CN114958847A - 一种rhob基因点突变的人源脑瘫兔模型及构建方法 - Google Patents
一种rhob基因点突变的人源脑瘫兔模型及构建方法 Download PDFInfo
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Abstract
本发明公开了一种RHOB基因点突变的人源脑瘫兔模型,涉及利用CRISPR‑CAS9构建人源脑瘫兔模型,同时和公开了其构建方法,能够有效模拟人RHOB S73F型脑瘫病理过程,有助于探究RHOB基因突变对于动物大脑及神经发育的具体调控作用及影响因素,增强对RHOB生物学功能的了解,为临床上预防和诊疗脑瘫疾病奠定相关的理论基础,提供新的思路和方法;能更有效地测试新药和新诊断标记物等在临床应用中的效果,同时大大降低新药研发的风险,为临床研究提供基础模型。
Description
技术领域
本发明公开了一种RHOB基因点突变的人源脑瘫兔模型,涉及利用CRISPR-CAS9构建人源脑瘫兔模型,同时和公开了其构建方法,属于人类病症模型构建技术领域。
背景技术
脑瘫是影响运动功能的主要神经发育障碍,全世界每1000名儿童中约有2到3人受到影响。运动障碍,包括痉挛、肌张力障碍、舞蹈症、共济失调,可能在出生后的最初几年内发作,并可见大脑发育紊乱。其致病因素可能与单基因突变有关,如RHOB基因的突变位点(p.S73F),其他一些因素,如早产、感染、缺氧缺血以及围产期前和围产期中风也可能导致疾病的发生。
RHOB,既Ras蛋白同源家族成员B,由196个氨基酸组成,大约20 kDa。其在调节细胞增殖、运动和迁移、细胞骨架重排、细胞极性、神经突起延伸和收缩、肿瘤发生等领域均具有重要作用,其信号的改变可导致恶性转化、神经异常和免疫性疾病。RHOB基因 (p.S73F)突变患者出现了包括脑室周围白质软化、痉挛性-张力障碍性双瘫、表达性语言障碍等异常。
发明内容
本发明提供一种RHOB基因点突变的人源脑瘫兔模型及构建方法,能够有效模拟人RHOB S73F型脑瘫病理过程。
本发明提供了一对用于构建RHOB基因点突变兔模型的寡聚核苷酸链,其特征在于:
在RHOB基因靶位点处设计1个sgRNA序列,合成一对寡聚核苷酸链制备sgRNA表达载体,寡聚核苷酸序列为:
sgRNA-F:TAGGGCTCTCCTACCCGGACACCGA;
sgRNA-R:AAACTCGGTGTCCGGGTAGGAGAGC;
本发明公开的一种RHOB基因点突变的人源脑瘫兔模型及构建方法,技术解决方案如下:
1)sgRNA表达载体的构建:
将上述合成的寡聚核苷酸链经退火形成双链,使用BbsⅠ限制性核酸内切酶将PUC57载体线性化,随后将酶切产物进行纯化回收,并将退火的sgRNA连接到PUC57载体上,进而完成PUC57-sgRNA载体的构建;
2)Cas9mRNA的合成: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;
酶切37℃3h,核酸电泳后,使用普通DNA琼脂糖胶回收试剂盒进行回收;
3)受精卵的获取和显微注射:注射***,之后注射人绒毛膜***,获取受精卵,通过显微注射仪器将预混好Cas9mRNA与sgRNA混合物注射到受精***质中,其中Cas9mRNA浓度为150ng/μl,sgRNA浓度为30ng/μl;
4)受精卵的培养和发育: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,待其发育至桑椹胚时期时,用吸卵针将单个胚胎转移到离心管中;
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:
利用CRISPR-CAS9构建RHOB基因点突变兔模型,能够有效模拟人RHOB S73F型脑瘫病理过程,有助于探究RHOB基因突变对于动物大脑及神经发育的具体调控作用及影响因素,增强对RHOB生物学功能的了解, 为临床上预防和诊疗脑瘫疾病奠定相关的理论基础,提供新的思路和方法;能更有效地测试新药和新诊断标记物等在临床应用中的效果,同时大大降低新药研发的风险,为临床研究提供基础模型。
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F0代RHOB点突变兔模型基因突变情况的sanger测序图;从DNA测序结果可得到:F0代个体均发生不同的突变情况;
图3是F0代RHOB点突变兔模型经鉴定后,将正常对照组和突变组分别记录并统计其在生长过程中体重变化情况;该图为正常对照组和突变组兔生长曲线,从图上可以看出突变组在生长发育过程中出现发育迟缓的现象;
图4-5是F0代RHOB点突变兔模型经鉴定后,将正常对照组和突变组拍摄步态足迹与行进姿势得到;可以看到突变组存在显著运动功能障碍;
图6是F0代RHOB点突变兔模型经鉴定后,将正常对照组和突变组拍核磁共振成像得到;可以看到突变组存在大脑发育异常。
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
(1)构建RHOB基因人源化点突变兔脑瘫疾病模型
1)CRISPR/Cas9***sgRNA设计和表达载体的构建
在RHOB基因靶位点处设计1个sgRNA序列,如图1、SEQNO.1及SEQNO.2所示,合成一对寡聚核苷酸链用于制备sgRNA:
sgRNA-F:TAGGGCTCTCCTACCCGGACACCGA;
sgRNA-R:AAACTCGGTGTCCGGGTAGGAGAGC;
该sgRNA的寡聚核苷酸链选取原则:选取突变碱基位置在5或6位的一条寡聚核苷酸链。合成的寡聚核苷酸经退火(95℃ 5min后置于室温冷却),连入经BbsⅠ酶切后回收的PUC57-sgRNA表达载体,完成sgRNA载体构建,如SEQNO.3所示,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备用,以作为体外转录模板。
酶切体系:质粒PUC57 20μl;
10×buffer 20μl;
BbsⅠ 1μl;
ddH2O 159μl;
酶切37℃ 3h,核酸电泳后,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,核酸电泳后,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***(FSH),之后注射人绒毛膜***(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与sgRNA混合物注射到受精***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,待其发育至桑椹胚时期时,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RHOB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,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,核酸电泳鉴定,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:GGTGAGCAGTGAGCGAAG;
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5min;95℃变性30s,58℃退火30s,72℃延伸30s;35个循环;72℃延伸5min;
③PCR产物进行测序,测序结果在RHOB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,样本则为基因突变。测序结果在RHOB基因引物设计的打靶位点附近出现双峰的情况,选择双峰的样品再次PCR,产物胶回收后连接至PGM-T载体,转化后挑取阳性克隆再次进行测序,测序结果中在RHOB基因靶位点附近发生碱基***或碱基缺失,导致阅读框移码突变,样本则为基因敲除;
5)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***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
RHOB基因人源化点突变兔脑瘫疾病模型基因型分析与表型鉴定
1)DNA测序鉴定兔脑瘫疾病模型的基因型
提取组织DNA,提取方法按照组织基因组提取试剂盒说明书进行操作(天根,北京,中国),进行PCR,核酸电泳鉴定,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:GGTGAGCAGTGAGCGAAG;
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5min;95℃变性30s,58℃退火30s,72℃延伸30s;35个循环;72℃延伸5min;
PCR产物进行测序,测序结果在RHOB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,样本则为基因突变。测序结果在RHOB基因引物设计的打靶位点附近出现双峰的情况,选择双峰的样品再次PCR,产物胶回收后连接至PGM-T载体,转化后挑取阳性克隆再次进行测序,测序结果中在RHOB基因靶位点附近发生碱基***或碱基缺失,导致阅读框移码突变,样本则为基因敲除;
如图2所示,得到了RHOBS73F/S73F、RHOBS73F/+、RHOBS73C/+、RHOBΔ1/+、RHOBΔ21/+等包括基因突变与基因敲除在内的多种F0代基因编辑兔;
2)兔体重结果采集
分别于出生后4-17周,每周测定正常兔与突变兔的体重,如图3所示,可见RHOBS73F /S73F兔存在发育迟缓;
3)兔健康状况监测
观察兔头部、骨骼、四肢等重要部位或组织是否发生病变;观察兔日常活动、行进过程是否存在运动功能障碍;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;如图4-5所示,可见RHOBS73F/S73F兔存在运动功能障碍;
