CN111471714A - Minos转座子系统介导的真核生物转基因细胞系及构建方法 - Google Patents
Minos转座子系统介导的真核生物转基因细胞系及构建方法 Download PDFInfo
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Abstract
本发明涉及一种Minos转座子系统介导的真核生物转基因细胞系及构建方法,首先设计并合成了包含Minos转座子的末端反向重复序列和标记基因表达框的Minos基础载体,命名为PUC57‑Mi‑puro;然后将外源基因表达序列用酶切连接的方式连接到Minos基础载体上,构建成转基因过表达载体;再将转基因过表达载体和Minos transposase表达载体Minos‑Helper混合后转染真核生物细胞,筛选获得真核生物转基因细胞系。本发明提供的转基因细胞系构建方法简单,成本低,周期短,能够广泛的用于真核生物转基因细胞系的构建。
Description
技术领域
本发明属于基因工程技术领域,涉及一种Minos转座子系统介导的真核生物转基因细胞系及构建方法。
背景技术
随着越来越多的生物完成了全基因组测序,生命科学研究步入了后基因组时代,功能基因组研究成为重中之重。遗传操作技术是研究功能基因组的重要技术手段,对于解析基因功能,探索生命奥妙至关重要。转基因技术是重要的遗传操作手段,转基因是指将人工分离和修饰过的基因,导入到生物体基因组中,从而达到改造生物的目的。主要是通过人为操作,实现基因跨个体甚至跨物种转移,在基础研究和生物工程领域都有广阔的应用前景。在基础科研领域,运用转基因技术,可以对特定功能基因在特定物种中快速过表达,从而研究该基因的功能。在生产上,跨物种表达的转基因抗虫作物,转基因抗除草剂作物等广泛种植,对解决人类温饱问题做出了巨大的贡献。
目前广泛应用的转基因递送系统主要有广泛应用于哺乳动物的慢病毒、腺病毒系统;广泛应用于植物的农杆菌介导的T-DNA插入法、原生质体融合、花粉管通道法;广泛应用于昆虫、不如动物的转座子介导的转基因方法等。其中转座子系统介导的转基因方法因其简单、高效、安全而被广泛应用。
Minos转座子系统是从果蝇D.hydei中得到的Ⅱ型转座子,在宿主细胞中按照剪切和粘贴模式实现转座,安全高效。此外,Minos转座子在基因组上的插入位点特征是TA碱基,主要是分布在编码基因的内含子区域,能够最大限度的减少插入位点对内源基因的影响,是合适的真核生物转基因系统。
发明内容
有鉴于此,本发明的目的在于提供一种Minos转座子系统介导的真核生物转基因细胞系及构建方法。
为达到上述目的,本发明提供如下技术方案:
1、Minos转座子系统介导的真核生物转基因细胞系的构建方法,具体步骤如下:
(1)合成Minos转座子系统基础载体PUC57-Mi-puro,其核苷酸序列如SEQ ID NO.1所示;
(2)将外源基因连接至PUC57-Mi-puro,构建得到转基因载体;
(3)将步骤(2)的转基因载体与Mino转座酶的表达载体混合后转染真核生物细胞,筛选,即得所述细胞系。
作为优选的技术方案之一,步骤(1)中,PUC57-Mi-puro包含Minos转座子的末端反向重复序列(inverted terminal repeat,ITR)和标记基因表达框。
作为进一步优选的技术方案之一,所述标记基因包括但不限于puromycin抗性筛选基因、Zeocin抗性筛选基因、Blast抗性筛选基因等药物抗性筛选基因,红色荧光蛋白、绿色荧光蛋白、蓝色荧光蛋白等荧光蛋白标记基因。
作为优选的技术方案之一,步骤(1)中,PUC57-Mi-puro包含:
Minos转座子的末端反向重复序列(inverted terminal repeat,ITR),其核苷酸序列如SEQ ID NO.3所示;
puromycin抗性筛选基因表达框(Hsp70-puro-SV40),其核苷酸序列如SEQ IDNO.4所示;
用于表达外源基因的框架,其核苷酸序列如SEQ ID NO.5所示;
IE2启动子-家蚕丝胶1终止信号Ser1PA,其核苷酸序列如SEQ ID NO.6所示。
作为优选的技术方案之一,步骤(2)中,所述外源基因包括但不限于蛋白编码基因,ncRNA表达基因,miRNA表达框,sgRNA表达框。
作为优选的技术方案之一,步骤(2)中,将外源基因的核苷酸序列按照5’到3’方向连接到Minos转座子系统基础载体PUC57-Mi-puro的多克隆位点,多克隆位点限制性酶切位点包括AgeI、AsiSI、BglII和KpnI,构建成Minos转基因载体。
作为优选的技术方案之一,步骤(2)中,采用酶切连接的方法将外源基因连接至PUC57-Mi-puro。
作为优选的技术方案之一,步骤(3)中,所述Mino转座酶的表达载体为Minos-Helper,其核苷酸序列如SEQ ID NO.2所示。
作为优选的技术方案之一,步骤(3)中,转基因载体与Mino转座酶的表达载体按照摩尔比1:1进行混合。
作为优选的技术方案之一,步骤(3)中,转染方法包括但不限于脂质体转染法、电穿孔转染法等。
作为优选的技术方案之一,步骤(3)中,利用含有嘌呤霉素(puromycin)的完全培养基细胞培养基连续筛选,直至所有细胞都整合了Minos转座子系统和外源基因表达框。
2、利用上述方法构建得到的Minos转座子系统介导的真核生物转基因细胞系。
本发明的有益效果在于:
本发明首先设计并合成了包含Minos转座子的末端反向重复序列(invertedterminal repeat,ITR)和标记基因表达框的Minos基础载体,命名为PUC57-Mi-puro;然后将外源基因表达序列用酶切连接的方式连接到Minos基础载体上,构建成转基因过表达载体;再将转基因过表达载体和Minos transposase表达载体Minos-Helper按照摩尔比1:1混合后转染真核生物细胞,筛选获得真核生物转基因细胞系。本发明提供的转基因细胞系构建方法简单,成本低,周期短,能够广泛的用于真核生物转基因细胞系的构建。由于Minos转座子是以“剪切-黏贴”的方式来实现来实现转座,因此可以通过控制转座子浓度等方式来控制Minos转座子系统携带的外源基因整合到宿主细胞的拷贝数。
附图说明
为了使本发明的目的、技术方案和有益效果更加清楚,本发明提供如下附图进行说明:
图1为Minos转座子系统基础载体PUC57-Mi-puro的图谱,包含:ITR,Minos转座子的末端反向重复序列(inverted terminal repeat,ITR);Hsp70,Hsp70启动子;puro,puromycin抗性筛选基因;SV40PA,SV40 polyA;IE2,IE2启动子;AgeI、AsiSI、BglII、KpnI,核酸内切酶的酶切位点;Ser1PA,家蚕丝胶1(ser1)polyA。
