CN116949097A - 一种sema4d人源化小鼠模型的构建方法及其应用 - Google Patents
一种sema4d人源化小鼠模型的构建方法及其应用 Download PDFInfo
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Abstract
本发明涉及一种SEMA4D人源化小鼠模型的构建方法，该构建方法：（1）构建表达人源化SEMA4D基因的打靶载体；（2）设计并获得针对小鼠Sema4d基因的sgRNA；（3）将打靶载体、sgRNA以及Cas9蛋白共注射或共电转至小鼠受精卵细胞质或细胞核中，并将该受精卵移植至假孕小鼠，对假孕生仔鼠进行基因型鉴定，筛选成功插入正确人源片段的阳性F0小鼠；（4）F0小鼠与背景鼠配繁获得F1小鼠，筛选出SEMA4D人源化小鼠模型。本发明构建的SEMA4D人源化小鼠在免疫学等领域具有应用价值。
Description
技术领域
本发明涉及动物基因工程领域，具体涉及一种SEMA4D人源化小鼠模型的构建方法及其应用。
背景技术
近年来肿瘤免疫疗法的概念已经越来越被大众熟知，但新型免疫治疗药物的研发仍面临诸多挑战。由于一般特异性人源蛋白的抗体不能识别小鼠内源蛋白，普通野生型小鼠无法作为测试这些药物的体内模型。而抗小鼠抗体制剂，由于物种特异性限制不能反映同类生物制剂在人体内的实际疗效。这些矛盾阻碍了免疫疗法的临床前评估，限制了新型肿瘤免疫药物研发的快速进展，因此研究者们开始致力于人源化小鼠模型的开发。
人源化小鼠是携带功能性人类基因、细胞、组织和/或器官、免疫系统或微生物的小鼠，其主要用于生物医学研究和临床治疗方案的开发。有些人源化小鼠携带人类细胞，有些则与人类具有某些一致的遗传和生理特性。因为小鼠基因组与人类基因组高度相似，且更易于操作和改变，所以小鼠可以方便地模拟人类的生物学特性，用于模拟人类疾病，从而帮助研究者破译致病原理和其分子机制，为药物的开发指引方向。
SEMA4D或CD100是一种来自信号素家族的蛋白质，在血管、神经和免疫系统中具有重要作用，它可以作为膜结合二聚体或作为蛋白水解切割产生的可溶性分子被发现。CD100由包括B淋巴细胞和T淋巴细胞、自然杀伤细胞和骨髓细胞以及内皮细胞在内的大多数造血细胞产生，根据细胞类型和生物体的不同，CD100通过与不同的受体结合发挥作用。细胞间粘附、血管生成、吞噬作用、T细胞启动和抗体产生是该分子许多功能的例子。值得注意的是，已在炎症和传染病中发现高CD100血清水平，但该蛋白质在这些疾病的发病机制中的作用仍有待阐明。CD100在无菌条件下以及动脉粥样硬化、自身免疫、肿瘤发生和抗肿瘤反应等多种疾病中都具有重要作用。
CD100属于信号蛋白4类，是所谓的“免疫信号蛋白”之一。已知它参与多种疾病的发病机制，例如动脉粥样硬化和多发性硬化、类风湿性关节炎、脑脊髓炎、多发性骨髓瘤、ANCA相关血管炎、系统性红斑狼疮和许多其他肿瘤。这些疾病共有的炎症和促血管生成成分表明该分子在免疫系统和内皮细胞功能中的重要作用。实际上，CD100由造血系统的大多数细胞表达，包括淋巴细胞，如B和T淋巴细胞和自然杀伤细胞，以及骨髓细胞（中性粒细胞、血小板和单核细胞）和内皮细胞，其表达通常在激活的T细胞表现出高水平的CD100。除了在膜中表达外，CD100还以可溶形式（sCD100）存在，它是通过蛋白水解切割以激活依赖性方式从膜CD100（mCD100）生成的。已知活化的T和B细胞会释放sCD100，用T细胞依赖性抗原免疫的小鼠或患有自身免疫性疾病的MRL/lpr小鼠的sCD100血清水平升高。
人源化小鼠模拟人类疾病，因此通常用于传染病，退行性和癌症疾病的研究。最近的模型还反映了造血，自然免疫，神经生物学和影响疾病病理生物学的分子途径。一系列免疫缺陷小鼠品系允许长寿命的人类祖细胞移植。先天性和适应性免疫的存在使高水平的人血淋巴重构成为可能，细胞对广泛的微生物感染具有敏感性。这些小鼠还有助于研究人类病理生物学，自然疾病过程和广泛人类疾病的治疗效果。因此，利用基因编辑的手段，将编码人类SEMA4D的基因敲入到其同源BALB/c小鼠位点，构建能表达人SEMA4D细胞因子的小鼠模型在研究免疫治疗、炎症、自身免疫疾病以及筛选评价SEMA4D靶点药物方面具有较高的应用价值。目前暂未见SEMA4D人源化小鼠模型构建方法及其在靶点药物应用方面的相关文献报道。
发明内容
针对目前存在的问题，本发明的第一方面提供一种SEMA4D人源化小鼠模型的构建方法，该构建方法包括如下步骤：
（1）构建表达人源化SEMA4D基因的打靶载体，用于人源化SEMA4D基因的插入；
（2）设计针对小鼠Sema4d基因翻译起始位点及其后编码区的sgRNA，利用体外转录技术获得上述sgRNA；
（3）将步骤（1）构建的打靶载体、步骤（2）获得的sgRNA以及Cas9蛋白共注射或共电转至小鼠受精卵细胞质或细胞核中，并将该受精卵移植至假孕小鼠，对假孕生仔鼠进行基因型鉴定，筛选成功插入正确人源片段的阳性F0小鼠；
（4）F0小鼠与背景鼠进行繁殖获得F1小鼠，对F1代鼠尾进行基因鉴定，筛选出SEMA4D人源化小鼠模型。
