CN114958848A - 一种人原发性小头畸形症兔模型及构建方法 - Google Patents
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Abstract
本发明公开了一种人原发性小头畸形症兔模型,同时还提供其建立方法,利用YIPF5基因突变构建模型,采用CRISPR‑CAS9基因突变技术构建兔YIPF5基因突变模拟人原发性小头畸形症的模型,该模型的获得能够有效地模拟人类疾病的病理过程,有助于探究YIPF5基因突变对于哺乳动物头部和神经发育的具体调控作用,能更有效地测试新药和新诊断试剂等在临床应用中的效果,为临床研究提供动物模型。
Description
技术领域
本发明公开了一种人原发性小头畸形症兔模型,同时还提供其建立方法,利用YIPF5基因突变构建模型,属于人类病症模型构建技术领域。
背景技术
人原发性小头畸形症是一种罕见的基于常染色体隐性遗传模式的神经发育障碍,其致病因素一般与单基因突变有关,其他一些因素,如Zika病毒感染、酒精刺激、孕期服用药物等也可能导致疾病的发生。目前的发病率从1.3-150/10万不等。患者临床表现为:患者大脑结构正常但头颅整体体积较小;患者出生时枕额头围比正常人低 2-3 个标准差;患者的大脑体积和大脑皮层的表面积明显较小。患者发病特点包括发育迟缓、智力障碍、癫痫及多动症等。
人类疾病的病症模型是疾病机理研究和新药研发的重要基础。因此建立人类疾病模型,有效地模拟人类疾病的病理过程,能更有效地测试新药和新诊断试剂等在临床应用中的效果,同时大大降低新药研发的风险。
发明内容
本发明提供一种人原发性小头畸形症兔模型的建立方法,是采用CRISPR-CAS9基因突变技术构建兔YIPF5基因突变模拟人原发性小头畸形症的方法。
本发明提供一种使YIPF5基因发生点突变的sgRNA,能够用于人原发性小头畸形症兔模型的建立,其特征在于:
所述sgRNA作用是将YIPF5基因的第218位W突变为R,制备具有YIPF5基因点突变的动物模型;其中,在所述的兔模型中,YIPF5基因点突变是纯合型。
所述sgRNA作用位点位于兔YIPF5基因的第6外显子,如序列表SEQNO.1所示。该sgRNA的核苷酸序列如序列表SEQNO.3下划线处所示。
在YIPF5基因第6个外显子处设计1个sgRNA序列作用靶点,序列如下:
sgRNA-F:ACACCATCCAATAATCCCAG;
sgRNA-R:CTGGGATTATTGGATGGTGT。
本发明提供的一种人原发性小头畸形症兔模型的方法,包括以下步骤:采用CRISPR-CAS9基因突变技术利用YIPF5基因突变构建而成,
1)CRISPR-CAS9和sgRNA表达载体的构建
将上述的一对寡聚核苷酸链,连接到酶切后的PUC57载体上,进而完成PUC57-sgRNA载体的构建;
CAS9表达质粒经酶切线性化,经酚氯仿抽提纯化后,溶于无核酸酶的水中作为模板,用于体外转录;CAS9mRNA的合成由试剂盒RNeasy Mini Kit(Qiagen,No.74104)在体外作用T7RNA聚合酶来完成,sgRNA的体外合成由试剂盒MiRNeasy Mini Kit(Qiasgen,No.217004)在体外利用T7RNA聚合酶完成;
2)受精卵的获取和显微注射
给母兔注射卵泡刺激素(FSH),之后注射人绒毛膜促性腺激素(HCG)(购于宁波第二激素厂),获取受精卵,通过显微注射仪器将预混好CAS9mRNA/sgRNA混合物注射到细胞质中 (CAS9mRNA终浓度为150ng/μl,sgRNA终浓度为30ng/μl);
3)受精卵的体外培养和发育
将显微注射的受精卵转移到培养液中,置于37℃恒温培养箱中培养,发育到桑椹胚时期时,用吸卵针将单个胚胎转移到离心管中;
4)胚胎YIPF5基因突变情况鉴定
(1)胚胎裂解
胚胎裂解试剂为NP40,裂解条件为:56℃ 1h; 95℃ 10min;
(2)DNA测序鉴定胚胎基因型情况
提取DNA,提取方法按照组织基因组提取试剂盒说明书进行操作(购于天根公司,北京,中国),进行PCR,电泳鉴定,并进行DNA测序,得到基因型鉴定结果;
5)胚胎移植和模型种群的获得
将胚胎移植到适龄母兔的输卵管内,待其自然生产,获得基因编辑动物模型。 利用PCR及测序方法进行遗传鉴定。筛选纯合突变个体,并对其遗传及表型稳定性进行监测鉴定,表型稳定的疾病模型进行集中扩繁,获得可稳定传代的模型种群。
本发明的积极效果在于:
采用CRISPR-CAS9基因突变技术构建兔YIPF5基因突变模拟人原发性小头畸形症的模型,该模型的获得能够有效地模拟人类疾病的病理过程,有助于探究YIPF5基因突变对于哺乳动物头部和神经发育的具体调控作用,能更有效地测试新药和新诊断试剂等在临床应用中的效果,为临床研究提供动物模型。
