CN111876418B - 先天性黑蒙模型犬的建立方法 - Google Patents
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Abstract
本发明涉及利用基因编辑技术制备先天性黑蒙模型犬的方法,以及获得的先天性黑蒙模型犬,先天性黑蒙模型犬的细胞和组织。所述方法包括如下步骤:(1)利用基因编辑技术获得RPE65基因编辑型先天性黑蒙模型犬受精卵;以及(2)将RPE65基因编辑型先天性黑蒙模型犬受精卵移植到双侧输卵管均进行了冲胚的母犬的一侧输卵管内,从而制备RPE65基因编辑型先天性黑蒙模型犬。
Description
技术领域
本发明涉及通过基因编辑技术建立先天性黑蒙疾病模型犬的方法,获得的先天性黑蒙疾病模型犬,以及先天性黑蒙模型犬的细胞和组织。
背景技术
犬是目前基础医学研究和教学中最常用的实验动物之一,尤其在生理、药理和病理生理学等实验研究中起着重要作用。通过对犬进行全基因组测序分析,共计确定约1.93万个基因,其中约1.8万个基因与人类已知基因相同,其基因组与人类的相似度高于小鼠等其他实验动物。犬在遗传性疾病方面与人类也极为相似,约有癌症、心脏病、聋哑、失明和免疫性神经系统疾病等360多种遗传疾病与人类相同,适于作为人类疾病研究的模式动物。而且,犬的遗传性疾病少,实验重复性好,血液循环和神经系统发达,消化系统及内脏与人相似,在毒理方面的反应与人类比较接近,尤其适合药理、循环生理、眼科、毒理、外科学等的研究。另外犬性格温顺容易调教,经短期训练能很好地配合实验,被国际医学、生物学界公认为较理想的实验用犬。
目前常用的制备犬疾病模型的方法主要包括:饲喂法、机械损伤法及免疫学方法等。由于饲喂法、机械损伤法和免疫学法均是在健康动物基础上,采用特殊的方法诱导其出现疾病表型,因此存在无法出现疾病表型、表型持续时间较短或无法模拟人类疾病症状等问题。而采用基因工程的方法对犬基因组进行基因敲除或转基因修饰,其疾病症状为原发症状,表型持续时间长,并且具有可遗传性。
德国眼科医师Theodor Leber于1869年首次提出先天性黑蒙症(Leber’scongenital amaurosis,LCA),并指出该疾病的特点为先天性盲合并眼球震颤及复杂多变的眼底表现。LCA属于一种严重的遗传致盲性视网膜病变,可导致婴儿在出生后一个月内完全丧失视力,约占遗传性视网膜疾病的5%,占学龄儿童盲的20%,全世界患病率为1/80000~1/30000。虽然该病发病率低,但我国人口基数大,临床上的患者数量并不少。由于缺乏对发病机制相应的认识,LCA目前仍然处于“无药可医”的阶段,该病在儿童早期发病并影响日常生活能力,严重影响患者的身心健康与生活质量,为患病家庭和社会带来极大的负担与压力。目前LCA患者治疗方式以造价高昂的基因治疗为主,未见其他治疗方法报道,另外,该病早期鉴别诊断困难,因此,进一步开展LCA致病机制和治疗方法的研究具有非常重要的临床意义。
1998年Redmond等构建了Rpe65基因敲除小鼠模型,并证明Rpe65在视黄醇视觉循环过程中发挥了重要作用。但小鼠缺乏以视锥细胞为主的视网膜中央凹,人类视锥细胞表达基因的突变在小鼠模型上可能有不同的表型结果,而且小鼠是夜间活动的动物,增加了光诱导视网膜变性的敏感性。鉴于以上生理解剖特征等方面的物种差异原因,小鼠模型虽然拥有较大优势,但是难以准确模拟人LCA疾病的发生和治疗过程。相比于小鼠,大动物模型能更准确模拟人类疾病,但目前缺乏LCA大动物模型,从而大大限制了LC A疾病发病机制和治疗方法的研究。
犬是药物临床前试验的最佳动物,也是理想的模式动物,构建犬LCA疾病模型将为开展LCA干细胞治疗、基因修复治疗和药物筛选提供动物模型,从而为人LCA的临床治疗提供重要的临床前试验依据,将对人LCA的治疗和研究具有非常重要的意义。
发明内容
