttaggata ctctaaatgt 14220
tcttttcaag gaataatgta tgcttatatt tttaaaaact aggaactggg gagaaggttg 14280
gggtttgatg ccttctgaca gagcccttgt gtttgtggac aaccatgaca atcagcgagg 14340
acatggtgct gggggagcat ccatcttgac attctgggat gctaggtagg taaagctatt 14400
ctttacattt atacatgttc tttactgata gtaaaattct agtttttttt attgtaatat 14460
tgttttgtaa tttccagact ctataaaatg gctgttggct ttatgttggc tcatccttat 14520
ggtttcacac gggtgatgtc aagttactat tggccaagaa atttccagaa tggaaaagta 14580
agtttaaata tacttttgaa gaaagagaaa gcagtcctgt tttttgtttt tgtttttttt 14640
tttttcattt tatgtatgta aaccacattt tctttcttat ttttcttttt tatattagat 14700
attttcttta tttacatttc aaatgtattc ccctttcctg gtcccccccc ccccactcca 14760
gaaagcccct atcccttccc cctcttcctg cttctatgag ggtgtgctcc aacccaccca 14820
cccactcctg cctccctgcc cttgaattcc cctacactgg ggaccaaggg cctcttcccc 14880
cattgatgtc caacaaggcc atcctctgct acaaataggg ctggagccat gggtccctcc 14940
atgtgtactc ctttgttgat ggtttagact ctgggaggtc tggcaggtct agttgattga 15000
tattgttgtt cttcttatgg ggttacaaac cccttcaact ccttcagtac tttctctaac 15060
tcatccactg gggatcccat gcttagtctg gtgattggct gcaagcatcc acgtctgtat 15120
ttgtcaggct ctggcagaac ctctcagcag acagctatat caggctcctg tcagcatgca 15180
gagactgcca caccttggga tccattccat atacagtaac caaatgcagt cctgttttaa 15240
tttcatatga aattgttaca caggagacat gccatcaaaa ttatttaata caatttaaaa 15300
tttttaagta tattatagaa ctatgaactg ctttaacact ttgaaacaat aacttgaaag 15360
ttagagaaca agaacacaca gtaaaacata tgagtctagt agttctcttg tcaatttgat 15420
gtggggattg tgtatagtag cacctgaaag gaggaactaa gcttttgttt atttaagcaa 15480
aataaatatt tgctaagtac ttttggaatt ttctgtggat tgtaaaaaaa acttttcagt 15540
ttaactgagg ttaccttaaa aaaatacatg aaataaatta tgttaaatta agcacataag 15600
tttgaagact tattcaagaa tagttaatgt tacaaaatat tttgcaatat attaatgtga 15660
acaatttcaa aaggcagcta agggtctggg gagatgattt agagagtaaa gctcctgtgg 15720
gaggaatagg agtttgagtc gctgggacct tgcaatgctg agctgacttg gtgtcttgga 15780
atgcaattcc ccagcaggtg gagacagcag atttctcaag tatcaaactg gccaattctt 15840
gcgttaaaaa cttagcctct atagagcaca gagagcttga catatacaga ggtggatgat 15900
cgcagccaac cattgaattg atcacggggt ccccaatgta caagttagag aaagaactga 15960
aggaactgaa ggggtttgca accccatagg aagaacaaca atatcaatca accagagctc 16020
tcagggtcta aacctccaac caaggagtgt gcatggaggg acttatggct ccagccctat 16080
gtgtagcaga ggatggcctt gtcaggcatc aatgggaaaa gaggcccttg gtcctgggaa 16140
ggcacgatac cccagtgtag ggggatttga gggtggggtg atgggattta gtgggtgggt 16200