附图说明
图1是本发明sgRNA的设计示意图;
图2是本发明PCR产物鉴定胚胎YIPF5基因突变情况的电泳图;
其中M:D2000为DNA分子标准量;1-9:显微注射后9个胚胎DNAPCR结果;10:阳性对照(正常胚胎);
图3是本发明PCR产物鉴定胚胎YIPF5基因突变情况的sanger测序图;
设计的YIPF5基因的鉴定引物为524bp,从DNA测序结果可得到:1,2,3,4胚胎均发生不同的突变情况;
图4是显微注射后得到的新生小兔经鉴定后,将对照组和突变组分别记录并统计其在生长过程中头围、体长和体重的情况;该图为正常组和突变组兔生长曲线,从图上可以发现突变组在生长发育过程中出现发育迟缓的现象;
图5是显微注射后得到的新生小兔经鉴定后,将对照组和突变组拍摄X光后得到;可以发现突变组头围明显低于正常组,并且发育迟缓。
具体实施方式
通过以下实施例进一步举例描述本发明,并不以任何方式限制本发明,在不背离本发明的技术解决方案的前提下,对本发明所作的本领域普通技术人员容易实现的任何改动或改变都将落入本发明的权利要求范围之内。
实施例1
本发明建立YIPF5基因突变原发性小头畸形症模型,前期准备和胚胎验证步骤如下:
1)CRISPR-CAS9系统sgRNA设计和表达载体的构建
设计在YIPF5基因第6个外显子处设计1个sgRNA序列作用靶点(如图1所示),合成一对寡聚核苷酸链:
sgRNA-F:ACACCATCCAATAATCCCAG;
sgRNA-R:CTGGGATTATTGGATGGTGT);
该sgRNA的寡聚核苷酸链选取原则:选取突变碱基位置在6位的一条寡聚核苷酸链;合成的寡聚核苷酸经退火(95℃ 5min后自然降至室温),连入经BbsⅠ酶切经回收PUC57-sgRNA表达载体,完成sgRNA载体构建,通过测序验证片段连接正确,进行克隆,扩大培养后提取质粒用于准备体外转录模板;
酶切体系:质粒PUC57:20μl、10×buffer:20μl、BbsⅠ:1μl、ddH2O:159μl;
酶切37℃ 3h,电泳跑胶后,使用普通DNA琼脂糖胶回收试剂盒(购于天根公司,北京,中国)进行回收,具体操作按说明书进行;
CAS9表达质粒(Addgene,实验室购买),经酶切线性化,经酚氯仿抽提纯化后,溶于无核酸酶的水中作为模板,用于体外转录。CAS9mRNA的合成由试剂盒RNeasy Mini Kit(Qiagen,No.74104)在体外作用T7RNA聚合酶来完成,sgRNA的体外合成由试剂盒MiRNeasyMini Kit(Qiasgen,No.217004)在体外利用T7RNA聚合酶完成;
酶切体系:NotⅠ:4μl、CAS9:50μl、BSA:30μl、Triton:30μl、10×H:30μl、ddH2O :156μl;
酶切37℃ 3h,电泳跑胶后,使用普通DNA琼脂糖胶回收试剂盒(购于天根公司,北京,中国)进行回收,具体操作按说明书进行;
2)受精卵的获取和显微注射:
注射卵泡刺激素(FSH),之后注射人绒毛膜促性腺激素(HCG)(购于宁波第二激素厂),获取受精卵,通过显微注射仪器将预混好CAS9mRNA/sgRNA混合物注射到细胞质中(CAS9mRNA终浓度为150ng/μl,sgRNA终浓度为30ng/μl);
3)受精卵的体外培养和发育
将显微注射的受精卵转移到培养液中,置于37℃恒温培养箱中培养,发育到桑椹胚时期时,用吸卵针将单个胚胎转移到离心管中,用于后面实验;
4)胚胎YIPF5基因突变情况鉴定
(1)胚胎裂解
胚胎裂解试剂为NP40,裂解条件为:56℃ 1h;95℃ 10min;
(2)DNA测序鉴定胚胎基因型突变情况:
提取DNA,提取方法按照组织基因组提取试剂盒说明书进行操作(购于天根公司,北京,中国),进行PCR,电泳鉴定(如图2所示),并进行DNA测序,得到基因型鉴定结果;
①胚胎裂解:胚胎裂解试剂为NP40,裂解条件为:56℃,1h;95℃,10min;
②DNA测序鉴定胚胎基因型突变情况:提取DNA,进行PCR,电泳鉴定,并进行DNA测序,得到基因型鉴定结果;
a、设计PCR引物如下:
上游引物:CACGGCTCCAATCGTAAAC;
下游引物:GAGAAACCCTGTATTCACAAAGA;
b、PCR反应体系如下:
模板DNA 1ul;
上游引物 1ul;
下游引物 1ul;
2×Taq plus 12.5ul;
ddH2O 9.5ul;
c、PCR反应条件:
95℃预变性7min;94℃变性30s,58℃退火30s,72℃延伸40s;30个循环;72℃延伸5min;
