CN114946766A - 一种构建先天性高胰岛素血症模型的方法及模型 - Google Patents
一种构建先天性高胰岛素血症模型的方法及模型 Download PDFInfo
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Abstract
本发明公开了一种构建先天性高胰岛素血症模型的方法及模型,通过对kcnj11基因P.H259R位点及P.H296R位点进行共同突变打靶,胚胎注射后进行胚胎移植,获得兔先天性高胰岛素血症模型,利用SpRY‑ABEmax基因编辑技术优势是没有识别PAM区域的序列限制,可以完成指定位点的突变顺利进行;克服了传统的CRISPR/Cas9基因编辑技术需要识别PAM区域为NGG碱基序列,大部分突变位点无法进行编辑;本发明所利用的SpRY‑ABEmax碱基编辑技术在家兔上进行应用,能够有助于基础医学研究及机制探究。
Description
技术领域
本发明公开了一种构建先天性高胰岛素血症模型的方法及模型,利用基因编辑技术进行kcnj11基因双位点共同突变方法,构建兔先天性高胰岛素血症模型,属于生物技术领域。
背景技术
先天性高胰岛素性低血糖血症(CHI)又称婴儿持续性高胰岛素血症性低血糖症等,是一种遗传异质性内分泌疾病。发病率为1/50000~1/30000,在近亲婚配的群体中发生率较高,约为1/2500。本病以胰岛素过量分泌或不受血糖调控和反复发作的严重低血糖为主要特征,是新生儿期和婴儿早期严重和持续性低血糖的常见原因。治疗以药物为主,如药物治疗无效可应用外科治疗。预后与分型及严重程度有关。且有一定的遗传倾向。ATP敏感性钾通道编码基因突变是关键病因,如KCNJ11基因。但尚有近50%CHI病因不明。CHH活产婴儿中的发病率为1/30 000~1/50 000,在血缘关系较密切的国家和地区(如***地区)CHI的发病率可能高至1/2 500 。因反复发生低血糖,可导致不可逆的中枢神经***损伤,故早期识别、诊断和合理处理十分重要。
kcnj11基因编码ATP敏感性钾通道(KATP)的Kir6.2亚基,是调节胰岛β细胞胰岛素分泌的重要基因。研究表明kcnj11基因P.H259R位点及P.H296R位点突变可导致婴儿持续性高胰岛素血症性低血糖症。然而前期单碱基基因编辑技术有PAM限制及设计要求,而这两个突变位点序列限制而无法完成,因此目前针对该突变位点还没有合适的动物模型。
发明内容
本发明针对现有技术存在的上述不足,提供一种构建先天性高胰岛素血症模型,利用SpRY-ABEmax基因编辑技术双位点共同编辑kcnj11基因P.H259R位点及P.H296R位点构建而成。
本发明公开的一种构建先天性高胰岛素血症模型,其特征在于:利用SpRY-ABEmax基因编辑***介导kcnj11基因P.H259R及P.H296R双位点共同突变构建而成,如序列表SEQNO.1和SEQNO.2所示。
本发明公开的一种构建先天性高胰岛素血症模型的方法,包括以下步骤:
1)sgRNA表达载体的构建:
针对基因P.H259R位点及P.H296R位点分别设计1个sgRNA序列作用靶点,合成两队对寡聚核苷酸链制备sgRNA,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,其寡聚核苷酸为:
sgRNA-1-F:TAGGCACCACGTCATCGACGCCAA;
sgRNA-1-R:AAACTTGGCGTCGATGACGTGGTG;
sgRNA-2-F:TAGGGGGGCATCACCACCCAGGCCC;
sgRNA-2-R:AAACGGGCCTGGGTGGTGATGCCCC;
两条合成的寡聚核苷酸链经退火形成双链,退火条件是:95℃ 5min后自然降至室温,分BbsⅠ限制性核酸内切酶对PUC57载体线性化,随后将酶切产物进行回收,并将退火的sgRNA连接到PUC57载体上,进而完成PUC57-sgRNA载体的构建,如序列表SEQNO.3和SEQNO.4所示;
其中: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进行回收;
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/sgRNA混合物注射到细胞质中,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,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。
本发明还提供了胚胎Kcnj11基因敲除情况鉴定方法:
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,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:ATCATCAGCGCCACCATC;
下游引物:GGAATCCGGAGAGATGCTAAAC;
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;
3、PCR产物进行测序,测序结果在kcnj11基因引物设计的打靶位点出现完全突变或不完全突变的情况,选择位点完全突变或者不完全突变的样品则为基因突变。
本发明的积极效果在于:
公开了一种先天性高胰岛素血症兔模型,通过对kcnj11基因P.H259R位点及P.H296R位点进行共同突变打靶,胚胎注射后进行胚胎移植,获得兔先天性高胰岛素血症模型,利用SpRY-ABEmax基因编辑技术优势是没有识别PAM区域的序列限制,可以完成指定位点的突变顺利进行;克服了传统的CRISPR/Cas9基因编辑技术需要识别PAM区域为NGG碱基序列,大部分突变位点无法进行编辑;本发明所利用的SpRY-ABEmax碱基编辑技术首次在家兔上进行应用,并首次实现双位点共同突变。
附图说明
图1是本发明kcnj11 sgRNA的设计示意图;
图2是本发明PCR产物鉴定胚胎kcnj11基因突变情况的sanger测序图;
图3是显微注射后得到的新生兔模型;
图4是新生兔模型基因型鉴定;
图5是新生兔模型血糖水平检测;
图6是新生兔模型胰岛素表达水平检测;
图7是新生兔模型胰腺组织胰岛素组化方法检测。
