CN113151276A - 一种il-4基因缺失斑马鱼 - Google Patents
一种il-4基因缺失斑马鱼 Download PDFInfo
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Abstract
本发明涉及一种IL‑4基因缺失斑马鱼的构建,具体步骤包括CRISPR/Cas9基因敲除靶位点设计,靶位点序列为:5’‑GACCTGAAGATCTCAACATC‑3’;构建sgRNA体外转录载体;sgRNA体外转录、纯化及鉴定;制备Cas9mRNA;制备F0代斑马鱼;将F0胚胎饲养至性成熟;与野生型成鱼外交,筛选F0;选取可产生有效突变的F0自交,筛选F1;从F1代突变体中挑选相同突变的雌鱼和雄鱼,杂交得到F2代;筛选IL‑4基因敲除纯合子即为稳定遗传的IL‑4基因缺失斑马鱼,本发明构建的IL‑4基因缺失斑马鱼可稳定遗传,IL‑4基因缺失斑马鱼可用于免疫相关疾病的研究。
Description
技术领域
本发明属于分子生物学领域,具体涉及一种IL-4基因缺失斑马鱼。
背景技术
IL-4是一种免疫调节因子,能够调节T细胞分化、免疫球蛋白E的类别转换、巨噬细胞的分化成熟等多个免疫应答反应。IL-4/IL-4R调节在Th2免疫体系中起重要作用,将IL-4基因缺失,可以获得与免疫相关疾病的动物模型。
斑马鱼一次产卵量大,且体外受精,比小鼠的受精卵容易获得,实验操作简单,成本低,且斑马鱼IL-4/IL-4R体系与人类体系作用相近,斑马鱼还具有个体小,饲养成本低、用药量少、可以高通量筛选药物的优点,因此,制作IL-4基因缺失斑马鱼模型,具有重大意义。
发明内容
本发明的目的是构建一种IL-4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’-GACCTGAAGATCTCAACATC-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’-ACCGGACCTGAAGATCTCAACATCG-3’(Seq No.2),5’-AAACGATGTTGAGATCTTCAGGTCG-3’(SeqNo.3),使用该试剂盒可以用于IL-4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。
根据本发明的又一个方面,本发明提供一种IL-4基因缺失斑马鱼的制备方法,所述制备方法包括:
1.将sgRNA和Cas9 mRNA共同导入斑马鱼中;
2.培养获得稳定遗传的IL-4基因缺失斑马鱼。
在本发明的具体实施方案中,所述sgRNA的获得步骤如下:
1.确定IL-4基因敲除的靶位点;
2.根据步骤1确定的靶位点序列设计扩增引物,PCR获得sgRNA的体外转录模板;
3.根据步骤2获得的模板进行体外转录。
作为本发明的具体实施例,所述sgRNA的获得步骤如下:
1.查询斑马鱼IL-4基因的基因序列,根据CRISPR/Cas9敲除原理,设计靶位点;
2.PCR法获得sgRNA的体外转录模板,正向引物序列为:5’-ACCGGACCTGAAGATCTCAACATCG-3’(Seq No.2),反向引物序列为:5’-AAACGATGTTGAGATCTTCAGGTCG-3’(Seq No.3),按照下列PCR条件进行扩增:预变性95℃3min,变性95℃30s,退火54℃30s,延伸72℃20s进行35个循环,72℃10min,PCR产物回收纯化;
3.以RNA体外转录试剂盒进行体外转录。
在本发明的具体实施方案中,所述Cas9 mRNA是按照以下步骤获得的:
以T7驱动的人源化的Cas9编码序列为模板,用T7 RNA聚合酶进行Cas9体外转录,然后带帽并加尾,RNA经无水乙醇法沉淀,用Nuclease-free Water重悬。
作为本发明的具体实施例,sgRNA和Cas9 mRNA共同导入斑马鱼中,包括:将sgRNA和Cas9 mRNA混合,sgRNA终浓度为50ng/μL、Cas9 mRNA终浓度为250ng/μL,在斑马鱼胚胎1-4细胞期进行显微注射,注射量为1nL。
在本发明的具体实施方案中,培养获得稳定遗传的IL-4基因缺失斑马鱼包括:
1.对经过注射的斑马鱼进行基因型鉴定,确定IL-4基因敲除的初建者F0;
2.IL-4基因敲除的初建者F0个体进行自交获得F1;
3.利用基因型鉴定的方法确定IL-4基因敲除的F1;
4.从F1代突变体中挑选相同突变的雌鱼和雄鱼,杂交得到F2代;
5.鉴定为F2代中IL-4基因敲除的纯合子即为稳定遗传的IL-4基因缺失斑马鱼。
根据本发明的另一方面,本发明提供了用于研究免疫相关疾病的斑马鱼模型。
本发明的有益效果和优点在于:
本发明构建的IL-4基因缺失斑马鱼为国内外首例。
本发明构建的IL-4基因缺失斑马鱼可稳定遗传,可用于免疫相关疾病的研究。
附图说明
图1:克隆骨架pGU6电泳图
图2:gRNA体外转录模板
图3:体外转录Cas9 mRNA
图4:PCR、酶切鉴定突变体
图5:IL-4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IL-4基因序列,根据CRISPR/Cas9敲除原理,在http://zifit.partners.org/ZiFiT/CSquare9Nuclease.aspx上设计IL-4靶位点,靶位点序列为:5’-GACCTGAAGATCTCAACATC-3’(Seq No.1);
3.构建gRNA体外转录载体
