CN113215199A - 一种Klotho+/-基因缺失斑马鱼 - Google Patents
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Abstract
本发明利用CRISPR/Cas9技术,构建成了Klotho+/‑基因缺失斑马鱼,具体步骤包括:1)根据Klotho基因序列,确定靶位点;2)制备gRNA;3)体外转录Cas9mRNA;4)将Cas9mRNA和gRNA混合,注射到单细胞期斑马鱼胚胎中;5)将F0胚胎饲养至性成熟;6)与野生型成鱼外交,筛选F0;7)选取可产生有效突变的F0自交,筛选F1。本发明构建的Klotho+/‑基因缺失斑马鱼可用于动脉硬化、骨质疏松症、高磷血症、血管和软组织钙化领域的研究。
Description
技术领域
本发明属于分子生物学领域,具体涉及一种Klotho+/-基因缺失斑马鱼。
背景技术
Klotho(KL)基因是日本研究者Kuro-o在做有关自发型高血压研究时发现的新型基因,随后大量的实验证实其与衰老有关,Klotho缺陷的小鼠出现多器官功能障碍和寿命缩短,显示出类似于人类衰老的症状,包括动脉硬化、骨质疏松症、高磷血症、血管和软组织钙化等。Klotho基因仅存在于脑、甲状旁腺、肾、唾液腺、生殖器官中,不在其他器官表达。斑马鱼Klotho蛋白具有与人和小鼠类似的结构。
斑马鱼模型具有很多优势,如:1.斑马鱼与人类基因同源性高、器官相似度高;2.斑马鱼体外受精,体外发育,胚胎透明,有利于活体观察内部结构和器官;3.斑马鱼一次产卵量大;4.斑马鱼繁殖发育快,生命周期短;5.饲养简单,可大规模诱变育种;6.形体娇小,饲养成本低,操作方便。
发明内容
本发明的目的是构建一种Klotho+/-基因缺失斑马鱼。
本发明的制备过程如下:
本发明靶位点DNA序列为:5’-GATAAGTTTATGTGGGCCGTCGG-3’(Seq No.1)。
本发明提供一种基因打靶试剂盒,所述试剂盒包括两条Oligo序列,其序列为5’-TAGGATAAGTTTATGTGGGCCGT-3’(Seq No.2),5’-AAACACGGCCCACATAAACTTAT-3’(Seq No.3),使用该试剂盒可以用于Klotho基因表达的沉默。
本发明提供了一种基因敲除方法,所述方法的步骤如下:利用所述的Oligo片段识别目的基因的靶位点,与Cas9结合并识别靶位点处PAM序列,引导核酸酶结合到目的基因靶位点处,并开始进行剪切,形成DSB缺口,随后细胞通过非同源末端连接修复机制修复目的基因双链,造成移码突变,最终目的基因被敲除。
进一步的,所述靶点为一个或以上。
进一步的,本发明提供Klotho+/-基因缺失斑马鱼的制备方法,所述制备方法包括:
1.根据Klotho基因序列,确定靶位点;
2.根据靶位点,设计Oligo序列;
3.构建gRNA体外转录载体;
4.PCR获得gRNA体外转录模板;
5.对步骤4获得的模板进行体外转录,得到gRNA;
6.制备Cas9 mRNA的体外转录模板;
7.体外转录Cas9 mRNA;
8.添加polyA序列、回收Cas9 mRNA;
9.将Cas9 mRNA和gRNA混合,注射到单细胞期斑马鱼胚胎中;
10.将F0胚胎饲养至性成熟;
11.与野生型成鱼外交,筛选F0;
12.选取可产生有效突变的F0自交,筛选F1;
本发明的有益效果和优点在于:
本发明构建的Klotho+/-基因缺失斑马鱼为国内外首例。
本发明构建的Klotho+/-基因缺失斑马鱼,可用于动脉硬化、骨质疏松症、高磷血症、血管和软组织钙化领域的研究。
附图说明
图1:克隆骨架pT7-gRNA-BbsⅠ电泳图
图2:菌液PCR鉴定
图3:gRNA体外转录模板
图4:体外转录Cas9 mRNA
图5:BbeⅠ酶切
图6:Klotho基因缺失斑马鱼与野生型序列对比
具体实施方式
下面通过实施例对本发明做进一步详细说明,实施例仅用来说明本发明,并不限制本发明的范围。
实施例1:本发明动物模型的制备
1.实验动物
按标准化方案养殖野生型斑马鱼(TU品系),水温28.5℃,光照/黑暗周期为14h/10h,成体斑马鱼产卵后收集胚胎,养殖于E3孵化液,以受精的小时数(hours postfertilization,hpf)或受精的天数(days post fertilization,dpf)表示胚胎和幼鱼的发育阶段。
2.CRISPR/Cas9基因敲除靶位点设计
在NCBI上查询斑马鱼Klotho基因序列(XM_685705.7,Seq No.4),根据CRISP R/Cas9敲除原理,在http://zifit.partners.org/ZiFiT/CSquare9Nuclease.aspx上设计Klotho靶位点,靶位点包含20个碱基,靶点的选择标准为:5’-GG-(N)18-NGG-3’;其中5’端的GG二核苷酸是T7启动子的一部分,靶位点3’端是NGG。
3.构建gRNA体外转录载体
3.1用BbsⅠ酶切pT7-gRNA、切胶回收(见图1),得到gRNA克隆骨架pT7-gRNA-BbsⅠ,酶切体系如下表1:
表1酶切体系:
3.2根据靶位点订购两条oligo,oligo1序列为5’-TAGGATAAGTTTATGTGGGCCGT-3’(Seq No.2),oligo2序列为5’-AAACACGGCCCACATAAACTTAT-3’(Seq No.3);
3.3用ddH2O分别将oligo溶解为10μM的溶液,退火,得到粘性末端小片段,退火程序见表2;
表2退火程序