③PCR产物进行测序,测序结果在YIPF5基因引物设计的打靶位点出现完全突变或者不完全突变的情况,选择位点完全突变或者不完全突变的样品则为基因突变。
实施例2
建立YIPF5基因突变原发性小头畸形症模型,步骤如下:
1)受精卵的获取和显微注射
注射卵泡刺激素(FSH),之后注射人绒毛膜促性腺激素(HCG)(购于宁波第二激素厂),获取受精卵,通过显微注射仪器将实施例1中构建并胚胎验证过确定浓度的CAS9mRNA/sgRNA混合物注射到细胞质中 (CAS9mRNA终浓度为150ng/μl,sgRNA终浓度为30ng/μl);
2)胚胎移植
将注射的受精卵移植到同期发情的适龄母兔两侧输卵管内,每侧约20枚,对代孕母兔进行规范化饲养,提供合理充足的饮食与稳定清洁的饲养环境,待其妊娠结束自然生产,获得F0代基因编辑动物模型。
验证例1
兔原发性小头畸形模型表型鉴定和基因型分析:
1)通过鉴定(如图3所示),获得了YIPF5-W218R点突变纯、杂合两种基因型的兔,以进一步研究YIPF5-W218R在小头畸形方面的表型:
2)兔头围、体长和体重结果采集
分别于出生后1周、2周、4周、6周、8周、10周、12周测定正常兔与突变兔的头围、体长和体重(如图4所示);
3)观察兔头部、骨骼、四肢等重要部位或组织是否发生病变。突变兔在生长过程中,出现死亡的个体,解剖观察各器官包括心脏、肝脏、脾脏、肾脏、肺脏病变情况,固定组织,做组织病理切片;
4)兔影像学分析:
在第9周,对兔子麻醉,进行X-射线,获取兔脑部影像学照片(如图5所示),进行相应结果分析。
结论:
本发明成功获得了YIPF5(p.W218R)基因突变兔模型,其从出生开始就表现出与WT相比头围较小、发育迟缓等一系列典型的人原发性小头畸形症症状,与人类临床病例结果相一致,本发明构建的模型准确可靠。
序列表
<110> 吉林大学
<120> 一种人原发性小头畸形症兔模型及构建方法
<160> 3
<170> SIPOSequenceListing 1.0
<210> 1
<211> 163
<212> DNA
<213> 兔(Oryctolagus cuniculus)
<400> 1
aggtatggta ggagtcattc tcaccgctgg gattattgga tggtgtagtt tttctgcttc 60
caaaattttt atttctgcgt tagccatgga aggacagcag cttttagtgg catatccatg 120
tgctttgtta tatggagtct ttgccctaat ttccgtcttt tga 163
<210> 2
<211> 524
<212> DNA
<213> 兔(Oryctolagus cuniculus)
<400> 2
cacggctcca atcgtaaacg taaatctgca tcagagtagc gcggcagcag cttgctggtc 60
caatttaaga gttcaaggtc ctttatgtgt atttggccca ctgatgtcca tgtcccaggt 120
aaattcagtt caaaagacgg aaattagggc aaagactcca tataacaaag cacatggata 180
tgccactaaa agctgctgtc cttccatggc taacgcagaa ataaaaattt tggaagcaga 240
aaaactacac catccaataa tcccagcggt gagaatgact cctaccatac ctctgaaacc 300
atcaaagaaa agggggagaa agaagagctg tgtaagaaca cttcaaaatc ctctttgggg 360
gaaaaaaagt attttaaatt cagatgtaga catatgcaga aataatcaag agggttttca 420
cacgtcaaaa tgcacctctc agtgacttca gggatgctga gatttcttgg gataaaaagc 480
tcactaacag tgacattgtg ctctttgtga atacagggtt tctc 524
<210> 3
<211> 848
<212> DNA
<213> 兔(Oryctolagus cuniculus)
<400> 3
catggtcata gctgtttcct gtgtgaaatt gttatccgct cacaattcca cacaacatac 60
gagccggaag cataaagtgt aaagcctggg gtgcctaatg agtgagctaa ctcacattaa 120