3.1用Bsa I酶切pGL3-U6-SgRNA-PGK-Puromycin(以下简称pGU6)、切胶回收(见图1),得到sgRNA克隆骨架(Seq No.4),约5000bp,反应体系见表1;
表1酶切体系:
3.2根据靶位点订购两条oligo,oligo1序列为5’-ACCGGACCTGAAGATCTCAACATCG-3’(Seq No.2),oligo2序列为5’-AAACGATGTTGAGATCTTCAGGTCG-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,退火反应体系见表2;
表2退火程序
3.4将退火后的片段与回收的上述sgRNA克隆骨架连接、转化,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,连接体系见表3;
表3连接体系
4.制备sgRNA
4.1订购引物序列T7-sgRNA-F:TTAATACGACTCACTCACTATAGGACCTGAAGATCTCAACATCG-3’(Seq No.5),sgRNA-R:AAAAGCACCGACTCGGTGCC(Seq No.6);按照以下反应和反应程序进行PCR扩增,反应体系为50μL,获得sgRNA体外转录的DNA模板,并用2%的琼脂糖凝胶电泳分析鉴定,约150bp(见图2),PCR反应体系见表4,PCR反应程序为95℃预变形5min,(94℃30s,52℃30s,72℃30s)重复30个循环,72℃5min,4℃1h;
表4 PCR反应体系
组分 | 加样量 |
10×PCR buffer | 5μL |
25mM MgSO<sub>4</sub> | 5μL |
2mM dNTPs | 5μL |
T4-sgRNA-F(10μM) | 1μL |
sgRNA-R(10μM) | 1μL |
pGU6-sgRNA | 10ng |
X5 High-Fidelity DNA polymerase | 1μL |
ddH<sub>2</sub>O | 50μL |
4.2纯化sgRNA的体外转录模板
4.2.1向上述PCR产物中加入等体积的氯仿/苯酚/异丙醇混合液,混合均匀;
4.2.2 4℃,16000g离心5min,小心吸取上清液到新的EP管中;
4.2.3加入1/10体积的3mM NaAC,混匀,加入上述总液体2倍体积的无水乙醇,混匀,-80℃放置至少1h,取出并4℃,16000g离心30min;
4.2.4小心弃掉上清液,用70%乙醇洗涤沉淀3次,每次加200μL洗涤,洗涤后4℃,16000g离心5min;
4.2.5用400μL无水乙醇洗涤,带走残留的盐溶液,吸尽液体,室温干燥5-10min使酒精挥发;
4.2.6视沉淀多少加入适量无RNA水溶解,测浓度;
4.3 sgRNA体外转录、纯化及鉴定
用MEGAshortscriptTMKit体外转录试剂盒按如下反应体系进行sgRNA的体外转录,反应体系见表5,反应条件为37℃,4h,体外转录结束后,加DNase 4.5μL,37℃反应20min,去除DNA模板,反应结束后,加入480μL Nuclease-free Water,用苯酚氯仿抽提法进行纯化,加RNase-free water溶解RNA沉淀,使浓度不低于500ng/μL,并分装,纯化后的RNA用2%琼脂糖凝胶进行电泳鉴定;
sgRNA体外转录体系见表5;
表5体外转录sgRNA反应体系
组分 | 加样量 |
NTPs | 8μL |
体外转录DNA模板 | 7μL |
T7 Enzyme Mix | 2μL |
10×T7 Reaction Buffer | 2μL |
Nuclease-free Water | To 20μl |
5.制备Cas9 mRNA
5.1制备Cas9 mRNA的体外转录模板:通过Xba I单酶切线性化pSP6-2sNLS-spCas9载体(37℃,4h以上);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6;
表6 Cas9 mRNA体外转录体系
5.3添加polyA序列,反应体系见表7,回收得到的mRNA(图3)可用于显微注射;
表7 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4);
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;
7.2 F1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;
7.3将能切开的PCR产物测序,测序确定突变类型;
8.从F1代突变体中挑选相同突变的雌鱼和雄鱼,杂交得到F2代,放置于28.5℃培养,于4dpf取部分胚胎,每个胚胎单独提取基因组DNA,再进行PCR和T7E1酶切检测,引物序列如下:ATGAAGACCTGAAGATCTCA(Seq No.7),CATTCACACTCTGGATGATT(Seq No.8);
9.通过PCR条带分析鉴定为纯合突变的效率,进一步进行测序鉴定(图5)。
序列表
<110> 广州博识生物科技有限公司
<120> 一种IL-4基因缺失斑马鱼
<160> 8
<170> SIPOSequenceListing 1.0
<210> 1
<211> 20
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 1
gacctgaaga tctcaacatc 20
