CN112852877A - 质粒载体及其在靶向猪col1a1位点定点整合外源基因中的应用 - Google Patents
质粒载体及其在靶向猪col1a1位点定点整合外源基因中的应用 Download PDFInfo
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Abstract
本发明涉及基因工程技术领域,尤其涉及质粒载体及其在靶向猪COL1A1基因下游序列(以下简称为:COL1A1位点)定点整合外源基因中的应用。本发明提供的质粒载体包括:COL1A1位点同源左臂,外源基因和COL1A1位点同源右臂,该载体是依托CRISPR/Cas9打靶***所设计,它能够将外源基因精确的引入到猪的COL1A1位点中,以解决传统转基因技术效率低下,外源基因随机整合,不同个体表达量差异大,整合位置检测难度大的问题。
Description
技术领域
本发明涉及基因工程技术领域,尤其涉及质粒载体及其在靶向猪COL1A1位点定点整合外源基因中的应用。
背景技术
转基因猪在农业与医学研究中发挥着重要的作用。一方面猪作为一种重要的畜牧用物种,另一方面,猪与人相对于人与小鼠等其他动物更为接近,医学研究上也发挥着不小的作用,比如猪与人的器官大小接近,这一点使异种移植成为了可能,再者猪与人的各器官的结构与血液生理生化指标相似,转基因猪成为更好的动物模型。
转基因猪在很多领域已经得到应用,然而却依然受到一定的限制,转基因的效率与整合位点的不确定性是主要原因。CRISPR/Cas***,广泛存在于真细菌和古生菌中,是一个用来保护宿主不受病毒二次侵害的获得性免疫***。该***由CRISPR体系转录出的RNA和Cas体系翻译出的蛋白相结合来发挥切割DNA的作用。目前在基因组编辑领域应用最为广泛的CRISPR/Cas***是II型的SpCas9***,它使用crRNAs与tracrRNAs嵌合形成的引导RNA与核酸酶Cas9组成复合体来识别、切割DNA,在基因组的特定位点造成DNA双链断裂。相较于ZFNs和TALENs,CRISPR/Cas9***以其极度简单、低构建成本等优势,迅速成为了基因组编辑的主流技术,并普及发展成为了现代生命科学实验室必不可少的研究工具之一。
基因组安全港湾是基因组中能够容纳新遗传物质整合的位点,以确保外源基因可预测地发挥作用。2014年赖良学课题组针对猪开发了Rosa26位点,在其中引入了loxp位点,并成功将RFP基因置换进入并成功表达,使外源基因能在该位点高效表达。但是Rosa26是一个全身性表达的友好性基因,具有自己的Rosa26启动子,在该位点***的基因往往会导致基因在全身性的广泛表达,然而转基因技术在实际运用中有时需要组织特异性表达,该位点很难实现。Col1a1位点是2006年由Caroline Beard首先使用,作为制备转基因鼠四环素诱导调控外源基因表达的整合位点,开发成为一个新的安全港湾。Col1A1位点位于猪第12号染色体的COL1A1基因3'UTR下游,并且不存在启动子,所以可以选择实验所需的启动子完成目的基因的时空特异性表达,更好的达到任务目标。在猪的基因组中定位COL1A1这样安全有效的基因修饰位点,将有利于稳定转基因猪培育的技术体系。
发明内容
有鉴于此,本发明要解决的技术问题在于提供质粒载体及其在靶向猪COL1A1位点定点整合外源基因中的应用。
本发明提供的质粒载体,其包括:Col1A1位点同源左臂,外源基因***位点和Col1A1位点;
所述同源左臂的核苷酸序列如SEQ ID NO:1所示;
所述同源右臂的核苷酸序列如SEQ ID NO:2所示。
本发明所述的质粒载体包括顺序连接的:Col1A1位点同源左臂,猪ICAM2启动子,外源基因、polyA1和Col1A1位点同源右臂。
一些实施例中,所述质粒载体包括顺序连接的:Col1A1位点同源左臂,猪ICAM2启动子,外源基因、polyA1、Col1A1位点同源右臂、PGK promoter、HSV-TK和poly A2。
一些具体实施例中,所述质粒载体的核苷酸序列如SEQ ID NO:3所示。
一些具体实施例中,所述质粒载体担载的外源基因为TM基因,其***方式通过Overlap PCR技术实现无缝连接。
本发明还提供了一种质粒载体组合,其包括定点整合载体和敲除载体:
所述定点整合载体为本发明所述的质粒载体;
所述敲除载体为含有sgRNA的pX458载体;所述sgRNA核苷酸序列为SEQ ID NO:4。
本发明所述的质粒载体组合,在靶向猪COL1A1位点定点整合外源基因中的应用。
本发明还提供了靶向猪COL1A1位点定点整合外源基因的方法,其包括:
将所述敲除载体和线性化的定点整合载体,转染猪体细胞,筛选获得阳性克隆。
在本发明中,所述猪体细胞为猪耳成纤维细胞。
转染猪耳成纤维细胞后,与***融合,获得整合外源基因的猪。
本发明提供的质粒载体包括:COL1A1位点同源左臂,外源基因和COL1A1位点同源右臂,该载体是依托CRISPR/Cas9打靶***所设计,它能够将外源基因精确的引入到猪的COL1A1位点中,以解决传统转基因技术效率低下,外源基因随机整合,不同个体表达量差异大,整合位置检测难度大的问题。
附图说明
图1示仔猪DNA水平上的定点整合鉴定。
图2示仔猪641的TM蛋白表达水平;
图3示COL1A1靶点的敲除载体;
图4示含有两侧同源臂的COL1A1位点的定点整合载体;
图5示构建原理。
具体实施方式
本发明提供了质粒载体及其在靶向猪COL1A1位点定点整合外源基因中的应用,本领域技术人员可以借鉴本文内容,适当改进工艺参数实现。特别需要指出的是,所有类似的替换和改动对本领域技术人员来说是显而易见的,它们都被视为包括在本发明。本发明的方法及应用已经通过较佳实施例进行了描述,相关人员明显能在不脱离本发明内容、精神和范围内对本文的方法和应用进行改动或适当变更与组合,来实现和应用本发明技术。
目前,在转基因猪的制备过程中,主要采用随机整合的方式使外源基因随机***猪基因组中。而本发明提供了一种借助定点切割***对猪COL1A1位点定点***的载体和方法,该方法依据CRISPR/Cas9***对猪COL1A1位点设计了一种打靶载体,克服了随机***技术中步骤繁琐、价格昂贵等缺陷,且更有利于稳定的转基因猪的培育。
本发明提供的质粒载体即打靶载体,又称为定点整合载体,是将两端连有被敲除基因的同源臂待***的外源基因引入到骨架载体中获得的。