优选的，所述步骤（1）中包括下述步骤：根据人源SEMA4D的结构及功能，在鼠源Sema4d翻译起始密码子（ATG）开始插入人源的SEMA4D基因、鼠源Sema4d和3’UTR-polyA，选取的人源SEMA4D基因氨基酸序列如SEQ ID No.1所示，被替换的鼠源Sema4d基因氨基酸序列如SEQ ID No.2所示。
优选的，所述步骤（1）中包括下述步骤：选取人源SEMA4D基因的1-636Aa，利用同源重组的技术替换小鼠Sema4d基因的1-636Aa，选取的人源SEMA4D基因序列如SEQ ID No.3所示。
优选的，所述步骤（1）中构建成功的打靶载体序列如SEQ ID No.4所示。
更优选的，所述步骤（2）中sgRNA的基因序列为SEQ ID NO.5和SEQ ID NO.6。
优选的，所述步骤（3）中提供受精卵的小鼠和假孕小鼠的品系为BALB/c。
优选的，所述步骤（3）中F0小鼠基因型鉴定使用的5’端鉴定引物如SEQ ID NO.7和SEQ ID NO.8所示，3’端鉴定引物如SEQ ID NO.9和SEQ ID NO.10所示。
优选的，所述步骤（3）中F0小鼠基因型鉴定使用的PCR反应体系如下：
。
优选的，所述步骤（3）中F0小鼠基因型鉴定使用的PCR反应条件如下：
。
本发明的第二方面提供上述构建方法得到的小鼠在研究SEMA4D基因相关功能和作用机制中的应用。
优选的，所述应用是非诊断和非治疗目的的。
本发明的第三方面提供上述构建方法得到的小鼠在筛选用于治疗与SEMA4D基因相关疾病的药物中的应用。
优选的，所述应用是非诊断和非治疗目的的。
本发明的有益效果：
本发明的动物模型设计了切割鼠源Sema4d基因的sgRNA，同时设计包含人源SEMA4D基因的Donor，将sgRNA、Donor和Cas9混样，注射到BALB/cJGpt背景小鼠的受精卵中，进行同源重组，获得阳性F0，F0小鼠与BALB/c小鼠配种获得稳定的遗传阳性F1小鼠模型。本发明构建的能表达人SEMA4D细胞因子的小鼠模型在研究免疫治疗、炎症、自身免疫疾病以及筛选评价SEMA4D靶点药物方面具有较高的应用价值。
附图说明
图1是本发明人源化SEMA4D小鼠模型策略图；
图2是SEMA4D-KI F0小鼠5’端和3’端基因鉴定结果电泳图；
图3是SEMA4D-KI F1小鼠5’端和3’端基因鉴定结果电泳图；
图4是BALB/c小鼠及BALB/c-hSEMA4D小鼠脾脏T细胞中hSEMA4D表达检测的流式细胞图；
图5是BALB/c小鼠及BALB/c-hSEMA4D小鼠脾脏B细胞中hSEMA4D表达检测的流式细胞图；
图6是BALB/c小鼠及BALB/c-hSEMA4D小鼠脾脏白细胞细胞亚群（T、B、NK和DC细胞）检测的流式细胞图；
图7是BALB/c小鼠及BALB/c-hSEMA4D小鼠脾脏白细胞细胞亚群（Neutrophils、Monocytes、Eosinophils和Macrophage细胞）检测的流式细胞图。
具体实施方式
通过实施例的方式对本发明作进一步的说明，但是本发明并不仅仅局限于以下实施例。
试验例1、SEMA4D人源化小鼠模型的建立
发明使用CRISPR/Cas9技术将小鼠Sema4d基因的1-636号氨基酸（Aa）替换为人类SEMA4D 1-636号Aa的相应区域，并和小鼠Sema4d 637-861号Aa构成嵌合的CDs，人SEMA4D基因序列将在内源性调控机制的指导下进行表达，从而构建可以表达人源SEMA4D的小鼠模型（策略图见图1），具体方法如下：
1、确定人源片段替换区域及插入的人源序列
根据人源SEMA4D基因的结构及功能，选取人源SEMA4D基因的信号肽及胞外区替换鼠源SEMA4D基因的信号肽及胞外区序列，选取的人源SEMA4D基因氨基酸序列如SEQ IDNo.1（Aa：1-636）所示，被替换的鼠源Sema4d基因氨基酸序列如SEQ ID No.2（Aa：1-636）所示。
MRMCTPIRGLLMALAVMFGTAMAFAPIPRITWEHREVHLVQFHEPDIYNYSALLLSEDKDTLYIGAREAVFAVNALNISEKQHEVYWKVSEDKKAKCAEKGKSKQTECLNYIRVLQPLSATSLYVCGTNAFQPACDHLNLTSFKFLGKNEDGKGRCPFDPAHSYTSVMVDGELYSGTSYNFLGSEPIISRNSSHSPLRTEYAIPWLNEPSFVFADVIRKSPDSPDGEDDRVYFFFTEVSVEYEFVFRVLIPRIARVCKGDQGGLRTLQKKWTSFLKARLICSRPDSGLVFNVLRDVFVLRSPGLKVPVFYALFTPQLNNVGLSAVCAYNLSTAEEVFSHGKYMQSTTVEQSHTKWVRYNGPVPKPRPGACIDSEARAANYTSSLNLPDKTLQFVKDHPLMDDSVTPIDNRPRLIKKDVNYTQIVVDRTQALDGTVYDVMFVSTDRGALHKAISLEHAVHIIEETQLFQDFEPVQTLLLSSKKGNRFVYAGSNSGVVQAPLAFCGKHGTCEDCVLARDPYCAWSPPTATCVALHQTESPSRGLIQEMSGDASVCPDKSKGSYRQHFFKHGGTAELKCSQKSNLARVFWKFQNGVLKAESPKYGLMGRKNLLIFNLSEGDSGVYQCLSEERVKNKTVF （SEQ ID No.1）。