gggggaagca ggatgagggg ggaagggata gggagctctt gggttgggag aaatcgggaa 16260
aggggtaaca tttgaaatgt aaataaacaa aatattcaat aaaaagaaag gacacagaga 16320
gaattttgtt ttagaaagat atttaaatgt gttgagaacc aggaaatgca tattcaattt 16380
ttggaaaatt tattacagaa tatcaggaca tatttattag cacctttatt caaaaggtgg 16440
cataaaacca attgatatta tatttaaata cttcacaggc attaatattt ttatattacc 16500
aaactattgt tctattttac aaataatgac acttgggcat aaagaactta catatattga 16560
atcagatttt aaaaaccatt ggtactggag agtttttaaa aaggagcttt tcattgattc 16620
attgtgattt tcatgtcatg ttccccaatc ccactcatct ccccattgct tcatatctgc 16680
ctactccctt gtaaccttcc tctcaaataa aaaaaataaa ataaaaatag gacaaggcaa 16740
acaaaaacac cctgttgtgt ttcacagtat accccttttg tccacatgtt tacttgcaaa 16800
tgttgacagt agtgagtcat tggtctggtt cgaggcctct ggcttctgat acactaccag 16860
tattgaatgc tctacagttt gctggggaga aaggggaaga ccatctctcc cttgcataag 16920
ccattgcaca gcagacgagg tgtggggcta gctctccagt gatcacatgc tggggccatg 16980
aactcctgcc atcaagacca gctcgattat gttatacaag tttggtacag ggtctgtgct 17040
ccagagtagg gcagtcagtg aggggcatgg ccttctcttt gtagcctgtg gacatctaca 17100
tggtcccagg tggcagtcca taccagggat gccctaatgg actttactgg taatatgagc 17160
catgggcatc aacacagacc cctgctgctg catggctatg gacccagaca ctgcccatag 17220
cagcagtatg agtcgacttc atcttggcct caggtggcag gacaagctac tcacataacg 17280
ctgttcctct ctactctcat gtctccagtt ctgtctcttc atagcgctca aaccagtcca 17340
tttctctccc atctccctac cacatacttg cacatcatag tggctcccac caagtgtggg 17400
ccatgaaaca tgcagagctc tgcctgcttt ctgcctgtca gcatcactga gcctgatctt 17460
aaatcctggt ctttctgatg ctaaagattt tgccatttta ctgacgttaa agaagagagc 17520
tattaagaaa ctgttgcatg gaagacactg aataaataaa aatgtataat tctgtgaaat 17580
atgacctaag ataactatgt tatatacact gaaatagtaa atactcatcg gtagttgtct 17640
gtataaatac tgaaggagtt ggatactaag tagatactat agacattcaa gtaacagaac 17700
actttgaatt tagtgtcatg gaagtcaaca tgcatgttaa aaacctgatg cttttccctt 17760
taattaagga tgtcaatgac tgggttggac caccaaataa caatggaaaa accaaagaag 17820
tgagcattaa cccagacagc acttgtggca atgactggat ctgtgaacat cgatggcgtc 17880
aaataaggtg gggatgtccc ctacagatct tgtgtaataa cattgtttaa ccttgtattt 17940
taaattgatg tttaagggta ccagcttttc acatgatact gtgtttttag gaacatggtt 18000
gccttcagaa atgtcgtcaa tggtcagcct tttgcaaact ggtgggataa tgacagcaac 18060
caggtagctt ttggcagagg aaacaaagga ttcattgtct ttaacaatga tgactggtaa 18120
gtggatatca cttaaagtag gattatatac tattatactt tttaatgaat ggatttaaga 18180