图2为显示了Minos转座子系统介导的家蚕胚胎细胞BmE转绿色荧光蛋白(EGFP)细胞系。
具体实施方式
下面将结合附图,对本发明的优选实施例进行详细的描述。
以下凡是未注明的具体实验方法,都按照公认的实验方法与条件实施,例如,按照试剂耗材厂商提供的说明书操作,或者按照经典实验书籍《分子克隆实验指南》(第三版,J.萨姆布鲁克等著))来完成实验。
实施例:
本实施例中所用到的家蚕胚胎细胞系(The Bombyx mori embryonic cell line,BmE)为生物实验中常用细胞系(PMID:17570024)。
构建Minos转座子介导的过表达绿色荧光蛋白(EGFP)的真核生物细胞系
1、Minos转座子系统基础载体PUC57-Mi-puro的构建
根据D.hydei中Minos转座子序列,设计并合成Minos转座子系统基础载体PUC57-Mi-puro,其核苷酸序列如SEQ ID NO.1所示,载体图谱如图1所示。
该基础载体包含:
Minos转座子的末端反向重复序列(inverted terminal repeat,ITR),其核苷酸序列如SEQ ID NO.3所示;
puromycin抗性筛选基因表达框(Hsp70-puro-SV40),其核苷酸序列如SEQ IDNO.4所示;
用于表达外源基因的框架,其核苷酸序列如SEQ ID NO.5所示;
IE2启动子-家蚕丝胶1终止信号Ser1PA,其核苷酸序列如SEQ ID NO.6所示。
2、绿色荧光蛋白(EGFP)的克隆
按照NCBI数据库提供的绿色荧光蛋白(EGFP)的基因序列(核苷酸序列如SEQ IDNO.7所示),设计引物:
>EGFP-F(AA),5’-ACCGGTGCGATCGCATGGTGAGCAAGGGCG-3’,其核苷酸序列如SEQ IDNO.8所示;
>EGFP-R(BK),5’-GGTACCAGATCTTTACTTGTACAGCTCGTCCATG-3’,其核苷酸序列如SEQ ID NO.9所示;
以载体piggyBac{3×P3-EGFP-SV40}为模板,扩增EGFP基因,然后连接到T载体,挑选单克隆,sanger测序后选择正确的单克隆,命名为T-EGFP(AABK)。PCR扩增酶选用高保真热启动酶(Max DNA Polymerase,Takara,Japan),反应总体系50μl,包括引物各1μl,模板1μl,2×酶预混液25μl,水22μl,反应条件如下:98℃预变性4min;98℃变性10s,55℃退火5s,72℃延伸5s;35个循环;72℃延伸10min;12℃保存。T克隆方案按照常规方法操作。
3、Minos介导的绿色荧光蛋白过表达载体PUC57-Mi-puro-EGFP的构建
将载体PUC57-Mi-puro用AgeI和KpnI双酶切,琼脂糖凝胶电泳后回收约5900bp大小的核酸作为骨架。将载体T-EGFP(AABK)用AgeI和KpnI双酶切,琼脂糖凝胶电泳后回收约740bp的核酸作为片段。将骨架和片段用T4 DNA连接酶连接后进行转化,挑选正确单克隆,成功构建Minos介导的绿色荧光蛋白过表达载体PUC57-Mi-puro-EGFP,核苷酸序列如SEQID NO.10所示。
1)酶切条件为50μl体系,包含1μg的载体{PUC57-Mi-puro或T-EGFP(AABK)},5μL的CutSmart缓冲液,AgeI和KpnI各1μl,用双蒸水补齐50μl,酶切条件为37℃过夜,限制性内切酶采购自NEB公司。
2)DNA连接酶为T4 DNA连接酶,连接总体系为50μL,其中骨架和片段按照摩尔比1:10添加,总质量为2μg,T4 DNA连接酶缓冲液5μL,T4 DNA连接酶1μL,用双蒸水补齐50μL,16℃连接4小时,T4 DNA连接酶采购自NEB公司。
3)转化所用的感受态为Trans1-T1感受态细胞,购买自全式金公司,按照公司说明书来执行转化实验。
4)正确单克隆的挑选。首先是挑选24个单克隆,用含有氨苄青霉素的LB液体培养基在37℃、220rmp摇10小时,然后用菌液电泳的方式挑选滞后克隆(对照载体为PUC57-Mi-puro),氨苄青霉素工作浓度为50μg/ml。然后将滞后克隆抽提质粒,酶切验证;最后,将酶切验证正确的单克隆执行sanger测序,最终挑选到正确的单克隆。
4、过表达绿色荧光蛋白(EGFP)的真核生物细胞系的构建。
将Minos介导的绿色荧光蛋白过表达载体PUC57-Mi-puro-EGFP和Minostransposase表达载体为Minos-Helper按照摩尔比1:1共转染家蚕胚胎细胞系BmE,转染方法为脂质体转染法(罗氏),转染方法按照试剂说明书操作。6-8小时后更完全培养基在27度条件下培养约一个月,完全培养基为包括体积浓度10%胎牛血清(fetal bovine serum,FBS,赛默飞世尔公司)和青霉素-链霉素(Penicillin-Streptomycin,20万单位/升,赛默飞世尔公司)的Grace’s培养基(赛默飞世尔公司),直至细胞状态恢复正常。然后更换含有puromycin的完全培养基连续筛选约2个月,puromycin工作浓度为0.5μg/ml,直至全部细胞都稳定整合绿色荧光蛋白(EGFP)表达框。
用流式细胞仪或者梯度稀释法分选单克隆细胞,扩大培养,可以得到过表达绿色荧光蛋白(EGFP)的真核生物细胞系。如图2所示。
最后说明的是,以上优选实施例仅用以说明本发明的技术方案而非限制,尽管通过上述优选实施例已经对本发明进行了详细的描述,但本领域技术人员应当理解,可以在形式上和细节上对其作出各种各样的改变,而不偏离本发明权利要求书所限定的范围。
序列表
<110> 西南大学
<120> Minos转座子系统介导的真核生物转基因细胞系及构建方法
<130> 2020
<160> 10
<170> SIPOSequenceListing 1.0
<210> 1
<211> 3169
<212> DNA
<213> Artificial
<400> 1
aagtgcttga aatgctaaat gttttcaatt tttcgccatt aagacaagcc tacacaaatg 60
cttctataaa ttatgccaag cacgttagca gcttctacga gccccaacca ctattaattc 120
gaacagcatg ttttttttgc agtgcgcaat gtttaacaca ctatattatc aatactacta 180
aagataacac ataccaatgc atttcgtctc aaagagaatt ttattctctt cacgacgaaa 240