本发明提供了一种RPE65基因编辑型先天性黑蒙模型犬的方法,所述方法包括针对犬RPE65基因序列选择打靶位点,构建表达载体,载体经验证有效后体外转录mRNA,然后采用胞质注射的方式将mRNA注射入犬受精卵中,再将胚胎移植入冲卵母犬体内,从而制备RPE65基因编辑型先天性黑蒙模型犬。本发明还涉及所建立的RPE65基因编辑型先天性黑蒙模型犬、及其组织和细胞。
第一方面,本发明提供了用于建立RPE65基因编辑型先天性黑蒙模型犬的方法,所述方法包括如下步骤:利用基因编辑技术获得RPE65基因编辑型先天性黑蒙模型犬受精卵或者RPE65基因编辑型先天性黑蒙模型犬体细胞。
所述基因编辑技术包括:BE3,CRISPR/Cas9,TALEN和ZFN。
在某些实施方案中,所述用于建立RPE65基因编辑型先天性黑蒙模型犬的方法包括以下步骤:(1)根据犬RPE65基因序列,针对外显子的序列确定打靶位点;(2)根据步骤(1)确定的打靶位点合成sgRNA序列,然后将合成的序列与骨架载体连接构建sgRNA打靶载体;(3)将sgRNA打靶载体体外转录获得sgRNA的mRNA,将CRISPR/Cas9体外转录为mRNA。
所述外显子可以为RPE65基因外显子2,RPE65基因外显子3和RPE65基因外显子5。优选地,所述外显子为SEQ ID NO:1所示的RPE65基因外显子5,针对RPE65基因外显子5中的序列进行插入、删除、置换和/或添加修饰。
优选地,所述打靶位点序列为5’-CCCTTGTTAACGTCTACCCAGT A-3’(SEQ ID NO:2)。
优选地,根据所述打靶位点序列合成的sgRNA序列及其互补序列为:
F:CACCGggatgcagagggagatggcg(SEQ ID NO:3);和
R:AAACcgccatctccctctgcatccC(SEQ ID NO:4)。
在某些实施方案中,所述用于建立RPE65基因编辑型先天性黑蒙模型犬的方法还包括将RPE65基因编辑型先天性黑蒙模型犬受精卵移植到双侧输卵管均进行了冲胚的母犬的一侧输卵管内,从而制备RPE65基因编辑型先天性黑蒙模型犬的步骤。优选地,将RPE65基因编辑型先天性黑蒙模型犬受精卵移植到双侧输卵管均进行了冲胚的母犬出血较少一侧输卵管内。
在某些实施方案中,所述用于建立RPE65基因编辑先天性黑蒙模型犬的方法还包括将RPE65基因编辑型先天性黑蒙模型犬体细胞核移植到犬去核卵母细胞中;以及将犬去核卵母细胞移植到双侧输卵管均进行了冲胚的母犬的一侧输卵管内,从而制备RPE65基因编辑型先天性黑蒙模型犬的步骤。优选地,将犬去核卵母细胞移植到双侧输卵管均进行了冲胚的母犬出血较少一侧输卵管内。
第二方面,本发明提供了犬RPE65基因编辑打靶载体,所述打靶载体是针对RPE65基因外显子2,3或5(SEQ ID NO:1所示的序列)中的打靶位点序列设计的sgRNA序列以及骨架载体构成。
优选地,针对RPE65基因外显子5(SEQ ID NO:1所示的序列)中的打靶位点序列设计的sgRNA序列。
优选地,所述打靶位点序列为5’-CCCTTGTTAACGTCTACCCAGTA-3’(SEQ ID NO:2)。
优选地,根据所述打靶位点序列合成的sgRNA序列及其互补序列为:
F:CACCGggatgcagagggagatggcg(SEQ ID NO:3);和
R:AAACcgccatctccctctgcatccC(SEQ ID NO:4)。
第三方面,本发明提供了由第一方面的方法获得的RPE65基因编辑型先天性黑蒙模型犬的体细胞、组织和器官。
优选地,所述体细胞、组织和器官包含SEQ ID NO:5所示的如下序列:CCCTTGTCAGTGCATAACGTCTACCCAGTA。
