CN108504693A - 利用Crispr技术敲除T合酶基因构建的O-型糖基化异常的结肠癌细胞系 - Google Patents

利用Crispr技术敲除T合酶基因构建的O-型糖基化异常的结肠癌细胞系 Download PDF

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CN108504693A
CN108504693A CN201810300881.7A CN201810300881A CN108504693A CN 108504693 A CN108504693 A CN 108504693A CN 201810300881 A CN201810300881 A CN 201810300881A CN 108504693 A CN108504693 A CN 108504693A
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温韬
安广宇
董希琛
刘健
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Beijing Chaoyang Hospital
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Abstract

本发明公开了利用Crispr技术敲除T合酶基因构建的O‑型糖基化异常的结肠癌细胞系。本发明的O‑型糖基化异常的结肠癌细胞系的制备方法包括如下步骤:利用CRISPR/Cas9系统对结肠癌细胞基因组中的C1GALT1基因进行编辑,进而使所述T‑synthase功能丧失,得到O‑型糖基化异常的结肠癌细胞系。本发明使用慢病毒做中间媒介敲除结肠癌细胞内C1GALT1基因,代替了质粒直接转化细胞,更高效地发挥Crispr/Cas9的敲除作用。为研究异常的O‑型糖基化对于结直肠癌发生发展的影响奠定基础。

Description

利用Crispr技术敲除T合酶基因构建的O-型糖基化异常的结 肠癌细胞系
技术领域
本发明属于生物技术领域,具体涉及利用Crispr/Cas9系统构建的敲除C1GALT1基因的结肠癌细胞系,特别涉及利用Crispr/Cas9技术靶向编辑C1GALT1基因制备O-型糖基化异常的HCT116结肠癌细胞的方法。
背景技术
糖链的修饰是重要的蛋白质翻译后修饰,黏蛋白O型糖基化是其中重要的类型之一。黏蛋白型O-聚糖通过一个在高尔基体发生的连续糖基转移作用合成,而合成的过程由一套糖基转移酶催化。T-synthase是合成O-聚糖核心1的唯一糖基转移酶,由C1GALT1基因编码,它主要的功能是将半乳糖添加到Tn抗原糖链上。T-synthase失活将导致机体细胞只能合成Tn抗原以及唾液酸化的sTn抗原。
已有研究发现在结直肠癌、肺癌、乳腺癌、宫颈癌、卵巢癌组织中高表达Tn抗原,Tn抗原的暴露与肿瘤的发生发展有密切联系。目前大部分结肠癌细胞系均为O-型糖基化正常的细胞系,为了研究O-型糖基化在肿瘤中的功能与机制,需要在体外构建可稳定遗传的异常型O-型糖基化细胞系。
发明内容
本发明的一个目的是提供一种O-型糖基化异常的结肠癌细胞系的制备方法。
本发明提供的O-型糖基化异常的结肠癌细胞系的制备方法包括如下步骤:利用CRISPR/Cas9系统对结肠癌细胞基因组中的C1GALT1基因进行编辑,进而使所述T-synthase功能丧失,得到O-型糖基化异常的结肠癌细胞系。
上述方法中,所述CRISPR/Cas9系统包括sgRNA;所述sgRNA的靶序列具体可为C1GALT1基因的第39-58位。
上述方法中,所述编辑的方法为向所述结肠癌细胞中导入C1GALT1基因编辑的慢病毒载体;所述C1GALT1基因编辑的慢病毒载体含有所述sgRNA的编码基因和Cas9蛋白的编码基因。
进一步的,所述C1GALT1基因编辑的慢病毒载体为将双链DNA分子插入慢病毒表达载体的酶切位点间得到的载体;所述双链DNA分子是将单链DNA分子甲和单链DNA分子乙退火制备得到的;
所述单链DNA分子甲的核苷酸序列如序列1所示;
所述单链DNA分子乙的核苷酸序列如序列2所示。
更进一步的,所述C1GALT1基因编辑的慢病毒载体为将所述双链DNA分子插入LentiCRISPRV2载体的BsmBI酶切位点间得到的载体。
上述方法中,所述结肠癌细胞为结肠癌细胞HCT116。
本发明的另一个目的是提供按照上述方法制备得到的O-型糖基化异常的结肠癌细胞系。
本发明还有一个目的是提供上述C1GALT1基因编辑的慢病毒载体。
上述C1GALT1基因编辑的慢病毒载体在制备O-型糖基化异常的结肠癌细胞系中的应用也属于本发明的保护范围。
上述方法或细胞系或应用中,所述O-型糖基化异常为T-synthase控制的O-型聚糖链延伸异常。
上述方法或细胞系或应用中,所述C1GALT1基因的核苷酸序列如序列3所示。
与现有技术相比,本发明具有如下优势:1)siRNA技术是从mRNA进行干预,瞬时敲降,不易稳定遗传;而crispr-cas9技术是从DNA水平对基因进行编辑,得到的O-型糖基化可以稳定遗传,即可以稳定传代;2)与siRNA技术相比,crispr-cas9技术敲除效率更高,可以获得90%以上纯度的细胞系;3)本发明经过对多个sgRNA序列进行验证,筛选得到敲除效率最高的sgRNA序列。
本发明利用Crispr/cas9技术靶向性敲除结肠癌细胞内C1GALT1基因,致使T-synthase表达缺失,从而使细胞发生异常的O-型糖基化,造成细胞表面Tn抗原暴露,从而形成Tn+性结直肠细胞。本发明使用慢病毒做中间媒介敲除结肠癌细胞内C1GALT1基因,代替了质粒直接转化细胞,更高效地发挥Crispr/Cas9的敲除作用。为研究异常的O-型糖基化对于结直肠癌发生发展的影响奠定基础。
附图说明
图1为培养过夜的Stbl3单菌落。
图2为筛选转染成功的细胞。
图3为westernblot验证结果。
图4为利用小鼠抗TnIgM抗体经流式验证T-synthase缺失细胞的Tn抗原表达情况。
具体实施方式
下述实施例中所使用的实验方法如无特殊说明,均为常规方法。
下述实施例中所用的材料、试剂等,如无特殊说明,均可从商业途径得到。
下述实施例中的定量试验,均设置三次重复实验,结果取平均值。
下述实施例中的mouse anti-Tn IgM mAb记载于文献“Wang Y,Ju T,Ding X etal.Cosmc is an essential chaperone for correct protein O-glycosylation.ProcNatl Acad Sci U S A 2010;107:9228-9233.”和“Ju T,Otto VI,Cummings RD.The Tnantigen-structural simplicity and biological complexity.Angew Chem Int EdEngl 2011;50:1770-1791.”中,公众可从申请人处获得,该生物材料只为重复本发明的相关实验所用,不可作为其它用途使用。
实施例1、敲除C1GALT1基因的HCT116结肠癌细胞的制备方法
一、待敲除C1GALT1基因的靶序列及sgRNA单链DNA序列的设计
1、待敲除C1GALT1基因的靶序列的设计
以C1GALT1基因(C1GALT1基因CDS序列如序列3所示)作为待敲除基因,并筛选合适的靶序列。本发明根据C1GALT1基因序列设计的靶序列为C1GALT1基因CDS序列的第39-58位。
2、sgRNA单链DNA序列的设计
根据步骤1确定的靶序列,共设计了三个sgRNA序列,分别将其命名为sgRNA1、sgRNA2和sgRNA3。将sgRNA1的两条单链DNA序列分别命名为Oligo1和Oligo2。将sgRNA2的两条单链DNA序列分别命名为Oligo3和Oligo4。将sgRNA3的两条单链DNA序列分别命名为Oligo5和Oligo6。序列分别如下:
Oligo1:5'-CACCGATCCTATTGCTGATCCACAG-3'(序列1);
Oligo2:5'-AAACCTGTGGATCAGCAATAGGATC-3'(序列2);
Oligo3:5’-CACCGGCAGATTCTAGCCAACATAA-3’;
Oligo4:3’-CCGTCTAAGATCGGTTGTATTCAAA-5’;
Oligo5:5’-CACCGGGATCATTATGAAGAACATT-3’;
Oligo6:3’-CCCTAGTAATACTTCTTGTAACAAA-5’。
二、表达sgRNA的重组质粒的构建
1、单链DNA退火
将Oligo1和Oligo2溶于纯水中,并加入T4PNK酶(NEB)和10×T4ligation Buffer(NEB)于PCR仪进行梯度降温退火,得到双链DNA甲。退火反应体系如表1所示。退火反应条件如下:37℃~30min,再升温至95℃~5min,然后以5℃/min的速率梯度降温至25℃。
表1、退火反应体系
Oligo1 1μL
Oligo2 1μL
10×T4 ligation Buffer(NEB) 1μL
H2O 6.5μL
T4PNK(NEB) 0.5μL
Total 10μL
3)将双链DNA稀释200倍,得到双链DNA溶液,待用。
分别将Oligo3和Oligo4、Oligo5和Oligo6按照上述方法进行退火,分别得到双链DNA乙和双链DNA丙。双链DNA甲编码sgRNA1、双链DNA乙编码sgRNA2、双链DNA丙编码sgRNA3。
2、酶切质粒