MRMCAPVRGLFLALVVVLRTAVAFAPVPRLTWEHGEVGLVQFHKPGIFNYSALLMSEDKDTLYVGAREAVFAVNALNISEKQHEVYWKVSEDKKSKCAEKGKSKQTECLNYIRVLQPLSSTSLYVCGTNAFQPTCDHLNLTSFKFLGKSEDGKGRCPFDPAHSYTSVMVGGELYSGTSYNFLGSEPIISRNSSHSPLRTEYAIPWLNEPSFVFADVIQKSPDGPEGEDDKVYFFFTEVSVEYEFVFKLMIPRVARVCKGDQGGLRTLQKKWTSFLKARLICSKPDSGLVFNILQDVFVLRAPGLKEPVFYAVFTPQLNNVGLSAVCAYTLATVEAVFSRGKYMQSATVEQSHTKWVRYNGPVPTPRPGACIDSEARAANYTSSLNLPDKTLQFVKDHPLMDDSVTPIDNRPKLIKKDVNYTQIVVDRTQALDGTFYDVMFISTDRGALHKAVILTKEVHVIEETQLFRDSEPVLTLLLSSKKGRKFVYAGSNSGVVQAPLAFCEKHGSCEDCVLARDPYCAWSPAIKACVTLHQEEASSRGWIQDMSGDTSSCLDKSKESFNQHFFKHGGTAELKCFQKSNLARVVWKFQNGELKAASPKYGFVGRKHLLIFNLSDGDSGVYQCLSEERVRNKTVS （SEQ ID No.2）。
2、注射获得阳性鼠
利用CRISPR Cas9技术将人源SEMA4D编码1-636 Aa的基因序列和小鼠Sema4d编码637-861 Aa的基因序列构成嵌合的CDs，建立SEMA4D基因人源化小鼠模型。以BALB/c为背景鼠，成功获得了SEMA4D人源化小鼠模型。
1）确定人源片段替换区域及插入的人源序列
根据人源SEMA4D蛋白胞外功能域及人鼠同源性比较，人源SEMA4D编码1-636 Aa的基因序列替换小鼠Sema4d编码1-636 Aa的基因序列，选取的人源SEMA4D基因替换的序列如SEQ ID No.3所示。
ATGAGGATGTGCACCCCCATTAGGGGGCTGCTCATGGCCCTTGCAGTGATGTTTGGGACAGCGATGGCATTTGCACCCATACCCCGGATCACCTGGGAGCACAGAGAGGTGCACCTGGTGCAGTTTCATGAGCCAGACATCTACAACTACTCAGCCTTGCTGCTGAGCGAGGACAAGGACACCTTGTACATAGGTGCCCGGGAGGCGGTCTTCGCTGTGAACGCACTCAACATCTCCGAGAAGCAGCATGAGGTGTATTGGAAGGTCTCAGAAGACAAAAAAGCAAAATGTGCAGAAAAGGGGAAATCAAAACAGACAGAGTGCCTCAACTACATCCGGGTGCTGCAGCCACTCAGCGCCACTTCCCTTTACGTGTGTGGGACCAACGCATTCCAGCCGGCCTGTGACCACCTGAACTTAACATCCTTTAAGTTTCTGGGGAAAAATGAAGATGGCAAAGGAAGATGTCCCTTTGACCCAGCACACAGCTACACATCCGTCATGGTTGATGGAGAACTTTATTCGGGGACGTCGTATAATTTTTTGGGAAGTGAACCCATCATCTCCCGAAATTCTTCCCACAGTCCTCTGAGGACAGAATATGCAATCCCTTGGCTGAACGAGCCTAGTTTCGTGTTTGCTGACGTGATCCGAAAAAGCCCAGACAGCCCCGACGGCGAGGATGACAGGGTCTACTTCTTCTTCACGGAGGTGTCTGTGGAGTATGAGTTTGTGTTCAGGGTGCTGATCCCACGGATAGCAAGAGTGTGCAAGGGGGACCAGGGCGGCCTGAGGACCTTGCAGAAGAAATGGACCTCCTTCCTGAAAGCCCGACTCATCTGCTCCCGGCCAGACAGCGGCTTGGTCTTCAATGTGCTGCGGGATGTCTTCGTGCTCAGGTCCCCGGGCCTGAAGGTGCCTGTGTTCTATGCACTCTTCACCCCACAGCTGAACAACGTGGGGCTGTCGGCAGTGTGCGCCTACAACCTGTCCACAGCCGAGGAGGTCTTCTCCCACGGGAAGTACATGCAGAGCACCACAGTGGAGCAGTCCCACACCAAGTGGGTGCGCTATAATGGCCCGGTACCCAAGCCGCGGCCTGGAGCGTGCATCGACAGCGAGGCACGGGCCGCCAACTACACCAGCTCCTTGAATTTGCCAGACAAGACGCTGCAGTTCGTTAAAGACCACCCTTTGATGGATGACTCGGTAACCCCAATAGACAACAGGCCCAGGTTAATCAAGAAAGATGTGAACTACACCCAGATCGTGGTGGACCGGACCCAGGCCCTGGATGGGACTGTCTATGATGTCATGTTTGTCAGCACAGACCGGGGAGCTCTGCACAAAGCCATCAGCCTCGAGCACGCTGTTCACATCATCGAGGAGACCCAGCTCTTCCAGGACTTTGAGCCAGTCCAGACCCTGCTGCTGTCTTCAAAGAAGGGCAACAGGTTTGTCTATGCTGGCTCTAACTCGGGCGTGGTCCAGGCCCCGCTGGCCTTCTGTGGGAAGCACGGCACCTGCGAGGACTGTGTGCTGGCGCGGGACCCCTACTGCGCCTGGAGCCCGCCCACAGCGACCTGCGTGGCTCTGCACCAGACCGAGAGCCCCAGCAGGGGTTTGATTCAGGAGATGAGCGGCGATGCTTCTGTGTGCCCGGATAAAAGTAAAGGAAGTTACCGGCAGCATTTTTTCAAGCACGGTGGCACAGCGGAACTGAAATGCTCCCAAAAATCCAACCTGGCCCGGGTCTTTTGGAAGTTCCAGAATGGCGTGTTGAAGGCCGAGAGCCCCAAGTACGGTCTTATGGGCAGAAAAAACTTGCTCATCTTCAACTTGTCAGAAGGAGACAGTGGGGTGTACCAGTGCCTGTCAGAGGAGAGGGTTAAGAACAAAACGGTCTTC（SEQ ID No.3）。
2）人源化打靶载体构建
将人源SEMA4D编码1-636 Aa的基因序列替换小鼠Sema4d编码1-636 Aa的基因序列，构建成功的打靶载体序列如SEQ ID No.4所示（斜体表示插入的片段，81903bp~83810bp表示插入的人源SEMA4D基因片段，83811bp~84488bp表示鼠源Sema4d exon17的部分序列，84489bp~85851bp表示鼠源的3’UTR）。
81301 ACATGT ATTCCC AGGGGA GTGCCC TGTAAC CGCAAA CCTCTG ATGTCT GGTAATATTCCT
81361 GGGTTA GCATGT CAGAAG TTGGGA AGTGAG CCCTCC AGGGAA ACTGAG ACCCGTACTGTT
81421 CCTTTC AGTTGT GGCATG TCACCA AGCACC TGGCTG TCCCTC TAGCTA GTTATTTCCACC
81481 TTGGTG GGTCCA TCCATC TTGGTG TGTTTT AGCCTT GTTGTC TTCTCT CTGTGGGATCTG
81541 TCTGTC TGGCAG CCCCAC AAATCC TGGATG CCTTCC ATGTCC CTCAGC AGGCAGGTTCAG
81601 ACAGGA TAGACA GTAGAA AGGGAT GGAGGG GACTGA GAACCA TCTGGA GCCGCTGGGACC
81661 TATAGT CTCGTC CTTTCC TTCTTT TGCAGG TCAGAC GGGAAC ACCGGC AGCCTTGGCATG
81721 ACGTCG TGAAGG TGGCCA TTGCTA ACCTGA CATGTG GGGACT CAGGAA CCCCACCCCTTA
81781 TGGGCT CCAGTC TGTGCT GCTGGC CCCAGC TCTGGG GCTCTA AGAGGT CCTTGCTGCTAC
81841 CCCACA GCAGCC TGCTGC CATCCA TGTGTG CCCGTT GCTGAA GGCCTC GGTGGCCCCTGC
81901 CCATGA GGATGT GCACCC CCATTA GGGGGC TGCTCA TGGCCC TTGCAG TGATGT TTGGGA
81961 CAGCGA TGGCAT TTGCAC CCATAC CCCGGA TCACCT GGGAGC ACAGAG AGGTGC ACCTGG
82021 TGCAGT TTCATG AGCCAG ACATCT ACAACT ACTCAG CCTTGC TGCTGA GCGAGG ACAAGG
82081 ACACCT TGTACA TAGGTG CCCGGG AGGCGG TCTTCG CTGTGA ACGCAC TCAACA TCTCCG
82141 AGAAGC AGCATG AGGTGT ATTGGA AGGTCT CAGAAG ACAAAA AAGCAA AATGTG CAGAAA
82201 AGGGGA AATCAA AACAGA CAGAGT GCCTCA ACTACA TCCGGG TGCTGC AGCCAC TCAGCG
82261 CCACTT CCCTTT ACGTGT GTGGGA CCAACG CATTCC AGCCGG CCTGTG ACCACC TGAACT
82321 TAACAT CCTTTA AGTTTC TGGGGA AAAATG AAGATG GCAAAG GAAGAT GTCCCT TTGACC