优选地,所述体细胞、组织和器官包含SEQ ID NO:9所示的如下序列:GTTTTTTTCTTACTTCCGAGGAGTGGAGGTCACTGACAATGCCCTTGTCAGTGCATAACGTCTACCCAGTAGGGGAAGATTACTATGCCTGCACGGAGACCAACTTCATTACAAAGATTAATCCTGA GACCCTGGAG ACAATTAAGC AG。
所述RPE65基因编辑型先天性黑蒙模型犬的体细胞、组织和器官钟所包含的特征序列是双等位基因7bp碱基序列插入后外显子5后获得的特征性序列,双等位基因7bp碱基序列的插入导致终止密码子TAA的出现,RPE65蛋白翻译提前终止,从而无法获得功能性蛋白。
优选地,所述体细胞、组织和器官包含SEQ ID NO:6所示的如下序列:CCCTTGTTTAACGTCTACCCAGTA。双等位基因1bp碱基插入导致Rpe65基因移码突变。和/或所述体细胞、组织和器官包含SEQ ID NO:7所示的如下序列:CCCTTGTAACGTCTACCCAGTA。双等位基因1bp碱基缺失导致Rpe65基因移码突变。优选地,所述体细胞、组织和器官包含SEQ IDNO:13所示的如下序列:GTTTTTTTCTTACTTCCGAGGAGTGGAGGTCACTGACAATGCCCTTGTTtAACGTCTACCCAGTAGGGGAAGATTACTATGCCTGCACGGAGACCAACTTCATTACAAAGATTAATCCTGAGACCCTGGAGACAATTAAGCAG;和/或SEQ ID NO:14所示的如下序列:GTTTTTTTCTTACTTCCGAGGAGTGGAGGTCACTGACAATGCCCTTGTAACGTCTACCCAGTAGGGGAAGATTACTATGCCTGCACGGAGACCAACTTCATTACAAAGATTAATCCTGAGACCCTGGAGACAATTAAGCAG。
优选地,所述体细胞、组织和器官包含SEQ ID NO:8所示的如下序列:CCCTTGCATTAACGTCTACCCAGTA。双等位基因2bp碱基插入导致Rpe65基因移码突变。优选地,所述体细胞、组织和器官包含SEQ ID NO:15所示的如下序列:GTTTTTTTCTTACTTCCGAGGAGTGGAGGTCACTGACAATGCCCTTGTAACGTCTACCCAGTAGGGGAAGATTACTATGCCTGCACGGAGACCAACTTCATTACAAAGATTAATCCTGAGACCCTGGAGACAATTAAGCAG。
优选地,所述体细胞为分类命名为比格犬成纤维细胞:Rpe65-KO-190815,其保藏在中国微生物菌种保藏管理委员会普通微生物中心(CGMCC),保藏地址为:北京市朝阳区北辰西路1号院3号(邮政编码100101),保藏号为CGMCC No.19945,保藏日期为2020年6月3日。
第四方面,本发明提供了用于检测具有SEQ ID NO:5、SEQ ID NO:6、SEQ ID NO:7、SEQ ID NO:8或SEQ ID NO:9所示序列的RPE65基因敲除型先天性黑蒙模型犬的引物对,所述引物对针对SEQ ID NO:10所示序列进行设计:
TTCATCCGCACCGATGCTTACGTCCGGGCAATGACCGAGAAAAGGATCGTCATAACGGAATTTGGCACCTGTGCGTTCCCAGATCCCTGCAAGAATATATTTTCCAGGTTATTGAAACTTGACTGCATGTTATTGAATCAAGACATTTTACATTAGCCCTTTTCTTTCACAGCTTGAAGGTTACTGGACTGAAAAACTCCGTTTGCTTCTGTAGGTTTTTTTCTTACTTCCGAGGAGTGGAGGTCACTGACAATGCCCTTGTTAACGTCTACCCAGTAGGGGAAGATTACTACGCCTGCACGGAGACCAACTTCATTACAAAGATTAATCCTGAGACCCTGGAGACAATTAAGCAGGTAGGACGAAATGCTCAGGCGACGTTGCTCAAGAATTTAGAATTTGCAGTTTAGATTTAACTGCAATTTTGGGGAAAGCTCATGAGGGCCAAATAGATTGTCTCGCTGCCTTGCTTTGTCATCAACTACTAGCCATGTGACACGAGGCACTCTTTATCTACAAAATTTTAAATATTTATTTAATATTATATTAATATATTATATTATTAATATATTGTTGATATATTATATATTAATATATTAATGTATATTATATATATTATATTAATATAATATTATATGGGTTGGAGGAAAACCTTATAAGATGCCTAACATGTTCATTCATTTATTTGTGACACAAATATTTATGGAGTCCCTTCCATGCATCAGAACTTCTACTAAAGGCAAAGGAAACATCAATAGGTAAGACAACACGGATAGGTCAAAAATTGGGAAGCTGTTTAGCTAATGGGAAGCTACTGAAACTTTTTACTCAGAGAAATGGTATAAGCAGAGCTCTGCTTTAGGATATTTGATCCGGAAGCCAAGCATAAGCCGTAGGGGAGGACGAAAGAGATAAGGGATAGTTGGAGACAGGTGAGG。
优选地,所述引物对的序列如下:
RPE65-F:5’-TTCATCCGCACCGATGCTTACGTC-3’(SEQ ID NO:11);
RPE65-R:5’-CCTCACCTGTCTCCAACTATCCCTT-3’(SEQ ID NO:12)。
第五方面,本发明涉及用于检测具有SEQ ID NO:5、SEQ ID NO:6、SEQ ID NO:7、SEQ ID NO:8、SEQ ID NO:9、SEQ ID NO:13、SEQ ID NO:14和/或SEQ ID NO:15所示序列的RPE65基因敲除型先天性黑蒙模型犬的试剂盒,所述试剂盒具有针对SEQ ID NO:10所示序列设计的引物对,优选地,所述引物对的序列如下:
RPE65-F:5’-TTCATCCGCACCGATGCTTACGTC-3’(SEQ ID NO:11);
RPE65-R:5’-CCTCACCTGTCTCCAACTATCCCTT-3’(SEQ ID NO:12)。
第六方面,本发明提供了由第一方面的方法建立的RPE65基因敲除型先天性黑蒙模型犬。
RPE65基因编码的RPE65蛋白是我们的视觉系统中一种非常重要的酶,可以催化11-顺-视网膜(11-cis-retinal)异构化成为全反式视黄醛(All-trans-retinal),从而触发光传导通路(Phototransduction Pathway),最终让大脑皮质的视觉中枢产生视觉。
附图说明
图1图示了编号190815、190828和190832的基因编辑犬的耳组织和尾组织与野生型犬相比Rpe65基因特征序列的比对。
图2:显示了编号为190815、190828和190832的Rpe65基因编辑犬的耳组织和尾组织与野生型犬的耳组织和尾组织的基因测序峰图的对比。
图3:编号为190815的Rpe65基因编辑犬出生30天照片。
图4:显示了190815的Rpe65基因编辑犬与野生型犬的视网膜电图结果。
具体实施方式
下面结合实施例及说明书附图对本发明的技术方案做进一步描述。这些实施例仅用于说明本发明而不用于限定本发明的保护范围。
实施例:
(1)Rpe65基因敲除犬的制备
Rpe65基因敲除犬的制备包括如下步骤:
1)根据犬Rpe65基因序列,针对外显子的序列确定打靶位点序列;
2)根据步骤(1)确定的打靶位点序列合成sgRNA序列及其互补序列,然后将合成的序列与骨架载体连接构建sgRNA打靶载体;