1)用BsmBI限制性内切酶(NEB)消化LentiCRISPRV2质粒载体(Addgene plasmid,货号为#52961),消化反应体系如表2所示,55℃反应20min后再向反应体系中加入6μLFastAP和6μL 10×APbuffer(NEB)使质粒脱磷酸化,得到消化产物。
2)将消化产物于10%琼脂糖凝胶进行电泳,并利用DNA胶提取试剂盒(BioFlux)回收长段质粒DNA。
表2、消化反应体系
BsmBIenzyme(NEB) 5μL
LentiCRISORV2 5μg
10×NEBBuffer(NEB) 5μL
H2O XμL
Total 50μL
3、质粒重组
将步骤2回收的长段质粒DNA与步骤1中制备的双链DNA用T4DNA快速连接酶(NEB)进行连接,得到重组质粒。重组质粒中包含sgRNA的编码基因和cas9的编码基因。连接反应体系如表3所示,连接条件:在室温条件下连接10min。
表3、连接反应体系
长段质粒DNA 50μg
双链DNA溶液 1μL
2×QuickLigaseBuffer(NEB) 5μL
H2O XμL
QuickLigase(NEB) 1μL
Total 11μL
4、质粒转化
取Stbl3感受态细菌(碧云天,D0378)置于冰上,将上述步骤3中的11μL体系(含有重组质粒)加到Stbl3感受态细菌中,冰置30min,42℃水浴60~90s热激感受态细菌,热激结束后立即置于冰上2min,期间勿挪动摇晃,得到转染重组质粒的Stbl3细菌。
5、细菌平板涂布
向转染重组质粒的Stbl3细菌中加入600μL的LB液体培养基,37℃,200rpm/min摇菌1h;然后将菌液1000rpm离心1min,弃部分上清液,留100μL;重悬菌液后将其均匀涂布在含氨苄青霉素(Amp)的LB琼脂培养板上,37℃孵育过夜。转染重组质粒的Stbl3细菌在Amp+琼脂LB培养基上过夜培养形成单菌落,培养过夜的Stbl3单菌落如图1所示,说明质粒转化成功。
6、挑菌扩增
选取独立生长菌落,用200μL枪头轻挑起,打入含6mL常温液体LB培养基(Amp+)的15mL离心管中,浮盖盖子勿拧紧,37℃空气浴200rpm/min摇菌14-16h。
7、质粒提取
将步骤6中摇好的菌液按1:1000比例接种于液体LB培养基(Amp+)中(200μL菌液:200mL LB培养基),37℃空气浴摇菌12-16h;其中5mL用于保菌,剩余菌液使用MachereyNagel质粒提取试剂盒(740410.50)提取质粒。
按照上述步骤4-7中的方法,将包装质粒PAX2和PMD2.g(addgene,货号分别为12260和12259)以同样方法进行转化和质粒提取。
8、慢病毒制备
1)将预先培养好的HEK293T细胞(ATCC,CRL-11268,USA)于六孔板,密度达到50%时,使用Lipofectamine3000(Invitrogen)将3种质粒(重组质粒、PAX2和PMD2.g)以4:3:1的比例共转染到HEK293T细胞中。具体步骤如下:将125μL的Opti-MEM(Gibco)和7.5μL的LipofectamineTM3000(Invitrogen)混匀,得到体系1;125μL Opti-MEM(Gibco)、2.5μg重组质粒、1.88μg包装质粒PAX2、0.63μg包装质粒PMD和5μL P3000TMReagent(Invitrogen)混匀,得到体系2;将体系1和体系2混匀,得到慢病毒反应体系(表4)。
2)将慢病毒反应体系在37℃,5%CO2培养48h后收取培养上清液,经0.45μm滤膜(Macherey Nagel)过滤,得到慢病毒溶液,于-80℃冰箱保存。
将步骤1)中的重组质粒替换为LentiCRISPRV2质粒载体,且保持其他步骤,得到转空载病毒溶液。
表4、慢病毒反应体系
三、敲除C1GALT1基因的HCT116结肠癌细胞的制备
1、慢病毒转染细胞
预先培养结肠癌细胞HCT116(ATCC,CCL-247,USA)于六孔板内,待密度达到70%时,以体积比为1:1的比例加入含10%(体积分数)FBS的McCoy’s 5A完全培养基(Gibco)和步骤二制备的慢病毒溶液(1mL完全培养基:1mL慢病毒溶液),再加入2μL 1000×polybrene转染增强剂(Solarbio);每隔24h按此方法换液转染一次,共需转染3次。
2、细胞筛选
在最后一次转染24h后,向完全培养基内加入嘌呤霉素(Gibco),使其终浓度为2μg/mL,筛选转染成功的细胞,至少筛选7天,使细胞系稳定生长。
转染了慢病毒的HCT116结肠癌细胞经过7天的嘌呤霉素(2μg/mL)的筛选,成功转染慢病毒的细胞(T-synthase敲除组细胞)存活下来(图2),并将成功转染慢病毒的T-synthase敲除组细胞命名为T-syn KO。