本发明提供的质粒载体包括:COL1A1位点同源左臂,外源基因和COL1A1位点同源右臂。所述的同源臂选取sgRNA上游约800bp作为左同源臂,下游约1200bp作为右同源臂。所述同源左臂的核苷酸序列如SEQ ID NO:1所示;所述同源右臂的核苷酸序列如SEQ ID NO:2所示。实验表明,相对于其他序列,在用本发明所述的左右同源臂,更容易将外源基因准确的整合入基因组的靶标位置。
本发明提供的质粒载体中还包括猪ICAM2启动子、polyA1、HSV-TK和poly A2。
其中,所述猪ICAM2是猪内皮特异性启动子,保证外源基因能够在猪的内皮细胞中特异性表达。本发明所述质粒载体中连接的猪ICAM2启动子的核苷酸序列为:SEQ ID NO:5;
所述HSV-TK(单纯疱疹病毒胸苷激酶基因)是一种***基因,当发生随机整合时,可产生细胞毒性物质,从而杀死细胞。本发明所述质粒载体中连接的HSV-TK片段的核苷酸序列为:SEQ ID NO:6;
PGKpromoter是HSV-TK的启动子,保证HSV-TK的表达。本发明所述质粒载体中连接的PGKpromoter片段的核苷酸序列为:SEQ ID NO:7;
本发明质粒载体中,PolyA片段的作用为稳定mRNA,同时有终止转录的作用,防止和下游基因产生融合mRNA。其中polyA1的序列为SEQ ID NO:8,polyA2的序列为SEQ ID NO:9。
顺序连接的COL1A1位点同源左臂,猪ICAM2启动子,外源基因、polyA1、COL1A1位点同源右臂、PGKpromoter、HSV-TK和poly A2的核苷酸序列为SEQ ID NO:10。
将上述片段连接入pCDNA3.1载体获得本发明所述的质粒载体,所述连接的位点为EcoR I和Not I。
所述外源基因,也称为目的基因。在本发明一些具体实施例中,所述外源基因为TM基因。TM基因编码的人血栓调节蛋白被认为是一个能够改善异种移植后凝血紊乱的蛋白,因此在猪的体内转入人TM基因可能是解决凝血***不兼容的有效途径。
在一些具体实施例中,各个功能原件、外源基因、COL1A1位点同源臂的连接采用Overlap PCR技术,各片段间无缝连接,减少了冗余序列,质粒转染后进一步提高了获取阳性细胞的效率。
本发明所述的质粒载体组合中,包括两个载体。其中一个是本发明提供的定点整合载体,用作将目的基因定点整合到宿主的基因组。另一个载体是敲除载体,用于靶向识别并打开宿主的COL1A1位点,提高定点整合的发生效率。本发明中,所述敲除载体为含有sgRNA的pX458载体;所述sgRNA核苷酸序列为SEQ ID NO:4。sgRNA连入位点为Bbs I酶切位点。
本发明所述的靶向猪COL1A1位点定点整合外源基因的方法中,转染猪体细胞、筛选、与***融合以及与***融合后获得整合外源基因猪的操作步骤皆采用本领域的常规手段。
本发明采用的试材皆为普通市售品,皆可于市场购得。下面结合实施例,进一步阐述本发明:
实施例1
猪COL1A1位点定点整合人血栓调节蛋白TM的转基因猪的制备
1、在猪基因组COL1A1位点处设计特异识别靶序列的sgRNA,并连入Bbs I酶切回收后的pX458载体,构建COL1A1靶点的敲除载体(如图3所示)。
通过Overlap PCR、酶切连接的方式获得外源基因片段,两端引入酶切位点EcoRI、NoT I,构建含有两侧同源臂的COL1A1位点的定点整合载体(图谱如图4)。
(1)构建pX458-Col1a1敲除载体:
a、sgRNA寡核苷酸链退火形成含粘性末端的双链片段
表1寡核苷酸序列信息
取表1中合成的sg-Col1a1-target-1正向、反向寡核苷酸链,分别加入ddH2O,调整终浓度为100μmol/L,取相同体积的正向、反向寡核苷酸链混匀,置于PCR仪内。反应条件:95℃10min,随后每分钟降低2℃,直到降至20℃取出保存在-20℃备用。
b、pX458载体酶切、胶回收
用BbsI限制性内切酶对pX458载体酶切,暴露粘性末端,37℃恒温金属浴,6h,反应体系如表2所示。配置1.0%的琼脂糖凝胶,对上述产物进行凝胶电泳分离,用DNA胶回收、纯化试剂盒将目的条带切胶回收、纯化,定浓度至30ng/μL,-20℃保存备用。
表2pX330-EGFP载体酶切体系
组分 | 体积(μL) |
pX458载体,1μg/μL | 2 |
BbsI限制性内切酶(NEB) | 2 |
10×Buffer(NEB,Buffer2) | 5 |
ddH<sub>2</sub>O | 41 |
总体积 | 50 |
c、连接
将a中退火形成的双链核苷酸与b中回收的pX458酶切产物混合,通过T4连接酶的作用,构建pX458-Col1a1敲除载体,反应体系如表3所示。
表3载体连接体系
组分 | 体积(μL) |
pX458酶切产物 | 1 |
退火形成的双链sg-Col1a1 | 1 |
T4连接酶 | 1 |
10×T4 Buffer | 1 |
ddH2O | 6 |
总体积 | 10 |
d、转化、测序、质粒大提
(2)构建定点整合载体:
a、TM-CDS片段
在NCBI中查找已有人TM基因(Gene ID:7056)的CDS序列,全长1728bp,送由苏州金唯智生物公司合成该片段,并连接在pUC57质粒上。
b、各片段扩增
在NCBI中查找已有猪COL1A1基因(Gene ID:100738123)的下游序列2600bp、猪ICAM2基因(Gene ID:414387),使用Primer Premier 5.0软件设计特异性引物,并用NCBI中的BLAST检测引物特异性,引物由苏州金唯智生物公司合成。
取猪肌肉组织提取DNA,分别加入表4中引物进行PCR,PCR产物进行胶回收,TA克隆连接至pClone007 Versatile Simple Vector载体。
表4引物序列信息
c、扩增各片段
以a、b的产物和我司制备的其他定点整合载体为模板,分别加入表5中的引物,PCR扩增依次获得各个片段。
表5引物序列信息
d、连接各个片段
通过Overlap PCR方式,对c中获得的6个片段进行无缝连接,经琼脂糖凝胶电泳,胶回收目的片段。
e、构建定点整合载体
将d中回收的目的片段和pCDNA3.1载体通过EcoR I、Not I限制性内切酶进行双酶切,通过T4连接酶的作用,构建定点整合载体。
2、对构建好的定点整合载体,转化大肠杆菌,完成质粒提取。对提取的质粒进行双酶切,胶回收目的片段并纯化和(1)中构建的敲除载体混合,转染猪耳成纤维细胞,筛选获得阳性克隆点。