<210> 2
<211> 25
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 2
accggacctg aagatctcaa catcg 25
<210> 3
<211> 25
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 3
aaacgatgtt gagatcttca ggtcg 25
<210> 4
<211> 4952
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 4
ggtaccgatt agtgaacgga tctcgacggt atcgatcacg agactagcct cgagcggccg 60
cccccttcac cgagggccta tttcccatga ttccttcata tttgcatata cgatacaagg 120
ctgttagaga gataattgga attaatttga ctgtaaacac aaagatatta gtacaaaata 180
cgtgacgtag aaagtaataa tttcttgggt agtttgcagt tttaaaatta tgttttaaaa 240
tggactatca tatgcttacc gtaacttgaa agtatttcga tttcttggct ttatatatct 300
tgtggaaagg acgaaacacc ggtgagaccg agagagggtc tcagttttag agctagaaat 360
agcaagttaa aataaggcta gtccgttatc aacttgaaaa agtggcaccg agtcggtgct 420
ttttttaaag aattctcgac ctcgagacaa atggcagtat tcatccacaa ttttaaaaga 480
aaagggggga ttggggggta cagtgcaggg gaaagaatag tagacataat agcaacagac 540
atacaaacta aagaattaca aaaacaaatt acaaaaattc aaaattttcg ggtttattac 600
agggacagca gagatccact ttggccgcgg ctcgaggggg ttggggttgc gccttttcca 660
aggcagccct gggtttgcgc agggacgcgg ctgctctggg cgtggttccg ggaaacgcag 720
cggcgccgac cctgggactc gcacattctt cacgtccgtt cgcagcgtca cccggatctt 780
cgccgctacc cttgtgggcc ccccggcgac gcttcctgct ccgcccctaa gtcgggaagg 840
ttccttgcgg ttcgcggcgt gccggacgtg acaaacggaa gccgcacgtc tcactagtac 900
cctcgcagac ggacagcgcc agggagcaat ggcagcgcgc cgaccgcgat gggctgtggc 960
caatagcggc tgctcagcag ggcgcgccga gagcagcggc cgggaagggg cggtgcggga 1020
ggcggggtgt ggggcggtag tgtgggccct gttcctgccc gcgcggtgtt ccgcattctg 1080
caagcctccg gagcgcacgt cggcagtcgg ctccctcgtt gaccgaatca ccgacctctc 1140
tccccagggg gatccaccgg agcttaccat gaccgagtac aagcccacgg tgcgcctcgc 1200
cacccgcgac gacgtcccca gggccgtacg caccctcgcc gccgcgttcg ccgactaccc 1260
cgccacgcgc cacaccgtcg atccggaccg ccacatcgag cgggtcaccg agctgcaaga 1320
actcttcctc acgcgcgtcg ggctcgacat cggcaaggtg tgggtcgcgg acgacggcgc 1380
cgcggtggcg gtctggacca cgccggagag cgtcgaagcg ggggcggtgt tcgccgagat 1440
cggcccgcgc atggccgagt tgagcggttc ccggctggcc gcgcagcaac agatggaagg 1500
cctcctggcg ccgcaccggc ccaaggagcc cgcgtggttc ctggccaccg tcggcgtctc 1560
gcccgaccac cagggcaagg gtctgggcag cgccgtcgtg ctccccggag tggaggcggc 1620
cgagcgcgcc ggggtgcccg ccttcctgga aacctccgcg ccccgcaacc tccccttcta 1680
cgagcggctc ggcttcaccg tcaccgccga cgtcgaggtg cccgaaggac cgcgcacctg 1740