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
本发明利用SpRY-ABEmax碱基编辑器成功构建kcnj11基因双位点共同突变先天性高胰岛素性低血糖血症兔模型:
1、SpRY-ABEmax***sgRNA设计和表达载体的构建
针对在kcnj11基因第设计2个sgRNA序列作用靶点,合成两对寡聚核苷酸链用于制备sgRNA;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 20ul;
10×buffer 20ul;
BbsⅠ 1ul;
ddH2O 159ul;
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;
SPRY-ABEmax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;SPRY-ABEmaxmRNA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;
酶切体系:NotⅠ 4 ul;
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***(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/sgRNA混合物注射到细胞质中(CAS9mRNA终浓度为150ng/ ul,sgRNA终浓度为30ng/ ul);
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kcnj11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;
(2)DNA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:TGGCTTGCCTGTCTTTCTT;
下游引物:ATGTCGTTGAGATGGAGGTATTC;
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;
(3)PCR产物进行测序,测序结果在kcnj11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。
实验例1
Kcnj11基因帕金森病兔模型表型鉴定和基因型分析
1)DNA测序鉴定Kcnj11基因帕金森病兔模型的基因型
提取出生兔模型组织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:TGGCTTGCCTGTCTTTCTT;
下游引物:ATGTCGTTGAGATGGAGGTATTC;
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EIF4G1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;
如图4所示,得到了Kcnj11基因突变兔模型;
2)兔模型血糖采集
分别于出生后3,4,5天测定正常兔与突变兔的血糖水平,如图5所示,出生3天后Kcnj11基因突变兔模型血糖值明显降低;
3)兔模型胰岛素表达水平检测
分别于出生后3,4,5天测定正常兔与突变兔的体重,如图6所示,出生3天后Kcnj11基因突变兔模型胰岛素表达水平明显升高;
4)兔模型组织学结果观察
观察兔身体重要部位或组织是否发生病变;兔模型在生长过程中若出现死亡的个体,立即解剖观察胰腺等病变情况,固定组织后进行组织病理切片;如图7所示,兔模型免疫组化结果显示存在胰岛组织存在明显的胰岛素阳性反应。
结论:
本发明成功构建kcnj11基因P.H259R位点及P.H296R位点双位点共同突变兔模型,其具有典型的先天性高胰岛素性低血糖血症症状,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> 一种构建先天性高胰岛素血症模型的方法及模型
<160> 4
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agcccaggta ccgcgcccgt gagcggaggg cccgcttcgt gtccaaaaaa gggaactgta 60
atgtggccca caagaacatc cgcgagcagg ggcgcttcct gcaggacgtc ttcaccacgc 120
tggtggacct caagtggacg cacacgctgc tcatcttcac catgtccttt ctctgcagct 180
ggctgctctt tgccatggtc tggtggctca tcgccttcgc ccacggtgac ctggcgcccg 240
gcgagggcgc cgccgtgccc tgcgtcacca gcatccactc cttttcctcc gccttccttt 300
tctccatcga ggtccaggtg accatcggct tcggcgggcg catggtgacg gaggagtgcc 360
ccctggccat cctgatcctc atcgtgcaga acatcgtagg gctcatgatc aacgccatca 420
tgctgggctg catcttcatg aagaccgccc aggcccaccg gcgcgccgag accctcatct 480
tcagcaagca tgccgtcatc gccctgcgcc aagggcgcct ctgtttcatg ctgcgcgtgg 540
gcgacctgcg caagagcatg atcatcagcg ccaccatcca catgcaggtg gtgcgcaaga 600
cgaccagccc cgagggcgag gtggtgccac tgcaccaggt ggacatcccc atggagaacg 660
gcgtgggcgg caacagcatc ttcctggtgg ccccgctcat catccaccac gtcatcgacg 720
ccaacagtcc actgtatgac ctggcgccca gcgacctgca ccaccaccag gacctggaga 780