aaaaaagttt tgctctattt ccaacaacaa caaaaatatg agtaatttat tcaaacggtt 300
tgcttaagag ataagaaaaa agtgaccact attaattcga acgcggcgta agcttacctt 360
aatctcaaga agagcaaaac aaaagcaact aatgtaacgg aatcattatc tagttatgat 420
ctgcaaataa tgctgcagcc taggcgagaa atttctctgg ccgttattcg ttattctctc 480
ttttcttttt gggtctctcc ctctctgcac taatgctctc tcactctgtc acacagtaaa 540
cggcatactg ctctcgttgg ttcgagagag cgcgcctcga atgttcgcga aaagagcgcc 600
ggagtataaa tagaggcgct tcgtctacgg agcgacaatt caattcaaac aagcaaagtg 660
aacacgtcgc taagcgaaag ctaagcaaat aaacaagcgc agctgaacaa gctaaacaat 720
ctgcagtaaa gtgcaagtta aagtgaatca attaaaagta accagcaacc aagtaaatca 780
actgcaacta ctgaaatctg ccaagaagta attattgaat acaagaagag aactctgggg 840
gatcatgacc gaatacaaac ccacagtgag actggccact agagacgatg ttcctagagc 900
tgtcagaact ttggctgccg ctttcgccga ttacccagct actagacaca ccgttgaccc 960
ggatagacac atcgaaagag tcaccgaatt gcaggaactc ttcctgacaa gagttggtct 1020
cgacattgga aaggtctggg tggccgacga tggagccgct gttgctgtct ggacaactcc 1080
cgaatcggtg gaagccggcg ctgttttcgc cgaaataggt cctagaatgg ctgaattgtc 1140
aggttctaga ctcgccgctc aacagcaaat ggaaggactg ttggcccctc acagaccaaa 1200
agaaccggcc tggttcctcg ctactgtggg agttagccca gatcaccagg gtaaaggact 1260
gggctccgct gtggttttgc caggagtcga agctgctgaa agagccggcg tgccggcttt 1320
cttggaaacc tcagccccaa gaaacctccc gttctacgaa agactgggct tcaccgtgac 1380
agctgacgtc gaagtgcccg aaggccctag aacatggtgc atgactagaa aacctggtgc 1440
tgactacaag gacgatgacg ataaagatta taaagacgat gacgataaag actataaaga 1500
tgacgacgat aaatacccct acgacgtgcc tgattacgct cggccgcgac tctagatcat 1560
aatcagccat gcggccgcga ctctagacca catttgtaga ggttttactt gctttaaaaa 1620
acctcccaca cctccccctg aacctgaaac ataaaatgaa tgcaattgtt gttgttaact 1680
tgtttattgc agcttataat ggttacaaat aaagcaatag catcacaaat ttcacaaata 1740
aagcattttt ttcactgcat tctagttgtg gtttgtccaa actcatcaat gtatcttaaa 1800
gcttatcgat acgcgtagtc gaccatgatg ataaacaatg tatggtgcta atgttgcttc 1860
aacaacaatt ctgttgaact gtgttttcat gtttgccaac aagcaccttt atactcggtg 1920
gcctccccac caccaacttt tttgcactgc aaaaaaacac gcttttgcac gcgggcccat 1980
acatagtaca aactctacgt ttcgtagact attttacata aatagtctac accgttgtat 2040
acgctccaaa tacactacca cacattgaac ctttttgcag tgcaaaaaag tacgtgtcgg 2100
cagtcacgta ggccggcctt atcgggtcgc gtcctgtcac gtacgaatca cattatcgga 2160
ccggacgagt gttgtcttat cgtgacagga cgccagcttc ctgtgttgct aaccgcagcc 2220
ggacgcaact ccttatcgga acaggacgcg cctccatatc agccgcgcgt tatctcatgc 2280
gcgtgaccgg acacgaggcg cccgtcccgc ttatcgcgcc tataaataca gcccgcaacg 2340
atctggtaaa cacagttgaa cagcatctgt tcgaaaccgg tgcgatcgca tcgatcgatc 2400
gagatctggt acctaaagct ttacaactaa acacgacttg gagtattcct tgtagtgttt 2460
aagattttaa atcttactta atgacttcga acgattttaa cgataacttt ctctttgttt 2520
aactttaatc agcatacata aaaagccccg gttttgtatc gggaagaaaa aaaatgtaat 2580
tgtgttgcct agataataaa cgtattatca aagtgtgtgg ttttccttta ccaaagaccc 2640
ctttaagatg ggcctaatgg gcttaagtcg agtcctttcc gatgtgttaa atacacattt 2700
