CN113355280A - 一种bmp7基因缺失外周血间充质干细胞的建立方法 - Google Patents
一种bmp7基因缺失外周血间充质干细胞的建立方法 Download PDFInfo
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Abstract
本发明涉及一种BMP7基因缺失外周血间充质干细胞的建立方法,具体步骤包括:1)根据BMP7基因序列,设计sgRNA序列;2)sgRNA活性检测,确定最优sgRNA,最优sgRNA序列为GGCGAGTGAGGAGGCGGGCG;3)将步骤2)中切割效率最高的sgRNA和嘌呤霉素抗性的PGK‑puro质粒电转到外周血间充质干细胞;4)基因敲除验证;5)将基因敲除质粒进行测序,本发明构建的BMP7基因缺失外周血间充质干细胞,可用于筛选促成骨形成药物。
Description
技术领域
本发明属于生物技术领域,具体涉及一种BMP7基因缺失外周血间充质干细胞的建立方法。
背景技术
间充质干细胞(mesenchymal stem cells,MSCs)属于成体干细胞的一种,主要来源于发育早期的中胚层和外胚层,具有自我更新、自我增殖和多向分化的能力,可分化为软骨细胞、骨细胞、脂肪细胞、肌肉细胞、神经细胞、施万细胞等,存在于外周血、骨髓、脐带血、皮肤、胎盘、羊水、牙髓等,其中骨髓组织中的含量最为丰富,外周血间充质干细胞(peripheral blood-derived mesenchymal stem cells,PBMSCs)相对于骨髓间充质干细胞具有以下优点:1.来源更加广泛;2.取材更加方便,临床上可操作性强;3.取材过程中减少组织损伤,降低局部组织感染的风险;4.降低医疗成本,减轻患者经济负担。
骨形成因子蛋白可以诱导骨形成,在组织胚胎发育、细胞的生长、分化、迁移凋亡中具有关键作用。骨形成因子7(BMP7)是TGF-β超家族的一员,其具有重要的生物学功能,不仅促进骨的再生,而且对肾脏高糖损伤和纤维化具有重要的保护功能,其在动物发育调控和器官生成、乳腺和***肿瘤的发生发展等过程中起着重要的调控功能。
将外周血充质干细胞的BMP7基因缺失,可以抑制其向成骨细胞分化,利用此模型,可以筛选促成骨形成药物,也可以研究BMP7的信号通路。
发明内容
本发明的目的是建立BMP7基因缺失的外周血间充质干细胞;
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ACACCGGCGAGTGAGGAGGCGGGCGG(Seq ID No.1)AAAACCGCCCGCCTCCTCACTCGCCG(Seq IDNo.2),使用该试剂盒可以用于BMP7基因表达的沉默。
进一步的,本发明提供一种BMP7基因缺失外周血间充质干细胞的建立方法,所述方法包括:
1.采集、冻存、传代外周血干细胞;
2.构建BMP7基因敲除外周血干细胞
2.1根据BMP7基因序列,设计sgRNA序列;
在NCBI上查询人BMP7基因序列(NM_001719,Seq ID No.3),根据CRISPR/Cas9敲除原理,在http://zifit.partners.org/ZiFiT/CSquare9Nuclease.aspx上设计BMP7靶位点;
2.2sgRNA活性检测,确定最优sgRNA;
根据sgRNA订购oligo1和oligo2;oligo1和oligo2退火形成寡核苷酸双链;将寡核苷酸双链连接到pX330载体上;再将连接产物转化至E coli DH5α感受态细胞中,在37℃的含氨苄抗性平板过夜生长,之后挑取单克隆至含氨苄抗性的LB培养基中,于37℃摇床过夜扩大培养,之后按照Tiangen小提质粒试剂盒的说明书提取质粒,最后将质粒测序;为检测sgRNA的切割效率,采用lipo3000脂质体转染法,将之前构建好的sgRNA与Cas9共表达的91个载体分别转染至293T细胞中,每6-8h换液,细胞转染36h后收集细胞,提取细胞总mRNA,反转录为cDNA,以cDNA为模板,用设计好的引物进行PCR,PrimerF:TTGGAAGAGGGTGGGTTG(SeqID No.4),PrimerR:TGTCTGAG TCAAGATGGAGA(Seq ID No.5),PCR条件如下:2×Phanta MaxBuffer 25μL,Phanta Max Super-Fidelity DNA Polymerase 1μL,dNTP Mix 1μL,PrimersF 2μL,PrimersR 2μL,cDNA 1μL,加ddH2O补至50μL,PCR程序如下:98℃预变形3min;(98℃变性15s;65℃退火15s;72℃延伸1min,2-4步共35个循环);72℃延伸5min;PCR产物大小3449bp,将PCR产物跑胶后按照Magen胶回收试剂盒说明书进行回收,得到回收产物,取800ng回收产物,2μL 10×NEB bufferⅡ,加ddH2O补至20μL,梯度退火:95℃,10min;95℃至85℃Ramp:2.0℃/s;85℃至25℃Ramp 0.2℃/s;16℃forever;向退火后的产物中加0.5μL T7核酸内切酶Ⅰ,37℃水浴30min,之后用2%琼脂糖凝胶电泳检测,通过是否有切开后的相对小的两条目的条带以及被切开的条带所占的比例选择切割效率最高的sgRNA;
2.3将步骤2.2中切割效率最高的sgRNA载体和嘌呤霉素抗性的PGK-puro质粒电转到外周血间充质干细胞;
待外周血间充质干细胞密度为70%-80%时,按照Lonza电转说明书,将sgRNA78和嘌呤霉素抗性的PGK-puro质粒共转染到外周血间充质干细胞中,待转染48h后,将细胞传代至10cm细胞培养皿培养,待细胞贴壁后加入嘌呤霉素(1μg/mL)进行筛选3d,经过2周的培养后挑选细胞克隆,并扩大培养;
3.基因敲除验证;
取BMP7基因敲除的外周血间充质干细胞,提取mRNA,反转录为cDNA,然后以cDNA为模板进行PCR扩增,PCR扩增引物、体系、程序同步骤2,将PCR产物跑胶后按照Magen胶回收试剂盒说明书进行回收,得到回收产物,取800ng回收产物,2μL 10×NEB bufferⅡ,加ddH2O补至20μL,梯度退火:95℃,10min;95℃至85℃Ramp:2.0℃/s;85℃至25℃Ramp 0.2℃/s;16℃forever;向退火后的产物中加0.5μL T7核酸内切酶Ⅰ,37℃水浴30min,之后用2%琼脂糖凝胶电泳检测;
4.将基因敲除细胞进行测序;
将PCR鉴定已敲除的细胞,提取mRNA,反转录为cDNA,交生物公司测序。
本发明的有益效果和优点在于:
本发明提供一种BMP7基因缺失外周血间充质干细胞的建立方法,可用于筛选促成骨形成药物。
附图说明
图1:外周血间充质干细胞
图2:基因敲除验证
图3:基因敲除序列与野生型序列比对
图4:外周血间充质干细胞向骨细胞分化
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,这些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:BMP7基因缺失外周血间充质干细胞的建立方法
1.采集、冻存、传代外周血干细胞;
1.1主要设备:生物安全柜、低温水平式离心机、血球计数板、显微镜、血细胞分离机;
1.2主要耗材:真空采血管(ACD/EDTA/肝素抗凝)、离心管、移液管、无菌吸头、冻存管、巴氏吸管、移液器、辅助移液器、注射器、无菌棉球、镊子、橡皮止血带;
1.3主要试剂:RPMI-1640培养基、肽牛血清、DMSO、淋巴细胞分离液、台盼蓝染色液、碘酒、75%酒精;
1.4实验步骤
1.4.1获得供体知情同意后,按照GB/T 38576-2020常规无菌抽取新鲜外周血至ACD/EDTA/肝素抗凝管中;
1.4.2获得供体知情同意后,使用血细胞分离机,按照操作手册规范地采集;采集到的血液或细胞在采集、处理和运输过程中避免冷冻和冷藏,并尽快分离PBMC。
1.4.3分离PBMC,取出无菌离心管,加入淋巴细胞分离液;同时将所得外周血样本用生理盐水/PBS等体积稀释,并充分混匀,形成外周血的稀释液;再将稀释液加入到淋巴细胞分离液中,应将稀释液缓慢匀速地加至淋巴细胞分离液面之上,确保血液稀释液和淋巴细胞分离液形成明显分层(上层为血液稀释液,下层为淋巴细胞分离液);
1.4.4室温低速离心(500×g,下同)20min;
1.4.5离心毕,可见离心管中液体形成明显分层,从上到下依次为血浆与生理盐水/PBC混合液层、单个核细胞白膜层、人淋巴细胞分离液层及红细胞层,用巴氏管或无菌吸头小心吸取白膜层,置于新的离心管中,注意不要吸到淋巴细胞分离液;补充适量的细胞培养基或缓冲液,用移液管吹打均匀;
1.4.6室温低速离心10min;
1.4.7吸弃上清液,保留细胞沉淀,补充适量的细胞培养基或缓冲液,轻柔且充分吹打均匀后,离心洗涤细胞:4℃低速离心5min;
1.4.8重复步骤1.4.7
1.4.9离心毕,吸弃上清液,进行活细胞计数;
1.4.10将PBMC按照一定密度种入培养板或培养瓶继续培养;
1.4.11进行活细胞计数,根据计算得到的PBMC总数,计算冻存所需管数;
将细胞沉淀用RPMI-1640培养基补充,充分吹打均匀后,用精确量程的移液器吸取适量细胞悬液,置于EP管中,随之在EP观众加入适量台盼蓝,使其终浓度为0.04%,充分混匀,台盼蓝染色3min,取适量细胞悬液匀速加入血细胞计数板,倒置相差显微镜下计数:分别计数四大格未染成蓝色的细胞总数和染成蓝色的细胞总数,将结果记录在专用计数本上,利用如下公式计算分离所得PBMC总数和存活率:
注:X:细胞总数;Y:四大格染成蓝色细胞总数;N:四大格细胞总数;n:细胞悬液稀释倍数;v:细胞悬液总体积;S:细胞存活率(%)
最终获得PBMC总数为:2.7×108,细胞存活率为73.8%。
1.4.12取出所需数量冻存管,标记好细胞编号、细胞总数和冻存日期;
1.4.13配制冻存液
1.4.14在PBMC细胞沉淀中加入一定体积配制好的细胞冻存液,轻柔且充分吹打均匀;将吹打均匀的细胞悬液分装入冻存管中,1ml冻存液可用于5×105-5×106个细胞的冻存;
1.4.15常规将细胞程序性降温;
1.4.16将经过程序性降温的冻存管转入液氮容器中,并记录好细胞冻存管放置的位置;
1.4.17 PBMC复苏时,提前将复苏设备预热至37℃,操作人员注意做好防护,迅速从液氮中取出所需管数细胞,将冻存管轻轻放入复苏设备,使细胞快速融化。
1.4.18在无菌离心管中加入适量的完全培养基,将细胞冻存管中细胞悬液吸出置于离心管中,室温低速离心10min,计数,按一定密度接种于培养板或培养瓶,见图1;
2.构建BMP7基因敲除外周血干细胞
2.1主要设备:PCR仪、超净工作台、核酸电泳仪、凝胶成像***、恒温摇床、生化培养箱、离心机、电子分析天平;
2.2主要耗材:离心管、无菌吸头、冻存管、移液器、移液管、培养板;
2.3主要试剂:琼脂糖凝胶DNA回收试剂盒、限制性内切酶、PCR试剂盒、E coli DH5α感受态细胞、293T细胞、考马斯亮蓝R-250、RPMI-1640培养基、肽牛血清、茜红素;
2.4在NCBI上查询人BMP7基因序列(NM_001719,Seq ID No.3),根据CRISPR/Cas9敲除原理,在http://zifit.partners.org/ZiFiT/CSquare9Nuclease.aspx上设计BMP7靶位点,见表1;
表1靶位点及Oligo序列
2.5根据sgRNA订购oligo1和oligo2;
2.6 oligo1和oligo2退火形成寡核苷酸双链;用ddH2O分别将oligo1和oligo2溶解为10μM的溶液,正链与负链各3μL,10×NEB BufferⅡ5μL,ddH2O补足至50μL,混匀后置于PCR仪中,设置退火程序:95℃10:00min;95℃到85℃Ramp:1.0℃/sec;85℃到25℃Ramp:0.1℃/sec;16℃forever;