4)兔影像学分析
在第12周,对兔子麻醉,进行核磁共振成像,获取兔脑部影像学照片,进行相应结果分析,如图6所示,可见RHOBS73F/S73F兔存在大脑发育异常。
结论:本发明成功构建RHOB(p.S73F)碱基编辑兔模型,其具有典型的脑瘫症状,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RHOB基因点突变的人源脑瘫兔模型及构建方法
<160> 3
<170> SIPOSequenceListing 1.0
<210> 1
<211> 591
<212> DNA
<213> 兔(Oryctolagus cuniculus)
<400> 1
atggcggcca tccgcaagaa gctggtggtg gtgggcgacg gcgcgtgcgg caagacgtgc 60
ctgctgatcg tgttcagcaa ggacgagttc cccgaggtgt acgtgcccac cgtcttcgag 120
aactatgtgg ccgacatcga ggtggacggc aagcaggtgg agctggcgct gtgggacaca 180
gcgggccagg aggactacga ccgcctgcgg ccgctctcct acccggacac cgacgtcatc 240
ctcatgtgct tctcggtgga cagcccggac tcgctggaga acatccccga gaagtgggtg 300
ccggaggtga aacacttttg ccccaacgtg cccatcatcc tggtggccaa caagaaggac 360
ctgcgcagcg acgagcacgt ccgcacagag ctggcgcgca tgaagcagga gcccgtgcgc 420
acggacgacg gccgcgccat ggccgtgcgc atccaagcct acgactacct cgagtgctcc 480
gccaagacca aggagggcgt gcgcgaggtc ttcgagacgg ccacgcgcgc cgcgctccag 540
aagcgctacg gctcgcagaa cggctgcatc aactgctgca aggtgctatg a 591
<210> 2
<211> 486
<212> DNA
<213> 兔(Oryctolagus cuniculus)
<400> 2
tcctgctgcc cgggcccggc cggctcatgg cggccatccg caagaagctg gtggtggtgg 60
gcgacggcgc gtgcggcaag acgtgcctgc tgatcgtgtt cagcaaggac gagttccccg 120
aggtgtacgt gcccaccgtc ttcgagaact atgtggccga catcgaggtg gacggcaagc 180
aggtggagct ggcgctgtgg gacacagcgg gccaggagga ctacgaccgc ctgcggccgc 240
tctcctaccc ggacaccgac gtcatcctca tgtgcttctc ggtggacagc ccggactcgc 300
tggagaacat ccccgagaag tgggtgccgg aggtgaaaca cttttgcccc aacgtgccca 360
tcatcctggt ggccaacaag aaggacctgc gcagcgacga gcacgtccgc acagagctgg 420
cgcgcatgaa gcaggagccc gtgcgcacgg acgacggccg cgccatggcc gtgcgcatcc 480
aagcct 486
<210> 3
<211> 1073
<212> DNA
<213> 兔(Oryctolagus cuniculus)
<400> 3
cagtgattgg agatcggtac ttcgcgaatg cgtcgagata ttgggtcttt aaaagcaccg 60
actcggtgcc actttttcaa gttgataacg gactagcctt attttaactt gctatttcta 120
gctctatcgg tgtccgggta ggagagccta tagtgagtcg tattaattgg gtatcggatg 180
ccgggaccga cgagtgcaga ggcgtgcaag cgagcttggc gtaatcatgg tcatagctgt 240
ttcctgtgtg aaattgttat ccgctcacaa ttccacacaa catacgagcc ggaagcataa 300
agtgtaaagc ctggggtgcc taatgagtga gctaactcac attaattgcg ttgcgctcac 360