82381 CAGCAC ACAGCT ACACAT CCGTCA TGGTTG ATGGAG AACTTT ATTCGG GGACGT CGTATA
82441 ATTTTT TGGGAA GTGAAC CCATCA TCTCCC GAAATT CTTCCC ACAGTC CTCTGA GGACAG
82501 AATATG CAATCC CTTGGC TGAACG AGCCTA GTTTCG TGTTTG CTGACG TGATCC GAAAAA
82561 GCCCAG ACAGCC CCGACG GCGAGG ATGACA GGGTCT ACTTCT TCTTCA CGGAGG TGTCTG
82621 TGGAGT ATGAGT TTGTGT TCAGGG TGCTGA TCCCAC GGATAG CAAGAG TGTGCA AGGGGG
82681 ACCAGG GCGGCC TGAGGA CCTTGC AGAAGA AATGGA CCTCCT TCCTGA AAGCCC GACTCA
82741 TCTGCT CCCGGC CAGACA GCGGCT TGGTCT TCAATG TGCTGC GGGATG TCTTCG TGCTCA
82801 GGTCCC CGGGCC TGAAGG TGCCTG TGTTCT ATGCAC TCTTCA CCCCAC AGCTGA ACAACG
82861 TGGGGC TGTCGG CAGTGT GCGCCT ACAACC TGTCCA CAGCCG AGGAGG TCTTCT CCCACG
82921 GGAAGT ACATGC AGAGCA CCACAG TGGAGC AGTCCC ACACCA AGTGGG TGCGCT ATAATG
82981 GCCCGG TACCCA AGCCGC GGCCTG GAGCGT GCATCG ACAGCG AGGCAC GGGCCG CCAACT
83041 ACACCA GCTCCT TGAATT TGCCAG ACAAGA CGCTGC AGTTCG TTAAAG ACCACC CTTTGA
83101 TGGATG ACTCGG TAACCC CAATAG ACAACA GGCCCA GGTTAA TCAAGA AAGATG TGAACT
83161 ACACCC AGATCG TGGTGG ACCGGA CCCAGG CCCTGG ATGGGA CTGTCT ATGATG TCATGT
83221 TTGTCA GCACAG ACCGGG GAGCTC TGCACA AAGCCA TCAGCC TCGAGC ACGCTG TTCACA
83281 TCATCG AGGAGA CCCAGC TCTTCC AGGACT TTGAGC CAGTCC AGACCC TGCTGC TGTCTT
83341 CAAAGA AGGGCA ACAGGT TTGTCT ATGCTG GCTCTA ACTCGG GCGTGG TCCAGG CCCCGC
83401 TGGCCT TCTGTG GGAAGC ACGGCA CCTGCG AGGACT GTGTGC TGGCGC GGGACC CCTACT
83461 GCGCCT GGAGCC CGCCCA CAGCGA CCTGCG TGGCTC TGCACC AGACCG AGAGCC CCAGCA
83521 GGGGTT TGATTC AGGAGA TGAGCG GCGATG CTTCTG TGTGCC CGGATA AAAGTA AAGGAA
83581 GTTACC GGCAGC ATTTTT TCAAGC ACGGTG GCACAG CGGAAC TGAAAT GCTCCC AAAAAT
83641 CCAACC TGGCCC GGGTCT TTTGGA AGTTCC AGAATG GCGTGT TGAAGG CCGAGA GCCCCA
83701 AGTACG GTCTTA TGGGCA GAAAAA ACTTGC TCATCT TCAACT TGTCAG AAGGAG ACAGTG
83761 GGGTGT ACCAGT GCCTGT CAGAGG AGAGGG TTAAGA ACAAAA CGGTCT TCCAGC TGCTGG
83821 CCAAGC ACGTTC TGGAAG TGAAGA TGGTAC CTCGGA CCCCCC CCTCAC CTACCT CAGAGG
83881 ATGCTC AGACAG AAGGTA GTAAGA TCACAT CCAAAA TGCCGG TTGCAT CTACCC AGGGGT
83941 CCTCTC CCCCTA CCCCGG CTCTGT GGGCAA CCTCCC CCAGAG CCGCCA CCCTAC CTCCCA
84001 AGTCCT CCTCCG GCACAT CCTGTG AACCAA AGATGG TCATCA ACACGG TCCCCC AGCTCC
84061 ACTCAG AGAAGA CGGTGT ATCTCA AGTCCA GTGACA ACCGCC TGCTCA TGTCTC TCCTCC
84121 TCTTCA TCTTTG TCCTCT TCCTCT GCCTCT TTTCCT ACAACT GCTACA AGGGCT ACCTGC