2.7将寡核苷酸双链连接到pX330载体上;BbsⅠ酶切pX330载体,琼脂糖凝胶电泳酶切产物,胶回收酶切载体,微量分光光度计测量回收后载体浓度,取回收后的载体50ng,与退火后的sgRNA寡核苷酸双链连接,再将连接产物转化至E coli DH5α感受态细胞中,经含氨苄抗性的平板37℃过夜生长,之后挑取单克隆至含氨苄抗性的LB培养基中,于37℃摇床过夜扩大培养,之后按照Tiangen小提质粒试剂盒的说明书提取质粒,最后将质粒测序;
2.8 sgRNA活性的检测
为检测sgRNA的切割效率,采用lipo3000脂质体转染法,将之前构建好的sgRNA与Cas9共表达的91个载体分别转染至293T细胞中,转染后6-8h换液,细胞转染36h后收集细胞,提取细胞总mRNA,反转录为cDNA,以cDNA为模板,用设计好的引物进行PCR,PrimerF:TTGGAAGAGGGTGGGTTG(Seq ID No.4),PrimerR:TGTCTGA GTCAAGATGGAGA(Seq ID No.5),PCR条件如下:2×Phanta Max Buffer 25μL,Phanta Max Super-Fidelity DNAPolymerase 1μL,dNTP Mix 1μL,PrimersF 2μL,PrimersR 2μL,cDNA 1μL,加ddH2O补至50μL,PCR程序如下:98℃预变形3min;(98℃变性15s,65℃退火15s,72℃延伸1min,2-4步共35个循环);72℃延伸5min;PCR产物大小3449bp,将PCR产物跑胶后按照Magen胶回收试剂盒说明书进行回收,得到回收产物,取800ng回收产物,2μL 10×NEB bufferⅡ,加ddH2O补至20μL,梯度退火:95℃,10min;95℃至85℃Ramp:2.0℃/s;85℃至25℃Ramp 0.2℃/s;16℃forever;向退火后的产物中加0.5μL T7核酸内切酶Ⅰ,37℃水浴30min,之后用2%琼脂糖凝胶电泳检测,通过是否有切开后的相对小的两条目的条带以及被切开的条带所占的比例选择切割效率最高的sgRNA,计算公式如下:
切割效率结果见表2,最终选择sgRNA78作为靶序列;
表2 sgRNA切割效率(%)
2.9将步骤2.8中切割效率最高的sgRNA载体(sgRAN78)和嘌呤霉素抗性的PGK-puro质粒电转到外周血间充质干细胞;待外周血间充质干细胞密度为70%-80%时,按照Lonza电转说明书,将sgRNA78和嘌呤霉素抗性的PGK-puro质粒共转染到外周血间充质干细胞中,待转染48h后,将细胞传代至10cm细胞培养皿培养,待细胞贴壁后加入嘌呤霉素(1μg/mL)进行筛选3d,经过2周的培养后挑选细胞克隆,并扩大培养;
3.基因敲除验证;取BMP7基因敲除的外周血间充质干细胞,提取mRNA,反转录为cDNA,然后以cDNA为模板进行PCR扩增,PCR扩增引物、体系、程序同步骤2,将PCR产物跑胶后按照Magen胶回收试剂盒说明书进行回收,得到回收产物,取800ng回收产物,2μL 10×NEBbufferⅡ,加ddH2O补至20μL,梯度退火:95℃,10min;95℃至85℃Ramp:2.0℃/s;85℃至25℃Ramp 0.2℃/s;16℃forever;向退火后的产物中加0.5μL T7核酸内切酶Ⅰ,37℃水浴30min,之后用2%琼脂糖凝胶电泳检测见图2;
4.将PCR鉴定已敲除的细胞,提取mRNA,反转录为cDNA,交生物公司测序,敲除序列与野生序列对边见图3。
实施例2外周血间充质干细胞诱导成骨分化
分别取野生型和基因缺失型第3代外周血来源间充质干细胞,按1×104密度接种于12孔板,培养24h细胞贴壁后换用含1×107μmol/L***、50mg/L维生素C、10mmol/Lβ-甘油磷酸钠、体积分数为0.07肽牛血清的成骨诱导液培养,每隔3d换液,第20天茜素红染色。
实验结果显示野生型外周血间充质干细胞在成骨诱导液的作用下,向骨细胞分化,但是基因缺失型外周血间充质干细胞则不分化(图4)。
序列表
<110> 北京大学第三医院
<120> 一种BMP7基因缺失外周血间充质干细胞的建立方法
<160> 276
<170> SIPOSequenceListing 1.0
<210> 1
<211> 26
<212> DNA
<213> 人工序列(Artificial sequence)
<400> 1
acaccggcga gtgaggaggc gggcgg 26
<210> 2
<211> 26
<212> DNA
<213> 人工序列(Artificial sequence)
<400> 2
aaaaccgccc gcctcctcac tcgccg 26
<210> 3
<211> 4021
<212> DNA
<213> Homo sapiens
<400> 3
actctggcgc tcccgaggcg gcctcttgtg cgatccaggg cgcacaaggc tgggagagcg 60
ccccggggcc cctgctatcc gcgccggagg ttggaagagg gtgggttgcc gccgcccgag 120
ggcgagagcg ccagaggagc gggaagaagg agcgctcgcc cgcccgcctg cctcctcgct 180
gcctccccgg cgttggctct ctggactcct aggcttgctg gctgctcctc ccacccgcgc 240
ccgcctcctc actcgccttt tcgttcgccg gggctgcttt ccaagccctg cggtgcgccc 300
gggcgagtgc ggggcgaggg gcccggggcc agcaccgagc agggggcggg ggtccgggca 360
gagcgcggcc ggccggggag gggccatgtc tggcgcgggc gcagcggggc ccgtctgcag 420
caagtgaccg agcggcgcgg acggccgcct gccccctctg ccacctgggg cggtgcgggc 480
ccggagcccg gagcccgggt agcgcgtaga gccggcgcga tgcacgtgcg ctcactgcga 540
gctgcggcgc cgcacagctt cgtggcgctc tgggcacccc tgttcctgct gcgctccgcc 600
ctggccgact tcagcctgga caacgaggtg cactcgagct tcatccaccg gcgcctccgc 660
agccaggagc ggcgggagat gcagcgcgag atcctctcca ttttgggctt gccccaccgc 720
ccgcgcccgc acctccaggg caagcacaac tcggcaccca tgttcatgct ggacctgtac 780
aacgccatgg cggtggagga gggcggcggg cccggcggcc agggcttctc ctacccctac 840
aaggccgtct tcagtaccca gggcccccct ctggccagcc tgcaagatag ccatttcctc 900
accgacgccg acatggtcat gagcttcgtc aacctcgtgg aacatgacaa ggaattcttc 960
cacccacgct accaccatcg agagttccgg tttgatcttt ccaagatccc agaaggggaa 1020
gctgtcacgg cagccgaatt ccggatctac aaggactaca tccgggaacg cttcgacaat 1080
gagacgttcc ggatcagcgt ttatcaggtg ctccaggagc acttgggcag ggaatcggat 1140
ctcttcctgc tcgacagccg taccctctgg gcctcggagg agggctggct ggtgtttgac 1200
atcacagcca ccagcaacca ctgggtggtc aatccgcggc acaacctggg cctgcagctc 1260
tcggtggaga cgctggatgg gcagagcatc aaccccaagt tggcgggcct gattgggcgg 1320
cacgggcccc agaacaagca gcccttcatg gtggctttct tcaaggccac ggaggtccac 1380
ttccgcagca tccggtccac ggggagcaaa cagcgcagcc agaaccgctc caagacgccc 1440
aagaaccagg aagccctgcg gatggccaac gtggcagaga acagcagcag cgaccagagg 1500
caggcctgta agaagcacga gctgtatgtc agcttccgag acctgggctg gcaggactgg 1560
atcatcgcgc ctgaaggcta cgccgcctac tactgtgagg gggagtgtgc cttccctctg 1620
aactcctaca tgaacgccac caaccacgcc atcgtgcaga cgctggtcca cttcatcaac 1680
ccggaaacgg tgcccaagcc ctgctgtgcg cccacgcagc tcaatgccat ctccgtcctc 1740
tacttcgatg acagctccaa cgtcatcctg aagaaataca gaaacatggt ggtccgggcc 1800
tgtggctgcc actagctcct ccgagaattc agaccctttg gggccaagtt tttctggatc 1860
ctccattgct cgccttggcc aggaaccagc agaccaactg ccttttgtga gaccttcccc 1920
tccctatccc caactttaaa ggtgtgagag tattaggaaa catgagcagc atatggcttt 1980
tgatcagttt ttcagtggca gcatccaatg aacaagatcc tacaagctgt gcaggcaaaa 2040
cctagcagga aaaaaaaaca acgcataaag aaaaatggcc gggccaggtc attggctggg 2100
aagtctcagc catgcacgga ctcgtttcca gaggtaatta tgagcgccta ccagccaggc 2160
cacccagccg tgggaggaag ggggcgtggc aaggggtggg cacattggtg tctgtgcgaa 2220
aggaaaattg acccggaagt tcctgtaata aatgtcacaa taaaacgaat gaatgaaaat 2280
ggttaggacg ttacagatat attttcctaa acaatttatc cccatttctc ggtttatcct 2340
gatgcgtaaa cagaagctgt gtcaagtgga gggcggggag gtccctctcc attccctaca 2400
gttttcatcc tgaggcttgc agaggcccag tgtttaccga ggtttgccca aatccaagat 2460
ctagtgggag gggaaagagc aaatgtctgc tccgaggagg gcggtgtgtt gatctttgga 2520
