CN110331164B - Lilra3基因敲入小鼠的打靶载体和lilra3基因敲入小鼠的构建方法 - Google Patents

Lilra3基因敲入小鼠的打靶载体和lilra3基因敲入小鼠的构建方法 Download PDF

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CN110331164B
CN110331164B CN201910716438.2A CN201910716438A CN110331164B CN 110331164 B CN110331164 B CN 110331164B CN 201910716438 A CN201910716438 A CN 201910716438A CN 110331164 B CN110331164 B CN 110331164B
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mouse
lilra3
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targeting vector
rosa
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郭建萍
栗占国
王宇轩
唐蕴荻
邹云东
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Peking University People's Hospital (peking University Second Clinical Medical College)
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Abstract

本发明提供了LILRA3基因敲入小鼠的打靶载体和LILRA3基因敲入小鼠的构建方法，属于基因工程技术领域，所述打靶载体的核苷酸序列如SEQ ID No.1所示，采用本发明提供的打靶载体能够将LILRA3基因构建到小鼠体内。

Description

LILRA3基因敲入小鼠的打靶载体和LILRA3基因敲入小鼠的构 建方法
技术领域
本发明属于基因工程技术领域，尤其涉及LILRA3基因敲入小鼠的打靶载体和LILRA3基因敲入小鼠的构建方法。
背景技术
在中国汉族人群中RA患者功能性LILRA3基因频率明显高于健康人，提示功能性LILRA3可能是RA的易感性因素，尤其在男性中有更强的相关性。早期ACPA阳性RA患者中，纯合型功能性LILRA3患者关节影像评分更高，功能性LILRA3可能加重疾病严重程度。已有体外实验证明LILRA3能够参与免疫调节，促进炎症发生。除MHC I类分子外，LILRA3的配体尚不明确，LILRA3功能研究受到限制。因此需要体内实验验证LILRA3在免疫调节中的作用。由于小鼠中不存在LILRA3同源基因，目前缺少基于动物实验的体内研究。
发明内容
有鉴于此，本发明的目的在于提供LILRA3基因敲入小鼠的打靶载体和LILRA3基因敲入小鼠的构建方法，采用本发明提供的打靶载体能够将LILRA3基因构建到小鼠体内。
为了实现上述发明目的，本发明提供了以下技术方案：
本发明提供了LILRA3基因敲入小鼠的打靶载体，所述打靶载体的核苷酸序列如SEQ ID No.1所示。
优选的，所述打靶载体的构建方法包括：将基因连入EGE载体中，得到打靶载体；所述基因具有SEQ ID No.2所示的核苷酸序列。
优选的，所述步骤3)连入的反应条件包括：将100ng EGE载体、500ng基因、2μL 10xDNA Ligase Buffer、1U DNA Ligase和18μL ddH2O混合后在22℃下孵育10min。
本发明还提供了LILRA3基因敲入小鼠的构建方法，包括：
将上述技术方案所述的打靶载体导入小鼠受精卵中，得到F0小鼠；
将得到的F0小鼠与野生型小鼠杂交，得到携带LILRA3基因的杂合型小鼠；
将携带LILRA3基因的杂合型小鼠进行杂交，得到的纯合型小鼠为LILRA3基因敲入小鼠。
优选的，所述打靶载体的使用量为5～10pL。
优选的，利用Rosa-GT-F、Rosa-GT-R和Rosa26-Test(L)-R3引物判定小鼠的基因型；
所述Rosa-GT-F引物的核苷酸序列如SEQ ID No.3所示；
所述Rosa-GT-R引物的核苷酸序列如SEQ ID No.4所示；
所述Rosa26-Test(L)-R3的核苷酸序列如SEQ ID No.5所示。
本发明提供了LILRA3基因敲入小鼠的打靶载体和LILRA3基因敲入小鼠的构建方法，所述打靶载体的核苷酸序列如SEQ ID No.1所示。打靶载体中的目的基因包含ROSA26基因的同源臂，通过同源重组的方式，将打靶载体上的目的基因定点转入ROSA26基因位点，使目的基因可稳定遗传。ROSA26基因几乎在所有组织中都能编码一种非必需的核RNA，且为外源基因的插入热点。ROSA26位点基因敲入技术可以非常有效地建立多用途的条件性转基因小鼠模型。采用本发明提供的打靶载体能够将LILRA3基因构建到小鼠体内。
附图说明
图1为打靶载体酶切验证结果；
图2为小鼠基因型的PCR鉴定结果；
图3为ELISA鉴定敲入基因在血清中表达结果；
图4为转基因打靶载体图谱；
图5为转基因打靶载体的酶切位点分布；
图6为小鼠交配策略图；
图7为引物设计策略图。
具体实施方式
本发明提供了LILRA3基因敲入小鼠的打靶载体，所述打靶载体的核苷酸序列如SEQ ID No.1所示，具体如下所示：
tcgcgcgtttcggtgatgacggtgaaaacctctgacacatgcagctcccggagacggtcacagcttgtctgtaagcggatgccgggagcagacaagcccgtcagggcgcgtcagcgggtgttggcgggtgtcggggctggcttaactatgcggcatcagagcagattgtactgagagtgcaccatatgcggtgtgaaataccgcacagatgcgtaaggagaaaataccgcatcaggcgccattcgccattcaggctgcgcaactgttgggaagggcgatcggtgcgggcctcttcgctattacgccagctggcgaaagggggatgtgctgcaaggcgattaagttgggtaacgccagggttttcccagtcacgacgttgtaaaacgacggccagtgaattgccgacgctttaggagtgaataccgaattcggaagagtcctgacccagggaagacattaaaaaggtagtggggtcgactagatgaaggagagcctttctctctgggcaagagcggtgcaatggtgtgtaaaggtagctgagaagacgaaaagggcaagcatcttcctgctaccaggctggggaggcccaggcccacgaccccgaggagagggaacgcagggagactgaggtgacccttctttcccccggggcccggtcgtgtggttcggtgtctcttttctgttggacccttaccttgacccaggcgctgccggggcctgggcccgggctgcggcgcacggcactcccgggaggcagcgagactcgagttaggcccaacgcggcgccacggcgtttcctggccgggaatggcccgtacccgtgaggtgggggtggggggcagaaaaggcggagcgagcccgaggcggggagggggagggccaggggcggagggggccggcactactgtgttggcggactggcgggactagggctgcgtgagtctctgagcgcaggcgggcggcggccgcccctcccccggcggcggcagcggcggcagcggcggcagctcactcagcccgctgcccgagcggaaacgccactgaccgcacggggattcccagtgccggcgccaggggcacgcgggacacgccccctcccgccgcgccattggcctctccgcccaccgccccacacttattggccggtgcgccgccaatcagcggaggctgccggggccgcctaaagaagaggctgtgctttggggctccggctcctcagagagcctcggctaggtaggggatcgggactctggcgggagggcggcttggtgcgtttgcggggatgggcggccgcggcaggccctccgagcgtggtggagccgttctgtgagacagccgggtacgagtcgtgacgctggaaggggcaagcgggtggtgggcaggaatgcggtccgccctgcagcaaccggagggggagggagaagggagcggaaaagtctccaccggacgcggccatggctcgggggggggggggcagcggaggagcgcttccggccgacgtctcgtcgctgattggcttcttttcctcccgccgtgtgtgaaaacacaaatggcgtgttttggttggcgtaaggcgcctgtcagttaacggcagccggagtgcgcagccgccggcagcctcgctctgcccactgggtggggcgggaggtaggtggggtgaggcgagctggacgtgcgggcgcggtcggcctctggcggggcgggggaggggagggagggtcagcgaaagtagctcgcgcgcgagcggccgcccaccctccccttcctctgggggagtcgttttacccgccgccggccgggcctcgtcgtctgattggctctcggggcccagaaaactggcccttgccattggctcgtgttcgtgcaagttgagtccatccgccggccagcgggggcggcgaggaggcgctcccaggttccggccctcccctcggccccgcgccgcagagtctggccgcgcgcccctgcgcaacgtggcaggaagcgcgcgctgggggcggggacgggcagtagggctgagcggctgcggggcgggtgcaagcacgtttccgacttgagttgcctcaagaggggcgtgctgagccagacctccatcgcgcactccggggagtggagggaaggagcgagggctcagttgggctgttttggaggcaggaagcacttgctctcccaaagtcgctctgagttgttatcagtaagggagctgcagtggagtaggcggggagaaggccgcacccttctcttaattaaccgtttaaacaattctgcaggaatctagttattaatagtaatcaattacggggtcattagttcatagcccatatatggagttccgcgttacataacttacggtaaatggcccgcctggctgaccgcccaacgacccccgcccattgacgtcaataatgacgtatgttcccatagtaacgccaatagggactttccattgacgtcaatgggtggagtatttacggtaaactgcccacttggcagtacatcaagtgtatcatatgccaagtacgccccctattgacgtcaatgacggtaaatggcccgcctggcattatgcccagtacatgaccttatgggactttcctacttggcagtacatctacgtattagtcatcgctattaccatggtcgaggtgagccccacgttctgcttcactctccccatctcccccccctccccacccccaattttgtatttatttattttttaattattttgtgcagcgatgggggcggggggggggggggggcgcgcgccaggcggggcggggcggggcgaggggcggggcggggcgaggcggagaggtgcggcggcagccaatcagagcggcgcgctccgaaagtttccttttatggcgaggcggcggcggcggcggccctataaaaagcgaagcgcgcggcgggcggggagtcgctgcgacgctgccttcgccccgtgccccgctccgccgccgcctcgcgccgcccgccccggctctgactgaccgcgttactcccacaggtgagcgggcgggacggcccttctcctccgggctgtaattagcgcttggtttaatgacggcttgtttcttttctgtggctgcgtgaaagccttgaggggctccgggagggccctttgtgcggggggagcggctcggggggtgcgtgcgtgtgtgtgtgcgtggggagcgccgcgtgcggctccgcgctgcccggcggctgtgagcgctgcgggcgcggcgcggggctttgtgcgctccgcagtgtgcgcgaggggagcgcggccgggggcggtgccccgcggtgcggggggggctgcgaggggaacaaaggctgcgtgcggggtgtgtgcgtgggggggtgagcagggggtgtgggcgcgtcggtcgggctgcaaccccccctgcacccccctccccgagttgctgagcacggcccggcttcgggtgcggggctccgtacggggcgtggcgcggggctcgccgtgccgggcggggggtggcggcaggtgggggtgccgggcggggcggggccgcctcgggccggggagggctcgggggaggggcgcggcggcccccggagcgccggcggctgtcgaggcgcggcgagccgcagccattgccttttatggtaatcgtgcgagagggcgcagggacttcctttgtcccaaatctgtgcggagccgaaatctgggaggcgccgccgcaccccctctagcgggcgcggggcgaagcggtgcggcgccggcaggaaggaaatgggcggggagggccttcgtgcgtcgccgcgccgccgtccccttctccctctccagcctcggggctgtccgcggggggacggctgccttcgggggggacggggcagggcggggttcggcttctggcgtgtgaccggcggctctagagcctctgctaaccatgttcatgccttcttctttttcctacagctcctgggcaacgtgctggttattgtgctgtctcatcattttggcaaagaattgatttgataccgcatttaaatataacttcgtataatgtatgctatacgaagttattcgcgatgaataaatgaaagcttgcaattaagggttccggatcctcggggacaccaaatatggcgatctcggccttttcgtttcttggagctgggacatgtttgccatcgatccatctaccaccagaacggccgttagatctgctgccaccgttgtttccaccgaagaaaccaccgttgccgtaaccaccacgacggttgttgctaaagaagctgccaccgccacggccaccgttgtagccgccgttgttgttattgtagttgctcatgttatttctggcacttcttggttttcctcttaagtgaggaggaacataaccattctcgttgttgtcgttgatgcttaaattttgcacttgttcgctcagttcagccataatatgaaatgcttttcttgttgttcttacggaataccacttgccacctatcaccacaactaactttttcccgttcctccatctcttttatattttttttctcgagggatctttgtgaaggaaccttacttctgtggtgtgacataattggacaaactacctacagagatttaaagctctaaggtaaatataaaatttttaagtgtataatgtgttaaactactgattctaattgtttgtgtattttagattccaacctatggaactgatgaatgggagcagtggtggaatgcctttaatgaggaaaacctgttttgctcagaagaaatgccatctagtgatgatgaggctactgctgactctcaacattctactcctccaaaaaagaagagaaaggtagaagaccccaaggactttccttcagaattgctaagttttttgagtcatgctgtgtttagtaatagaactcttgcttgctttgctatttacaccacaaaggaaaaagctgcactgctatacaagaaaattatggaaaaatattctgtaacctttataagtaggcataacagttataatcataacatactgttttttcttactccacacaggcatagagtgtctgctattaataactatgctcaaaaattgtgtacctttagctttttaatttgtaaaggggttaataaggaatatttgatgtatagtgccttgactagagatcataatcagccataccacatttgtagaggttttacttgctttaaaaaacctcccacacctccccctgaacctgaaacataaaatgaatgcaattgttgttgttaacttgtttattgcagcttataatggttacaaataaagcaatagcatcacaaatttcacaaataaagcatttttttcactgcattctagttgtggtttgtccaaactcatcaatgtatcttatcatgtctggatctgacatggtaagtaagcttgggctgcaggtcgagggacctacccatcaagctgatccggaacccttaatataacttcgtataatgtatgctatacgaagttattaggtccctcgacctgcagcccaagctagatcgaattcaggcgcgccccgccaccatgacccccatcctcacggtcctgatctgtctcgggctgagcctggaccccaggacccacgtgcaggcagggcccctccccaagcccaccctctgggctgagccaggctctgtgatcacccaagggagtcctgtgaccctcaggtgtcaggggagcctggagacgcaggagtaccatctatatagagaaaagaaaacagcactctggattacacggatcccacaggagcttgtgaagaagggccagttccccatcctatccatcacctgggaacatgcagggcggtattgctgtatctatggcagccacactgcaggcctctcagagagcagtgaccccctggagctggtggtgacaggagcctacagcaaacccaccctctcagctctgcccagccctgtggtgacctcaggagggaatgtgaccatccagtgtgactcacaggtggcatttgatggcttcattctgtgtaaggaaggagaagatgaacacccacaatgcctgaactcccattcccatgcccgtgggtcatcccgggccatcttctccgtgggccccgtgagcccaagtcgcaggtggtcgtacaggtgctatggttatgactcgcgcgctccctatgtgtggtctctacccagtgatctcctggggctcctggtcccaggtgtttctaagaagccatcactctcagtgcagccgggtcctgtcgtggcccctggggagaagctgaccttccagtgtggctctgatgccggctacgacagatttgttctgtacaaggagtggggacgtgacttcctccagcgccctggccggcagccccaggctgggctctcccaggccaacttcaccctgggccctgtgagccgctcctacgggggccagtacacatgctccggtgcatacaacctctcctccgagtggtcggcccccagcgaccccctggacatcctgatcacaggacagatccgtgccagacccttcctctccgtgcggccgggccccacagtggcctcaggagagaacgtgaccctgctgtgtcagtcacagggagggatgcacactttccttttgaccaaggagggggcagctgattccccgctgcgtctaaaatcaaagcgccaatctcataagtaccaggctgaattccccatgagtcctgtgacctcggcccacgcggggacctacaggtgctacggctcactcagctccaacccctacctgctgactcaccccagtgaccccctggagctcgtggtctcaggagcagctgagaccctcagcccaccacaaaacaagtccgactccaaggctggtgagtgagatatcaagcttatcgataatcaacctctggattacaaaatttgtgaaagattgactggtattcttaactatgttgctccttttacgctatgtggatacgctgctttaatgcctttgtatcatgctattgcttcccgtatggctttcattttctcctccttgtataaatcctggttgctgtctctttatgaggagttgtggcccgttgtcaggcaacgtggcgtggtgtgcactgtgtttgctgacgcaacccccactggttggggcattgccaccacctgtcagctcctttccgggactttcgctttccccctccctattgccacggcggaactcatcgccgcctgccttgcccgctgctggacaggggctcggctgttgggcactgacaattccgtggtgttgtcggggaaatcatcgtcctttccttggctgctcgcctgtgttgccacctggattctgcgcgggacgtccttctgctacgtcccttcggccctcaatccagcggaccttccttcccgcggcctgctgccggctctgcggcctcttccgcgtcttcgccttcgccctcagacgagtcggatctccctttgggccgcctccccgcatcgataccgtcgacctcgacctcgactgtgccttctagttgccagccatctgttgtttgcccctcccccgtgccttccttgaccctggaaggtgccactcccactgtcctttcctaataaaatgaggaaattgcatcgcattgtctgagtaggtgtcattctattctggggggtggggtggggcaggacagcaagggggaggattgggaagacaatggcaggcatgctggggaactagtacgcgtcggaggggggaggggagtgttgcaatacctttctgggagttctctgctgcctcctggcttctgaggaccgccctgggcctgggagaatcccttccccctcttccctcgtgatctgcaactccagtctttctagaagatgggcgggagtcttctgggcaggcttaaaggctaacctggtgtgtgggcgttgtcctgcaggggaattgaacaggtgtaaaattggagggacaagacttcccacagattttcggttttgtcgggaagttttttaataggggcaaataaggaaaatgggaggataggtagtcatctggggttttatgcagcaaaactacaggttattattgcttgtgatccgcctcggagtattttccatcgaggtagattaaagacatgctcacccgagttttatactctcctgcttgagatccttactacagtatgaaattacagtgtcgcgagttagactatgtaagcagaattttaatcatttttaaagagcccagtacttcatatccatttctcccgctccttctgcagccttatcaaaaggtattttagaacactcattttagccccattttcatttattatactggcttatccaacccctagacagagcattggcattttccctttcctgatcttagaagtctgatgactcatgaaaccagacagattagttacatacaccacaaatcgaggctgtagctggggcctcaacactgcagttcttttataactccttagtacactttttgttgatcctttgccttgatccttaattttcagtgtctatcacctctcccgtcaggtggtgttccacatttgggcctattctcagtccagggagttttacaacaatagatgtattgagaatccaacctaaagcttaactttccactcccatgaatgcctctctcctttttctccatttataaactgagctattaaccattaatggtttccaggtggatgtctcctcccccaatattacctgatgtatcttacatattgccaggctgatattttaagacattaaaaggtatatttcattattgagccacatggtattgattactgcttactaaaattttgtcattgtacacatctgtaaaaggtggttccttttggaatgcaaagttcaggtgtttgttgtctttcctgacctaaggtcttgtgagcttgtattttttctatttaagcagtgctttctcttggactggcttgactcatggcattctacacgttattgctggtctaaatgtgattttgccaagcttcttcaggacctataattttgcttgacttgtagccaaacacaagtaaaatgattaagcaacaaatgtatttgtgaagcttggtttttaggttgttgtgttgtgtgtgcttgtgctctataataatactatccaggggctggagaggtggctcggagttcaagagcacagactgctcttccagaagtcctgagttcaattcccagcaaccacatggtggctcacaaccatctgtaatgggatctgatgccctcttctggtgtgtctgaagaccacaagtgtattcacattaaataaataaatcctccttcttcttcttttttttttttttaaagagaatactgtctccagtagaatttactgaagtaatgaaatactttgtgtttgttccaatatggtagccaataatcaaattactctttaagcactggaaatgttaccaaggaactaatttttatttgaagtgtaactgtggacagaggagccataactgcagactggatcctcgaagcttccgacgctttaggagtgaataccagcttggcgtaatcatggtcatagctgtttcctgtgtgaaattgttatccgctcacaattccacacaacatacgagccggaagcataaagtgtaaagcctggggtgcctaatgagtgagctaactcacattaattgcgttgcgctcactgcccgctttccagtcgggaaacctgtcgtgccagctgcattaatgaatcggccaacgcgcggggagaggcggtttgcgtattgggcgctcttccgcttcctcgctcactgactcgctgcgctcggtcgttcggctgcggcgagcggtatcagctcactcaaaggcggtaatacggttatccacagaatcaggggataacgcaggaaagaacatgtgagcaaaaggccagcaaaaggccaggaaccgtaaaaaggccgcgttgctggcgtttttccataggctccgcccccctgacgagcatcacaaaaatcgacgctcaagtcagaggtggcgaaacccgacaggactataaagataccaggcgtttccccctggaagctccctcgtgcgctctcctgttccgaccctgccgcttaccggatacctgtccgcctttctcccttcgggaagcgtggcgctttctcatagctcacgctgtaggtatctcagttcggtgtaggtcgttcgctccaagctgggctgtgtgcacgaaccccccgttcagcccgaccgctgcgccttatccggtaactatcgtcttgagtccaacccggtaagacacgacttatcgccactggcagcagccactggtaacaggattagcagagcgaggtatgtaggcggtgctacagagttcttgaagtggtggcctaactacggctacactagaaggacagtatttggtatctgcgctctgctgaagccagttaccttcggaaaaagagttggtagctcttgatccggcaaacaaaccaccgctggtagcggtggtttttttgtttgcaagcagcagattacgcgcagaaaaaaaggatctcaagaagatcctttgatcttttctacggggtctgacgctcagtggaacgaaaactcacgttaagggattttggtcatgagattatcaaaaaggatcttcacctagatccttttaaattaaaaatgaagttttaaatcaatctaaagtatatatgagtaaacttggtctgacagttaccaatgcttaatcagtgaggcacctatctcagcgatctgtctatttcgttcatccatagttgcctgactccccgtcgtgtagataactacgatacgggagggcttaccatctggccccagtgctgcaatgataccgcgagacccacgctcaccggctccagatttatcagcaataaaccagccagccggaagggccgagcgcagaagtggtcctgcaactttatccgcctccatccagtctattaattgttgccgggaagctagagtaagtagttcgccagttaatagtttgcgcaacgttgttgccattgctacaggcatcgtggtgtcacgctcgtcgtttggtatggcttcattcagctccggttcccaacgatcaaggcgagttacatgatcccccatgttgtgcaaaaaagcggttagctccttcggtcctccgatcgttgtcagaagtaagttggccgcagtgttatcactcatggttatggcagcactgcataattctcttactgtcatgccatccgtaagatgcttttctgtgactggtgagtactcaaccaagtcattctgagaatagtgtatgcggcgaccgagttgctcttgcccggcgtcaatacgggataataccgcgccacatagcagaactttaaaagtgctcatcattggaaaacgttcttcggggcgaaaactctcaaggatcttaccgctgttgagatccagttcgatgtaacccactcgtgcacccaactgatcttcagcatcttttactttcaccagcgtttctgggtgagcaaaaacaggaaggcaaaatgccgcaaaaaagggaataagggcgacacggaaatgttgaatactcatactcttcctttttcaatattattgaagcatttatcagggttattgtctcatgagcggatacatatttgaatgtatttagaaaaataaacaaataggggttccgcgcacatttccccgaaaagtgccacctgacgtctaagaaaccattattatcatgacattaacctataaaaataggcgtatcacgaggccctttcgtc。
在本发明中，所述打靶载体的构建方法优选包括：将基因连入EGE载体中，得到打靶载体；所述基因具有SEQ ID No.2所示的核苷酸序列，所述基因委托苏州金唯智生物科技有限公司合成；具体如下所示：
ggaagagtcctgacccagggaagacattaaaaaggtagtggggtcgactagatgaaggagagcctttctctctgggcaagagcggtgcaatggtgtgtaaaggtagctgagaagacgaaaagggcaagcatcttcctgctaccaggctggggaggcccaggcccacgaccccgaggagagggaacgcagggagactgaggtgacccttctttcccccggggcccggtcgtgtggttcggtgtctcttttctgttggacccttaccttgacccaggcgctgccggggcctgggcccgggctgcggcgcacggcactcccgggaggcagcgagactcgagttaggcccaacgcggcgccacggcgtttcctggccgggaatggcccgtacccgtgaggtgggggtggggggcagaaaaggcggagcgagcccgaggcggggagggggagggccaggggcggagggggccggcactactgtgttggcggactggcgggactagggctgcgtgagtctctgagcgcaggcgggcggcggccgcccctcccccggcggcggcagcggcggcagcggcggcagctcactcagcccgctgcccgagcggaaacgccactgaccgcacggggattcccagtgccggcgccaggggcacgcgggacacgccccctcccgccgcgccattggcctctccgcccaccgccccacacttattggccggtgcgccgccaatcagcggaggctgccggggccgcctaaagaagaggctgtgctttggggctccggctcctcagagagcctcggctaggtaggggatcgggactctggcgggagggcggcttggtgcgtttgcggggatgggcggccgcggcaggccctccgagcgtggtggagccgttctgtgagacagccgggtacgagtcgtgacgctggaaggggcaagcgggtggtgggcaggaatgcggtccgccctgcagcaaccggagggggagggagaagggagcggaaaagtctccaccggacgcggccatggctcgggggggggggggcagcggaggagcgcttccggccgacgtctcgtcgctgattggcttcttttcctcccgccgtgtgtgaaaacacaaatggcgtgttttggttggcgtaaggcgcctgtcagttaacggcagccggagtgcgcagccgccggcagcctcgctctgcccactgggtggggcgggaggtaggtggggtgaggcgagctggacgtgcgggcgcggtcggcctctggcggggcgggggaggggagggagggtcagcgaaagtagctcgcgcgcgagcggccgcccaccctccccttcctctgggggagtcgttttacccgccgccggccgggcctcgtcgtctgattggctctcggggcccagaaaactggcccttgccattggctcgtgttcgtgcaagttgagtccatccgccggccagcgggggcggcgaggaggcgctcccaggttccggccctcccctcggccccgcgccgcagagtctggccgcgcgcccctgcgcaacgtggcaggaagcgcgcgctgggggcggggacgggcagtagggctgagcggctgcggggcgggtgcaagcacgtttccgacttgagttgcctcaagaggggcgtgctgagccagacctccatcgcgcactccggggagtggagggaaggagcgagggctcagttgggctgttttggaggcaggaagcacttgctctcccaaagtcgctctgagttgttatcagtaagggagctgcagtggagtaggcggggagaaggccgcacccttctc。
在本发明中，所述连入的反应条件优选包括：将100ng EGE载体、500ng基因、2μL10x DNA Ligase Buffer、1U DNA Ligase和18μL ddH2O混合后在22℃下孵育10min。
本发明还提供了LILRA3基因敲入小鼠的构建方法，包括：
将上述技术方案所述的打靶载体导入小鼠受精卵中，得到F0小鼠；
将得到的F0小鼠与野生型小鼠杂交，得到携带LILRA3基因的杂合型小鼠；
将携带LILRA3基因的杂合型小鼠进行杂交，得到的纯合型小鼠为LILRA3基因敲入小鼠。
在本发明中，所述打靶载体导入小鼠受精卵的量优选为5～10pL。在本发明中，所述打靶载体导入的方法优选采用显微注射受精卵的方法进行，本发明对所述显微注射受精卵的方法没有特殊限定，采用本领域常规使用的方法即可。
本发明优选利用Rosa-GT-F、Rosa-GT-R和Rosa26-Test(L)-R3引物判定小鼠的基因型；所述Rosa-GT-F引物的核苷酸序列如SEQ ID No.3所示，具体如下所示：
agtcgctctgagttgttatcag；
所述Rosa-GT-R引物的核苷酸序列如SEQ ID No.4所示，具体如下所示：
tgagcatgtctttaatctacctcgatg；
所述Rosa26-Test(L)-R3的核苷酸序列如SEQ ID No.5所示，具体如下所示：
gtcaatggaaagtccctattggcgt。
在本发明中，所述引物Rosa26-Test(L)-R3设计在外源基因序列中，引物Rosa26-GT-F设计在野生型基因序列中，使用Rosa-GT-F和Rosa26-Test(L)-R3这对引物进行PCR时，不能扩增出野生型等位基因的产物，只能扩增出突变型等位基因的产物。
引物对Rosa-GT-F和Rosa-GT-R设计在野生基因序列中，野生型等位基因的PCR产物比较短，容易扩增出来，突变型等位基因的PCR产物比较长，无法扩增出来。所以Rosa-GT-F和Rosa-GT-R这对引物用于鉴定野生型等位基因的存在，所以根据Rosa-GT-F、Rosa-GT-R和Rosa26-Test(L)-R3三条引物的PCR结果判断小鼠的具体基因型(纯合/杂合/野生)。
下面结合实施例对本发明提供的技术方案进行详细的说明，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
打靶载体的构建：
将基因(SEQ IDNo.2)连入EGE载体中，得到打靶载体，打靶载体的核苷酸序列如SEQ ID No.1所示；
连入的反应条件包括：将100ng EGE载体、500ng基因、2μL 10x DNA LigaseBuffer、1U DNA Ligase和18μL ddH2O混合后在22℃下孵育10min。
打靶载体的验证：
EcoRV和AscI为打靶载体上的唯一酶切位点，分别位于目的基因两侧。通过使用EcoRV和AscI两个限制性内切酶验证打靶载体构建完成，预期酶切产物为两条，1336bp(目的基因片段)和10424bp(载体剩余片段)，结果见图1。
反应条件如表1：
表1反应条件
Figure BDA0002155587150000101
Figure BDA0002155587150000111
实施例2
LILRA3基因敲入小鼠的构建：
将实施例1构建得到的打靶载体采用显著注射受精卵的方法注射到小鼠受精卵中，得到F0小鼠；由于胚胎早期卵裂速度很快，因此得到的F0小鼠为嵌合体；
将得到的F0小鼠与野生型小鼠杂交，得到携带LILRA3基因的杂合型小鼠，能够稳定遗传；
将携带LILRA3基因的杂合型小鼠进行杂交，得到的纯合型小鼠为LILRA3基因敲入小鼠。
引物Rosa26-Test(L)-R3设计在外源基因序列中，引物Rosa26-GT-F设计在野生型基因序列中，使用Rosa-GT-F和Rosa26-Test(L)-R3这对引物进行PCR时，不能扩增出野生型等位基因的产物，只能扩增出突变型等位基因的产物。