attacactga tgcgtcgaat gtacactttt aataggatag ctccactaaa aattatttta 2760
tttatttaat ttgttgcacc aaaactgata cattgacgaa acgcgtatgg cgcgccatta 2820
attaaattat tgttttaagt atgatagtaa atcacattac gccgcgttcg aattaatagt 2880
ggtcactttt ttcttatctc ttaagcaaac cgtttgaata aattactcat atttttgttg 2940
ttgttggaaa tagagcaaaa cttttttttt cgtcgtgaag agaataaaat tctctttgag 3000
acgaaatgca ttggtatgtg ttatctttag tagtattgat aatatagtgt gttaaacatt 3060
gcgcactgca aaaaaaacat gctgttcgaa ttaatagtgg ttggggctcg tagaaaacga 3120
aaaatatctt aagctagcat agagaatgga gcaaaactca atttgatgc 3169
<210> 2
<211> 2595
<212> DNA
<213> Artificial
<400> 2
cgagaaattt ctctggccgt tattcgttat tctctctttt ctttttgggt ctctccctct 60
ctgcactaat gctctctcac tctgtcacac agtaaacggc atactgctct cgttggttcg 120
agagagcgcg cctcgaatgt tcgcgaaaag agcgccggag tataaataga ggcgcttcgt 180
ctacggagcg acaattcaat tcaaacaagc aaagtgaaca cgtcgctaag cgaaagctaa 240
gcaaataaac aagcgcagct gaacaagcta aacaatctgc agtaaagtgc aagttaaagt 300
gaatcaatta aaagtaacca gcaaccaagt aaatcaactg caactactga aatctgccaa 360
gaagtaatta ttgaatacaa gaagagaact ctgggggatc tgatcgataa tggttcgtgg 420
taaacctatt tctaaagaaa tcagagtatt gattagggat tattttaaat ctggaaagac 480
acttacggag ataagcaagc aattaaattt gcctaagtcg tctgtgcatg gggtgataca 540
aattttcaaa aaaaatggga atattgaaaa taacattgcg aatagaggcc gaacatcagc 600
aataacaccc cgcgacaaaa gacaactggc caaaattgtt aaggctgatc gtcgccaatc 660
tttgagaaat ttggcttcta agtggtcgca gacaattggc aaaactgtca agcgagagtg 720
gacgcgacag caattaaaaa gtattggata tggtttttat aaagccaagg aaaaaccctt 780
gcttacgctt cgtcaaaaaa agaagcgttt gcaatgggct cgggaaagga tgtcttggac 840
tcaaaggcaa tgggatacca tcatattcag cgatgaagct aaatttgatg ttagtgtcgg 900
cgatacgaga aaacgcgtca tccgtaagag gtcagaaaca taccataaag actgccttaa 960
aagaacaaca aagtttcctg cgagcactat ggtatgggga tgtatgtctg ccaaaggatt 1020
aggaaaactt catttcattg aagggacagt taatgctgaa aaatatatta atattttaca 1080
agatagtttg ttgccatcaa taccaaaact atcagattgc ggtgaattca cttttcagca 1140
ggacggagca tcatcgcaca cagccaagcg aaccaaaaat tggctgcaat ataatcaaat 1200
ggaggtttta gattggccat caaatagtcc agatctaagc ccaattgaaa atatttggtg 1260
gctaatgaaa aaccagcttc gaaatgagcc acaaaggaat atttctgact tgaaaatcaa 1320
gttgcaagag atgtgggact caatttctca agagcattgc aaaaatttgt taagctcaat 1380
gccaaaacga gttaaatgcg taatgcaggc caagggcgac gttacacaat tctaatctag 1440
agatcttcca tacctaccag ttctgcgcct gcagcaatgg caacaacgtt gcccggatcg 1500
aggtcgacta aggccaaaga gtctaatttt tgttcatcaa tgggttataa catatgggtt 1560
atattataag tttgttttaa gtttttgaga ctgataagaa tgtttcgatc gaatattcca 1620
tagaacaaca atagtattac ctaattacca agtcttaatt tagcaaaaat gttattgctt 1680
atagaaaaaa taaattattt atttgaaatt taaagtcaac ttgtcattta atgtcttgta 1740
gacttttgaa agtcttacga tacaattagt atctaatata catgggttca ttctacattc 1800
tatattagtg atgatttctt tagctagtaa tacattttaa ttatattcgg ctttgatgat 1860
tttctgattt tttccgaacg gattttcgta gaccctttcg atctcataat ggctcatttt 1920