ggaaaaatat gttctgttgt tcagctggat ttgccgtggc agaaatgaaa ctaggtgtgt 2580
gaaatacccg cagacatttg ggattggctt ttcacctcgc cccagtggta gtaaatccat 2640
gtgaaattgc agaggggaca aggacagcaa gtaggatgga acttgcaact caaccctgtt 2700
gttaagaagc accaatgggc cgggcacagt agctcccacc tgtaatccca gcactttggg 2760
aggctgaggt gggcggatca tttgaggtca ggagttcgag accagcctgg ccaacatggt 2820
gaaaccccat ctctactaaa aatacaaaaa ttagccgggc atggtggcac gcacctgtaa 2880
tcccagctac tctggaggct gaggcaggag aattgcttga accccagagg tggaggttgc 2940
agtgagccaa gatcgtccca ctgcactcca gcttgggtga caaaacaaga ctccatctca 3000
aaagaaaaaa aaaacagcac caatgaagcc tagttctcca cgggagtggg gtgagcagga 3060
gcactgcaca tcgccccagt ggaccctctg gtctttgtct gcagtggcat tccaaggctg 3120
ggccctggca agggcacccg tggctgtctc ttcatttgca gaccctgatc agaagtctct 3180
gcaaacaaat ttgctccttg aattaagggg gagatggcat aataggaggt ctgatgggtg 3240
caggatgtgc tggacttaca ttgcaaatag aagccttgtt gagggtgaca tcctaaccaa 3300
gtgtcccgat ttggaggtgg catttctgac gtggctcttg gtgtaagcct gccttgcctt 3360
ggctggtgag tcccataaat agtatgcact cagcctccgg ccacaaacac aaggcctagg 3420
ggagggctag actgtctgca aacgttttct gcatctgtaa agaaaacaag gtgatcgaaa 3480
actgtggcca tgtggaaccc ggtcttgtgg gggactgttt ctccatcttg actcagacag 3540
ttcctggaaa caccggggct ctgtttttat tttctttgat gtttttcttc tttagtagct 3600
tgggctgcag cctccactct ctagtcactg gggaggagta ttttttgtta tgtttggttt 3660
catttgctgg cagagctggg gctttttgtg tgatccctct tggtgtgagt tttctgaccc 3720
aaccagcctc tggttagcat catttgtaca tttaaacctg taaatagttg ttacaaagca 3780
aagagattat ttatttccat ccaaagctct tttgaacacc cccccccctt taatccctcg 3840
ttcaggacga tgagcttgct ttccttcaac ctgtttgttt tcttatttaa gactatttat 3900
taatggttgg accaatgtac tcacagctgt tgcgtcgagc agtccttagt gaaaattctg 3960
tataaataga caaaatgaaa agggtttgac cttgcaataa aaggagacgt ttggttctgg 4020
c 4021
<210> 4
<211> 18
<212> DNA
<213> 人工序列(Artificial sequence)
<400> 4
ttggaagagg gtgggttg 18
<210> 5
<211> 20
<212> DNA
<213> 人工序列(Artificial sequence)
<400> 5
tgtctgagtc aagatggaga 20
<210> 6
<211> 20
<212> DNA
<213> 人工序列(Artificial sequence)
<400> 6
ggcggcctct tgtgcgatcc 20
<210> 7
<211> 26
<212> DNA
<213> 人工序列(Artificial sequence)
<400> 7
acaccggcgg cctcttgtgc gatccg 26
<210> 8
<211> 26
<212> DNA
<213> 人工序列(Artificial sequence)
<400> 8
aaaacggatc gcacaagagg ccgccg 26
<210> 9
<211> 20
<212> DNA
<213> 人工序列(Artificial sequence)
<400> 9
gcggcctctt gtgcgatcca 20
<210> 10
<211> 26
<212> DNA
<213> 人工序列(Artificial sequence)
<400> 10
acaccgcggc ctcttgtgcg atccag 26
<210> 11
<211> 26
<212> DNA
<213> 人工序列(Artificial sequence)
<400> 11
aaaactggat cgcacaagag gccgcg 26
<210> 12
<211> 20
<212> DNA
<213> 人工序列(Artificial sequence)
<400> 12
gatccagggc gcacaaggct 20
<210> 13
<211> 26
<212> DNA
<213> 人工序列(Artificial sequence)
<400> 13
acaccgatcc agggcgcaca aggctg 26
<210> 14
<211> 26
<212> DNA
<213> 人工序列(Artificial sequence)
<400> 14
aaaacagcct tgtgcgccct ggatcg 26
<210> 15
<211> 20
<212> DNA
<213> 人工序列(Artificial sequence)
<400> 15
ggggcccctg ctatccgcgc 20
<210> 16
<211> 26
<212> DNA
<213> 人工序列(Artificial sequence)
<400> 16
acaccggggc ccctgctatc cgcgcg 26
<210> 17
<211> 26
<212> DNA
<213> 人工序列(Artificial sequence)
<400> 17
aaaacgcgcg gatagcaggg gccccg 26
<210> 18
<211> 20
<212> DNA
<213> 人工序列(Artificial sequence)
<400> 18
gcccctgcta tccgcgccgg 20
<210> 19
<211> 26
<212> DNA
<213> 人工序列(Artificial sequence)
<400> 19
acaccgcccc tgctatccgc gccggg 26
<210> 20
<211> 26
<212> DNA
<213> 人工序列(Artificial sequence)
<400> 20
aaaacccggc gcggatagca ggggcg 26
<210> 21
<211> 20
<212> DNA
<213> 人工序列(Artificial sequence)
<400> 21
gccggaggtt ggaagagggt 20
<210> 22
<211> 26
<212> DNA
<213> 人工序列(Artificial sequence)
<400> 22
acaccgccgg aggttggaag agggtg 26
<210> 23
<211> 26
<212> DNA
<213> 人工序列(Artificial sequence)
<400> 23
aaaacaccct cttccaacct ccggcg 26
<210> 24
<211> 20
<212> DNA
<213> 人工序列(Artificial sequence)
<400> 24
gggtgggttg ccgccgcccg 20
<210> 25
<211> 26
<212> DNA
<213> 人工序列(Artificial sequence)
<400> 25
acaccgggtg ggttgccgcc gcccgg 26
<210> 26
<211> 26
<212> DNA
<213> 人工序列(Artificial sequence)
<400> 26
aaaaccgggc ggcggcaacc cacccg 26
<210> 27
<211> 20
<212> DNA
<213> 人工序列(Artificial sequence)
<400> 27
ggtgggttgc cgccgcccga 20
<210> 28
<211> 26
<212> DNA
<213> 人工序列(Artificial sequence)
<400> 28
acaccggtgg gttgccgccg cccgag 26
<210> 29
<211> 26
<212> DNA
<213> 人工序列(Artificial sequence)
<400> 29
aaaactcggg cggcggcaac ccaccg 26
<210> 30
<211> 20
<212> DNA
<213> 人工序列(Artificial sequence)
<400> 30
gggcgagagc gccagaggag 20
<210> 31
<211> 26
<212> DNA
<213> 人工序列(Artificial sequence)
<400> 31
acaccgggcg agagcgccag aggagg 26
<210> 32
<211> 26
<212> DNA
<213> 人工序列(Artificial sequence)
<400> 32
aaaacctcct ctggcgctct cgcccg 26
<210> 33
<211> 20
<212> DNA
<213> 人工序列(Artificial sequence)
<400> 33
ggcgagagcg ccagaggagc 20
<210> 34
<211> 26
<212> DNA
<213> 人工序列(Artificial sequence)
<400> 34
acaccggcga gagcgccaga ggagcg 26
<210> 35
<211> 26
<212> DNA
<213> 人工序列(Artificial sequence)
<400> 35
aaaacgctcc tctggcgctc tcgccg 26
<210> 36
<211> 20
<212> DNA
<213> 人工序列(Artificial sequence)
<400> 36
gcgccagagg agcgggaaga 20
<210> 37
<211> 26
<212> DNA