引物对Rosa-GT-F和Rosa-GT-R设计在野生基因序列中，野生型等位基因的PCR产物比较短，容易扩增出来，突变型等位基因的PCR产物比较长，无法扩增出来。所以Rosa-GT-F和Rosa-GT-R这对引物用于鉴定野生型等位基因的存在，所以根据Rosa-GT-F、Rosa-GT-R和Rosa26-Test(L)-R3三条引物的PCR结果判断小鼠的具体基因型(纯合/杂合/野生)。鉴定结果见图2。
ELISA鉴定敲入基因在血清中表达：
实验步骤：
在平底96孔板Nunc(Thermo Fisher Scientific，MA，US)每孔加入100μL的0.5μg/ml抗LILRA3单克隆抗体(M01；Novus，CO，US)，放置过夜。
然后将板用含0.05％Tween20的PBS洗涤3次。
每孔加入2％BSA的PBS在室温下封闭2小时。
洗涤3次后，每孔加入100μL的标准品或测试血清(在PBS中1：100稀释)在室温下温育1.5小时。
洗涤3次后，每孔加入100μL的0.5μg/ml抗LILRA3多克隆抗体(AF2574；Novus，CO，US)在室温下孵育1.5小时。
洗涤3次后，每孔加入100μL二抗(1：5000稀释；HAF109，R&D System，MN，US)在室温下孵育1小时。
洗涤3次后，每孔加入100μL的TMB底物溶液(Thermo Fisher Scientific，MA，US)并在黑暗中温育30分钟，随后每孔加入50μL的1M H2SO4终止反应。
使用酶标仪(BioTek，VT，US)在450/540nm波长下测量光密度然后计算浓度。结果见图3。从图3中可以得出转基因小鼠血清中可检测到LILRA3蛋白，证明LILRA3基因在小鼠体内表达。
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> 北京大学人民医院（北京大学第二临床医学院）
<120> LILRA3基因敲入小鼠的打靶载体和LILRA3基因敲入小鼠的构建方法
<160> 5
<170> SIPOSequenceListing 1.0
<210> 1
<211> 11766
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 1
tcgcgcgttt cggtgatgac ggtgaaaacc tctgacacat gcagctcccg gagacggtca 60
cagcttgtct gtaagcggat gccgggagca gacaagcccg tcagggcgcg tcagcgggtg 120
ttggcgggtg tcggggctgg cttaactatg cggcatcaga gcagattgta ctgagagtgc 180
accatatgcg gtgtgaaata ccgcacagat gcgtaaggag aaaataccgc atcaggcgcc 240
attcgccatt caggctgcgc aactgttggg aagggcgatc ggtgcgggcc tcttcgctat 300
tacgccagct ggcgaaaggg ggatgtgctg caaggcgatt aagttgggta acgccagggt 360
tttcccagtc acgacgttgt aaaacgacgg ccagtgaatt gccgacgctt taggagtgaa 420
taccgaattc ggaagagtcc tgacccaggg aagacattaa aaaggtagtg gggtcgacta 480
gatgaaggag agcctttctc tctgggcaag agcggtgcaa tggtgtgtaa aggtagctga 540
gaagacgaaa agggcaagca tcttcctgct accaggctgg ggaggcccag gcccacgacc 600
ccgaggagag ggaacgcagg gagactgagg tgacccttct ttcccccggg gcccggtcgt 660
gtggttcggt gtctcttttc tgttggaccc ttaccttgac ccaggcgctg ccggggcctg 720
ggcccgggct gcggcgcacg gcactcccgg gaggcagcga gactcgagtt aggcccaacg 780
cggcgccacg gcgtttcctg gccgggaatg gcccgtaccc gtgaggtggg ggtggggggc 840
agaaaaggcg gagcgagccc gaggcgggga gggggagggc caggggcgga gggggccggc 900
actactgtgt tggcggactg gcgggactag ggctgcgtga gtctctgagc gcaggcgggc 960
ggcggccgcc cctcccccgg cggcggcagc ggcggcagcg gcggcagctc actcagcccg 1020
ctgcccgagc ggaaacgcca ctgaccgcac ggggattccc agtgccggcg ccaggggcac 1080
gcgggacacg ccccctcccg ccgcgccatt ggcctctccg cccaccgccc cacacttatt 1140
ggccggtgcg ccgccaatca gcggaggctg ccggggccgc ctaaagaaga ggctgtgctt 1200
tggggctccg gctcctcaga gagcctcggc taggtagggg atcgggactc tggcgggagg 1260
gcggcttggt gcgtttgcgg ggatgggcgg ccgcggcagg ccctccgagc gtggtggagc 1320
cgttctgtga gacagccggg tacgagtcgt gacgctggaa ggggcaagcg ggtggtgggc 1380
aggaatgcgg tccgccctgc agcaaccgga gggggaggga gaagggagcg gaaaagtctc 1440
caccggacgc ggccatggct cggggggggg ggggcagcgg aggagcgctt ccggccgacg 1500
tctcgtcgct gattggcttc ttttcctccc gccgtgtgtg aaaacacaaa tggcgtgttt 1560
tggttggcgt aaggcgcctg tcagttaacg gcagccggag tgcgcagccg ccggcagcct 1620
cgctctgccc actgggtggg gcgggaggta ggtggggtga ggcgagctgg acgtgcgggc 1680
gcggtcggcc tctggcgggg cgggggaggg gagggagggt cagcgaaagt agctcgcgcg 1740
cgagcggccg cccaccctcc ccttcctctg ggggagtcgt tttacccgcc gccggccggg 1800
cctcgtcgtc tgattggctc tcggggccca gaaaactggc ccttgccatt ggctcgtgtt 1860
cgtgcaagtt gagtccatcc gccggccagc gggggcggcg aggaggcgct cccaggttcc 1920
ggccctcccc tcggccccgc gccgcagagt ctggccgcgc gcccctgcgc aacgtggcag 1980
gaagcgcgcg ctgggggcgg ggacgggcag tagggctgag cggctgcggg gcgggtgcaa 2040
gcacgtttcc gacttgagtt gcctcaagag gggcgtgctg agccagacct ccatcgcgca 2100
ctccggggag tggagggaag gagcgagggc tcagttgggc tgttttggag gcaggaagca 2160
cttgctctcc caaagtcgct ctgagttgtt atcagtaagg gagctgcagt ggagtaggcg 2220
gggagaaggc cgcacccttc tcttaattaa ccgtttaaac aattctgcag gaatctagtt 2280
attaatagta atcaattacg gggtcattag ttcatagccc atatatggag ttccgcgtta 2340
cataacttac ggtaaatggc ccgcctggct gaccgcccaa cgacccccgc ccattgacgt 2400
caataatgac gtatgttccc atagtaacgc caatagggac tttccattga cgtcaatggg 2460
tggagtattt acggtaaact gcccacttgg cagtacatca agtgtatcat atgccaagta 2520
cgccccctat tgacgtcaat gacggtaaat ggcccgcctg gcattatgcc cagtacatga 2580
ccttatggga ctttcctact tggcagtaca tctacgtatt agtcatcgct attaccatgg 2640
tcgaggtgag ccccacgttc tgcttcactc tccccatctc ccccccctcc ccacccccaa 2700
ttttgtattt atttattttt taattatttt gtgcagcgat gggggcgggg gggggggggg 2760
ggcgcgcgcc aggcggggcg gggcggggcg aggggcgggg cggggcgagg cggagaggtg 2820
cggcggcagc caatcagagc ggcgcgctcc gaaagtttcc ttttatggcg aggcggcggc 2880
ggcggcggcc ctataaaaag cgaagcgcgc ggcgggcggg gagtcgctgc gacgctgcct 2940
tcgccccgtg ccccgctccg ccgccgcctc gcgccgcccg ccccggctct gactgaccgc 3000