aatataatat attatgacct aaaaactgta gaaaaaaatg cttgctcttg atgtgttcaa 18240
ttttgtagaa tttgttttga gttttgatat tgctgttgtc cttgatgcat ttttacactg 18300
tcatgtgcat atattctcat tcatatactc atgtgacacc tatataaaag acataaacat 18360
taaataataa tcttaaaaat cagtgaaagg atgggcagta ttagtttcaa attatcacac 18420
cctaactcta tatctcaact atttattttt ctatttcttt ggtatacaat cttaaaatgt 18480
ctgcctctgt gaaataattc tctttttctt tgtgattaat cttttcaata catttgtata 18540
catgactatg gctgatggtt tttctctgga gacagacgtg gattggttct acaatctcca 18600
atgactggcc ttcaatttga gatgcatgca tctatatctt ctcctttaat aatttcatcc 18660
tgtatacaac tatacttaga gaatctcttt gcatgaatct gagttatctt ttgctcgggt 18720
tatctcttgc tcatttagag gattgtatta gtttctgtct tgatgctatg attaaacaac 18780
atgaccaagg gaacttatag aagaaagaat ttatatgtgc ttgcaattac agagttttga 18840
ctccatgaag gaaaggtatg aaggtggaaa ctgggaggtg atgactcaca ttttgaacca 18900
aaggcacaat acagaaagat ctgactagaa aaacatgcaa gtgttattat tctcaaagct 18960
gccttcagtg aaaaaatttc tccaaataat ccacatcacc cacttcccca aactgtgcca 19020
tgatctagaa accaagtttt ttttttaatt tattattttt taattaggta ttttcttcat 19080
ttacatttca aatgctatct caaaagtccc ccagatcccc cccactcccc tacccaccca 19140
ctcccactac ttggccctgg cgttcccccg tactgaggta tataacgttt gcaagaccaa 19200
agggcctctc ttcccaatga tggttgacta ggccatcttc tttttttctt tttttaatat 19260
tttttaatta ggtattttct tcatttacgt ttcaaaagct atccccaaag tcccccagac 19320
atcttctgat acatatgcag ctagacacac gagctcttag aattgggaac aaaacactca 19380
tggaaggagt tacagagaca aagtttggag ctgagatgaa aggatgggtc acctagagac 19440
tgccataccc ggagatccat cccataatca gcctccaaac gttgacacca ttgcatacac 19500
cagcaagatt ttgctgaaag gacccagata tagctgtctc ttgtgagact atgcaggtgc 19560
ctggcaaaca aagaagtgga tgctcacagt cagctattgg atggaacaca gcgtccccaa 19620
tggaggagct agagaaagta cccaaggagc taaaggggtc tgcagcccta taggtggaac 19680
aacaatatga actaaccagt accctcggag ctcatgtctc tagaaaacaa gttttaaact 19740
tttcatgaac tctctaactc tctctgtctc tatctatcta tctatctatc tatctatcta 19800
tctatctatc tatctatcta cccatatata tgcatatgtg tgtgcttttg tatatcatat 19860
aatatatgat atatatatat atataatgta aaaatcattt attaggtcat tggccattaa 19920
atcataattt ctatttatag agcataatag acaagtattc attttggaga tagtttgatt 19980
tcttacattt gtctgtgttg aatttgatta cagaattaac tttttacttt ttcagggctt 20040
tgtcagaaac tttacagact ggtcttcctg ctggcacata ctgtgatgtc atttctggag 20100
ataaagtcga tggcaattgc actggaataa aagtctatgt tggcaatgat ggcaaagctc 20160
acttttctat tagtaactct gccgaagacc catttattgc aatccatgca gagtcaaaaa 20220