<213> 人工序列(Artificial sequence)
<400> 37
acaccgcgcc agaggagcgg gaagag 26
<210> 38
<211> 26
<212> DNA
<213> 人工序列(Artificial sequence)
<400> 38
aaaactcttc ccgctcctct ggcgcg 26
<210> 39
<211> 20
<212> DNA
<213> 人工序列(Artificial sequence)
<400> 39
gggctgcttt ccaagccctg 20
<210> 40
<211> 26
<212> DNA
<213> 人工序列(Artificial sequence)
<400> 40
acaccgggct gctttccaag ccctgg 26
<210> 41
<211> 26
<212> DNA
<213> 人工序列(Artificial sequence)
<400> 41
aaaaccaggg cttggaaagc agcccg 26
<210> 42
<211> 20
<212> DNA
<213> 人工序列(Artificial sequence)
<400> 42
gcggtgcgcc cgggcgagtg 20
<210> 43
<211> 26
<212> DNA
<213> 人工序列(Artificial sequence)
<400> 43
acaccgcggt gcgcccgggc gagtgg 26
<210> 44
<211> 26
<212> DNA
<213> 人工序列(Artificial sequence)
<400> 44
aaaaccactc gcccgggcgc accgcg 26
<210> 46
<211> 20
<212> DNA
<213> 人工序列(Artificial sequence)
<400> 46
ggtgcgcccg ggcgagtgcg 20
<210> 46
<211> 26
<212> DNA
<213> 人工序列(Artificial sequence)
<400> 46
acaccggtgc gcccgggcga gtgcgg 26
<210> 47
<211> 26
<212> DNA
<213> 人工序列(Artificial sequence)
<400> 47
aaaaccgcac tcgcccgggc gcaccg 26
<210> 48
<211> 20
<212> DNA
<213> 人工序列(Artificial sequence)
<400> 48
gcccgggcga gtgcggggcg 20
<210> 49
<211> 26
<212> DNA
<213> 人工序列(Artificial sequence)
<400> 49
acaccgcccg ggcgagtgcg gggcgg 26
<210> 50
<211> 26
<212> DNA
<213> 人工序列(Artificial sequence)
<400> 50
aaaaccgccc cgcactcgcc cgggcg 26
<210> 51
<211> 20
<212> DNA
<213> 人工序列(Artificial sequence)
<400> 51
gagtgcgggg cgaggggccc 20
<210> 52
<211> 26
<212> DNA
<213> 人工序列(Artificial sequence)
<400> 52
acaccgagtg cggggcgagg ggcccg 26
<210> 53
<211> 26
<212> DNA
<213> 人工序列(Artificial sequence)
<400> 53
aaaacgggcc cctcgccccg cactcg 26
<210> 54
<211> 20
<212> DNA
<213> 人工序列(Artificial sequence)
<400> 54
gcccggggcc agcaccgagc 20
<210> 55
<211> 26
<212> DNA
<213> 人工序列(Artificial sequence)
<400> 55
acaccgcccg gggccagcac cgagcg 26
<210> 56
<211> 26
<212> DNA
<213> 人工序列(Artificial sequence)
<400> 56
aaaacgctcg gtgctggccc cgggcg 26
<210> 57
<211> 20
<212> DNA
<213> 人工序列(Artificial sequence)
<400> 57
ggccagcacc gagcaggggg 20
<210> 58
<211> 26
<212> DNA
<213> 人工序列(Artificial sequence)
<400> 58
acaccggcca gcaccgagca gggggg 26
<210> 59
<211> 26
<212> DNA
<213> 人工序列(Artificial sequence)
<400> 59
aaaacccccc tgctcggtgc tggccg 26
<210> 60
<211> 20
<212> DNA
<213> 人工序列(Artificial sequence)
<400> 60
gccagcaccg agcagggggc 20
<210> 61
<211> 26
<212> DNA
<213> 人工序列(Artificial sequence)
<400> 61
acaccgccag caccgagcag ggggcg 26
<210> 62
<211> 26
<212> DNA
<213> 人工序列(Artificial sequence)
<400> 62
aaaacgcccc ctgctcggtg ctggcg 26
<210> 63
<211> 20
<212> DNA
<213> 人工序列(Artificial sequence)
<400> 63
gcgggggtcc gggcagagcg 20
<210> 64
<211> 26
<212> DNA
<213> 人工序列(Artificial sequence)
<400> 64
acaccgcggg ggtccgggca gagcgg 26
<210> 65
<211> 26
<212> DNA
<213> 人工序列(Artificial sequence)
<400> 65
aaaaccgctc tgcccggacc cccgcg 26
<210> 66
<211> 20
<212> DNA
<213> 人工序列(Artificial sequence)
<400> 66
gggtccgggc agagcgcggc 20
<210> 67
<211> 26
<212> DNA
<213> 人工序列(Artificial sequence)
<400> 67
acaccgggtc cgggcagagc gcggcg 26
<210> 68
<211> 26
<212> DNA
<213> 人工序列(Artificial sequence)
<400> 68
aaaacgccgc gctctgcccg gacccg 26
<210> 69
<211> 20
<212> DNA
<213> 人工序列(Artificial sequence)
<400> 69
gggcagagcg cggccggccg 20
<210> 70
<211> 26
<212> DNA
<213> 人工序列(Artificial sequence)
<400> 70
acaccgggca gagcgcggcc ggccgg 26
<210> 71
<211> 26
<212> DNA
<213> 人工序列(Artificial sequence)
<400> 71
aaaaccggcc ggccgcgctc tgcccg 26
<210> 72
<211> 20
<212> DNA
<213> 人工序列(Artificial sequence)
<400> 72
gagcgcggcc ggccggggag 20
<210> 73
<211> 26
<212> DNA
<213> 人工序列(Artificial sequence)
<400> 73
acaccgagcg cggccggccg gggagg 26
<210> 74
<211> 26
<212> DNA
<213> 人工序列(Artificial sequence)
<400> 74
aaaacctccc cggccggccg cgctcg 26
<210> 75
<211> 20
<212> DNA
<213> 人工序列(Artificial sequence)
<400> 75
ggccggggag gggccatgtc 20
<210> 76
<211> 26
<212> DNA
<213> 人工序列(Artificial sequence)
<400> 76
acaccggccg gggaggggcc atgtcg 26
<210> 77
<211> 26
<212> DNA
<213> 人工序列(Artificial sequence)
<400> 77
aaaacgacat ggcccctccc cggccg 26
<210> 78
<211> 20
<212> DNA
<213> 人工序列(Artificial sequence)
<400> 78
gggaggggcc atgtctggcg 20
<210> 79
<211> 26
<212> DNA
<213> 人工序列(Artificial sequence)
<400> 79
acaccgggag gggccatgtc tggcgg 26
<210> 80
<211> 26
<212> DNA
<213> 人工序列(Artificial sequence)
<400> 80
aaaaccgcca gacatggccc ctcccg 26
<210> 81
<211> 20
<212> DNA
<213> 人工序列(Artificial sequence)
<400> 81
ggaggggcca tgtctggcgc 20
<210> 82
<211> 26
<212> DNA
<213> 人工序列(Artificial sequence)
<400> 82
acaccggagg ggccatgtct ggcgcg 26
<210> 83
<211> 26
<212> DNA
<213> 人工序列(Artificial sequence)
<400> 83
aaaacgcgcc agacatggcc cctccg 26
<210> 84
<211> 20
<212> DNA
<213> 人工序列(Artificial sequence)
<400> 84
gtctgcagca agtgaccgag 20
<210> 85
<211> 26
<212> DNA
<213> 人工序列(Artificial sequence)
<400> 85
acaccgtctg cagcaagtga ccgagg 26
<210> 86
<211> 26
<212> DNA
<213> 人工序列(Artificial sequence)
<400> 86
aaaacctcgg tcacttgctg cagacg 26
<210> 87
<211> 20
<212> DNA
<213> 人工序列(Artificial sequence)
<400> 87
gcctgccccc tctgccacct 20
<210> 88
<211> 26
<212> DNA
<213> 人工序列(Artificial sequence)
<400> 88