3)将sgRNA打靶载体体外转录获得sgRNA的mRNA,以及将CRISPR/Cas9体外转录为mRNA;
4)将步骤(3)获得的sgRNA的mRNA和CRISPR/Cas9的mRNA混合后胞质内注射到犬受精卵中;以及
5)将犬受精卵移植到双侧输卵管均进行了冲胚的母犬出血较少的一侧输卵管内。
可以针对Rpe65基因序列的外显子2(caagaagctgtttgaaaccgtgg)、外显子3(agtctcctccgatgcggaccggg)或者外显子5(TTGTTAACGTCTACCCAGTAGGG)序列确定打靶位点序列。在本实施例中的打靶位点序列为针对外显子5确定的如下序列:5'-ttgttaacgtctacccagtaGGG-3'。
在本实施例中步骤2)中合成的sgRNA序列及其互补序列为:
sgRNA序列:CACCGTTGTTAACGTCTACCCAGTAGGG;以及
sgRNA互补序列:AAAC CCCTACTGGGTAGACGTTAACAA C。
共进行胚胎移植25次,移植受体25只,出生幼犬合计39只(参见下表1),其中编号为190815的母犬Rpe65基因突变类型为在外显子5双等位基因7bp碱基序列插入,导致终止密码子TAA的出现,RPE65蛋白翻译提前终止,无法获得功能性蛋白(具有SEQ ID NO:5所示的如下特征序列:CCCTTGTCAGTGCATAACGTCTACCCAGTA;以及SEQ ID NO:9所示的如下特征序列:GTTTTTTTCTTACTTCCGAGGAGTGGAGGTCACTGACAATGCCCTTGTCAGTGCATAACGTCTACCCAGTAGGGGAAGATTACTATGCCTGCACGGAGACCAACTTCATTACAAAGATTAATCCTGAGACCCTGGAG ACAATTAAGCAG);编号为190828的犬为双等位基因1bp碱基插入和1bp碱基缺失的嵌合体,均导致Rpe65基因移码突变,在耳组织(具有SEQ ID NO:6所示的如下特征序列:CCCTTGTTTAACGTCTACCCAGTA)和尾组织(具有SEQ ID NO:7所示的如下特征序列:CCCTTGTAACGTCTACCCAGTA)显示不同的移码突变;190832为双等位基因2bp碱基插入导致Rpe65基因移码突变(具有SEQ ID NO:8所示的如下特征序列:CCCTTGCATTAACGTCTACCCAGTA)。参见附图1,其中显示了编号为190815、190828、190832的基因编辑犬的耳组织和尾组织与野生型犬相比Rpe65基因编辑的结果。可以看出,编号为190815的Rpe65基因编辑犬的耳组织和尾组织在Rpe65基因第5号外显子存在7个碱基的插入序列:CAGTGCA;编号为190828的Rpe65基因编辑犬的耳组织和尾组织在Rpe65基因第5号外显子存在1个碱基的插入序列:T和1个碱基序列的缺失:T;编号为190832的Rpe65基因编辑犬的耳组织和尾组织在Rpe65基因第5号外显子存在2个碱基的插入序列:CA。参见附图2,显示了编号为190815、190828和190832的Rpe65基因基因编辑犬耳组织和尾组织与野生型犬基因测序峰图的对比。
表1:胚胎移植结果
编号为190815的基因编辑犬体细胞的分类命名为比格犬成纤维细胞:Rpe65-KO-190815,保藏在中国微生物菌种保藏管理委员会普通微生物中心(CGMCC),保藏地址为:北京市朝阳区北辰西路1号院3号(邮政编码100101),保藏号为CGMCC No.19945,保藏日期为2020年6月3日。
(2)Rpe65基因编辑犬的视网膜电图检测:
Rpe65基因编辑犬190815分别在2月龄和4月龄时进行了视网膜电图(ERG)分析:野生型家犬的a波、b波振幅随着刺激强度的增加而增加,而Rpe65基因突变犬随着刺激强度的增加无视网膜电位变化(图4)。可以看出,编辑Rpe65基因造成基因编辑犬视网膜对光刺激反应出现障碍,这一结果与Rpe65基因自然突变家犬ERG结果一致从而进一步验证了获得了Rpe65基因编辑犬。