3、通过体细胞移植技术,将阳性克隆点与去核的***进行融合,获得人血栓调节蛋白TM转基因猪,对出生的仔猪进行DNA和蛋白水平的验证。
4、克隆出生的仔猪记为:639、640、641、642和643。
5、出生仔猪鉴定
(1)DNA水平鉴定
经PCR鉴定(图1),五头仔猪均在猪Col1a1位点整合人TM基因。
(2)蛋白水平鉴定
对出生的仔猪其中一头641采集主动脉,内皮细胞建系,流式染色鉴定蛋白表达量,HUEC是人主动脉内皮细胞,156-EC是现有GTKO猪主动脉内皮细胞,结果显示:641的TM蛋白表达和人主动脉内皮细胞水平相近(图2)。
以上仅是本发明的优选实施方式,应当指出,对于本技术领域的普通技术人员来说,在不脱离本发明原理的前提下,还可以做出若干改进和润饰,这些改进和润饰也应视为本发明的保护范围。
序列表
<110> 成都中科奥格生物科技有限公司
<120> 质粒载体及其在靶向猪COL1A1位点定点整合外源基因中的应用
<130> MP2032453
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aaagcagttt tcccctgggg tgggaggaag caagagactt tgtacctatt ttgtatgtgt 240
ataataattt gagatgtttt taattatttt gattgctgga ataaagcatg tggaaatgac 300
ccaaaccaat cttgcactgg cctcctgatt tccttccttg gagacggagg gagggggaga 360
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ttcccagcca cctgcaggcc actggctgtg gagcccggcg ccgcggctgc cgccgtctcg 2280
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tgctccgggg tgtgccacaa cctccccggt accttcgagt gcatctgcgg gcccgactcg 3120
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gagtacaagt gcgcggcccc ttccaaggag gtagtgctgc agcacgtgcg gaccgagcgg 3420
acgccgcaga gactctgact gtgccttcta gttgccagcc atctgttgtt tgcccctccc 3480
ccgtgccttc cttgaccctg gaaggtgcca ctcccactgt cctttcctaa taaaatgagg 3540
aaattgcatc aatgcttcct gctccttctt ctggggcccc aggagagcct tcccagggcc 3600
ttggagagtt gctgtccagg gactaaccct gtgctctagg aaggctgcag gccctgacca 3660
gctgggcagg tcctgggtcc ctcctggcct tctaagttcc ccaaacatga gacctctggg 3720
tgtggggtgg cctggggagg tcattttgcc caggccctac ctcctgccca ttcctaaccc 3780
tttttaaaaa tctgtgcgtc ctcttcttcc ttcttctccc tcccttccct tttcgctcac 3840
cctctgctgc tggcctgaga gccggaggcc cccaggggga aggcgactgg tctcctcccc 3900
agtctcaggg aagggagaca gagaatccag gaagccagaa ctcagcagac gaagcaccca 3960
gggacctaga gatgggttga aaagttgaca gctgtcccac ctgcctccca aggtctcagg 4020
gcctaaacct ccaaggcagg aaaggcccct gtccctccct ggggtccata gaaagaggga 4080
caagtctgca cggaccattt gctgtaatat taacaccttg gctgtcatta ggtagtcttg 4140
gctgttaatt atgtcctgtg ataatgtatt attagcacgc cgaccacata gggtagggaa 4200
ctgcagctag taaacaaaag tttgttccta tgcaaattac ccaaagttag caggtgatcc 4260
ttggatcagt gcattgatgg gtgatgtctg cctgacaccg tctttggtga ctctcatccg 4320
cccaggacct cctgctcccc gggcccttgg ctggcttggc cccatccccc tccgccccca 4380
tgggggatcc agaagcatcc tgagcctggc atccaaggct ttacctggcc gctactcatt 4440
tgttccttct atcgcctccc ctgcccacac cagtcacact ggcaaattca acacgctcct 4500
caccttccag gcctttctta tatttcaaag gtctcattgt tttctcatgc tcctgcccaa 4560
ggcagggctg agtttgcctt tgagtttgca aatctctccg tgcctgcggg attcacctca 4620
gccttaagac cagatattgt tgccatttat atgctgtcct gttttcaccc tgttcttctg 4680
tccagaccaa gcggggctgg ctcaggccag aggctaagtg ctgtgcaatg cagggctggg 4740
tgcagggttc aggtggaga 4759
<210> 4
<211> 20
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 4
gcccaaggag acctggagaa 20
<210> 5