acaccgcctg ccccctctgc cacctg 26
<210> 89
<211> 26
<212> DNA
<213> 人工序列(Artificial sequence)
<400> 89
aaaacaggtg gcagaggggg caggcg 26
<210> 90
<211> 20
<212> DNA
<213> 人工序列(Artificial sequence)
<400> 90
gccccctctg ccacctgggg 20
<210> 91
<211> 26
<212> DNA
<213> 人工序列(Artificial sequence)
<400> 91
acaccgcccc ctctgccacc tggggg 26
<210> 92
<211> 26
<212> DNA
<213> 人工序列(Artificial sequence)
<400> 92
aaaaccccca ggtggcagag ggggcg 26
<210> 93
<211> 20
<212> DNA
<213> 人工序列(Artificial sequence)
<400> 93
ggcggtgcgg gcccggagcc 20
<210> 94
<211> 26
<212> DNA
<213> 人工序列(Artificial sequence)
<400> 94
acaccggcgg tgcgggcccg gagccg 26
<210> 95
<211> 26
<212> DNA
<213> 人工序列(Artificial sequence)
<400> 95
aaaacggctc cgggcccgca ccgccg 26
<210> 96
<211> 20
<212> DNA
<213> 人工序列(Artificial sequence)
<400> 96
gggcccggag cccggagccc 20
<210> 97
<211> 26
<212> DNA
<213> 人工序列(Artificial sequence)
<400> 97
acaccgggcc cggagcccgg agcccg 26
<210> 98
<211> 26
<212> DNA
<213> 人工序列(Artificial sequence)
<400> 98
aaaacgggct ccgggctccg ggcccg 26
<210> 99
<211> 20
<212> DNA
<213> 人工序列(Artificial sequence)
<400> 99
gcccgggtag cgcgtagagc 20
<210> 100
<211> 26
<212> DNA
<213> 人工序列(Artificial sequence)
<400> 100
acaccgcccg ggtagcgcgt agagcg 26
<210> 101
<211> 26
<212> DNA
<213> 人工序列(Artificial sequence)
<400> 101
aaaacgctct acgcgctacc cgggcg 26
<210> 102
<211> 20
<212> DNA
<213> 人工序列(Artificial sequence)
<400> 102
gcacagcttc gtggcgctct 20
<210> 103
<211> 26
<212> DNA
<213> 人工序列(Artificial sequence)
<400> 103
acaccgcaca gcttcgtggc gctctg 26
<210> 104
<211> 26
<212> DNA
<213> 人工序列(Artificial sequence)
<400> 104
aaaacagagc gccacgaagc tgtgcg 26
<210> 105
<211> 20
<212> DNA
<213> 人工序列(Artificial sequence)
<400> 105
gttcctgctg cgctccgccc 20
<210> 106
<211> 26
<212> DNA
<213> 人工序列(Artificial sequence)
<400> 106
acaccgttcc tgctgcgctc cgcccg 26
<210> 107
<211> 26
<212> DNA
<213> 人工序列(Artificial sequence)
<400> 107
aaaacgggcg gagcgcagca ggaacg 26
<210> 108
<211> 20
<212> DNA
<213> 人工序列(Artificial sequence)
<400> 108
ggcgcctccg cagccaggag 20
<210> 109
<211> 26
<212> DNA
<213> 人工序列(Artificial sequence)
<400> 109
acaccggcgc ctccgcagcc aggagg 26
<210> 110
<211> 26
<212> DNA
<213> 人工序列(Artificial sequence)
<400> 110
aaaacctcct ggctgcggag gcgccg 26
<210> 111
<211> 20
<212> DNA
<213> 人工序列(Artificial sequence)
<400> 111
gcctccgcag ccaggagcgg 20
<210> 112
<211> 26
<212> DNA
<213> 人工序列(Artificial sequence)
<400> 112
acaccgcctc cgcagccagg agcggg 26
<210> 113
<211> 26
<212> DNA
<213> 人工序列(Artificial sequence)
<400> 113
aaaacccgct cctggctgcg gaggcg 26
<210> 114
<211> 21
<212> DNA
<213> 人工序列(Artificial sequence)
<400> 114
gcgcgagatc ctctccattt s 21
<210> 115
<211> 26
<212> DNA
<213> 人工序列(Artificial sequence)
<400> 115
acaccgcgcg agatcctctc catttg 26
<210> 116
<211> 26
<212> DNA
<213> 人工序列(Artificial sequence)
<400> 116
aaaacaaatg gagaggatct cgcgcg 26
<210> 117
<211> 20
<212> DNA
<213> 人工序列(Artificial sequence)
<400> 117
gctggacctg tacaacgcca 20
<210> 118
<211> 26
<212> DNA
<213> 人工序列(Artificial sequence)
<400> 118
acaccgctgg acctgtacaa cgccag 26
<210> 119
<211> 26
<212> DNA
<213> 人工序列(Artificial sequence)
<400> 119
aaaactggcg ttgtacaggt ccagcg 26
<210> 120
<211> 20
<212> DNA
<213> 人工序列(Artificial sequence)
<400> 120
ggacctgtac aacgccatgg 20
<210> 121
<211> 26
<212> DNA
<213> 人工序列(Artificial sequence)
<400> 121
acaccggacc tgtacaacgc catggg 26
<210> 122
<211> 26
<212> DNA
<213> 人工序列(Artificial sequence)
<400> 122
aaaacccatg gcgttgtaca ggtccg 26
<210> 123
<211> 20
<212> DNA
<213> 人工序列(Artificial sequence)
<400> 123
gtacaacgcc atggcggtgg 20
<210> 124
<211> 26
<212> DNA
<213> 人工序列(Artificial sequence)
<400> 124
acaccgtaca acgccatggc ggtggg 26
<210> 125
<211> 26
<212> DNA
<213> 人工序列(Artificial sequence)
<400> 125
aaaacccacc gccatggcgt tgtacg 26
<210> 126
<211> 20
<212> DNA
<213> 人工序列(Artificial sequence)
<400> 126
gccatggcgg tggaggaggg 20
<210> 127
<211> 26
<212> DNA
<213> 人工序列(Artificial sequence)
<400> 127
acaccgccat ggcggtggag gagggg 26
<210> 128
<211> 26
<212> DNA
<213> 人工序列(Artificial sequence)
<400> 128
aaaacccctc ctccaccgcc atggcg 26
<210> 129
<211> 20
<212> DNA
<213> 人工序列(Artificial sequence)
<400> 129
gtggaggagg gcggcgggcc 20
<210> 130
<211> 26
<212> DNA
<213> 人工序列(Artificial sequence)
<400> 130
acaccgtgga ggagggcggc gggccg 26
<210> 131
<211> 26
<212> DNA
<213> 人工序列(Artificial sequence)
<400> 131
aaaacggccc gccgccctcc tccacg 26
<210> 132
<211> 20
<212> DNA
<213> 人工序列(Artificial sequence)
<400> 132
gaggagggcg gcgggcccgg 20
<210> 133
<211> 26
<212> DNA
<213> 人工序列(Artificial sequence)
<400> 133
acaccgagga gggcggcggg cccggg 26
<210> 134
<211> 26
<212> DNA
<213> 人工序列(Artificial sequence)
<400> 134
aaaacccggg cccgccgccc tcctcg 26
<210> 135
<211> 20
<212> DNA
<213> 人工序列(Artificial sequence)
<400> 135
gggcggcggg cccggcggcc 20
<210> 136
<211> 26
<212> DNA
<213> 人工序列(Artificial sequence)
<400> 136
acaccgggcg gcgggcccgg cggccg 26
<210> 137
<211> 26
<212> DNA
<213> 人工序列(Artificial sequence)
<400> 137
aaaacggccg ccgggcccgc cgcccg 26
<210> 138
<211> 20
<212> DNA
<213> 人工序列(Artificial sequence)
<400> 138
ggcggcgggc ccggcggcca 20
<210> 139
<211> 26
<212> DNA