tcatcgtcat cctcgagggg gtggtggaaa ccacgggcat caccacccag gcccgcacct 840
cctacctggc agacgagatc ctgtgggggc agcgcttcgt gcccattgtg gccgaggagg 900
acgggcgcta ctccgtggac tactccaagt ttggcaacac cgtgaaagtg cccacgcccc 960
tctgcacggc ccgccagctg gacgaggacc gcagcctgct ggacgccctg acgctcacct 1020
ccgcccgagg acccctacgc aagcgcagcg tgcccgtcgc caaggccaag cccaagttta 1080
gcatctctcc agattccttg tcctga 1106
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atgctgtccc gaaaggggat catcccggag gagtatgtgc tgacgcggct ggccgaggac 60
cccgccgatg ctgtcccgaa aggggatcat cccggaggag tatgtgctga cgcggctggc 120
cgaggacccc gccgagccca ggtaccgcgc ccgtgagcgg agggcccgct tcgtgtccaa 180
aaaagggaac tgtaatgtgg cccacaagaa catccgcgag caggggcgct tcctgcagga 240
cgtcttcacc acgctggtgg acctcaagtg gacgcacacg ctgctcatct tcaccatgtc 300
ctttctctgc agctggctgc tctttgccat ggtctggtgg ctcatcgcct tcgcccacgg 360
tgacctggcg cccggcgagg gcgccgccgt gccctgcgtc accagcatcc actccttttc 420
ctccgccttc cttttctcca tcgaggtcca ggtgaccatc ggcttcggcg ggcgcatggt 480
gacggaggag tgccccctgg ccatcctgat cctcatcgtg cagaacatcg tagggctcat 540
gatcaacgcc atcatgctgg gctgcatctt catgaagacc gcccaggccc accggcgcgc 600
cgagaccctc atcttcagca agcatgccgt catcgccctg cgccaagggc gcctctgttt 660
catgctgcgc gtgggcgacc tgcgcaagag catgatcatc agcgccacca tccacatgca 720
ggtggtgcgc aagacgacca gccccgaggg cgaggtggtg ccactgcacc aggtggacat 780
ccccatggag aacggcgtgg gcggcaacag catcttcctg gtggccccgc tcatcatcca 840
ccgcgtcatc gacgccaaca gtccactgta tgacctggcg cccagcgacc tgcaccacca 900
ccaggacctg gagatcatcg tcatcctcga gggggtggtg gaaaccacgg gcgtcaccac 960
ccaggcccgc acctcctacc tggcagacga gatcctgtgg gggcagcgct tcgtgcccat 1020
tgtggccgag gaggacgggc gctactccgt ggactactcc aagtttggca acaccgtgaa 1080
agtgcccacg cccctctgca cggcccgcca gctggacgag gaccgcagcc tgctggacgc 1140
cctgacgctc acctccgccc gaggacccct acgcaagcgc agcgtgcccg tcgccaaggc 1200
caagcccaag tttagcatct ctccagattc cttgtcctga 1240
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cagtgattgg agatcggtac ttcgcgaatg cgtcgagata ttgggtcttt aaaagcaccg 60
actcggtgcc actttttcaa gttgataacg gactagcctt attttaactt gctatttcta 120
gctctaccta tagtgagtcg tattaattgg gtatcggatg ccgggaccga cgagtgcaga 180
ggcgtgcaag cgagcttggc gtaatcatgg tcatagctgt ttcctgtgtg aaattgttat 240
ccgctcacaa ttccacacaa catacgagcc ggaagcataa agtgtaaagc ctggggtgcc 300
taatgagtga gctaactcac attaattgcg ttgcgctcac tgcccgcttt ccagtcggga 360
aacctgtcgt gccagctgca ttaatgaatc ggccaacgcg cggggagagg cggtttgcgt 420
attgggcgct cttccgcttc ctcgctcact gactcgctgc gctcggtcgt tcggctgcgg 480
cgagcggtat cagctcactc aaaggcggta atacggttat ccacagaatc aggggataac 540
gcaggaaaga acatgtgagc aaaaggccag caaaaggcca ggaaccgtaa aaaggccgcg 600