<211> 903
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 5
tctagaaaca cactgcaagt cctcagccct accccccccc atcaaggccc cctgtctgga 60
gtgggggtgc accctaagct agcccaggaa gcctccccga gttacttctg tctgcgcctg 120
ctggggacta tgctggggct gctggggact atgctgagtg cgtttccctc aagttgtctt 180
tttttttttt tttttttttg tcttttgtcc ttttatggag gtccccaagc taggggtcta 240
atcggagctg tagctgccgg cctacaccac agccacagca acgccggatc cttatcccac 300
tgagcaaggc cagggatcga acctcaacct catggttcct agtcagattc atttccgctg 360
cgccatgatg ggagtgccgc cctcaagttg tcttgtatca gggagtcaga ggctggctga 420
gattgaaagc ggagtgtcca ggtttaagca gacttgtgcc aggcagttga ctaaagcacc 480
agcagttcct gcccgggttt ctctgcccac ttagtcatgg tgactgcatg ccctggccgg 540
gtcctgactt tggatagtcc cttcccagag gggaccctga cagtgtgtcc cctgggattt 600
ggggctcccc agatctgggg cttgtgggca tcactgtcct ccgtgcacat gcagcactgt 660
cacatgtgtc ccctgatggg agcatgccag gatgacaagg ttatccctgc ttatcagctc 720
tttcccagct tcctctgcct gttgcttata gacctgagcc ctgggcgcca cgtcttcctc 780
aaataatgag aggaagtgtg gtcatcggca cacagatggc tgggggttcc ggattgcggc 840
tcccggccac cactccctga cctgcgcccc catctctcgc caggcggtcc tcagctcttt 900
cct 903
<210> 6
<211> 1188
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 6
gatcttggtg gcgtgaaact cccgcacctc ttcggcaagc gccttgtaga agcgcgtatg 60
gcttcgtacc cctgccatca acacgcgtct gcgttcgacc aggctgcgcg ttctcgcggc 120
catagcaacc gacgtacggc gttgcgccct cgccggcagc aagaagccac ggaagtccgc 180
ctggagcaga aaatgcccac gctactgcgg gtttatatag acggtcctca cgggatgggg 240
aaaaccacca ccacgcaact gctggtggcc ctgggttcgc gcgacgatat cgtctacgta 300
cccgagccga tgacttactg gcaggtgctg ggggcttccg agacaatcgc gaacatctac 360
accacacaac accgcctcga ccagggtgag atatcggccg gggacgcggc ggtggtaatg 420
acaagcgccc agataacaat gggcatgcct tatgccgtga ccgacgccgt tctggctcct 480
catatcgggg gggaggctgg gagctcacat gccccgcccc cggccctcac cctcatcttc 540
gaccgccatc ccatcgccgc cctcctgtgc tacccggccg cgcgatacct tatgggcagc 600
atgacccccc aggccgtgct ggcgttcgtg gccctcatcc cgccgacctt gcccggcaca 660
aacatcgtgt tgggggccct tccggaggac agacacatcg accgcctggc caaacgccag 720
cgccccggcg agcggcttga cctggctatg ctggccgcga ttcgccgcgt ttacgggctg 780
cttgccaata cggtgcggta tctgcagggc ggcgggtcgt ggcgggagga ttggggacag 840
ctttcgggga cggccgtgcc gccccagggt gccgagcccc agagcaacgc gggcccacga 900
ccccatatcg gggacacgtt atttaccctg tttcgggccc ccgagttgct ggcccccaac 960
ggcgacctgt acaacgtgtt tgcctgggcc ttggacgtct tggccaaacg cctccgtccc 1020
atgcacgtct ttatcctgga ttacgaccaa tcgcccgccg gctgccggga cgccctgctg 1080
caacttacct ccgggatggt ccagacccac gtcaccaccc ccggctccat accgacgatc 1140
tgcgacctgg cgcgcacgtt tgcccgggag atgggggagg ctaactga 1188
<210> 7
<211> 500
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 7
gggtagggga ggcgcttttc ccaaggcagt ctggagcatg cgctttagca gccccgctgg 60