3.4将退火后的片段与回收的上述gRNA克隆骨架pT7-gRNA-BbsⅠ连接、转化,挑取克隆,用RV-M与Oligo2作为引物进行菌液PCR鉴定(58℃退火,延伸30sec,30cycle),目的条带约为130bp(见图2),挑取阳性克隆送去测序,选取序列正确的克隆甘油保菌、提质粒,RV-M序列:5’-AGCGGATAACAATTTCACACAGGA-3’(Seq No.5),连接体系见表3;
表3连接体系
4.制备gRNA
4.1用T7-cr fwd和tracr rev引物对,以构建好的gRNA体外转录载体为模板,使用高保真酶PCR,得到gRNA体外转录模板(58℃退火,延伸30sec,40cycle,40μl体系),取1μlPCR产物电泳,确认为单一条带(125bp)后直接回收PCR产物(见图3),用于后续的实验,T7-cr fwd序列:5’-GAAATTAATACGACTCACTATA-3’(Seq No.6),tracr rev序列:5’-AAAAAAAGCACCGACTCGGTGCCAC-3’(Seq No.7);
4.2体外转录gRNA
gRNA体外转录体系见表4;
表4体外转录mRNA反应体系
4.3回收gRNA
4.3.1用RNase-free water将gRNA转录体系稀释到300μl,加入330μl无水乙醇;
4.3.2将溶液加到回收柱中,10000g/min离心15s;
4.3.3加入700μl的miRNA Wash SolutionⅠ,离心5-10s;
4.3.4加入500μl的Wash SolutionⅡ,离心5-10s,重复一次;
4.3.5弃去收集管中的液体,离心1min,去除残余的液体;
4.3.6加入适量95℃预热的RNase-free water,最大转速离心20-30s,收集得到gRN A溶液;
5.制备Cas9 mRNA
5.1制备Cas9 mRNA的体外转录模板:通过XbaⅠ单酶切线性化pSP6-2sNLS-spC as9载体;取少量电泳确认线性化完全后,直接回收线性化产物;
5.2体外转录Cas9 mRNA,mRNA体外转录体系见表5;
表5 Cas9 mRNA体外转录体系
5.3添加polyA序列,回收得到的mRNA(图4)可用于显微注射;
表6 mRNA加polyA的反应体系
6.制备F0代斑马鱼
6.1将Cas9 mRNA和gRNA混合,注射到单细胞斑马鱼胚胎中,同时将未注射的同批胚胎作为对照,Cas9 mRNA 300-500pg,gRNA 25-200pg;
6.2取2-4dpf注射后表型正常的胚胎,提取基因组DNA,PCR、T7E1酶切检测靶位点突变效率(图5);
6.3将能切开的PCR产物测序,检测突变类型;
6.4选取突变效率较高、存活率较高的同批次注射的F0胚胎饲养至性成熟;
6.5与野生型成鱼外交,将3-5个1dpf的F1胚胎混和为一组提取基因组DNA;
6.6通过PCR和酶切检测靶位点突变情况;
6.7将能切开的PCR产物测序,确定突变类型;
6.8选取可产生有效突变的F0鱼交配,大量饲养F1。
7.筛选携带靶位点突变的F1成鱼
7.1将F1饲养至足够大,直至适合剪尾鳍;
7.2F1成鱼剪尾鳍、提基因组,通过PCR和酶切逐条进行基因检测,筛选出F1杂合子,PCR引物为AAGCCTTCCTCTATGACAC(Seq No.8),CCGATAATGACTGACTCCTA(Seq No.9)。
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<120> 一种Klotho+/-基因缺失斑马鱼
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<210> 3
<211> 23
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 3
aaacacggcc cacataaact tat 23
<210> 4
<211> 5246
<212> DNA
<213> Danio rerio
<400> 4
attcagctca ctcctttaac atttacaaca cagtccagtt ctctgacacc atgaaagtga 60
catggattcc tctgccgctc gttttgttct gtcaatttca gggcacagca tctgatccag 120
gagcaggaca gcacacatgg gacacattca gcaaacttcc atatcccgac gacaaagcct 180
tcctctatga cacctttcct gataagttta tgtgggccgt cggcaccgct gcatattcag 240
tggaaggagc ctgggaaaaa gacgggaagg gtaagtcaat ctgggacact tttactcgag 300
gagggacccg agtgtccaga ggagacgttg gtagcgatag ttatcataac atccctggag 360
accttcgagc tcttcagcaa ctaggagtca gtcattatcg gttctccttg tcctggccac 420
gcattttctc caatggcact aaggaaagct acaacgacaa aggtgtggag tattataaaa 480
atctgatccg gggtttgaag gacattaagg tgcagcctgt tgtcactttg tatcattggg 540
acctgcctga cagtttgcag acgctctttg gaggctggag taattcggtt atggtggagc 600