attgcgatgg acggtcagga gagctccact tttgaatttc tgttcgcaga caccgcattt 1980
gtagcacata gccgggacat ccggtttggg gagattttcc agtctctgtt gcaattggtt 2040
ttcgggaatg cgttgcaggc gcatacgctc tatatcctcc gaacggcgct ggttgaccct 2100
agcatttaca taaggatcag cagcaaaatt tgcctctact tcattgcccg gaatcacagc 2160
aatcagatgt ccctttcggt tacgatggat attcaggtgc gaaccgcaca caaagctctc 2220
gccgcacact ccacactgat atggtcgctc gcccgtgtgg cgccgcatat ggatcttaag 2280
gtcgttggac tgcacaaagc tcttgctgca cattttgcag gagtacggcc tttgacccgt 2340
gtgcaatcgc atgtgtcgcg ccagcttgtt ctgcgaaata aactaacggg aattcctgca 2400
gcccggggga tccgcggccg cataggccac tagtggatct ggatcctcta gagtcgacct 2460
cgaacgttaa cgttaacgta acgttaactc gaggcctcga ggtcgacctg cagccaagct 2520
ttgcgtactc gcaaattatt aaaaataaaa ctttaaaaat aatttcgtct aattaatatt 2580
atgagttaat tcaaa 2595
<210> 3
<211> 405
<212> DNA
<213> Artificial
<400> 3
aagtgcttga aatgctaaat gttttcaatt tttcgccatt aagacaagcc tacacaaatg 60
cttctataaa ttatgccaag cacgttagca gcttctacga gccccaacca ctattaattc 120
gaacagcatg ttttttttgc agtgcgcaat gtttaacaca ctatattatc aatactacta 180
aagataacac ataccaatgc atttcgtctc aaagagaatt ttattctctt cacgacgaaa 240
aaaaaagttt tgctctattt ccaacaacaa caaaaatatg agtaatttat tcaaacggtt 300
tgcttaagag ataagaaaaa agtgaccact attaattcga acgcggcgta agcttacctt 360
aatctcaaga agagcaaaac aaaagcaact aatgtaacgg aatca 405
<210> 4
<211> 341
<212> DNA
<213> Artificial
<400> 4
attgttttaa gtatgatagt aaatcacatt acgccgcgtt cgaattaata gtggtcactt 60
ttttcttatc tcttaagcaa accgtttgaa taaattactc atatttttgt tgttgttgga 120
aatagagcaa aacttttttt ttcgtcgtga agagaataaa attctctttg agacgaaatg 180
cattggtatg tgttatcttt agtagtattg ataatatagt gtgttaaaca ttgcgcactg 240
caaaaaaaac atgctgttcg aattaatagt ggttggggct cgtagaaaac gaaaaatatc 300
ttaagctagc atagagaatg gagcaaaact caatttgatg c 341
<210> 5
<211> 1373
<212> DNA
<213> Artificial
<400> 5
cgagaaattt ctctggccgt tattcgttat tctctctttt ctttttgggt ctctccctct 60
ctgcactaat gctctctcac tctgtcacac agtaaacggc atactgctct cgttggttcg 120
agagagcgcg cctcgaatgt tcgcgaaaag agcgccggag tataaataga ggcgcttcgt 180
ctacggagcg acaattcaat tcaaacaagc aaagtgaaca cgtcgctaag cgaaagctaa 240
gcaaataaac aagcgcagct gaacaagcta aacaatctgc agtaaagtgc aagttaaagt 300
gaatcaatta aaagtaacca gcaaccaagt aaatcaactg caactactga aatctgccaa 360
gaagtaatta ttgaatacaa gaagagaact ctgggggatc atgaccgaat acaaacccac 420
agtgagactg gccactagag acgatgttcc tagagctgtc agaactttgg ctgccgcttt 480
cgccgattac ccagctacta gacacaccgt tgacccggat agacacatcg aaagagtcac 540
cgaattgcag gaactcttcc tgacaagagt tggtctcgac attggaaagg tctgggtggc 600
cgacgatgga gccgctgttg ctgtctggac aactcccgaa tcggtggaag ccggcgctgt 660
tttcgccgaa ataggtccta gaatggctga attgtcaggt tctagactcg ccgctcaaca 720
gcaaatggaa ggactgttgg cccctcacag accaaaagaa ccggcctggt tcctcgctac 780
tgtgggagtt agcccagatc accagggtaa aggactgggc tccgctgtgg ttttgccagg 840
agtcgaagct gctgaaagag ccggcgtgcc ggctttcttg gaaacctcag ccccaagaaa 900