<213> 人工序列(Artificial sequence)
<400> 139
acaccggcgg cgggcccggc ggccag 26
<210> 140
<211> 26
<212> DNA
<213> 人工序列(Artificial sequence)
<400> 140
aaaactggcc gccgggcccg ccgccg 26
<210> 141
<211> 20
<212> DNA
<213> 人工序列(Artificial sequence)
<400> 141
gggcttctcc tacccctaca 20
<210> 142
<211> 26
<212> DNA
<213> 人工序列(Artificial sequence)
<400> 142
acaccgggct tctcctaccc ctacag 26
<210> 143
<211> 26
<212> DNA
<213> 人工序列(Artificial sequence)
<400> 143
aaaactgtag gggtaggaga agcccg 26
<210> 144
<211> 20
<212> DNA
<213> 人工序列(Artificial sequence)
<400> 144
gctaccacca tcgagagttc 20
<210> 145
<211> 26
<212> DNA
<213> 人工序列(Artificial sequence)
<400> 145
acaccgctac caccatcgag agttcg 26
<210> 158
<211> 26
<212> DNA
<213> 人工序列(Artificial sequence)
<400> 158
aaaacgaact ctcgatggtg gtagcg 26
<210> 146
<211> 20
<212> DNA
<213> 人工序列(Artificial sequence)
<400> 146
gacgaagctc atgaccatgt 20
<210> 147
<211> 26
<212> DNA
<213> 人工序列(Artificial sequence)
<400> 147
acaccgacga agctcatgac catgtg 26
<210> 148
<211> 26
<212> DNA
<213> 人工序列(Artificial sequence)
<400> 148
aaaacacatg gtcatgagct tcgtcg 26
<210> 149
<211> 20
<212> DNA
<213> 人工序列(Artificial sequence)
<400> 149
gctcatgacc atgtcggcgt 20
<210> 150
<211> 26
<212> DNA
<213> 人工序列(Artificial sequence)
<400> 150
acaccgctca tgaccatgtc ggcgtg 26
<210> 151
<211> 26
<212> DNA
<213> 人工序列(Artificial sequence)
<400> 151
aaaacacgcc gacatggtca tgagcg 26
<210> 152
<211> 20
<212> DNA
<213> 人工序列(Artificial sequence)
<400> 152
gaaatggcta tcttgcaggc 20
<210> 153
<211> 26
<212> DNA
<213> 人工序列(Artificial sequence)
<400> 153
acaccgaaat ggctatcttg caggcg 26
<210> 154
<211> 26
<212> DNA
<213> 人工序列(Artificial sequence)
<400> 154
aaaacgcctg caagatagcc atttcg 26
<210> 155
<211> 20
<212> DNA
<213> 人工序列(Artificial sequence)
<400> 155
ggctggccag aggggggccc 20
<210> 156
<211> 26
<212> DNA
<213> 人工序列(Artificial sequence)
<400> 156
acaccggctg gccagagggg ggcccg 26
<210> 157
<211> 26
<212> DNA
<213> 人工序列(Artificial sequence)
<400> 157
aaaacgggcc cccctctggc cagccg 26
<210> 159
<211> 20
<212> DNA
<213> 人工序列(Artificial sequence)
<400> 159
gctggccaga ggggggccct 20
<210> 160
<211> 26
<212> DNA
<213> 人工序列(Artificial sequence)
<400> 160
acaccgctgg ccagaggggg gccctg 26
<210> 161
<211> 26
<212> DNA
<213> 人工序列(Artificial sequence)
<400> 161
aaaacagggc ccccctctgg ccagcg 26
<210> 162
<211> 20
<212> DNA
<213> 人工序列(Artificial sequence)
<400> 162
ggggccctgg gtactgaaga 20
<210> 163
<211> 26
<212> DNA
<213> 人工序列(Artificial sequence)
<400> 163
acaccggggc cctgggtact gaagag 26
<210> 164
<211> 26
<212> DNA
<213> 人工序列(Artificial sequence)
<400> 164
aaaactcttc agtacccagg gccccg 26
<210> 165
<211> 20
<212> DNA
<213> 人工序列(Artificial sequence)
<400> 165
ggtactgaag acggccttgt 20
<210> 166
<211> 26
<212> DNA
<213> 人工序列(Artificial sequence)
<400> 166
acaccggtac tgaagacggc cttgtg 26
<210> 167
<211> 26
<212> DNA
<213> 人工序列(Artificial sequence)
<400> 167
aaaacacaag gccgtcttca gtaccg 26
<210> 168
<211> 20
<212> DNA
<213> 人工序列(Artificial sequence)
<400> 168
gtactgaaga cggccttgta 20
<210> 169
<211> 26
<212> DNA
<213> 人工序列(Artificial sequence)
<400> 169
acaccgtact gaagacggcc ttgtag 26
<210> 170
<211> 26
<212> DNA
<213> 人工序列(Artificial sequence)
<400> 170
aaaactacaa ggccgtcttc agtacg 26
<210> 171
<211> 20
<212> DNA
<213> 人工序列(Artificial sequence)
<400> 171
gaagacggcc ttgtaggggt 20
<210> 172
<211> 26
<212> DNA
<213> 人工序列(Artificial sequence)
<400> 172
acaccgaaga cggccttgta ggggtg 26
<210> 173
<211> 26
<212> DNA
<213> 人工序列(Artificial sequence)
<400> 173
aaaacacccc tacaaggccg tcttcg 26
<210> 174
<211> 20
<212> DNA
<213> 人工序列(Artificial sequence)
<400> 174
gtaggagaag ccctggccgc 20
<210> 175
<211> 26
<212> DNA
<213> 人工序列(Artificial sequence)
<400> 175
acaccgtagg agaagccctg gccgcg 26
<210> 176
<211> 26
<212> DNA
<213> 人工序列(Artificial sequence)
<400> 176
aaaacgcggc cagggcttct cctacg 26
<210> 177
<211> 20
<212> DNA
<213> 人工序列(Artificial sequence)
<400> 177
gccgccctcc tccaccgcca 20
<210> 178
<211> 26
<212> DNA
<213> 人工序列(Artificial sequence)
<400> 178
acaccgccgc cctcctccac cgccag 26
<210> 179
<211> 26
<212> DNA
<213> 人工序列(Artificial sequence)
<400> 179
aaaactggcg gtggaggagg gcggcg 26
<210> 180
<211> 20
<212> DNA
<213> 人工序列(Artificial sequence)
<400> 180
gtacaggtcc agcatgaaca 20
<210> 181
<211> 26
<212> DNA
<213> 人工序列(Artificial sequence)
<400> 181
acaccgtaca ggtccagcat gaacag 26
<210> 182
<211> 26
<212> DNA
<213> 人工序列(Artificial sequence)
<400> 182
aaaactgttc atgctggacc tgtacg 26
<210> 183
<211> 20
<212> DNA
<213> 人工序列(Artificial sequence)
<400> 183
gtgccgagtt gtgcttgccc 20
<210> 184
<211> 26
<212> DNA
<213> 人工序列(Artificial sequence)
<400> 184
acaccgtgcc gagttgtgct tgcccg 26
<210> 185
<211> 26
<212> DNA
<213> 人工序列(Artificial sequence)
<400> 185
aaaacgggca agcacaactc ggcacg 26
<210> 186
<211> 20
<212> DNA
<213> 人工序列(Artificial sequence)
<400> 186
ggaggtgcgg gcgcgggcgg 20
<210> 187
<211> 26
<212> DNA
<213> 人工序列(Artificial sequence)
<400> 187
acaccggagg tgcgggcgcg ggcggg 26
<210> 188
<211> 26
<212> DNA
<213> 人工序列(Artificial sequence)
<400> 188
aaaacccgcc cgcgcccgca cctccg 26
<210> 189
<211> 20
<212> DNA
<213> 人工序列(Artificial sequence)