84181 CCGGAC AGTGCT TAAAAT TCCGCT CAGCCC TGCTGC TTGGAA AGAAAA CACCCA AGTCAG
84241 ACTTCT CTGACC TGGAGC AGAGTG TGAAGG AGACAC TGGTCG AGCCTG GGAGCT TCTCCC
84301 AGCAGA ACGGCG ACCACC CCAAGC CAGCCC TGGATA CGGGCT ATGAAA CGGAGC AGGACA
84361 CCATCA CCAGCA AAGTCC CCACGG ATCGTG AGGACT CGCAAC GGATCG ATGAAC TCTCTG
84421 CCCGGG ACAAAC CGTTTG ATGTCA AGTGTG AACTGA AGTTTG CAGATT CGGATG CTGACG
84481 GGGACT GAGGCC AGCGTG TCCCAG CCCATG CCCCTC TGTCTT CGTGGA GAGTGT TGTGTT
84541 GAGCCC ATTCAG TAGCCG AGTCTT GTCACT CTGTGC CAGCCT CAGTCC TGTGTC CCTTTT
84601 TCTCTT GGGTTG AGCCTG TGGCTC ATCCCC TTTGTC CTTTTG GGAAGC AAGTAT CTATTC
84661 CAGTCT CAAGTC CTGCAG TTGCTG GAGCGC TTACGC ACCTGA GCCCTT TGTGTC CTGGGG
84721 GAGAGA TGGCCA CCTCCG TGGGCT GCGAAG AGCCAC CCCTTC CTCTTC CGATTC TCCTAG
84781 CAGCCA CTCAGA GATAAT TTAATT CCAGAT TGGAAA CGCCCT TTTAGT TTATCA GATTGG
84841 TAACTT ACATCC TGCTGC CCAGAT GGCACG GACAGT TTTCTT TCACTT AATTAT TATTTT
84901 TTTTTT AAGGAT TTTCGC TCCTAT TGTGTT GATGTC TTAGGT CATTTT CTTTTT TTCTTT
84961 CTCTTT TTTTAT TACCAG AGGAGA TGTTTT AATATT CATGAG AAGAGG AACATT TTCTAG
85021 ATTTTT TTGTTG TTATAT ATTGAG ATATAA AATATG GCTATG TTGCTT AAGATT CTCAGG
85081 GATAGA CTTATT TTTGTT AACTTC ATTCTT TCCTGC TGTTAG GAACAT AGGCCT AAAATT
85141 GTCTCT TGAGTT TGCTCA CCCTTT TGTTTT GGTAGG GTTTTT TTGTTG TTGCTG TTATTG
85201 TTTCTA GTTTTT AATCTT ATTCAT TTTGAA GGATTT TTCTTT CTGAAC TTTTTA AATTTT
85261 TATATT TTCCTG CCATAC ATCTAC AAAGTG GGTTTT GAGTGA GGGCAG GTGGCC CAGTGG
85321 CTTTGG GTGGCG ACTGAG CTGGTC CCACGA GGGGAG GAGGGT TTATAT ACCCCA TGACCC
85381 TGCGGC TTCTTG GCGCCT CCTGCC CATGAG GATCAC ATCCTG TCTCTC CTTGCT TCCATC
85441 TCTCAT CACTGC CCTTGG ACTTCC GCCTTG ACTGTC CATGAA AGACAG AAATGG GTTGGG
85501 TAGTTG GGCTCC CAACCT CGGATG GTGACC GCAACA TCCCGT GTGGGC GGCCGG CCGCTC
85561 CTGCAG CCCGAC TCTCCT GCCAGT GTCTTT CAGGAT GTCAAC GGGTGG TACGAT TCTGGC
85621 ATTTGT TTCTCG CTCACC GTGTGT GGAACA CTCATT CCATGT AGAGGG TGACGA ACTCTG
85681 GATTCC CCCCCA CCCCCA CCCCGT GCCGTG TAGACA CTCATC TTCTGC ATGACA TGATCT
85741 ACCATT CGGTGT AAACAT TTGTGT TTATAA GATTTA CTTTGT TTTTAT TTTTCT ACTTGG
85801 AACTGT ACACAT TTGAAA AGTACC CAAATA AACCAG AAGCTT TATCGT TGAGGG CCAACA
85861 CTAGAG CTCGCT GATCAG CCTCGA CTGTGC CTTCTA GTTGCC AGCCAT CTGTTG TTTGCC