gttactccca caggtgagcg ggcgggacgg cccttctcct ccgggctgta attagcgctt 3060
ggtttaatga cggcttgttt cttttctgtg gctgcgtgaa agccttgagg ggctccggga 3120
gggccctttg tgcgggggga gcggctcggg gggtgcgtgc gtgtgtgtgt gcgtggggag 3180
cgccgcgtgc ggctccgcgc tgcccggcgg ctgtgagcgc tgcgggcgcg gcgcggggct 3240
ttgtgcgctc cgcagtgtgc gcgaggggag cgcggccggg ggcggtgccc cgcggtgcgg 3300
ggggggctgc gaggggaaca aaggctgcgt gcggggtgtg tgcgtggggg ggtgagcagg 3360
gggtgtgggc gcgtcggtcg ggctgcaacc ccccctgcac ccccctcccc gagttgctga 3420
gcacggcccg gcttcgggtg cggggctccg tacggggcgt ggcgcggggc tcgccgtgcc 3480
gggcgggggg tggcggcagg tgggggtgcc gggcggggcg gggccgcctc gggccgggga 3540
gggctcgggg gaggggcgcg gcggcccccg gagcgccggc ggctgtcgag gcgcggcgag 3600
ccgcagccat tgccttttat ggtaatcgtg cgagagggcg cagggacttc ctttgtccca 3660
aatctgtgcg gagccgaaat ctgggaggcg ccgccgcacc ccctctagcg ggcgcggggc 3720
gaagcggtgc ggcgccggca ggaaggaaat gggcggggag ggccttcgtg cgtcgccgcg 3780
ccgccgtccc cttctccctc tccagcctcg gggctgtccg cggggggacg gctgccttcg 3840
ggggggacgg ggcagggcgg ggttcggctt ctggcgtgtg accggcggct ctagagcctc 3900
tgctaaccat gttcatgcct tcttcttttt cctacagctc ctgggcaacg tgctggttat 3960
tgtgctgtct catcattttg gcaaagaatt gatttgatac cgcatttaaa tataacttcg 4020
tataatgtat gctatacgaa gttattcgcg atgaataaat gaaagcttgc aattaagggt 4080
tccggatcct cggggacacc aaatatggcg atctcggcct tttcgtttct tggagctggg 4140
acatgtttgc catcgatcca tctaccacca gaacggccgt tagatctgct gccaccgttg 4200
tttccaccga agaaaccacc gttgccgtaa ccaccacgac ggttgttgct aaagaagctg 4260
ccaccgccac ggccaccgtt gtagccgccg ttgttgttat tgtagttgct catgttattt 4320
ctggcacttc ttggttttcc tcttaagtga ggaggaacat aaccattctc gttgttgtcg 4380
ttgatgctta aattttgcac ttgttcgctc agttcagcca taatatgaaa tgcttttctt 4440
gttgttctta cggaatacca cttgccacct atcaccacaa ctaacttttt cccgttcctc 4500
catctctttt atattttttt tctcgaggga tctttgtgaa ggaaccttac ttctgtggtg 4560
tgacataatt ggacaaacta cctacagaga tttaaagctc taaggtaaat ataaaatttt 4620
taagtgtata atgtgttaaa ctactgattc taattgtttg tgtattttag attccaacct 4680
atggaactga tgaatgggag cagtggtgga atgcctttaa tgaggaaaac ctgttttgct 4740
cagaagaaat gccatctagt gatgatgagg ctactgctga ctctcaacat tctactcctc 4800
caaaaaagaa gagaaaggta gaagacccca aggactttcc ttcagaattg ctaagttttt 4860
tgagtcatgc tgtgtttagt aatagaactc ttgcttgctt tgctatttac accacaaagg 4920
aaaaagctgc actgctatac aagaaaatta tggaaaaata ttctgtaacc tttataagta 4980
ggcataacag ttataatcat aacatactgt tttttcttac tccacacagg catagagtgt 5040
ctgctattaa taactatgct caaaaattgt gtacctttag ctttttaatt tgtaaagggg 5100
ttaataagga atatttgatg tatagtgcct tgactagaga tcataatcag ccataccaca 5160
tttgtagagg ttttacttgc tttaaaaaac ctcccacacc tccccctgaa cctgaaacat 5220
aaaatgaatg caattgttgt tgttaacttg tttattgcag cttataatgg ttacaaataa 5280
agcaatagca tcacaaattt cacaaataaa gcattttttt cactgcattc tagttgtggt 5340
ttgtccaaac tcatcaatgt atcttatcat gtctggatct gacatggtaa gtaagcttgg 5400
gctgcaggtc gagggaccta cccatcaagc tgatccggaa cccttaatat aacttcgtat 5460
aatgtatgct atacgaagtt attaggtccc tcgacctgca gcccaagcta gatcgaattc 5520
aggcgcgccc cgccaccatg acccccatcc tcacggtcct gatctgtctc gggctgagcc 5580
tggaccccag gacccacgtg caggcagggc ccctccccaa gcccaccctc tgggctgagc 5640
caggctctgt gatcacccaa gggagtcctg tgaccctcag gtgtcagggg agcctggaga 5700
cgcaggagta ccatctatat agagaaaaga aaacagcact ctggattaca cggatcccac 5760
aggagcttgt gaagaagggc cagttcccca tcctatccat cacctgggaa catgcagggc 5820
ggtattgctg tatctatggc agccacactg caggcctctc agagagcagt gaccccctgg 5880
agctggtggt gacaggagcc tacagcaaac ccaccctctc agctctgccc agccctgtgg 5940
tgacctcagg agggaatgtg accatccagt gtgactcaca ggtggcattt gatggcttca 6000
ttctgtgtaa ggaaggagaa gatgaacacc cacaatgcct gaactcccat tcccatgccc 6060
gtgggtcatc ccgggccatc ttctccgtgg gccccgtgag cccaagtcgc aggtggtcgt 6120
acaggtgcta tggttatgac tcgcgcgctc cctatgtgtg gtctctaccc agtgatctcc 6180
tggggctcct ggtcccaggt gtttctaaga agccatcact ctcagtgcag ccgggtcctg 6240
tcgtggcccc tggggagaag ctgaccttcc agtgtggctc tgatgccggc tacgacagat 6300
ttgttctgta caaggagtgg ggacgtgact tcctccagcg ccctggccgg cagccccagg 6360
ctgggctctc ccaggccaac ttcaccctgg gccctgtgag ccgctcctac gggggccagt 6420
acacatgctc cggtgcatac aacctctcct ccgagtggtc ggcccccagc gaccccctgg 6480
acatcctgat cacaggacag atccgtgcca gacccttcct ctccgtgcgg ccgggcccca 6540
cagtggcctc aggagagaac gtgaccctgc tgtgtcagtc acagggaggg atgcacactt 6600
tccttttgac caaggagggg gcagctgatt ccccgctgcg tctaaaatca aagcgccaat 6660
ctcataagta ccaggctgaa ttccccatga gtcctgtgac ctcggcccac gcggggacct 6720
acaggtgcta cggctcactc agctccaacc cctacctgct gactcacccc agtgaccccc 6780