gtgcatgacc cgcaagcccg gtgcctgacg cccgccccac gacccgcagc gcccgaccga 1800
aaggagcgca cgaccccatg catcggtacc tttaagacca atgacttaca aggcagctgt 1860
agatcttagc cactttctag agtcggggcg gccggccgct tcgagcagac atgataagat 1920
acattgatga gtttggacaa accacaacta gaatgcagtg aaaaaaatgc tttatttgtg 1980
aaatttgtga tgctattgct ttatttgtaa ccattataag ctgcaataaa caagttaaca 2040
acaacaattg cattcatttt atgtttcagg ttcaggggga ggtgtgggag gttttttaaa 2100
gcaagtaaaa cctctacaaa tgtggtaaaa tcgataagga tccgtcgacc gatgcccttg 2160
agagccttca acccagtcag ctccttccgg tgggcgcggg gcatgactat cgtcgccgca 2220
cttatgactg tcttctttat catgcaactc gtaggacagg tgccggcagc gctcttccgc 2280
ttcctcgctc actgactcgc tgcgctcggt cgttcggctg cggcgagcgg tatcagctca 2340
ctcaaaggcg gtaatacggt tatccacaga atcaggggat aacgcaggaa agaacatgtg 2400
agcaaaaggc cagcaaaagg ccaggaaccg taaaaaggcc gcgttgctgg cgtttttcca 2460
taggctccgc ccccctgacg agcatcacaa aaatcgacgc tcaagtcaga ggtggcgaaa 2520
cccgacagga ctataaagat accaggcgtt tccccctgga agctccctcg tgcgctctcc 2580
tgttccgacc ctgccgctta ccggatacct gtccgccttt ctcccttcgg gaagcgtggc 2640
gctttctcaa tgctcacgct gtaggtatct cagttcggtg taggtcgttc gctccaagct 2700
gggctgtgtg cacgaacccc ccgttcagcc cgaccgctgc gccttatccg gtaactatcg 2760
tcttgagtcc aacccggtaa gacacgactt atcgccactg gcagcagcca ctggtaacag 2820
gattagcaga gcgaggtatg taggcggtgc tacagagttc ttgaagtggt ggcctaacta 2880
cggctacact agaaggacag tatttggtat ctgcgctctg ctgaagccag ttaccttcgg 2940
aaaaagagtt ggtagctctt gatccggcaa acaaaccacc gctggtagcg gtggtttttt 3000
tgtttgcaag cagcagatta cgcgcagaaa aaaaggatct caagaagatc ctttgatctt 3060
ttctacgggg tctgacgctc agtggaacga aaactcacgt taagggattt tggtcatgag 3120
attatcaaaa aggatcttca cctagatcct tttaaattaa aaatgaagtt ttaaatcaat 3180
ctaaagtata tatgagtaaa cttggtctga cagttaccaa tgcttaatca gtgaggcacc 3240
tatctcagcg atctgtctat ttcgttcatc catagttgcc tgactccccg tcgtgtagat 3300
aactacgata cgggagggct taccatctgg ccccagtgct gcaatgatac cgcgggaccc 3360
acgctcaccg gctccagatt tatcagcaat aaaccagcca gccggaaggg ccgagcgcag 3420
aagtggtcct gcaactttat ccgcctccat ccagtctatt aattgttgcc gggaagctag 3480
agtaagtagt tcgccagtta atagtttgcg caacgttgtt gccattgcta caggcatcgt 3540
ggtgtcacgc tcgtcgtttg gtatggcttc attcagctcc ggttcccaac gatcaaggcg 3600
agttacatga tcccccatgt tgtgcaaaaa agcggttagc tccttcggtc ctccgatcgt 3660
tgtcagaagt aagttggccg cagtgttatc actcatggtt atggcagcac tgcataattc 3720
tcttactgtc atgccatccg taagatgctt ttctgtgact ggtgagtact caaccaagtc 3780