tgttcagaga ctatgccgat ttttgcttta agacatttgg atcggatgtg aaattttgga 660
tcactatcga caatcctttt gtcgtggcct ggcatggata tggaactggg gttgtggctc 720
cgggtatcaa gaatgactct gatttgcctt tcagagtcgg acacaatctg ctgaaggctc 780
acgctgcagc ctggcacctg tatgacgagc gatatcgtgc cgctcagggt gggagagtgt 840
ccatggctct gggatctcac tggatcaaac ccagcagaac cagacaggag agccgcaaag 900
cctgccagcg ctctctaaac tttgtgcttg gctggttcgc ccgtcccttg tttgtagatg 960
gagattatcc accctgcatg aaggacaatc tgacccacag actgccgtcc ttcaccgagg 1020
ccgagagcgc gtatgtgaac ggaaccgccg acttctttgc tctgtctcat ggacctgctc 1080
ttagttttca gctgatcaat gacagtctgc gcttcggcca gactgaagat ttgggtctga 1140
gaatgctttt gtactgggtc cgtgcagagt acaacaatcc tcccatcttt gtggtggaga 1200
gcggctggta tggaagtggc aacaccaaaa caaaggatgc caagcatatg tactatttaa 1260
agcgcttcat tatggagacg ctaaaagcaa tccatgttga cagggtgaat gtgattggct 1320
acacggcctg gtctctgctg gatggatatg agtggtaccg tgaatatgcg atacgcagag 1380
ggctgttcta tgtggatttc aacactcctg atctgaagag agagccgaag gcatctgcca 1440
ctttctatag caaactcata gagaagaatg gcttccctca gctgccagag aaccgccctg 1500
cacacggtgc atttccctgt gactttgctt ggggagtggc tgccaactcc atacaggttg 1560
acactacccc tacgcagttc accgacacta atgtttacat ctggaacatt tctggaaacg 1620
gagagctgaa aaagctccct gggctgcagg cgccccatct gcgcaggaca ccccactgcg 1680
ccgactacgg cagcatccgt cagcaggtgt ctgatctact ccgaatgcaa gtctcccatt 1740
tccatttctc cctcaactgg tcctccatcg tccccacagg ccacgtgtct gatgctaacg 1800
aaacattgct aagatactat tattgttttg tcagcgagct acaaaaagtg aacataaccc 1860
ctgtggtgac tctttggcac catacaggga aactgtccag ccttccagca ccaatggagg 1920
ccagtgatgg ctggcagagc gagaagactg tccaggcctt tgtggactat gctaggttgt 1980
gtttccatcg actaggagct catgtcaagc tctggatcac actcaatgag cccaacgatg 2040
aggatcttga atacactgta ggtcaccagc tgctacgtgc acatgctcta gcttggcacg 2100
tctacgacag agaattccgc aaggcccaag gtggcaaagc atcactggtt cttcatatgg 2160
attgggtcga acctgcattt tcattcaaca gggaggacgt ggcccctgca gaccgagtcc 2220
tggactttcg agtgggatgg tttgcggagc ctattttcgg taaaggcgat tacccggcgg 2280
tgatgcgtag ctggctccaa cagaggaaca ctattgatct tttcaactac cacttaccca 2340
cgttcagcga agaggatcgg ctgttggtga aaggaaccta cgatttcttc gccatcagcc 2400
attttactac ctcgatggtg tacgatggag tggaggataa atacaccttt aaggacaagc 2460
tgcaggttca gcttatatct gacgtgacct ggatcatgtc cccaagacgc aactctcctg 2520
tggttccctg gggtctacgc aaggcattga actgggtgaa ctcgcgatac aaaggtgttc 2580
ccatttacgt gatggccaat ggtgttcaag aggataccgc taggtttaag gatagcttgc 2640
gcagctatta cctttacaac tatgtgaatg aggccttgaa agcatacatg ctggatgcag 2700