tggagctcgt ggtctcagga gcagctgaga ccctcagccc accacaaaac aagtccgact 6840
ccaaggctgg tgagtgagat atcaagctta tcgataatca acctctggat tacaaaattt 6900
gtgaaagatt gactggtatt cttaactatg ttgctccttt tacgctatgt ggatacgctg 6960
ctttaatgcc tttgtatcat gctattgctt cccgtatggc tttcattttc tcctccttgt 7020
ataaatcctg gttgctgtct ctttatgagg agttgtggcc cgttgtcagg caacgtggcg 7080
tggtgtgcac tgtgtttgct gacgcaaccc ccactggttg gggcattgcc accacctgtc 7140
agctcctttc cgggactttc gctttccccc tccctattgc cacggcggaa ctcatcgccg 7200
cctgccttgc ccgctgctgg acaggggctc ggctgttggg cactgacaat tccgtggtgt 7260
tgtcggggaa atcatcgtcc tttccttggc tgctcgcctg tgttgccacc tggattctgc 7320
gcgggacgtc cttctgctac gtcccttcgg ccctcaatcc agcggacctt ccttcccgcg 7380
gcctgctgcc ggctctgcgg cctcttccgc gtcttcgcct tcgccctcag acgagtcgga 7440
tctccctttg ggccgcctcc ccgcatcgat accgtcgacc tcgacctcga ctgtgccttc 7500
tagttgccag ccatctgttg tttgcccctc ccccgtgcct tccttgaccc tggaaggtgc 7560
cactcccact gtcctttcct aataaaatga ggaaattgca tcgcattgtc tgagtaggtg 7620
tcattctatt ctggggggtg gggtggggca ggacagcaag ggggaggatt gggaagacaa 7680
tggcaggcat gctggggaac tagtacgcgt cggagggggg aggggagtgt tgcaatacct 7740
ttctgggagt tctctgctgc ctcctggctt ctgaggaccg ccctgggcct gggagaatcc 7800
cttccccctc ttccctcgtg atctgcaact ccagtctttc tagaagatgg gcgggagtct 7860
tctgggcagg cttaaaggct aacctggtgt gtgggcgttg tcctgcaggg gaattgaaca 7920
ggtgtaaaat tggagggaca agacttccca cagattttcg gttttgtcgg gaagtttttt 7980
aataggggca aataaggaaa atgggaggat aggtagtcat ctggggtttt atgcagcaaa 8040
actacaggtt attattgctt gtgatccgcc tcggagtatt ttccatcgag gtagattaaa 8100
gacatgctca cccgagtttt atactctcct gcttgagatc cttactacag tatgaaatta 8160
cagtgtcgcg agttagacta tgtaagcaga attttaatca tttttaaaga gcccagtact 8220
tcatatccat ttctcccgct ccttctgcag ccttatcaaa aggtatttta gaacactcat 8280
tttagcccca ttttcattta ttatactggc ttatccaacc cctagacaga gcattggcat 8340
tttccctttc ctgatcttag aagtctgatg actcatgaaa ccagacagat tagttacata 8400
caccacaaat cgaggctgta gctggggcct caacactgca gttcttttat aactccttag 8460
tacacttttt gttgatcctt tgccttgatc cttaattttc agtgtctatc acctctcccg 8520
tcaggtggtg ttccacattt gggcctattc tcagtccagg gagttttaca acaatagatg 8580
tattgagaat ccaacctaaa gcttaacttt ccactcccat gaatgcctct ctcctttttc 8640
tccatttata aactgagcta ttaaccatta atggtttcca ggtggatgtc tcctccccca 8700
atattacctg atgtatctta catattgcca ggctgatatt ttaagacatt aaaaggtata 8760
tttcattatt gagccacatg gtattgatta ctgcttacta aaattttgtc attgtacaca 8820
tctgtaaaag gtggttcctt ttggaatgca aagttcaggt gtttgttgtc tttcctgacc 8880
taaggtcttg tgagcttgta ttttttctat ttaagcagtg ctttctcttg gactggcttg 8940
actcatggca ttctacacgt tattgctggt ctaaatgtga ttttgccaag cttcttcagg 9000
acctataatt ttgcttgact tgtagccaaa cacaagtaaa atgattaagc aacaaatgta 9060
tttgtgaagc ttggttttta ggttgttgtg ttgtgtgtgc ttgtgctcta taataatact 9120
atccaggggc tggagaggtg gctcggagtt caagagcaca gactgctctt ccagaagtcc 9180
tgagttcaat tcccagcaac cacatggtgg ctcacaacca tctgtaatgg gatctgatgc 9240
cctcttctgg tgtgtctgaa gaccacaagt gtattcacat taaataaata aatcctcctt 9300
cttcttcttt tttttttttt taaagagaat actgtctcca gtagaattta ctgaagtaat 9360
gaaatacttt gtgtttgttc caatatggta gccaataatc aaattactct ttaagcactg 9420
gaaatgttac caaggaacta atttttattt gaagtgtaac tgtggacaga ggagccataa 9480
ctgcagactg gatcctcgaa gcttccgacg ctttaggagt gaataccagc ttggcgtaat 9540
catggtcata gctgtttcct gtgtgaaatt gttatccgct cacaattcca cacaacatac 9600
gagccggaag cataaagtgt aaagcctggg gtgcctaatg agtgagctaa ctcacattaa 9660
ttgcgttgcg ctcactgccc gctttccagt cgggaaacct gtcgtgccag ctgcattaat 9720
gaatcggcca acgcgcgggg agaggcggtt tgcgtattgg gcgctcttcc gcttcctcgc 9780
tcactgactc gctgcgctcg gtcgttcggc tgcggcgagc ggtatcagct cactcaaagg 9840
cggtaatacg gttatccaca gaatcagggg ataacgcagg aaagaacatg tgagcaaaag 9900
gccagcaaaa ggccaggaac cgtaaaaagg ccgcgttgct ggcgtttttc cataggctcc 9960
gcccccctga cgagcatcac aaaaatcgac gctcaagtca gaggtggcga aacccgacag 10020
gactataaag ataccaggcg tttccccctg gaagctccct cgtgcgctct cctgttccga 10080
ccctgccgct taccggatac ctgtccgcct ttctcccttc gggaagcgtg gcgctttctc 10140
atagctcacg ctgtaggtat ctcagttcgg tgtaggtcgt tcgctccaag ctgggctgtg 10200
tgcacgaacc ccccgttcag cccgaccgct gcgccttatc cggtaactat cgtcttgagt 10260
ccaacccggt aagacacgac ttatcgccac tggcagcagc cactggtaac aggattagca 10320
gagcgaggta tgtaggcggt gctacagagt tcttgaagtg gtggcctaac tacggctaca 10380
ctagaaggac agtatttggt atctgcgctc tgctgaagcc agttaccttc ggaaaaagag 10440
ttggtagctc ttgatccggc aaacaaacca ccgctggtag cggtggtttt tttgtttgca 10500
agcagcagat tacgcgcaga aaaaaaggat ctcaagaaga tcctttgatc ttttctacgg 10560