tgcccgcttt ccagtcggga aacctgtcgt gccagctgca ttaatgaatc ggccaacgcg 420
cggggagagg cggtttgcgt attgggcgct cttccgcttc ctcgctcact gactcgctgc 480
gctcggtcgt tcggctgcgg cgagcggtat cagctcactc aaaggcggta atacggttat 540
ccacagaatc aggggataac gcaggaaaga acatgtgagc aaaaggccag caaaaggcca 600
ggaaccgtaa aaaggccgcg ttgctggcgt ttttccatag gctccgcccc cctgacgagc 660
atcacaaaaa tcgacgctca agtcagaggt ggcgaaaccc gacaggacta taaagatacc 720
aggcgtttcc ccctggaagc tccctcgtgc gctctcctgt tccgaccctg ccgcttaccg 780
gatacctgtc cgcctttctc ccttcgggaa gcgtggcgct ttctcatagc tcacgctgta 840
ggtatctcag ttcggtgtag gtcgttcgct ccaagctggg ctgtgtgcac gaaccccccg 900
ttcagcccga ccgctgcgcc ttatccgggt aactatcgtc ttgagtccaa cccggtaaga 960
cacgacttat cgccactggc agcagccact ggtacaggat tagcagagcg agtatgtaag 1020
cgttgctaca gagttcttga agtggtgcct aactacgggc ttaccactaa gga 1073
Claims (3)
1.一对用于构建RHOB基因点突变的人源脑瘫兔模型的寡聚核苷酸链,其特征在于:
在RHOB基因靶位点处设计1个sgRNA序列,合成一对寡聚核苷酸链制备sgRNA表达载体,寡聚核苷酸序列为:
sgRNA-F:TAGGGCTCTCCTACCCGGACACCGA;
sgRNA-R:AAACTCGGTGTCCGGGTAGGAGAGC。
2.一种RHOB基因点突变的人源脑瘫兔模型,其特征在于:利用CRISPR/Cas9技术突变RHOB基因p.S73F位点构建而成。
3.一种RHOB基因点突变的人源脑瘫兔模型的构建方法,包括以下步骤:
1)sgRNA表达载体的构建:
将权利要求1合成的寡聚核苷酸链经退火形成双链,使用BbsⅠ限制性核酸内切酶将PUC57载体线性化,随后将酶切产物进行纯化回收,并将退火的sgRNA连接到PUC57载体上,进而完成PUC57-sgRNA载体的构建;
2)Cas9mRNA的合成:
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;酶切37℃3h,核酸电泳后,使用普通DNA琼脂糖胶回收试剂盒进行回收;
3)受精卵的获取和显微注射:
注射***,之后注射人绒毛膜***,获取受精卵,通过显微注射仪器将预混好Cas9mRNA与sgRNA混合物注射到受精***质中;
4)受精卵的培养和发育:
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,待其发育至桑椹胚时期时,用吸卵针将单个胚胎转移到离心管中;
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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Non-Patent Citations (4)
Title |
---|
SHENG CHIH JIN1: "Mutations disrupting neuritogenesis genes confer risk for cerebral palsy", NAT GENET., vol. 52, no. 10, 31 October 2020 (2020-10-31), pages 3 * |
ZHANG D等: "NCBI Reference Sequence: NP_004031.1", Retrieved from the Internet <URL:https://www.ncbi.nlm.nih.gov/protein/4757764?sat=50&satkey=72349677> * |
刘亭君: "DMP1 敲除兔低血磷性佝偻病模型的建立", 中国优秀硕士学位论文全文数据库,医药卫生科技辑, 15 January 2019 (2019-01-15), pages 1 * |
孙晗笑: "药物分子毒理学", 30 November 2012, 暨南大学出版社, pages: 185 - 187 * |
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