gcacttggcg ctacacaagt ggcctctggc ctcgcacaca ttccacatcc accggtaggc 120
gccaaccggc tccgttcttt ggtggcccct tcgcgccacc ttctactcct cccctagtca 180
ggaagttccc ccccgccccg cagctcgcgt cgtgcaggac gtgacaaatg gaagtagcac 240
gtctcactag tctcgtgcag atggacagca ccgctgagca atggaagcgg gtaggccttt 300
ggggcagcgg ccaatagcag ctttgctcct tcgctttctg ggctcagagg ctgggaaggg 360
gtgggtccgg gggcgggctc aggggcgggc tcaggggcgg ggcgggcgcc cgaaggtcct 420
ccggaggccc ggcattctgc acgcttcaaa agcgcacgtc tgccgcgctg ttctcctctt 480
cctcatctcc gggcctttcg 500
<210> 8
<211> 112
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 8
ctgtgccttc tagttgccag ccatctgttg tttgcccctc ccccgtgcct tccttgaccc 60
tggaaggtgc cactcccact gtcctttcct aataaaatga ggaaattgca tc 112
<210> 9
<211> 90
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 9
aacacggaag gagacaatac cggaaggaac ccgcgctatg acggcaataa aaagacagaa 60
taaaacgcac gggtgttggg tcgtttgttc 90
<210> 10
<211> 6578
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 10
ctttcttctt ccccctcggc cgcctctcac cccctgaggc ctctctcccc ccacgacctc 60
ctctctctcc tctgaaaccc tctcctcctc agctgcatcc caccctcgtg gcctctctct 120
ctctctgtct gtcctgtgtc ctctctcact gggtttcaga gcacagatgc ccaaagcaca 180
aaagcagttt tcccctgggg tgggaggaag caagagactt tgtacctatt ttgtatgtgt 240
ataataattt gagatgtttt taattatttt gattgctgga ataaagcatg tggaaatgac 300
ccaaaccaat cttgcactgg cctcctgatt tccttccttg gagacggagg gagggggaga 360
cctgggggag ggcgcttggg ggggggtggg ctctcttctt tctgcgctcc ccccccccac 420
ctccaacacc ttgacgaccc ctcctgcttc cgcttgcctt tctcaggctt taacactttc 480
tcctcgccct ctcagcatgc gcatgcgcgt gcctctacct cccccgcaca tcctggcctg 540
cccaccctga atgtcctggc ccagcgatgc caccaactct ctcgctccgt ccacggctgg 600
ggaggggggc actctgcagg gttggggggc actgggaggc tgggttgggt gagggagggg 660
tgcctgggcc cccacccccc agcaagttct ctccctaggc gaactggagg gtcgtctggc 720
ctcttgagcc ttgttgctgg ctctgagctc taccaagaga gtgaccagca ggaccgcacc 780
atcagtggtt gctgagactg cgtggggtct agaaacacac tgcaagtcct cagccctacc 840
cccccccatc aaggccccct gtctggagtg ggggtgcacc ctaagctagc ccaggaagcc 900
tccccgagtt acttctgtct gcgcctgctg gggactatgc tggggctgct ggggactatg 960
ctgagtgcgt ttccctcaag ttgtcttttt tttttttttt ttttttgtct tttgtccttt 1020
tatggaggtc cccaagctag gggtctaatc ggagctgtag ctgccggcct acaccacagc 1080
cacagcaacg ccggatcctt atcccactga gcaaggccag ggatcgaacc tcaacctcat 1140
ggttcctagt cagattcatt tccgctgcgc catgatggga gtgccgccct caagttgtct 1200
tgtatcaggg agtcagaggc tggctgagat tgaaagcgga gtgtccaggt ttaagcagac 1260
ttgtgccagg cagttgacta aagcaccagc agttcctgcc cgggtttctc tgcccactta 1320
gtcatggtga ctgcatgccc tggccgggtc ctgactttgg atagtccctt cccagagggg 1380
accctgacag tgtgtcccct gggatttggg gctccccaga tctggggctt gtgggcatca 1440
ctgtcctccg tgcacatgca gcactgtcac atgtgtcccc tgatgggagc atgccaggat 1500
gacaaggtta tccctgctta tcagctcttt cccagcttcc tctgcctgtt gcttatagac 1560