SEQUENCE LISTING
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Claims (8)
1.用于建立RPE65基因编辑型先天性黑蒙模型犬的方法,其特征在于,所述方法包括如下步骤:(1)根据犬RPE65基因序列,针对外显子的序列确定打靶位点;(2)根据步骤(1)确定的打靶位点合成sgRNA序列,然后将合成的序列与骨架载体连接构建sgRNA打靶载体;(3)将sgRNA打靶载体体外转录获得sgRNA的mRNA,并且将CRISPR/Cas9体外转录为mRNA;(4)将步骤(3)获得的sgRNA的mRNA和CRISPR/Cas9的mRNA混合后胞质内注射到犬受精卵中;以及(5)将犬受精卵移植到双侧输卵管均进行了冲胚的母犬出血较少的一侧输卵管内;
所述打靶位点序列为SEQ ID NO:1所示的外显子5的如下序列:
5’-CCCTTGTTAACGTCTACCCAGT A-3’(SEQ ID NO:2),
根据所述打靶位点序列合成的sgRNA序列及其互补序列为:
F:CACCGTTGTTAACGTCTACCCAGTAGGG(SEQ ID NO:3);和
R:AAAC CCCTACTGGGTAGACGTTAACAAC(SEQ ID NO:4)。
2.犬RPE65基因编辑打靶载体,其特征在于所述打靶载体由针对RPE65基因外显子5中的打靶位点序列设计的sgRNA序列以及骨架载体构成,
所述打靶位点序列为SEQ ID NO:2所示的5’-CCCTTGTTAACGTCTACCCAGTA-3’,
根据所述打靶位点序列合成的sgRNA序列及其互补序列为:
F:CACCGTTGTTAACGTCTACCCAGTAGGG(SEQ ID NO:3);和
R:AAAC CCCTACTGGGTAGACGTTAACAAC(SEQ ID NO:4)。
3.由权利要求1的方法获得的RPE65基因编辑型先天性黑蒙模型犬的体细胞、组织和器官。
4.根据权利要求3的体细胞、组织和器官,其特征在于,所述体细胞、组织和器官包含:SEQ ID NO:5所示的如下序列:CCCTTGTCAGTGCATAACGTCTACCCAGTA。
5.根据权利要求4的体细胞、组织和器官,其特征在于,所述体细胞、组织和器官包含:SEQ ID NO:9所示的如下序列:GTTTTTTTCTTACTTCCGAGGAGTGGAGGTCACTGACAATGCCCTTGTCAGTGCATAACGTCTACCCAGTAGGGGAAGATTACTATGCCTGCACGGAGACCAACTTCATTACAAAGATTAATCCTGAGACCCTGGAGACAATTAAGCAG。
6.根据权利要求3的体细胞、组织和器官,其特征在于所述体细胞、组织和器官包含:SEQ ID NO:6所示的如下序列:CCCTTGTTTAACGTCTACCCAGTA,和/或SEQ ID NO:7所示的如下序列:CCCTTGTAACGTCTACCCAGTA。
7.根据权利要求3的体细胞、组织和器官,其特征在于,所述体细胞、组织和器官包含:SEQ ID NO:8所示的如下序列:CCCTTGCATTAACGTC TACCCAGTA。
8.根据权利要求3的体细胞、组织和器官,其特征在于所述体细胞为分类命名为比格犬成纤维细胞:Rpe65-KO-190815,其保藏在中国微生物菌种保藏管理委员会普通微生物中心(CGMCC),保藏地址为:北京市朝阳区北辰西路1号院3号(邮政编码100101),保藏号为CGMCCNo.19945,保藏日期为2020年6月3日。
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