85921 CCTCCC CCGTGC CTTCCT TGACCC TGGAAG GTGCCA CTCCCA CTGTCC TTTCCT AATAAA
85981 ATGAGG AAATTG CATCGC ATTGTC TGAGTA GGTGTC ATTCTA TTCTGG GGGGTG GGGTGG
86041 GGCAGG ACAGCA AGGGGG AGGATT GGGAAG ACAATA GCAGGC ATGCTG GGGAATGCCGCG
86101 GTGGCA TTTGCA CCTGTG CCTCGG CTCACC TGGGAA CATGGA GGTAAG TGAGTGTGAGCC
86161 ATGAGT GAGTGT GAGTGT GGGTGC AGCAGC TCTGGA GAGTGT GGGGTC CCTGCTTTGGAT
86221 GCTCTG TGGGGT CTGTCA GGGCAG GCTTAA GCCGCT GGGATG TCAAAG GATGCTGTGTGA
86281 CCAGGA GACCCC ACAGCA AGCGCC AAGCTT AGGTTG GACGGT GTATGT GACCTCAGTGGC
86341 TCACGG CTTACT TAATGC TGACGA CATCCC AGGTAC TTATGC TGCACT GTTGAGATTAGC
86401 TCAGCC TCAGAG AGGTCT CCAGGC CCGATT TCCAGG ATGGGG ACACAC TGGCTGTCCCAC
86461 ATTCCA GGGAGG GAACTT TGTGAA GACAGA AGCCGG GAAGCA AAGTGC ACTGAGAATGTC
86521 CAACTC TGAAGC TACCTG GACACT CCGGGT GCTGAC TTGTAG GCAAGT TCAGATGAAGTT
86581 CATTGA TGGGGG AGGGGG CAGAAT GCTGAG CTCAGC ATCACA GCAGGA ACCTCCACACTC
（SEQ ID No.4）。
3）sgRNA的构建
（1）分别合成sgRNA上下游引物，引物纯化方式为PAGE；
（2）sgRNA上下游引物分别稀释至100umol/μl，以1:1的配比混匀，室温自行缓慢退火；
（3）退火形成的双链与Puc57-sgRNA-NEO-Amp （Bsa I）连接1h，转化，涂布Amp+平板；
（4）挑取单克隆，进行PCR鉴定；
（5）PCR阳性的单克隆进一步测序确认，测序引物为pUC57-T7-F；
（6）以测序正确的克隆为模板，然后用引物PCR扩增sgRNA转录的DNA产物；
（7）以sgRNA转录的DNA产物为模板，转录sgRNA并进一步纯化。然后将构建好的打靶载体通过转录然后再逆转录，获得可用于注射的ssDNA donor。
4）SEMA4D人源化小鼠制备的sgRNA筛选
设计并合成了sgRNA（SEMA4D-S1+SEMA4D-S2，具体序列信息见表1），将5’端靶点位和3’靶点位的sgRNA配对，然后将sgRNA与Cas9蛋白进行孵育后，注射到0.5天的受精卵中，进行培养至囊胚后，通过对小鼠SEMA4D基因的KO阳性率进行鉴定，以此对sgRNA切割活性进行验证。
sgRNA切割实验鉴定方法：将收集的囊胚进行PCR扩增，PCR的方案如表3-4所示，将扩增的条带进行二代测序，结果与WT条带进行对比，统计发生突变的概率。
表1 sgRNA信息
5）SEMA4D人源化小鼠模型建立
将筛选得到的高切割效率sgRNA（SEMA4D-S1+SEMA4D-S2）设计并构建携带人源序列的ssDNA donor，将ssDNA donor及Cas9/sgRNA系统注射至0.5 d的小鼠受精卵中，移植至0.5d假孕雌鼠体内，等小鼠出生后，经基因鉴定筛选出中靶小鼠（F0）。
6）人源化F0小鼠基因型鉴定
对获得的F0小鼠的鼠尾基因组DNA分别使用表2所示的两对引物进行中靶后的两端PCR鉴定，PCR反应条件和反应程序如表3和表4所示。引物GGPT0X0296-01-mSema4d-5tF1/GPT0X0296-01-hSEMA4D-5tR1分别位于5’端同源臂外及ssDNA donor的人源片段内，如该对引物扩增产生PCR产物，说明目标donor在小鼠基因组5’端进行了有效插入；GPT0X0296-01-hSEMA4D-3tF1/GPT0X0296-01-mSema4d-3tR1分别位于ssDNA donor的人源片段内及3’端同源臂外，如该对引物扩增产生PCR引物，说明目标donor在小鼠基因组3’端进行了有效插入。
表2 F0鉴定引物
表3 PCR反应体系
表4 PCR反应条件
本试验例注射获得F0小鼠，采用上述鉴定方案检测阳性F0小鼠。hSEMA4D F0鼠尾的基因组PCR鉴定见图2所示。其中8#、9#、11#和18#号小鼠的人源SEMA4D基因5’及3’端鉴定均为阳性，但8#和9#测序未通过，所以采用11#和18#号小鼠进行繁育。此外，通过上述鉴定方法对其他多批F0小鼠进行鉴定，均得到可进行繁育的F0阳性小鼠。