cctcccgttc tacgaaagac tgggcttcac cgtgacagct gacgtcgaag tgcccgaagg 960
ccctagaaca tggtgcatga ctagaaaacc tggtgctgac tacaaggacg atgacgataa 1020
agattataaa gacgatgacg ataaagacta taaagatgac gacgataaat acccctacga 1080
cgtgcctgat tacgctcggc cgcgactcta gatcataatc agccatgcgg ccgcgactct 1140
agaccacatt tgtagaggtt ttacttgctt taaaaaacct cccacacctc cccctgaacc 1200
tgaaacataa aatgaatgca attgttgttg ttaacttgtt tattgcagct tataatggtt 1260
acaaataaag caatagcatc acaaatttca caaataaagc atttttttca ctgcattcta 1320
gttgtggttt gtccaaactc atcaatgtat cttaaagctt atcgatacgc gta 1373
<210> 6
<211> 985
<212> DNA
<213> Artificial
<400> 6
catgatgata aacaatgtat ggtgctaatg ttgcttcaac aacaattctg ttgaactgtg 60
ttttcatgtt tgccaacaag cacctttata ctcggtggcc tccccaccac caactttttt 120
gcactgcaaa aaaacacgct tttgcacgcg ggcccataca tagtacaaac tctacgtttc 180
gtagactatt ttacataaat agtctacacc gttgtatacg ctccaaatac actaccacac 240
attgaacctt tttgcagtgc aaaaaagtac gtgtcggcag tcacgtaggc cggccttatc 300
gggtcgcgtc ctgtcacgta cgaatcacat tatcggaccg gacgagtgtt gtcttatcgt 360
gacaggacgc cagcttcctg tgttgctaac cgcagccgga cgcaactcct tatcggaaca 420
ggacgcgcct ccatatcagc cgcgcgttat ctcatgcgcg tgaccggaca cgaggcgccc 480
gtcccgctta tcgcgcctat aaatacagcc cgcaacgatc tggtaaacac agttgaacag 540
catctgttcg aaaccggtgc gatcgcatcg atcgatcgag atctggtacc taaagcttta 600
caactaaaca cgacttggag tattccttgt agtgtttaag attttaaatc ttacttaatg 660
acttcgaacg attttaacga taactttctc tttgtttaac tttaatcagc atacataaaa 720
agccccggtt ttgtatcggg aagaaaaaaa atgtaattgt gttgcctaga taataaacgt 780
attatcaaag tgtgtggttt tcctttacca aagacccctt taagatgggc ctaatgggct 840
taagtcgagt cctttccgat gtgttaaata cacatttatt acactgatgc gtcgaatgta 900
cacttttaat aggatagctc cactaaaaat tattttattt atttaatttg ttgcaccaaa 960
actgatacat tgacgaaacg cgtat 985
<210> 7
<211> 720
<212> DNA
<213> Artificial
<400> 7
atggtgagca agggcgagga gctgttcacc ggggtggtgc ccatcctggt cgagctggac 60
ggcgacgtaa acggccacaa gttcagcgtg tccggcgagg gcgagggcga tgccacctac 120
ggcaagctga ccctgaagtt catctgcacc accggcaagc tgcccgtgcc ctggcccacc 180
ctcgtgacca ccctgaccta cggcgtgcag tgcttcagcc gctaccccga ccacatgaag 240
cagcacgact tcttcaagtc cgccatgccc gaaggctacg tccaggagcg caccatcttc 300
ttcaaggacg acggcaacta caagacccgc gccgaggtga agttcgaggg cgacaccctg 360
gtgaaccgca tcgagctgaa gggcatcgac ttcaaggagg acggcaacat cctggggcac 420
aagctggagt acaactacaa cagccacaac gtctatatca tggccgacaa gcagaagaac 480
ggcatcaagg tgaacttcaa gatccgccac aacatcgagg acggcagcgt gcagctcgcc 540
gaccactacc agcagaacac ccccatcggc gacggccccg tgctgctgcc cgacaaccac 600
tacctgagca cccagtccgc cctgagcaaa gaccccaacg agaagcgcga tcacatggtc 660
ctgctggagt tcgtgaccgc cgccgggatc actctcggca tggacgagct gtacaagtaa 720
<210> 8
<211> 30
<212> DNA
<213> Artificial
<400> 8
accggtgcga tcgcatggtg agcaagggcg 30
<210> 9
<211> 34
<212> DNA
<213> Artificial
<400> 9
ggtaccagat ctttacttgt acagctcgtc catg 34
<210> 10
<211> 3877
<212> DNA
<213> Artificial