按照上述方法,将慢病毒溶液替换为转空载病毒溶液,得到转空载病毒的对照组细胞(Control)。
3、WesternBlot检测T-synthase的表达情况
采用WesternBlot检测敲除组细胞T-syn KO和转空载病毒的对照组细胞Control中的T-synthase的表达情况。具体步骤如下:预先在100mm的大皿中培养T-synthase敲除组细胞T-syn KO和对照组细胞密度至90%,用预冷的PBS清洗2次细胞。向每个大皿中加入300μL RIPA细胞裂解液(碧云天,含1%蛋白酶抑制剂和1%PMSF),冰上静置15min,用细胞刮刮下细胞于EP管内,在冰上进行超声破碎细胞,每次15s共超声4次。4℃,12000rpm/min离心10min,上清液为提取的细胞总蛋白。经过BCA方法蛋白定量,按比例加入5×loadingbuffer(碧云天,P0015L),并在95℃加热10min制成蛋白上样液。SDS-PAGE电泳分离蛋白转PVDF膜(BD,ISEQ00010),5%脱脂牛奶(BD,232100)室温下封闭1h。TBST清洗膜上残余牛奶,每次5min,共3次。一抗稀释液(碧云天,P0023A)1:500稀释T-synthase一抗(Santa Cruz,sc-100745)并在4℃过夜孵育一抗。TBST清洗膜4次,每次5min,5%脱脂牛奶1:8000稀释辣根酶标记的山羊抗小鼠IgG二抗(中杉金桥,ZB-5305),室温孵育1h。TBST清洗膜4次,每次5min,Chemiluminescent HRP Substrate(Millipore,WBKLS0100)浸泡膜5min,在Bio-Radimaging system(Bio-Rad ChemiDoc MP,1708195)曝光显影。
WesternBlot检测结果图3所示。经过westernblot验证表明:与只转染了空载病毒的对照组细胞相比,T-synthase敲除组细胞T-syn KO中的T-synthase几乎被完全敲除。
4、流式验证T-synthase敲除效率
采用流式细胞技术检测敲除组细胞T-syn KO和转空载病毒的对照组细胞Control中T-synthase敲除效率。利用小鼠抗Tn IgM抗体标记细胞表面Tn抗原,经过流式分选纯化敲除组细胞T-syn KO。具体步骤如下:将1×105细胞重悬于预冷的PBS中,PBS清洗2遍后,加入mouse anti-Tn IgM mAb,使其终浓度为10μg/mL,同型对照以同样浓度加入mouse IgMisotype-antibody(Santa Cruz,sc-3881),4℃孵育2小时。再用PBS清洗细胞3次,加入PE-labeled goat anti-mouse IgM secondary antibody(BD,562033),4℃孵育1小时。最后用PBS清洗3次后,上流式机(Canto II,BD Bioscience)分析。
结果如图4所示。结果表明:Western blot结果说明T-synthase已被敲除,经流式检测,以sgRNA1作为sgRNA进行编辑的结肠癌细胞HCT116中得到的Tn+结肠癌细胞比例为90.3%,而以sgRNA2或sgRNA3作为sgRNA进行编辑的结肠癌细胞HCT116中得到的Tn+结肠癌细胞比例不足45%。说明以sgRNA1作为sgRNA对C1GALT1基因进行编辑具有更高的敲除效率。敲除C1GALT1基因后的结肠癌细胞HCT116为O-型糖基化异常的细胞,利用此细胞系,可以研究异常O-型糖基化对结肠癌细胞的生物学行为影响。
序列表
<110>首都医科大学附属北京朝阳医院
<120>利用Crispr技术敲除T合酶基因构建的O-型糖基化异常的结肠癌细胞系
<160>3
<170>PatentIn version 3.5
<210>1
<211>25
<212>DNA
<213>人工序列(Artificial Sequence)
<400>1
caccgatcct attgctgatc cacag 25
<210>2
<211>25
<212>DNA
<213>人工序列(Artificial Sequence)
<400>2
aaacctgtgg atcagcaata ggatc 25
<210>3
<211>1092
<212>DNA
<213>人工序列(Artificial Sequence)
<400>3
atggcctcta aatcctggct gaatttttta accttcctct gtggatcagc aataggattt 60
cttttatgtt ctcagctatt tagtattttg ttgggagaaa aggttgacac ccagcctaat 120
gttcttcata atgatcctca tgcaaggcat tcagatgata atggacagaa tcatctagaa 180