<400> 189
gaggtgcggg cgcgggcggt 20
<210> 190
<211> 26
<212> DNA
<213> 人工序列(Artificial sequence)
<400> 190
acaccgaggt gcgggcgcgg gcggtg 26
<210> 191
<211> 26
<212> DNA
<213> 人工序列(Artificial sequence)
<400> 191
aaaacaccgc ccgcgcccgc acctcg 26
<210> 192
<211> 20
<212> DNA
<213> 人工序列(Artificial sequence)
<400> 192
gcggtggggc aagcccaaaa 20
<210> 193
<211> 26
<212> DNA
<213> 人工序列(Artificial sequence)
<400> 193
acaccgcggt ggggcaagcc caaaag 26
<210> 194
<211> 26
<212> DNA
<213> 人工序列(Artificial sequence)
<400> 194
aaaacttttg ggcttgcccc accgcg 26
<210> 195
<211> 20
<212> DNA
<213> 人工序列(Artificial sequence)
<400> 195
ggggcaagcc caaaatggag 20
<210> 196
<211> 26
<212> DNA
<213> 人工序列(Artificial sequence)
<400> 196
acaccggggc aagcccaaaa tggagg 26
<210> 197
<211> 26
<212> DNA
<213> 人工序列(Artificial sequence)
<400> 197
aaaacctcca ttttgggctt gccccg 26
<210> 198
<211> 20
<212> DNA
<213> 人工序列(Artificial sequence)
<400> 198
gctgcatctc ccgccgctcc 20
<210> 199
<211> 26
<212> DNA
<213> 人工序列(Artificial sequence)
<400> 199
acaccgctgc atctcccgcc gctccg 26
<210> 200
<211> 26
<212> DNA
<213> 人工序列(Artificial sequence)
<400> 200
aaaacggagc ggcgggagat gcagcg 26
<210> 201
<211> 20
<212> DNA
<213> 人工序列(Artificial sequence)
<400> 201
gctcctggct gcggaggcgc 20
<210> 202
<211> 26
<212> DNA
<213> 人工序列(Artificial sequence)
<400> 202
acaccgctcc tggctgcgga ggcgcg 26
<210> 203
<211> 26
<212> DNA
<213> 人工序列(Artificial sequence)
<400> 203
aaaacgcgcc tccgcagcca ggagcg 26
<210> 204
<211> 20
<212> DNA
<213> 人工序列(Artificial sequence)
<400> 204
gtccaggctg aagtcggcca 20
<210> 205
<211> 26
<212> DNA
<213> 人工序列(Artificial sequence)
<400> 205
acaccgtcca ggctgaagtc ggccag 26
<210> 206
<211> 26
<212> DNA
<213> 人工序列(Artificial sequence)
<400> 206
aaaactggcc gacttcagcc tggacg 26
<210> 207
<211> 20
<212> DNA
<213> 人工序列(Artificial sequence)
<400> 207
gggcggagcg cagcaggaac 20
<210> 208
<211> 26
<212> DNA
<213> 人工序列(Artificial sequence)
<400> 208
acaccgggcg gagcgcagca ggaacg 26
<210> 209
<211> 26
<212> DNA
<213> 人工序列(Artificial sequence)
<400> 209
aaaacgttcc tgctgcgctc cgcccg 26
<210> 210
<211> 20
<212> DNA
<213> 人工序列(Artificial sequence)
<400> 210
ggcggagcgc agcaggaaca 20
<210> 211
<211> 26
<212> DNA
<213> 人工序列(Artificial sequence)
<400> 211
acaccggcgg agcgcagcag gaacag 26
<210> 212
<211> 26
<212> DNA
<213> 人工序列(Artificial sequence)
<400> 212
aaaactgttc ctgctgcgct ccgccg 26
<210> 213
<211> 20
<212> DNA
<213> 人工序列(Artificial sequence)
<400> 213
gcggagcgca gcaggaacag 20
<210> 214
<211> 26
<212> DNA
<213> 人工序列(Artificial sequence)
<400> 214
acaccgcgga gcgcagcagg aacagg 26
<210> 215
<211> 26
<212> DNA
<213> 人工序列(Artificial sequence)
<400> 215
aaaacctgtt cctgctgcgc tccgcg 26
<210> 216
<211> 20
<212> DNA
<213> 人工序列(Artificial sequence)
<400> 216
gccggctcta cgcgctaccc 20
<210> 217
<211> 26
<212> DNA
<213> 人工序列(Artificial sequence)
<400> 217
acaccgccgg ctctacgcgc tacccg 26
<210> 218
<211> 26
<212> DNA
<213> 人工序列(Artificial sequence)
<400> 218
aaaacgggta gcgcgtagag ccggcg 26
<210> 219
<211> 20
<212> DNA
<213> 人工序列(Artificial sequence)
<400> 219
gctacccggg ctccgggctc 20
<210> 220
<211> 26
<212> DNA
<213> 人工序列(Artificial sequence)
<400> 220
acaccgctac ccgggctccg ggctcg 26
<210> 221
<211> 26
<212> DNA
<213> 人工序列(Artificial sequence)
<400> 221
aaaacgagcc cggagcccgg gtagcg 26
<210> 222
<211> 20
<212> DNA
<213> 人工序列(Artificial sequence)
<400> 222
gcaccgcccc aggtggcaga 20
<210> 223
<211> 26
<212> DNA
<213> 人工序列(Artificial sequence)
<400> 223
acaccgcacc gccccaggtg gcagag 26
<210> 224
<211> 26
<212> DNA
<213> 人工序列(Artificial sequence)
<400> 224
aaaactctgc cacctggggc ggtgcg 26
<210> 225
<211> 20
<212> DNA
<213> 人工序列(Artificial sequence)
<400> 225
gctcggtcac ttgctgcaga 20
<210> 226
<211> 26
<212> DNA
<213> 人工序列(Artificial sequence)
<400> 226
acaccgctcg gtcacttgct gcagag 26
<210> 227
<211> 26
<212> DNA
<213> 人工序列(Artificial sequence)
<400> 227
aaaactctgc agcaagtgac cgagcg 26
<210> 228
<211> 20
<212> DNA
<213> 人工序列(Artificial sequence)
<400> 228
ggcgaacgaa aaggcgagtg 20
<210> 229
<211> 26
<212> DNA
<213> 人工序列(Artificial sequence)
<400> 229
acaccggcga acgaaaaggc gagtgg 26
<210> 230
<211> 26
<212> DNA
<213> 人工序列(Artificial sequence)
<400> 230
aaaaccactc gccttttcgt tcgccg 26
<210> 231
<211> 20
<212> DNA
<213> 人工序列(Artificial sequence)
<400> 231
gaacgaaaag gcgagtgagg 20
<210> 232
<211> 26
<212> DNA
<213> 人工序列(Artificial sequence)
<400> 232
acaccgaacg aaaaggcgag tgaggg 26
<210> 233
<211> 26
<212> DNA
<213> 人工序列(Artificial sequence)
<400> 233
aaaaccctca ctcgcctttt cgttcg 26
<210> 234
<211> 20
<212> DNA
<213> 人工序列(Artificial sequence)
<400> 234
gaaaaggcga gtgaggaggc 20
<210> 235
<211> 26
<212> DNA
<213> 人工序列(Artificial sequence)
<400> 235
acaccgaaaa ggcgagtgag gaggcg 26
<210> 236
<211> 26
<212> DNA
<213> 人工序列(Artificial sequence)
<400> 236
aaaacgcctc ctcactcgcc ttttcg 26
<210> 237
<211> 20
<212> DNA
<213> 人工序列(Artificial sequence)
<400> 237
ggcgagtgag gaggcgggcg 20
<210> 240
<211> 20
<212> DNA
<213> 人工序列(Artificial sequence)
<400> 240
gcgagtgagg aggcgggcgc 20
<210> 241
<211> 26
<212> DNA
<213> 人工序列(Artificial sequence)
<400> 241
acaccgcgag tgaggaggcg ggcgcg 26
<210> 242