ctgagccctg ggcgccacgt cttcctcaaa taatgagagg aagtgtggtc atcggcacac 1620
agatggctgg gggttccgga ttgcggctcc cggccaccac tccctgacct gcgcccccat 1680
ctctcgccag gcggtcctca gctctttcct atgcttgggg tcctggtcct tggcgcgctg 1740
gccctggccg gcctggggtt ccccgcaccc gcagagccgc agccgggtgg cagccagtgc 1800
gtcgagcacg actgcttcgc gctctacccg ggccccgcga ccttcctcaa tgccagtcag 1860
atctgcgacg gactgcgggg ccacctaatg acagtgcgct cctcggtggc tgccgatgtc 1920
atttccttgc tactgaacgg cgacggcggc gttggccgcc ggcgcctctg gatcggcctg 1980
cagctgccac ccggctgcgg cgaccccaag cgcctcgggc ccctgcgcgg cttccagtgg 2040
gttacgggag acaacaacac cagctatagc aggtgggcac ggctcgacct caatggggct 2100
cccctctgcg gcccgttgtg cgtcgctgtc tccgctgctg aggccactgt gcccagcgag 2160
ccgatctggg aggagcagca gtgcgaagtg aaggccgatg gcttcctctg cgagttccac 2220
ttcccagcca cctgcaggcc actggctgtg gagcccggcg ccgcggctgc cgccgtctcg 2280
atcacctacg gcaccccgtt cgcggcccgc ggagcggact tccaggcgct gccggtgggc 2340
agctccgccg cggtggctcc cctcggctta cagctaatgt gcaccgcgcc gcccggagcg 2400
gtccaggggc actgggccag ggaggcgccg ggcgcttggg actgcagcgt ggagaacggc 2460
ggctgcgagc acgcgtgcaa tgcgatccct ggggctcccc gctgccagtg cccagccggc 2520
gccgccctgc aggcagacgg gcgctcctgc accgcatccg cgacgcagtc ctgcaacgac 2580
ctctgcgagc acttctgcgt tcccaacccc gaccagccgg gctcctactc gtgcatgtgc 2640
gagaccggct accggctggc ggccgaccaa caccggtgcg aggacgtgga tgactgcata 2700
ctggagccca gtccgtgtcc gcagcgctgt gtcaacacac agggtggctt cgagtgccac 2760
tgctacccta actacgacct ggtggacggc gagtgtgtgg agcccgtgga cccgtgcttc 2820
agagccaact gcgagtacca gtgccagccc ctgaaccaaa ctagctacct ctgcgtctgc 2880
gccgagggct tcgcgcccat tccccacgag ccgcacaggt gccagatgtt ttgcaaccag 2940
actgcctgtc cagccgactg cgaccccaac acccaggcta gctgtgagtg ccctgaaggc 3000
tacatcctgg acgacggttt catctgcacg gacatcgacg agtgcgaaaa cggcggcttc 3060
tgctccgggg tgtgccacaa cctccccggt accttcgagt gcatctgcgg gcccgactcg 3120
gcccttgccc gccacattgg caccgactgt gactccggca aggtggacgg tggcgacagc 3180
ggctctggcg agcccccgcc cagcccgacg cccggctcca ccttgactcc tccggccgtg 3240
gggctcgtgc attcgggctt gctcataggc atctccatcg cgagcctgtg cctggtggtg 3300
gcgcttttgg cgctcctctg ccacctgcgc aagaagcagg gcgccgccag ggccaagatg 3360
gagtacaagt gcgcggcccc ttccaaggag gtagtgctgc agcacgtgcg gaccgagcgg 3420
acgccgcaga gactctgact gtgccttcta gttgccagcc atctgttgtt tgcccctccc 3480
ccgtgccttc cttgaccctg gaaggtgcca ctcccactgt cctttcctaa taaaatgagg 3540
aaattgcatc aatgcttcct gctccttctt ctggggcccc aggagagcct tcccagggcc 3600
ttggagagtt gctgtccagg gactaaccct gtgctctagg aaggctgcag gccctgacca 3660
gctgggcagg tcctgggtcc ctcctggcct tctaagttcc ccaaacatga gacctctggg 3720
tgtggggtgg cctggggagg tcattttgcc caggccctac ctcctgccca ttcctaaccc 3780
tttttaaaaa tctgtgcgtc ctcttcttcc ttcttctccc tcccttccct tttcgctcac 3840
cctctgctgc tggcctgaga gccggaggcc cccaggggga aggcgactgg tctcctcccc 3900