ggtctgacgc tcagtggaac gaaaactcac gttaagggat tttggtcatg agattatcaa 10620
aaaggatctt cacctagatc cttttaaatt aaaaatgaag ttttaaatca atctaaagta 10680
tatatgagta aacttggtct gacagttacc aatgcttaat cagtgaggca cctatctcag 10740
cgatctgtct atttcgttca tccatagttg cctgactccc cgtcgtgtag ataactacga 10800
tacgggaggg cttaccatct ggccccagtg ctgcaatgat accgcgagac ccacgctcac 10860
cggctccaga tttatcagca ataaaccagc cagccggaag ggccgagcgc agaagtggtc 10920
ctgcaacttt atccgcctcc atccagtcta ttaattgttg ccgggaagct agagtaagta 10980
gttcgccagt taatagtttg cgcaacgttg ttgccattgc tacaggcatc gtggtgtcac 11040
gctcgtcgtt tggtatggct tcattcagct ccggttccca acgatcaagg cgagttacat 11100
gatcccccat gttgtgcaaa aaagcggtta gctccttcgg tcctccgatc gttgtcagaa 11160
gtaagttggc cgcagtgtta tcactcatgg ttatggcagc actgcataat tctcttactg 11220
tcatgccatc cgtaagatgc ttttctgtga ctggtgagta ctcaaccaag tcattctgag 11280
aatagtgtat gcggcgaccg agttgctctt gcccggcgtc aatacgggat aataccgcgc 11340
cacatagcag aactttaaaa gtgctcatca ttggaaaacg ttcttcgggg cgaaaactct 11400
caaggatctt accgctgttg agatccagtt cgatgtaacc cactcgtgca cccaactgat 11460
cttcagcatc ttttactttc accagcgttt ctgggtgagc aaaaacagga aggcaaaatg 11520
ccgcaaaaaa gggaataagg gcgacacgga aatgttgaat actcatactc ttcctttttc 11580
aatattattg aagcatttat cagggttatt gtctcatgag cggatacata tttgaatgta 11640
tttagaaaaa taaacaaata ggggttccgc gcacatttcc ccgaaaagtg ccacctgacg 11700
tctaagaaac cattattatc atgacattaa cctataaaaa taggcgtatc acgaggccct 11760
ttcgtc 11766
<210> 2
<211> 1812
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 2
ggaagagtcc tgacccaggg aagacattaa aaaggtagtg gggtcgacta gatgaaggag 60
agcctttctc tctgggcaag agcggtgcaa tggtgtgtaa aggtagctga gaagacgaaa 120
agggcaagca tcttcctgct accaggctgg ggaggcccag gcccacgacc ccgaggagag 180
ggaacgcagg gagactgagg tgacccttct ttcccccggg gcccggtcgt gtggttcggt 240
gtctcttttc tgttggaccc ttaccttgac ccaggcgctg ccggggcctg ggcccgggct 300
gcggcgcacg gcactcccgg gaggcagcga gactcgagtt aggcccaacg cggcgccacg 360
gcgtttcctg gccgggaatg gcccgtaccc gtgaggtggg ggtggggggc agaaaaggcg 420
gagcgagccc gaggcgggga gggggagggc caggggcgga gggggccggc actactgtgt 480
tggcggactg gcgggactag ggctgcgtga gtctctgagc gcaggcgggc ggcggccgcc 540
cctcccccgg cggcggcagc ggcggcagcg gcggcagctc actcagcccg ctgcccgagc 600
ggaaacgcca ctgaccgcac ggggattccc agtgccggcg ccaggggcac gcgggacacg 660
ccccctcccg ccgcgccatt ggcctctccg cccaccgccc cacacttatt ggccggtgcg 720
ccgccaatca gcggaggctg ccggggccgc ctaaagaaga ggctgtgctt tggggctccg 780
gctcctcaga gagcctcggc taggtagggg atcgggactc tggcgggagg gcggcttggt 840
gcgtttgcgg ggatgggcgg ccgcggcagg ccctccgagc gtggtggagc cgttctgtga 900
gacagccggg tacgagtcgt gacgctggaa ggggcaagcg ggtggtgggc aggaatgcgg 960
tccgccctgc agcaaccgga gggggaggga gaagggagcg gaaaagtctc caccggacgc 1020
ggccatggct cggggggggg ggggcagcgg aggagcgctt ccggccgacg tctcgtcgct 1080
gattggcttc ttttcctccc gccgtgtgtg aaaacacaaa tggcgtgttt tggttggcgt 1140
aaggcgcctg tcagttaacg gcagccggag tgcgcagccg ccggcagcct cgctctgccc 1200
actgggtggg gcgggaggta ggtggggtga ggcgagctgg acgtgcgggc gcggtcggcc 1260
tctggcgggg cgggggaggg gagggagggt cagcgaaagt agctcgcgcg cgagcggccg 1320
cccaccctcc ccttcctctg ggggagtcgt tttacccgcc gccggccggg cctcgtcgtc 1380
tgattggctc tcggggccca gaaaactggc ccttgccatt ggctcgtgtt cgtgcaagtt 1440
gagtccatcc gccggccagc gggggcggcg aggaggcgct cccaggttcc ggccctcccc 1500
tcggccccgc gccgcagagt ctggccgcgc gcccctgcgc aacgtggcag gaagcgcgcg 1560
ctgggggcgg ggacgggcag tagggctgag cggctgcggg gcgggtgcaa gcacgtttcc 1620
gacttgagtt gcctcaagag gggcgtgctg agccagacct ccatcgcgca ctccggggag 1680
tggagggaag gagcgagggc tcagttgggc tgttttggag gcaggaagca cttgctctcc 1740
caaagtcgct ctgagttgtt atcagtaagg gagctgcagt ggagtaggcg gggagaaggc 1800
cgcacccttc tc 1812
<210> 3
<211> 22
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 3
agtcgctctg agttgttatc ag 22
<210> 4
<211> 27
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 4
tgagcatgtc tttaatctac ctcgatg 27
<210> 5
<211> 25
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 5
gtcaatggaa agtccctatt ggcgt 25

Claims (4)

1.LILRA3基因敲入小鼠的打靶载体，其特征在于，所述打靶载体的核苷酸序列如SEQID No.1所示。
2.LILRA3基因敲入小鼠的构建方法，其特征在于，包括：
将权利要求1所述的打靶载体导入小鼠受精卵中，得到F0小鼠；
将得到的F0小鼠与野生型小鼠杂交，得到携带LILRA3基因的杂合型小鼠；
将携带LILRA3基因的杂合型小鼠进行杂交，得到的纯合型小鼠为LILRA3基因敲入小鼠。
3.根据权利要求2所述的构建方法，其特征在于，所述打靶载体的使用量为5～10pL。
4.根据权利要求2所述的构建方法，其特征在于，利用Rosa-GT-F、Rosa-GT-R和Rosa26-Test(L)-R3引物判定小鼠的基因型；
所述Rosa-GT-F引物的核苷酸序列如SEQ ID No.3所示；
所述Rosa-GT-R引物的核苷酸序列如SEQ ID No.4所示；
所述Rosa26-Test(L)-R3的核苷酸序列如SEQ ID No.5所示。
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