attctgagaa tagtgtatgc ggcgaccgag ttgctcttgc ccggcgtcaa tacgggataa 3840
taccgcgcca catagcagaa ctttaaaagt gctcatcatt ggaaaacgtt cttcggggcg 3900
aaaactctca aggatcttac cgctgttgag atccagttcg atgtaaccca ctcgtgcacc 3960
caactgatct tcagcatctt ttactttcac cagcgtttct gggtgagcaa aaacaggaag 4020
gcaaaatgcc gcaaaaaagg gaataagggc gacacggaaa tgttgaatac tcatactctt 4080
cctttttcaa tattattgaa gcatttatca gggttattgt ctcatgagcg gatacatatt 4140
tgaatgtatt tagaaaaata aacaaatagg ggttccgcgc acatttcccc gaaaagtgcc 4200
acctgacgcg ccctgtagcg gcgcattaag cgcggcgggt gtggtggtta cgcgcagcgt 4260
gaccgctaca cttgccagcg ccctagcgcc cgctcctttc gctttcttcc cttcctttct 4320
cgccacgttc gccggctttc cccgtcaagc tctaaatcgg gggctccctt tagggttccg 4380
atttagtgct ttacggcacc tcgaccccaa aaaacttgat tagggtgatg gttcacgtag 4440
tgggccatcg ccctgataga cggtttttcg ccctttgacg ttggagtcca cgttctttaa 4500
tagtggactc ttgttccaaa ctggaacaac actcaaccct atctcggtct attcttttga 4560
tttataaggg attttgccga tttcggccta ttggttaaaa aatgagctga tttaacaaaa 4620
atttaacgcg aattttaaca aaatattaac gtttacaatt tcccattcgc cattcaggct 4680
gcgcaactgt tgggaagggc gatcggtgcg ggcctcttcg ctattacgcc agcccaagct 4740
accatgataa gtaagtaata ttaaggtacg ggaggtactt ggagcggccg caataaaata 4800
tctttatttt cattacatct gtgtgttggt tttttgtgtg aatcgatagt actaacatac 4860
gctctccatc aaaacaaaac gaaacaaaac aaactagcaa aataggctgt ccccagtgca 4920
agtgcaggtg ccagaacatt tctctatcga ta 4952
<210> 5
<211> 44
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 5
ttaatacgac tcactcacta taggacctga agatctcaac atcg 44
<210> 6
<211> 20
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 6
aaaagcaccg actcggtgcc 20
<210> 7
<211> 20
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 7
atgaagacct gaagatctca 20
<210> 8
<211> 20
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 8
cattcacact ctggatgatt 20
Claims (1)
1.一种IL-4基因缺失斑马鱼的应用,其特征在于,将IL-4基因缺失斑马鱼用于免疫相关疾病的研究,所述的IL-4基因缺失斑马鱼制备过程包括以下步骤:
1)CRISPR/Cas9基因敲除靶位点设计
在NCBI上查询斑马鱼IL-4基因序列,根据CRISPR/Cas9敲除原理设计靶位点,靶位点序列为:5’-GACCTGAAGATCTCAACATC-3;
2)设计Oligo序列,根据靶位点设计Oligo序列,所述的Oligo序列为5’-ACCGGACCTGAAGATCTCAACATCG-3’,5’-AAACGATGTTGAGATCTTCAGGTCG-3’;
3)构建sgRNA体外转录载体;
4)sgRNA体外转录、纯化及鉴定;
5)制备Cas9 mRNA;
6)制备F0代斑马鱼;
7)将F0胚胎饲养至性成熟;
8)与野生型成鱼外交,筛选F0;
9)选取可产生有效突变的F0自交,筛选F1;
10)从F1代突变体中挑选相同突变的雌鱼和雄鱼,杂交得到F2代;
11)筛选IL-4基因敲除纯合子即为稳定遗传的IL-4基因缺失斑马鱼。
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