ttgctggcgt ttttccatag gctccgcccc cctgacgagc atcacaaaaa tcgacgctca 660
agtcagaggt ggcgaaaccc gacaggacta taaagatacc aggcgtttcc ccctggaagc 720
tccctcgtgc gctctcctgt tccgaccctg ccgcttaccg gatacctgtc cgcctttctc 780
ccttcgggaa gcgtggcgct ttctcatagc tcacgctgta ggtatctcag ttcggtgtag 840
gtcgttcgct ccaagctggg ctgtgtgcac gaaccccccg ttcagcccga ccgctgcgcc 900
ttatccgggt aactatcgtc ttgagtccaa cccggtaaga cacgacttat cgccactggc 960
agcagccact ggtacaggat tagcagagcg agtatgtaag cgttgctaca gagttcttga 1020
agtggtgcct aactacgggc ttaccactaa gga 1053
<210> 4
<211> 1053
<212> DNA
<213> 兔(Oryctolagus cuniculus)
<400> 4
cagtgattgg agatcggtac ttcgcgaatg cgtcgagata ttgggtcttt aaaagcaccg 60
actcggtgcc actttttcaa gttgataacg gactagcctt attttaactt gctatttcta 120
gctctaccta tagtgagtcg tattaattgg gtatcggatg ccgggaccga cgagtgcaga 180
ggcgtgcaag cgagcttggc gtaatcatgg tcatagctgt ttcctgtgtg aaattgttat 240
ccgctcacaa ttccacacaa catacgagcc ggaagcataa agtgtaaagc ctggggtgcc 300
taatgagtga gctaactcac attaattgcg ttgcgctcac tgcccgcttt ccagtcggga 360
aacctgtcgt gccagctgca ttaatgaatc ggccaacgcg cggggagagg cggtttgcgt 420
attgggcgct cttccgcttc ctcgctcact gactcgctgc gctcggtcgt tcggctgcgg 480
cgagcggtat cagctcactc aaaggcggta atacggttat ccacagaatc aggggataac 540
gcaggaaaga acatgtgagc aaaaggccag caaaaggcca ggaaccgtaa aaaggccgcg 600
ttgctggcgt ttttccatag gctccgcccc cctgacgagc atcacaaaaa tcgacgctca 660
agtcagaggt ggcgaaaccc gacaggacta taaagatacc aggcgtttcc ccctggaagc 720
tccctcgtgc gctctcctgt tccgaccctg ccgcttaccg gatacctgtc cgcctttctc 780
ccttcgggaa gcgtggcgct ttctcatagc tcacgctgta ggtatctcag ttcggtgtag 840
gtcgttcgct ccaagctggg ctgtgtgcac gaaccccccg ttcagcccga ccgctgcgcc 900
ttatccgggt aactatcgtc ttgagtccaa cccggtaaga cacgacttat cgccactggc 960
agcagccact ggtacaggat tagcagagcg agtatgtaag cgttgctaca gagttcttga 1020
agtggtgcct aactacgggc ttaccactaa gga 1053
Claims (3)
1.一种构建先天性高胰岛素血症模型,其特征在于:利用kcnj11基因P.H259R及P.H296R双位点共同突变构建而成,如序列表SEQNO.1所示和SEQNO.2所示。
2.一种构建先天性高胰岛素血症模型的两对寡聚核苷酸链,其特征在于针对基因P.H259R位点及P.H296R位点分别设计1个sgRNA序列作用靶点,合成两队对寡聚核苷酸链制备sgRNA,其寡聚核苷酸为:
sgRNA-1-F:TAGGCACCACGTCATCGACGCCAA;
sgRNA-1-R:AAACTTGGCGTCGATGACGTGGTG;
sgRNA-2-F:TAGGGGGGCATCACCACCCAGGCCC;
sgRNA-2-R:AAACGGGCCTGGGTGGTGATGCCCC。
3.一种构建先天性高胰岛素血症模型的方法,包括以下步骤:
1)sgRNA表达载体的构建:
针对基因P.H259R位点及P.H296R位点分别设计1个sgRNA序列作用靶点,合成两队对寡聚核苷酸链如权利要求2所述制备sgRNA,两条合成的寡聚核苷酸链经退火形成双链,分BbsⅠ限制性核酸内切酶对PUC57载体线性化,随后将酶切产物进行回收,并将退火的sgRNA连接到PUC57载体上,进而完成PUC57-sgRNA载体的构建,酶切37℃ 3h,电泳跑胶后,使用普通DNA琼脂糖胶回收试剂盒进行回收;
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,电泳跑胶后,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/sgRNA混合物注射到细胞质中;
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,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。
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