agtctcaggg aagggagaca gagaatccag gaagccagaa ctcagcagac gaagcaccca 3960
gggacctaga gatgggttga aaagttgaca gctgtcccac ctgcctccca aggtctcagg 4020
gcctaaacct ccaaggcagg aaaggcccct gtccctccct ggggtccata gaaagaggga 4080
caagtctgca cggaccattt gctgtaatat taacaccttg gctgtcatta ggtagtcttg 4140
gctgttaatt atgtcctgtg ataatgtatt attagcacgc cgaccacata gggtagggaa 4200
ctgcagctag taaacaaaag tttgttccta tgcaaattac ccaaagttag caggtgatcc 4260
ttggatcagt gcattgatgg gtgatgtctg cctgacaccg tctttggtga ctctcatccg 4320
cccaggacct cctgctcccc gggcccttgg ctggcttggc cccatccccc tccgccccca 4380
tgggggatcc agaagcatcc tgagcctggc atccaaggct ttacctggcc gctactcatt 4440
tgttccttct atcgcctccc ctgcccacac cagtcacact ggcaaattca acacgctcct 4500
caccttccag gcctttctta tatttcaaag gtctcattgt tttctcatgc tcctgcccaa 4560
ggcagggctg agtttgcctt tgagtttgca aatctctccg tgcctgcggg attcacctca 4620
gccttaagac cagatattgt tgccatttat atgctgtcct gttttcaccc tgttcttctg 4680
tccagaccaa gcggggctgg ctcaggccag aggctaagtg ctgtgcaatg cagggctggg 4740
tgcagggttc aggtggagag ggtaggggag gcgcttttcc caaggcagtc tggagcatgc 4800
gctttagcag ccccgctggg cacttggcgc tacacaagtg gcctctggcc tcgcacacat 4860
tccacatcca ccggtaggcg ccaaccggct ccgttctttg gtggcccctt cgcgccacct 4920
tctactcctc ccctagtcag gaagttcccc cccgccccgc agctcgcgtc gtgcaggacg 4980
tgacaaatgg aagtagcacg tctcactagt ctcgtgcaga tggacagcac cgctgagcaa 5040
tggaagcggg taggcctttg gggcagcggc caatagcagc tttgctcctt cgctttctgg 5100
gctcagaggc tgggaagggg tgggtccggg ggcgggctca ggggcgggct caggggcggg 5160
gcgggcgccc gaaggtcctc cggaggcccg gcattctgca cgcttcaaaa gcgcacgtct 5220
gccgcgctgt tctcctcttc ctcatctccg ggcctttcga cctgcagcga cccgcttaac 5280
agcgtcaaca gcgtgccgca gatcttggtg gcgtgaaact cccgcacctc ttcggcaagc 5340
gccttgtaga agcgcgtatg gcttcgtacc cctgccatca acacgcgtct gcgttcgacc 5400
aggctgcgcg ttctcgcggc catagcaacc gacgtacggc gttgcgccct cgccggcagc 5460
aagaagccac ggaagtccgc ctggagcaga aaatgcccac gctactgcgg gtttatatag 5520
acggtcctca cgggatgggg aaaaccacca ccacgcaact gctggtggcc ctgggttcgc 5580
gcgacgatat cgtctacgta cccgagccga tgacttactg gcaggtgctg ggggcttccg 5640
agacaatcgc gaacatctac accacacaac accgcctcga ccagggtgag atatcggccg 5700
gggacgcggc ggtggtaatg acaagcgccc agataacaat gggcatgcct tatgccgtga 5760
ccgacgccgt tctggctcct catatcgggg gggaggctgg gagctcacat gccccgcccc 5820
cggccctcac cctcatcttc gaccgccatc ccatcgccgc cctcctgtgc tacccggccg 5880
cgcgatacct tatgggcagc atgacccccc aggccgtgct ggcgttcgtg gccctcatcc 5940
cgccgacctt gcccggcaca aacatcgtgt tgggggccct tccggaggac agacacatcg 6000
accgcctggc caaacgccag cgccccggcg agcggcttga cctggctatg ctggccgcga 6060
ttcgccgcgt ttacgggctg cttgccaata cggtgcggta tctgcagggc ggcgggtcgt 6120
ggcgggagga ttggggacag ctttcgggga cggccgtgcc gccccagggt gccgagcccc 6180
agagcaacgc gggcccacga ccccatatcg gggacacgtt atttaccctg tttcgggccc 6240