ggacaaatga acttcaatgc agattctagc caacataaag atgagaacac agacattgct 240
gaaaacctct atcagaaagt tagaattctt tgctgggtta tgaccggccc tcaaaaccta 300
gagaaaaagg ccaaacacgt caaagctact tgggcccagc gttgtaacaa agtgttgttt 360
atgagttcag aagaaaataa agacttccct gctgtgggac tgaaaaccaa agaaggcaga 420
gatcaactat actggaaaac aattaaagct tttcagtatg ttcatgaaca ttatttagaa 480
gatgctgatt ggtttttgaa agcagatgat gacacgtatg tcatactaga caatttgagg 540
tggcttcttt caaaatacga ccctgaagaa cccatttact ttgggagaag atttaagcct 600
tatgtaaagc agggctacat gagtggagga gcaggatatg tactaagcaa agaagccttg 660
aaaagatttg ttgatgcatt taaaacagac aagtgtacac atagttcctc cattgaagac 720
ttagcactgg ggagatgcat ggaaattatg aatgtagaag caggagattc cagagatacc 780
attggaaaag aaacttttca tccctttgtg ccagaacacc atttaattaa aggttatcta 840
cctagaacgt tttggtactg gaattacaac tattatcctc ctgtagaggg tcctggttgc 900
tgctctgatc ttgcagtttc ttttcactat gttgattcta caaccatgta tgagttagaa 960
tacctcgttt atcatcttcg tccatatggt tatttataca gatatcaacc taccttacct 1020
gaacgtatac taaaggaaat tagtcaagca aacaaaaatg aagatacaaa agtgaagtta 1080
ggaaatcctt ga 1092

Claims (10)

1.一种O-型糖基化异常的结肠癌细胞系的制备方法,包括如下步骤:利用CRISPR/Cas9系统对结肠癌细胞基因组中的C1GALT1基因进行编辑,进而使所述T-synthase功能丧失,得到O-型糖基化异常的结肠癌细胞系。
2.根据权利要求1所述的方法,其特征在于:所述CRISPR/Cas9系统包括sgRNA;
或,所述sgRNA的靶序列为C1GALT1基因的第39-58位。
3.根据权利要求1或2所述的方法,其特征在于:所述编辑的方法为向所述结肠癌细胞中导入C1GALT1基因编辑的慢病毒载体;
或,所述C1GALT1基因编辑的慢病毒载体含有所述sgRNA的编码基因和Cas9蛋白的编码基因。
4.根据权利要求1-3任一所述的方法,其特征在于:所述C1GALT1基因编辑的慢病毒载体为将双链DNA分子插入慢病毒表达载体的酶切位点间得到的载体;
所述双链DNA分子是将单链DNA分子甲和单链DNA分子乙退火制备得到的;
所述单链DNA分子甲的核苷酸序列如序列1所示;
所述单链DNA分子乙的核苷酸序列如序列2所示。
5.根据权利要求4所述的方法,其特征在于:所述C1GALT1基因编辑的慢病毒载体为将所述双链DNA分子插入LentiCRISPRV2载体的BsmBI酶切位点间得到的载体。
6.根据权利要求1-5任一所述的方法,其特征在于:所述结肠癌细胞为结肠癌细胞HCT116。
7.按照权利要求1-6中任一所述的方法制备得到的O-型糖基化异常的结肠癌细胞系。
8.权利要求3中所述的C1GALT1基因编辑的慢病毒载体。
9.权利要求3中所述的C1GALT1基因编辑的慢病毒载体在制备O-型糖基化异常的结肠癌细胞系中的应用。
10.根据权利要求1-6任一所述的方法或权利要求7所述的细胞系或权利要求9所述的应用,其特征在于:所述O-型糖基化异常为T-synthase控制的O-型聚糖链延伸异常。
CN201810300881.7A 2018-04-04 2018-04-04 利用Crispr技术敲除T合酶基因构建的O-型糖基化异常的结肠癌细胞系 Pending CN108504693A (zh)

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