<400> 10
aagtgcttga aatgctaaat gttttcaatt tttcgccatt aagacaagcc tacacaaatg 60
cttctataaa ttatgccaag cacgttagca gcttctacga gccccaacca ctattaattc 120
gaacagcatg ttttttttgc agtgcgcaat gtttaacaca ctatattatc aatactacta 180
aagataacac ataccaatgc atttcgtctc aaagagaatt ttattctctt cacgacgaaa 240
aaaaaagttt tgctctattt ccaacaacaa caaaaatatg agtaatttat tcaaacggtt 300
tgcttaagag ataagaaaaa agtgaccact attaattcga acgcggcgta agcttacctt 360
aatctcaaga agagcaaaac aaaagcaact aatgtaacgg aatcattatc tagttatgat 420
ctgcaaataa tgctgcagcc taggcgagaa atttctctgg ccgttattcg ttattctctc 480
ttttcttttt gggtctctcc ctctctgcac taatgctctc tcactctgtc acacagtaaa 540
cggcatactg ctctcgttgg ttcgagagag cgcgcctcga atgttcgcga aaagagcgcc 600
ggagtataaa tagaggcgct tcgtctacgg agcgacaatt caattcaaac aagcaaagtg 660
aacacgtcgc taagcgaaag ctaagcaaat aaacaagcgc agctgaacaa gctaaacaat 720
ctgcagtaaa gtgcaagtta aagtgaatca attaaaagta accagcaacc aagtaaatca 780
actgcaacta ctgaaatctg ccaagaagta attattgaat acaagaagag aactctgggg 840
gatcatgacc gaatacaaac ccacagtgag actggccact agagacgatg ttcctagagc 900
tgtcagaact ttggctgccg ctttcgccga ttacccagct actagacaca ccgttgaccc 960
ggatagacac atcgaaagag tcaccgaatt gcaggaactc ttcctgacaa gagttggtct 1020
cgacattgga aaggtctggg tggccgacga tggagccgct gttgctgtct ggacaactcc 1080
cgaatcggtg gaagccggcg ctgttttcgc cgaaataggt cctagaatgg ctgaattgtc 1140
aggttctaga ctcgccgctc aacagcaaat ggaaggactg ttggcccctc acagaccaaa 1200
agaaccggcc tggttcctcg ctactgtggg agttagccca gatcaccagg gtaaaggact 1260
gggctccgct gtggttttgc caggagtcga agctgctgaa agagccggcg tgccggcttt 1320
cttggaaacc tcagccccaa gaaacctccc gttctacgaa agactgggct tcaccgtgac 1380
agctgacgtc gaagtgcccg aaggccctag aacatggtgc atgactagaa aacctggtgc 1440
tgactacaag gacgatgacg ataaagatta taaagacgat gacgataaag actataaaga 1500
tgacgacgat aaatacccct acgacgtgcc tgattacgct cggccgcgac tctagatcat 1560
aatcagccat gcggccgcga ctctagacca catttgtaga ggttttactt gctttaaaaa 1620
acctcccaca cctccccctg aacctgaaac ataaaatgaa tgcaattgtt gttgttaact 1680
tgtttattgc agcttataat ggttacaaat aaagcaatag catcacaaat ttcacaaata 1740
aagcattttt ttcactgcat tctagttgtg gtttgtccaa actcatcaat gtatcttaaa 1800
gcttatcgat acgcgtagtc gaccatgatg ataaacaatg tatggtgcta atgttgcttc 1860
aacaacaatt ctgttgaact gtgttttcat gtttgccaac aagcaccttt atactcggtg 1920
gcctccccac caccaacttt tttgcactgc aaaaaaacac gcttttgcac gcgggcccat 1980
acatagtaca aactctacgt ttcgtagact attttacata aatagtctac accgttgtat 2040
acgctccaaa tacactacca cacattgaac ctttttgcag tgcaaaaaag tacgtgtcgg 2100
cagtcacgta ggccggcctt atcgggtcgc gtcctgtcac gtacgaatca cattatcgga 2160
ccggacgagt gttgtcttat cgtgacagga cgccagcttc ctgtgttgct aaccgcagcc 2220
ggacgcaact ccttatcgga acaggacgcg cctccatatc agccgcgcgt tatctcatgc 2280
gcgtgaccgg acacgaggcg cccgtcccgc ttatcgcgcc tataaataca gcccgcaacg 2340
atctggtaaa cacagttgaa cagcatctgt tcgaaaccgg tgcgatcgca tggtgagcaa 2400
gggcgaggag ctgttcaccg gggtggtgcc catcctggtc gagctggacg gcgacgtaaa 2460