tgaatctaaa aggctatttt gcatacgcat tcagtgacca gcgggaccca ggctttggca 2760
tgtatggtca cgtgcaggaa gaggtgattt caaaatcctc actgggccat tacaaaaaca 2820
ttatccgcca caatggcttc ccagccccag gcacatctca gcatcagtgt ccccatgctc 2880
cagctcaagg atcgggccga tatgtcctca ccaagaagcc tgtggtcggc tttctctctc 2940
tggtgagctc ctgcatgctc atcactatgt gccttgttat ctactatgca tttaagaggc 3000
acaagttaac caccaaaaaa tgattgatac agctctgctg acaattttaa tttattaagt 3060
ggttttctca ttactgtagg tctcttcaca ctgtgcttaa ccctgtgttt ttgttgtatt 3120
aaatcggcac gtgatgataa ccagtttatc caagttaaag tcaaggaaaa gtcaaggttt 3180
taaaacccct aaaatgttct gcctttacag gttaaaaaat ctttatgttt ttgaaactat 3240
tgaagaccac agaagtcaat catttatttt aatcatttaa cctgacatta attattttaa 3300
cctgtttact gttttaaaac attataaatg cttcttaata taaaacttac tgtgtttaat 3360
ggggggaaaa gattgttgtt ttttaccctg acatttaaaa aaaaaaatta gggcagtaat 3420
tacaatacag taaatccgat gtttttatgt gaggttatca tataatcaga atcttatacc 3480
agcccatgcc ttgtactaaa ccccagtttt aaccgcaggt aaagaagtgt ttcatatttg 3540
tgatttagaa gcaccagttc tgtgcttaac cctaaacatt cagcctgaaa ttatatggcc 3600
tttgaatgtt acattaagac cactcgtcac ttatttatgt cacagaaacg gcataaacta 3660
tatagaaatt ggtgttttag tgtttaaagg caataatgac aattgtgatt aaatataaaa 3720
atgcatcaga tggagtaaac aggattatat atcacaggat tacatttatc cagagtaaaa 3780
atgacccagg gttaactaat tcaggtgtga aaagccctaa tgtatgctat ttataaaacg 3840
taaatctgca caaattgggt tttacccctt gccattctca agctaaagat ttggactttg 3900
gactggtgct catttaaaga gatagttcac ctgaaaaaat gatagaagga tttgtcataa 3960
tgtactcact ctcaagtggt tccaaacctt tgttaaagtt tctttaatct gttgaacact 4020
aaataagata ttttgaagaa tgtttgaaac tggcagcctt tgtcacggac tactagtttc 4080
taacataaat atcttctttt gttcaacaga agtaagaacc aaaaacaggt ttagatcaaa 4140
tggcgggtga gtacatgatg agaaaagttt tgttttttgg gtgaactatc ccctttaatt 4200
gggggagttt gtaaagtggc aatctaaaac gttcatttct ctgaattaaa tgtgtataaa 4260
tgaatacaca tttttataaa cgagtcccca ggatagttat gtaatggcta gctttaaaat 4320
ccttatttat ttgtaaaaag taagatattt gcattgtttt gtcaagtttg cacggcagtt 4380
tggtttttgc acgataaaac atttataagg gaaatgtatt gtatttgttt ggcatgttat 4440
agaggttctt agtatgatta gtctaaataa gtgatgttaa agggatagtt cacccaaaaa 4500
tgaaagcgta ttcactattt accaaactaa aaatatctat atcaggggtg tccaaactct 4560
gtcctggagg gccaatgtcc tgctgagttt agctgcaact tgcttcaaca caccagcaag 4620
taagtttcta gtacagggtt gttcagatcc ggtcctggaa ggcgagcgtt cagcatagtt 4680
tagctgtatc cccaatcaga cacacccaag ccagtaatca agcacttact ttgctcactt 4740