阳性F0小鼠与背景鼠配繁获得F1，对F1代鼠尾进行基因鉴定，F1代小鼠基因鉴定结果见图3所示（WT对照为BALB/c基因组DNA；N为空白对照；M为DNA Marker：8000bp\5000bp\3000bp\2000bp\1000bp\750bp\500bp\250bp\100bp），20#、22#、28#-31#小鼠人源SEMA4D基因5’及3’端鉴定均为阳性，未发生串联，同时鼠源检测也为阳性，表明获得的小鼠为正确进行基因重组的杂合阳性小鼠。F1小鼠大量扩繁后进行互配，获得纯合子小鼠。
试验例2、BALB/c-hSEMA4D人源化纯合小鼠蛋白表达验证
1、BALB/c-hSEMA4D小鼠脾脏T细胞中hSEMA4D表达检测
收集BALB/c-hSEMA4D纯合小鼠和BALB/c背景小鼠的脾脏细胞用物种特异性的抗SEMA4D抗体对CD3+ T细胞进行流式细胞仪分析，检测结果如图4所示。检测结果表明，鼠Sema4d只在BALB/c小鼠中检测到，人SEMA4D只在BALB/c-hSEMA4D小鼠中检测到而在BALB/c小鼠中检测不到，表明BALB/c-hSEMA4D纯合小鼠中只表达人源SEMA4D蛋白。
2、BALB/c-hSEMA4D小鼠脾脏B细胞中hSEMA4D表达检测
收集BALB/c-hSEMA4D纯合小鼠和BALB/c背景小鼠的脾脏细胞用物种特异性的抗SEMA4D抗体对CD19+ B细胞进行流式细胞仪分析，检测结果如图5所示。试验结果表明，鼠Sema4d只在BALB/c小鼠中检测到，人SEMA4D只在BALB/c-hSEMA4D小鼠中检测到而在BALB/c小鼠中检测不到，表明BALB/c-hSEMA4D纯合小鼠中只表达人源SEMA4D蛋白。
3、小鼠脾脏白细胞细胞亚群的分析
取6周龄BALB/c小鼠和BALB/c-hSEMA4D纯合小鼠的脾脏细胞进行流式检测，以确定其免疫细胞组分的比例，检测结果如图6和图7所示。检测结果表明，BALB/c小鼠和BALB/c-hSEMA4D小鼠相比，它们的T、B、NK、DC、Neutrophils、Monocytes、Eosinophils和Macrophage细胞组分几乎一致。
上述试验结果表明，本发明通过将小鼠SEMA4D基因进行人源化基因的替换，成功构建得到BALB/c-hSEMA4D小鼠模型，预示着该模型在肿瘤学、免疫治疗、自身免疫等领域的研究中具有广阔的应用前景。
尽管已经对本方法实施步骤进行详细说明，但是对于本领域的技术人员来讲，依然可以在本发明范围内对方法中部分参数及整体方案进行修改。因此，凡在本发明精神和原则范围之内做的更改、替换、调整等行为均应在该发明所涵盖范围内。
Claims (10)
1.一种SEMA4D人源化小鼠模型的构建方法，其特征在于，所述构建方法包括如下步骤：
（1）构建表达人源化SEMA4D基因的打靶载体，用于人源化SEMA4D基因的插入；
（2）设计针对小鼠Sema4d基因翻译起始位点及其后编码区的sgRNA，利用体外转录技术获得上述sgRNA；
（3）将步骤（1）构建的打靶载体、步骤（2）获得的sgRNA以及Cas9蛋白共注射或共电转至小鼠受精卵细胞质或细胞核中，并将该受精卵移植至假孕小鼠，对假孕生仔鼠进行基因型鉴定，筛选成功插入正确人源片段的阳性F0小鼠；
（4）F0小鼠与背景鼠进行繁殖获得F1小鼠，对F1代鼠尾进行基因鉴定，筛选出SEMA4D人源化小鼠模型。
2. 根据权利要求1所述的构建方法，其特征在于，所述步骤（1）中包括下述步骤：根据人源SEMA4D的结构及功能，在鼠源Sema4d翻译起始密码子（ATG）开始插入人源的SEMA4D基因、鼠源Sema4d和3’UTR-polyA，选取的人源SEMA4D基因氨基酸序列如SEQ ID No.1所示，被替换的鼠源Sema4d基因氨基酸序列如SEQ ID No.2所示。
3. 根据权利要求1所述的构建方法，其特征在于，所述步骤（1）中包括下述步骤：选取人源SEMA4D基因的1-636Aa，利用同源重组的技术替换小鼠Sema4d基因的1-636Aa，选取的人源SEMA4D基因序列如SEQ ID No.3所示。
4. 根据权利要求1所述的构建方法，其特征在于，所述步骤（1）中构建成功的打靶载体序列如SEQ ID No.4所示。
5. 根据权利要求1所述的构建方法，其特征在于，所述步骤（2）中sgRNA的基因序列为SEQ ID NO.5和SEQ ID NO.6。
6.根据权利要求1所述的构建方法，其特征在于，所述步骤（3）中提供受精卵的小鼠和假孕小鼠的品系为BALB/c。
7. 根据权利要求1所述的构建方法，其特征在于，所述步骤（3）中F0小鼠基因型鉴定使用的5’端鉴定引物如SEQ ID NO.7和SEQ ID NO.8所示，3’端鉴定引物如SEQ ID NO.9和SEQID NO.10所示。
8.根据权利要求1所述的构建方法，其特征在于，所述步骤（3）中F0小鼠基因型鉴定使用的PCR反应条件如下：
。
9.权利要求1-8任一项所述的SEMA4D人源化小鼠模型的构建方法得到的小鼠在研究SEMA4D基因相关功能和作用机制中的应用。
10.权利要求1-8任一项所述的SEMA4D人源化小鼠模型的构建方法得到的小鼠在筛选用于治疗与SEMA4D基因相关疾病的药物中的应用。
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