<211> 26
<212> DNA
<213> 人工序列(Artificial sequence)
<400> 242
aaaacgcgcc cgcctcctca ctcgcg 26
<210> 243
<211> 20
<212> DNA
<213> 人工序列(Artificial sequence)
<400> 243
gtgaggaggc gggcgcgggt 20
<210> 244
<211> 26
<212> DNA
<213> 人工序列(Artificial sequence)
<400> 244
acaccgtgag gaggcgggcg cgggtg 26
<210> 245
<211> 26
<212> DNA
<213> 人工序列(Artificial sequence)
<400> 245
aaaacacccg cgcccgcctc ctcacg 26
<210> 246
<211> 20
<212> DNA
<213> 人工序列(Artificial sequence)
<400> 246
gaggagcagc cagcaagcct 20
<210> 247
<211> 26
<212> DNA
<213> 人工序列(Artificial sequence)
<400> 247
acaccgagga gcagccagca agcctg 26
<210> 248
<211> 26
<212> DNA
<213> 人工序列(Artificial sequence)
<400> 248
aaaacaggct tgctggctgc tcctcg 26
<210> 247
<211> 20
<212> DNA
<213> 人工序列(Artificial sequence)
<400> 247
ggagtccaga gagccaacgc 20
<210> 248
<211> 26
<212> DNA
<213> 人工序列(Artificial sequence)
<400> 248
acaccggagt ccagagagcc aacgcg 26
<210> 249
<211> 26
<212> DNA
<213> 人工序列(Artificial sequence)
<400> 249
aaaacgcgtt ggctctctgg actccg 26
<210> 250
<211> 20
<212> DNA
<213> 人工序列(Artificial sequence)
<400> 250
gagtccagag agccaacgcc 20
<210> 251
<211> 26
<212> DNA
<213> 人工序列(Artificial sequence)
<400> 251
acaccgagtc cagagagcca acgccg 26
<210> 252
<211> 26
<212> DNA
<213> 人工序列(Artificial sequence)
<400> 252
aaaacggcgt tggctctctg gactcg 26
<210> 253
<211> 20
<212> DNA
<213> 人工序列(Artificial sequence)
<400> 253
ggaggcagcg aggaggcagg 20
<210> 254
<211> 26
<212> DNA
<213> 人工序列(Artificial sequence)
<400> 254
acaccggagg cagcgaggag gcaggg 26
<210> 255
<211> 26
<212> DNA
<213> 人工序列(Artificial sequence)
<400> 255
aaaaccctgc ctcctcgctg cctccg 26
<210> 256
<211> 20
<212> DNA
<213> 人工序列(Artificial sequence)
<400> 256
gaggcagcga ggaggcaggc 20
<210> 257
<211> 26
<212> DNA
<213> 人工序列(Artificial sequence)
<400> 257
acaccgaggc agcgaggagg caggcg 26
<210> 258
<211> 26
<212> DNA
<213> 人工序列(Artificial sequence)
<400> 258
aaaacgcctg cctcctcgct gcctcg 26
<210> 259
<211> 20
<212> DNA
<213> 人工序列(Artificial sequence)
<400> 259
gcagcgagga ggcaggcggg 20
<210> 260
<211> 27
<212> DNA
<213> 人工序列(Artificial sequence)
<400> 260
acaccgcagc gaggaggcag gcggggs 27
<210> 261
<211> 26
<212> DNA
<213> 人工序列(Artificial sequence)
<400> 261
aaaaccccgc ctgcctcctc gctgcg 26
<210> 262
<211> 20
<212> DNA
<213> 人工序列(Artificial sequence)
<400> 262
gctccttctt cccgctcctc 20
<210> 263
<211> 26
<212> DNA
<213> 人工序列(Artificial sequence)
<400> 263
acaccgctcc ttcttcccgc tcctcg 26
<210> 264
<211> 26
<212> DNA
<213> 人工序列(Artificial sequence)
<400> 264
aaaacgagga gcgggaagaa ggagcg 26
<210> 265
<211> 20
<212> DNA
<213> 人工序列(Artificial sequence)
<400> 265
gcgctctcgc cctcgggcgg 20
<210> 266
<211> 26
<212> DNA
<213> 人工序列(Artificial sequence)
<400> 266
acaccgcgct ctcgccctcg ggcggg 26
<210> 267
<211> 26
<212> DNA
<213> 人工序列(Artificial sequence)
<400> 267
aaaacccgcc cgagggcgag agcgcg 26
<210> 268
<211> 20
<212> DNA
<213> 人工序列(Artificial sequence)
<400> 268
ggcgcggata gcaggggccc 20
<210> 269
<211> 26
<212> DNA
<213> 人工序列(Artificial sequence)
<400> 269
acaccggcgc ggatagcagg ggcccg 26
<210> 270
<211> 26
<212> DNA
<213> 人工序列(Artificial sequence)
<400> 270
aaaacgggcc cctgctatcc gcgccg 26
<210> 271
<211> 20
<212> DNA
<213> 人工序列(Artificial sequence)
<400> 271
gcgcggatag caggggcccc 20
<210> 272
<211> 26
<212> DNA
<213> 人工序列(Artificial sequence)
<400> 272
acaccgcgcg gatagcaggg gccccg 26
<210> 273
<211> 26
<212> DNA
<213> 人工序列(Artificial sequence)
<400> 273
aaaacggggc ccctgctatc cgcgcg 26
<210> 274
<211> 20
<212> DNA
<213> 人工序列(Artificial sequence)
<400> 274
gatcgcacaa gaggccgcct 20
<210> 275
<211> 26
<212> DNA
<213> 人工序列(Artificial sequence)
<400> 275
acaccgatcg cacaagaggc cgcctg 26
<210> 276
<211> 26
<212> DNA
<213> 人工序列(Artificial sequence)
<400> 276
aaaacaggcg gcctcttgtg cgatcg 26
Claims (6)
1.一种BMP7基因缺失外周血间充质干细胞的建立方法,其特征在于,包括以下步骤:
1)采集、冻存、传代外周血干细胞;
2)根据BMP7基因序列,设计sgRNA序列;
3)sgRNA活性检测,确定最优sgRNA,最优sgRNA序列为GGCGAGTGAGGAGGCGGGCG,具体步骤包括:
a.将寡核苷酸双链连接到pX330载体上,再将连接产物转化至E coli DH5α感受态细胞中,扩大培养,提取质粒;
b.将之前构建好的sgRNA与Cas9共表达的91个载体分别转染至293T细胞中;
c.提取步骤b细胞的mRNA,反转录为cDNA,用设计好的引物进行PCR;
d.酶切步骤c中的PCR产物;
e.通过是否有切开后的相对小的两条目的条带以及被切开的条带所占的比例选择切割效率最高的sgRNA;
4)将步骤3)中切割效率最高的sgRNA和嘌呤霉素抗性的PGK-puro质粒电转到外周血间充质干细胞;
5)基因敲除验证;
6)将基因敲除细胞进行测序。
2.根据权利要求1所述的一种BMP7基因缺失外周血间充质干细胞的建立方法,其特征在于,步骤3)中所述PCR引物序列为PrimerF:TTGGAAGAGGGTGGGTTG,Prime rR:TGTCTGAGTCAAGATGGAGA。
3.根据权利要求1所述的一种BMP7基因缺失外周血间充质干细胞的建立方法,其特征在于,步骤3)中所述PCR反应体系为2×Phanta Max Buffer 25μL,Phanta Max Su per-Fidelity DNA Polymerase 1μL,dNTP Mix 1μL,PrimersF 2μL,PrimersR 2μL,cD NA 1μL,ddH2O补足50μL。
4.根据权利要求1所述的一种BMP7基因缺失外周血间充质干细胞的建立方法,其特征在于,步骤3)中所述PCR反应程序为:98℃预变形3min;(98℃变性15s;65℃退火15s;72℃延伸1min;2-4步共35个循环);72℃延伸5min。
5.根据权利要求1所述的一种BMP7基因缺失外周血间充质干细胞的建立方法,其特征在于,所述基因敲除验证使用T7核酸内切酶Ⅰ进行。
6.根据权利要求1所述的一种BMP7基因缺失外周血间充质干细胞的应用,其征在于,所述的BMP7基因缺失外周血间充质干细胞,用于筛选促成骨形成药物。
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CN109777780A (zh) * | 2019-01-31 | 2019-05-21 | 上海拉德钫斯生物科技有限公司 | 一种构建rbm17基因敲除的间充质干细胞细胞系的方法 |
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CN112852819A (zh) * | 2021-03-12 | 2021-05-28 | 江苏和泽干细胞基因工程有限公司 | 一种ptch1基因缺失间充质干细胞的建立方法 |
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