gaaatgtcct ggcaggtgtt ttgaggagag ttggagctaa atttttaagg acaccggccc 4800
tcccggactg agtttggaca cctgttttag tatatctagt aagagcttgg ttagctggtt 4860
caggtgtctt ggattgggtt ggagctaaac tctccaggac accggccctc caggagtgca 4920
agtggacacc cctgatctat atcacacctt catgtgtttc gtgtcaaaca tgaaagaaga 4980
tattttaaaa aacgctaaaa acctgtaact attgacttcc atagtaggaa acacaaatat 5040
tgtcaatggt ttcaggtttc ctgcattctt cataatatct cctttcatgt ttaacacaag 5100
aaaagaaggt taacgggtaa agttgagtat gtgatgacag aattttcatt tctggatgaa 5160
gcgtctcttt agaacaggtc atgtattttc tgatgtgtaa attacatgat cacatgggta 5220
ttaaataatt gttaactgtc taattg 5246
<210> 5
<211> 24
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 5
agcggataac aatttcacac agga 24
<210> 6
<211> 22
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 6
gaaattaata cgactcacta ta 22
<210> 7
<211> 25
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 7
aaaaaaagca ccgactcggt gccac 25
<210> 8
<211> 19
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 8
aagccttcct ctatgacac 19
<210> 9
<211> 20
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 9
ccgataatga ctgactccta 20
Claims (2)
1.一种Klotho+/-基因缺失斑马鱼,其特征在于,其构建方法包括以下步骤:
1)根据Klotho基因序列,确定靶位点;
所述的靶位点为:5’-GATAAGTTTATGTGGGCCGTCGG-3’
2)根据靶位点,设计Oligo序列,所述的Oligo序列为5’-TAGGATAAGTTTATGTGGGCCGT-3’,5’-AAACACGGCCCACATAAACTTAT-3’;
3)构建gRNA体外转录载体;
4)PCR获得gRNA体外转录模板;
5)对步骤4获得的模板进行体外转录,得到gRNA;
6)制备Cas9 mRNA的体外转录模板;
7)体外转录Cas9 mRNA;
8)添加polyA序列、回收Cas9 mRNA;
9)将Cas9 mRNA和gRNA混合,注射到单细胞期斑马鱼胚胎中;
10)将F0胚胎饲养至性成熟;
11)与野生型成鱼外交,筛选F0;
12)选取可产生有效突变的F0自交,筛选F1。
2.如权利要求1所述的Klotho+/-基因缺失斑马鱼的应用,其特征在于,将所述的Klotho+/-基因缺失斑马鱼用于动脉硬化、骨质疏松症、高磷血症、血管和软组织钙化领域的研究。
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Citations (1)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN109219663A (zh) * | 2016-06-02 | 2019-01-15 | 克洛索治疗有限公司 | 治疗性重组klotho蛋白及其组合物和方法 |
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2021
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Patent Citations (1)
Publication number | Priority date | Publication date | Assignee | Title |
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CN109219663A (zh) * | 2016-06-02 | 2019-01-15 | 克洛索治疗有限公司 | 治疗性重组klotho蛋白及其组合物和方法 |
Non-Patent Citations (1)
Title |
---|
SINGH等: "aKlotho Regulates Age-Associated Vascular Calcification and Lifespan in Zebrafish", CELL REPORTS, vol. 28, pages 2767 - 2776 * |
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