ttgcgttgcg ctcactgccc gctttccagt cgggaaacct gtcgtgccag ctgcattaat 180
gaatcggcca acgcgcgggg agaggcggtt tgcgtattgg gcgctcttcc gcttcctcgc 240
tcactgactc gctgcgctcg gtcgttcggc tgcggcgagc ggtatcagct cactcaaagg 300
cggtaatacg gttatccaca gaatcagggg ataacgcagg aaagaacatg tgagcaaaag 360
gccagcaaaa ggccaggaac cgtaaaaagg ccgcgttgct ggcgtttttc cataggctcc 420
gcccccctga cgagcatcac aaaaatcgac gctcaagtca gaggtggcga aacccgacag 480
gactataaag ataccaggcg tttccccctg gaagctccct cgtgcgctct cctgttccga 540
ccctgccgct taccggatac ctgtccgcct ttctcccttc gggaagcgtg gcgctttctc 600
atagctcacg ctgtaggtat ctcagttcgg tgtaggtcgt tcgctccaag ctgggctgtg 660
tgcacgaacc ccccgttcag cccgaccgct gcgccttatc cgggtaacta tcgtcttgag 720
tccaacccgg taagacacga cttatcgcca ctggcagcag ccactggtac aggattagca 780
gagcgagtat gtaagcgttg ctacagagtt cttgaagtgg tgcctaacta cgggcttacc 840
actaagga 848
Claims (4)
1.一种使YIPF5基因发生点突变的sgRNA在人原发性小头畸形症兔模型的建立方法中的应用,其特征在于:
所述sgRNA作用是将YIPF5基因的第218位W突变为R,制备具有YIPF5基因点突变的动物模型;其中,在所述的兔模型中,YIPF5基因点突变是纯合型。
2.根据权利要求1所述的应用,其特征在于:
所述sgRNA作用位点位于兔YIPF5基因的第6外显子,如序列表SEQNO.1所示;
该sgRNA的核苷酸序列如序列表SEQNO.3下划线处所示。
3.一种用于建立人原发性小头畸形症兔模型的方法的一对寡聚核苷酸链,其特征在于:
在YIPF5基因第6个外显子处设计1个sgRNA序列作用靶点,序列如下:
sgRNA-F:ACACCATCCAATAATCCCAG;
sgRNA-R:CTGGGATTATTGGATGGTGT。
4. 一种人原发性小头畸形症兔模型的方法,其特征在于采用CRISPR-CAS9基因突变技术利用YIPF5基因突变构建而成,包括以下步骤:
1)CRISPR-CAS9和sgRNA表达载体的构建
将权利要求3所说的一对寡聚核苷酸链,连接到酶切后的PUC57载体上,进而完成PUC57-sgRNA载体的构建;
CAS9表达质粒经酶切线性化,经酚氯仿抽提纯化后,溶于无核酸酶的水中作为模板,用于体外转录;CAS9mRNA的合成由试剂盒RNeasy Mini Kit在体外作用T7RNA聚合酶来完成,sgRNA的体外合成由试剂盒MiRNeasy Mini Kit在体外利用T7RNA聚合酶完成;
2)受精卵的获取和显微注射
给母兔注射卵泡刺激素,之后注射人绒毛膜促性腺激素,获取受精卵,通过显微注射仪器将预混好CAS9mRNA/sgRNA混合物注射到细胞质中;
3)受精卵的体外培养和发育
将显微注射的受精卵转移到培养液中,置于37℃恒温培养箱中培养,发育到桑椹胚时期时,用吸卵针将单个胚胎转移到离心管中;
4)胚胎YIPF5基因突变情况鉴定
(1)胚胎裂解
胚胎裂解试剂为NP40,裂解条件为:56℃ 1h; 95℃ 10min;
(2)DNA测序鉴定胚胎基因型情况
提取DNA,提取方法按照组织基因组提取试剂盒说明书进行操作,进行PCR,电泳鉴定,并进行DNA测序,得到基因型鉴定结果;
5)胚胎移植和模型种群的获得:将胚胎移植到适龄母兔的输卵管内,待其自然生产,获得基因编辑动物模型; 利用PCR及测序方法进行遗传鉴定;筛选纯合突变个体,并对其遗传及表型稳定性进行监测鉴定,表型稳定的疾病模型进行集中扩繁,获得可稳定传代的模型种群。
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