ccgagttgct ggcccccaac ggcgacctgt acaacgtgtt tgcctgggcc ttggacgtct 6300
tggccaaacg cctccgtccc atgcacgtct ttatcctgga ttacgaccaa tcgcccgccg 6360
gctgccggga cgccctgctg caacttacct ccgggatggt ccagacccac gtcaccaccc 6420
ccggctccat accgacgatc tgcgacctgg cgcgcacgtt tgcccgggag atgggggagg 6480
ctaactgaaa cacggaagga gacaataccg gaaggaaccc gcgctatgac ggcaataaaa 6540
agacagaata aaacgcacgg gtgttgggtc gtttgttc 6578
Claims (10)
1.质粒载体,其包括:COL1A1位点同源左臂,外源基因和COL1A1位点同源右臂;
所述同源左臂的核苷酸序列如SEQ ID NO:1所示;
所述同源右臂的核苷酸序列如SEQ ID NO:2所示。
2.根据权利要求1所述的质粒载体,其特征在于,包括顺序连接的:COL1A1位点同源左臂,猪ICAM2启动子,外源基因、polyA-1和COL1A1位点同源右臂。
3.根据权利要求1或2所述的质粒载体,其特征在于,包括顺序连接的:COL1A1位点同源左臂,猪ICAM2启动子,外源基因、polyA-1、COL1A1位点同源右臂、PGK启动子、HSV-TK和polyA-2。
4.根据权利要求1或2所述的质粒载体,其特征在于,其核苷酸序列如SEQ ID NO:3所示。
5.根据权利要求4所述的质粒载体,其特征在于,所述外源基因为TM基因,其构建方式利用Overlap PCR,与猪ICAM2启动子和polyA-1无缝连接。
6.一种质粒载体组合,其特征在于,包括定点整合载体和敲除载体:
所述定点整合载体为权利要求1~5任一项所述的质粒载体;
所述敲除载体为含有sgRNA的pX458载体;所述sgRNA核苷酸序列为SEQ ID NO:4。
7.权利要求5所述的质粒载体组合,在靶向猪COL1A1位点定点整合外源基因中的应用。
8.靶向猪COL1A1位点定点整合外源基因的方法,其包括:
将权利要求6所述的敲除载体,和线性化的权利要求5所述定点整合载体,转染猪体细胞,筛选获得阳性克隆。
9.根据权利要求8所述的方法,其特征在于,所述猪体细胞为猪耳成纤维细胞。
10.根据权利要求8所述的方法,其特征在于,转染猪耳成纤维细胞后,与***融合,获得获得整合外源基因的猪。
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Cited By (4)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
EP3545079B1 (en) | 2016-11-24 | 2022-04-06 | Cambridge Enterprise Limited | Controllable transcription |
CN114958760A (zh) * | 2021-02-23 | 2022-08-30 | 南京启真基因工程有限公司 | 一种构建阿尔兹海默症模型猪的基因编辑技术及其应用 |
CN116694683A (zh) * | 2023-06-02 | 2023-09-05 | 中国农业科学院北京畜牧兽医研究所 | 靶向猪COL1A1基因下游序列的CRISPR-Cas9***及其应用 |
CN116790603A (zh) * | 2023-08-18 | 2023-09-22 | 成都中科奥格生物科技有限公司 | 一种sgRNA、CRISPR/Cas9载体及其构建方法和用途 |
Citations (2)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN109112161A (zh) * | 2018-08-17 | 2019-01-01 | 华中农业大学 | 制备rosa26基因突变及肌内脂肪提高的动物的方法 |
CN111647604A (zh) * | 2020-06-29 | 2020-09-11 | 中国农业科学院北京畜牧兽医研究所 | 特异性识别猪COL1A1基因的gRNA及其生物材料、试剂盒和应用 |
-
2021
- 2021-04-07 CN CN202110371942.0A patent/CN112852877A/zh active Pending
Patent Citations (2)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN109112161A (zh) * | 2018-08-17 | 2019-01-01 | 华中农业大学 | 制备rosa26基因突变及肌内脂肪提高的动物的方法 |
CN111647604A (zh) * | 2020-06-29 | 2020-09-11 | 中国农业科学院北京畜牧兽医研究所 | 特异性识别猪COL1A1基因的gRNA及其生物材料、试剂盒和应用 |
Non-Patent Citations (2)
Title |
---|
RIEDER,M.J.等: "Homo sapiens thrombomodulin (THBD) gene, complete cds", 《GENBANK DATABASE》 * |
未知: "Sus scrofa isolate TJ Tabasco breed Duroc chromosome 12, Sscrofa11.1, whole genome shotgun sequence", 《GENBANK DATABASE》 * |
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CN116694683A (zh) * | 2023-06-02 | 2023-09-05 | 中国农业科学院北京畜牧兽医研究所 | 靶向猪COL1A1基因下游序列的CRISPR-Cas9***及其应用 |
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