tataaaattt aaaataaaca catattgaga gcatcaataa tttggatttc ctgtctttta 20280
tggaatttaa tttttttatt tcaatcacat ttgataaaat gtataggcaa taaacttgaa 20340
aaggtaaaaa aaagttcatt aaaatcatac ataattatga gaaaattaca cccaggattt 20400
tgatacattt tagaagatta tctttaaatt atttgtcaaa agaaattctc atatttgtga 20460
agccatcctg acaaatatat tgtcttatca cttatataat ttgtttcgtg gtctcctaat 20520
ttctcattac tttaattata tttactccat tgtcttctat tatttcctca tgctttcact 20580
agatcctctc tttttcccag caaagccctc catcccacat tttcatgtaa tttttgcttt 20640
gatttttgtg atgatggttt catgtttcac ctaccagttt tgtaaaccct caaccaaata 20700
ttgactatta ttgatcttca tttcatactg gctttctata tatgatctat ctttatctgt 20760
ccttctatcc atccatccat ccatccatcc atccatccat ccatccatct acacatccat 20820
ccacccatcc atccaccatc tatctatcta tctatctatc tatctatcta tctatctatc 20880
tatctatcta tctatctata tttgggaccc cattgctctt aagtagggtt gcctacatga 20940
ccatggctgg aaagttacta cttgaaccaa ggcagcttac cagtggataa aacactaaga 21000
agtatatttt tccccacctt agcacccata aatttcttat agctcattag gacgggattg 21060
accttcgggt gtctccccaa attcctggta gaatcttaa 21099
Claims (10)
1.一种Amy1基因敲除小鼠动物模型的构建方法，其特征在于，所述构建方法包括如下步骤：
S1，确定Amy1基因的特异性靶位点sgRNA1和sgRNA4，并与Cas9核酸酶体外转录成mRNA；
S2，将有活性的sgRNA1、sgRNA4和Cas9RNA显微注射入小鼠受精卵中，获得Amy1基因敲除小鼠；
所述sgRNA1如SEQ ID NO:1所示，所述sgRNA4如SEQ ID NO:2所示。
2.根据权利要求1所述的Amy1基因敲除小鼠动物模型的构建方法，其特征在于，步骤S2包括以下步骤：
S21，小鼠促排卵和体外受精，培育受精卵；
S22，将有活性的sgRNA1、sgRNA4和Cas9RNA显微注射到小鼠受精卵中；
S22，受精卵体外培养、植入受体及靶向基因修饰动物的培育。
3.根据权利要求1或2所述的Amy1基因敲除小鼠动物模型的构建方法，其特征在于，步骤S22包括以下步骤：
S221，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代小鼠；
S222，提取F0代小鼠尾部DNA，PCR扩增并将产物送测序；
S223，将阳性F0代小鼠与野生型异性小鼠交配获得F1代杂合子小鼠；
S224，将F1代杂合子小鼠杂交获得F2代纯合子小鼠，即为小鼠动物模型。
4.根据权利要求3所述的Amy1基因敲除小鼠动物模型的构建方法，其特征在于，所述阳性F0代小鼠Amy1基因删除了3.1kb个碱基。
5.根据权利要求4所述的Amy1基因敲除小鼠动物模型的构建方法，其特征在于，所述阳性F0代小鼠Amy1基因删除的碱基序列如SEQ ID NO.8所示。
6.根据权利要求3所述的Amy1基因敲除小鼠动物模型的构建方法，其特征在于，所述PCR扩增采用Amy1-seq-F+Amy1-seq-R2nn引物对；引物Amy1-seq-F的序列如SEQ ID NO.3所示；引物Amy1-seq-R2nn的序列如SEQ ID NO.4所示。
7.根据权利要求3所述的Amy1基因敲除小鼠动物模型的构建方法，其特征在于，所述F2代纯合子小鼠基因组序列如SEQ ID NO.9。
8.根据权利要求3所述的Amy1基因敲除小鼠动物模型的构建方法，其特征在于，用Amy1-seq-F+Amy1-seq-R2nn和Amy1-WT-F+Amy1-seq-R2nn两对引物对F2代小鼠进行PCR检测，鉴定出F2代纯合子小鼠；引物Amy1-seq-F的序列如SEQ ID NO.3所示；引物Amy1-seq-R2nn的序列如SEQ ID NO.4所示；引物Amy1-WT-F的序列如SEQ ID NO.5所示。
9.一种Amy1基因打靶载体，其特征在于，所述载体是基于CRISPR/Cas9系统的sgRNA表达载体，sgRNA的作用位点的序列如SEQ ID NO:1和SEQ ID NO:2所示。
10.一种如权利要求9所述的sgRNA在构建糖尿病或代谢综合征相关的基因敲除小鼠动物模型中的用途。
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