cggccacaag ttcagcgtgt ccggcgaggg cgagggcgat gccacctacg gcaagctgac 2520
cctgaagttc atctgcacca ccggcaagct gcccgtgccc tggcccaccc tcgtgaccac 2580
cctgacctac ggcgtgcagt gcttcagccg ctaccccgac cacatgaagc agcacgactt 2640
cttcaagtcc gccatgcccg aaggctacgt ccaggagcgc accatcttct tcaaggacga 2700
cggcaactac aagacccgcg ccgaggtgaa gttcgagggc gacaccctgg tgaaccgcat 2760
cgagctgaag ggcatcgact tcaaggagga cggcaacatc ctggggcaca agctggagta 2820
caactacaac agccacaacg tctatatcat ggccgacaag cagaagaacg gcatcaaggt 2880
gaacttcaag atccgccaca acatcgagga cggcagcgtg cagctcgccg accactacca 2940
gcagaacacc cccatcggcg acggccccgt gctgctgccc gacaaccact acctgagcac 3000
ccagtccgcc ctgagcaaag accccaacga gaagcgcgat cacatggtcc tgctggagtt 3060
cgtgaccgcc gccgggatca ctctcggcat ggacgagctg tacaagtaaa gatctggtac 3120
ctaaagcttt acaactaaac acgacttgga gtattccttg tagtgtttaa gattttaaat 3180
cttacttaat gacttcgaac gattttaacg ataactttct ctttgtttaa ctttaatcag 3240
catacataaa aagccccggt tttgtatcgg gaagaaaaaa aatgtaattg tgttgcctag 3300
ataataaacg tattatcaaa gtgtgtggtt ttcctttacc aaagacccct ttaagatggg 3360
cctaatgggc ttaagtcgag tcctttccga tgtgttaaat acacatttat tacactgatg 3420
cgtcgaatgt acacttttaa taggatagct ccactaaaaa ttattttatt tatttaattt 3480
gttgcaccaa aactgataca ttgacgaaac gcgtatggcg cgccattaat taaattattg 3540
ttttaagtat gatagtaaat cacattacgc cgcgttcgaa ttaatagtgg tcactttttt 3600
cttatctctt aagcaaaccg tttgaataaa ttactcatat ttttgttgtt gttggaaata 3660
gagcaaaact ttttttttcg tcgtgaagag aataaaattc tctttgagac gaaatgcatt 3720
ggtatgtgtt atctttagta gtattgataa tatagtgtgt taaacattgc gcactgcaaa 3780
aaaaacatgc tgttcgaatt aatagtggtt ggggctcgta gaaaacgaaa aatatcttaa 3840
gctagcatag agaatggagc aaaactcaat ttgatgc 3877
Claims (10)
1.Minos转座子系统介导的真核生物转基因细胞系的构建方法,其特征在于,具体步骤如下:
(1)合成Minos转座子系统基础载体PUC57-Mi-puro,其核苷酸序列如SEQ ID NO.1所示;
(2)将外源基因连接至PUC57-Mi-puro,构建得到转基因载体;
(3)将步骤(2)的转基因载体与Mino转座酶的表达载体混合后转染真核生物细胞,筛选,即得所述细胞系。
2.根据权利要求1所述的构建方法,其特征在于,步骤(1)中,PUC57-Mi-puro包含Minos转座子的末端反向重复序列和标记基因表达框。
3.根据权利要求2所述的构建方法,其特征在于,所述标记基因包括但不限于puromycin抗性筛选基因、Zeocin抗性筛选基因、Blast抗性筛选基因等药物抗性筛选基因,红色荧光蛋白、绿色荧光蛋白、蓝色荧光蛋白等荧光蛋白标记基因。
4.根据权利要求1所述的构建方法,其特征在于,步骤(1)中,PUC57-Mi-puro包含:
Minos转座子的末端反向重复序列(inverted terminal repeat,ITR),其核苷酸序列如SEQ ID NO.3所示;
puromycin抗性筛选基因表达框(Hsp70-puro-SV40),其核苷酸序列如SEQ ID NO.4所示;
用于表达外源基因的框架,其核苷酸序列如SEQ ID NO.5所示;
IE2启动子-家蚕丝胶1终止信号Ser1PA,其核苷酸序列如SEQ ID NO.6所示。
5.根据权利要求1所述的构建方法,其特征在于,步骤(2)中,所述外源基因包括但不限于蛋白编码基因,ncRNA表达基因,miRNA表达框,sgRNA表达框。
6.根据权利要求1所述的构建方法,其特征在于,步骤(2)中,将外源基因的核苷酸序列按照5’到3’方向连接到Minos转座子系统基础载体PUC57-Mi-puro的多克隆位点,多克隆位点限制性酶切位点包括AgeI、AsiSI、BglII和KpnI,构建成Minos转基因载体。
7.根据权利要求1所述的构建方法,其特征在于,步骤(2)中,采用酶切连接的方法将外源基因连接至PUC57-Mi-puro。
8.根据权利要求1所述的构建方法,其特征在于,步骤(3)中,所述Mino转座酶的表达载体为Minos-Helper,其核苷酸序列如SEQ ID NO.2所示。
9.根据权利要求1所述的构建方法,其特征在于,步骤(3)中,转基因载体与Mino转座酶的表达载体按照摩尔比1:1进行混合。
10.利用权利要求1~9中任一项所述方法构建得到的Minos转座子系统介导的真核生物转基因细胞系。
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