CN115216492B - 一种小鼠原发神经胶质瘤模型的制备方法及其应用 - Google Patents
一种小鼠原发神经胶质瘤模型的制备方法及其应用 Download PDFInfo
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Abstract
本发明公开了一种小鼠原发神经胶质瘤模型的制备方法及其应用，包括下列步骤：1)PB‑CAG‑MCS‑IRES‑EGFP质粒构建，2)PB‑CAG‑Pdgfa‑HA‑IRES‑EGFP质粒的构建，将Pdgfa基因插入到所述PB‑CAG‑MCS‑IRES‑EGFP质粒的多克隆位点，表达携带HA标签的PDGFA蛋白与用于示踪的绿色荧光蛋白EGFP；3)原发胶质瘤小鼠模型的制备，获得原发胶质瘤小鼠模型。本发明将为神经胶质瘤的发生和治疗等相关研究提供重要的研究模型。
Description
技术领域
本发明涉及生物技术领域，尤其涉及一种小鼠原发神经胶质瘤模型的制备方法及其应用。
背景技术
神经胶质瘤(Glioma)是中枢神经系统常见肿瘤。胶质母细胞瘤(Glioblastoma,GBM)更是恶性程度极高的神经系统肿瘤。胶质母细胞瘤GBM根据其基因表达等特征，又可以分为神经前体型(proneural,PN)、间充质型(mesenchymal,MES)(MES),经典型(classical，CL)和神经型(neural,NL)。不同的肿瘤亚型往往与不同的基因突变相关。比如，超过90％否认CL-GBM具有增强的EGFR活性和CDKN2A缺失，MES-GBM常与NF1/PTEN基因突变有关，而绝大部分PN-GBM中可以观察到PDGFRA扩增和IDH1基因突变。目前对胶质瘤的研究模型主要有体外培养的胶质瘤细胞，比如A172,U-251等；或者通过向小鼠脑中接种体外培养的肿瘤细胞，进而形成的移植瘤。但是，这些模型都只能研究肿瘤形成后的进展、治疗效果等问题，无法研究肿瘤发生的问题，且这些细胞系状态下的肿瘤细胞与体内原发性胶质瘤往往存在很大的差别。因此，原发胶质瘤模型是胶质瘤研究以及药物筛选的必不可少的工具。
目前原发胶质瘤模型的制备主要依赖几种遗传修饰小鼠的使用，制备难度较大。常见的方法有如下几种：(1)通过NG2-CreER、hGFAP-CreER、Nestin-Cre等表达Cre重组酶的工具鼠，结合Ptenfx/fx或者p53fx/fx，LSL-EGFRviii等小鼠品系，在少突胶质细胞前体细胞(oligodendrocyte progenitor cells,OPCs)、神经前体细胞中条件性敲除p53、Nf1和Pten等基因或者激活EGFR、PDGFRa信号通路的活性；(2)借助表达禽类病毒受体A(avian tumorvirus receptor A,TVA)的Olig2-tva,hGFAP-tva,Nestin-tva工具鼠，通过向小鼠脑中注射携带外源基因的禽类病毒RCAS质粒，实现禽类病毒在小鼠神经细胞中的复制和感染，介导外源基因的持续表达，最终诱发神经胶质瘤；(3)借助表达Cas9的转基因小鼠，通过电转表达针对p53、Nf1和Pten基因的small guide RNAs的质粒，实现这些抑癌基因的失活，从而诱发小鼠原发性胶质瘤；(4)转座子系统也是近年来被采用的方法，可以通过将外源基因转座整合到特定的遗传修饰小鼠脑部神经细胞中，起到激活或者敲除特定基因的目的，从而诱发胶质瘤。但是，以上这些原发胶质瘤模型都借助了遗传修饰小鼠，因而在实施上收到原材料的限制。因此，需要开发一种可在普通野生型小鼠中实现的原发胶质瘤模型。
piggyBac转座子系统也被证明可以有效实现外源基因在小鼠细胞中的稳定表达，并且比其他转座子系统具有更高的效率，是在小鼠组织中表达外源基因或者进行基因敲除的理想方法。已有研究表明，单独激活Pdgfa基因的持续表达，即可在小鼠脑中诱发前述PN-GBM样神经胶质瘤。
发明内容
本发明提供了一种小鼠原发神经胶质瘤模型的制备方法及其应用，以解决现有技术的上述问题。
本发明的方案是：
一种小鼠原发神经胶质瘤模型的制备方法，包括下列步骤：
1)PB-CAG-MCS-IRES-EGFP质粒构建，所述PB-CAG-MCS-IRES-EGFP质粒包含启动子与下游的IRES-EGFP序列，在启动子与IRES-EGFP序列之间加入多克隆位点；
2)PB-CAG-Pdgfa-HA-IRES-EGFP质粒的构建，将Pdgfa基因插入到所述PB-CAG-MCS-IRES-EGFP质粒的多克隆位点，表达携带HA标签的PDGFA蛋白与用于示踪的绿色荧光蛋白EGFP；
3)原发胶质瘤小鼠模型的制备，通过将质粒PB-CAG-Pdgfa-HA-IRES-EGFP与表达转座酶的质粒pCAGGS-piggyBac Transposase共同使用，诱导小鼠，获得原发胶质瘤小鼠模型。
作为优选的技术方案，所述启动子为CAG启动子或其他哺乳动物细胞可用的启动子。
作为优选的技术方案，所述PB-CAG-Pdgfa-HA-IRES-EGFP质粒在piggyBac转座酶存在时被整合入细胞基因组中，实现目标基因在转入细胞及其子代细胞中的稳定表达。
本发明还公开了一种小鼠原发神经胶质瘤模型的制备方法用piggyBac转座子质粒PB-CAG-MCS-IRES-EGFP，所述PB-CAG-MCS-IRES-EGFP的核苷酸序列如SEQ ID No.1所示。
本发明还公开了一种小鼠原发神经胶质瘤模型制备方法的应用，为神经胶质瘤的发生与治疗研究提供重要的研究模型。
由于采用了上述技术方案一种小鼠原发神经胶质瘤模型的制备方法，包括下列步骤：1)PB-CAG-MCS-IRES-EGFP质粒构建，所述PB-CAG-MCS-IRES-EGFP质粒包含启动子与下游的IRES-EGFP序列，在启动子与IRES-EGFP序列之间加入多克隆位点；2)PB-CAG-Pdgfa-HA-IRES-EGFP质粒的构建，将Pdgfa基因插入到所述PB-CAG-MCS-IRES-EGFP质粒的多克隆位点，表达携带HA标签的PDGFA蛋白与用于示踪的绿色荧光蛋白EGFP；3)原发胶质瘤小鼠模型的制备，通过将质粒PB-CAG-Pdgfa-HA-IRES-EGFP与表达转座酶的质粒pCAGGS-piggyBacTransposase共同使用，诱导小鼠，获得原发胶质瘤小鼠模型。
发明优点：
本发明构建了一个携带EGFP的piggyBac转座子质粒，通过将Pdgfa基因插入该质粒得到PB-CAG-Pdgfa-HA-IRES-EGFP质粒，并通过小鼠胚胎质粒电转技术将该质粒转入胚胎期14.5(E14.5)的小鼠大脑皮层，并在小鼠出生后诱发原发性胶质瘤。利用该发明所构建的质粒系统和所描述的方法，可在普通的野生型小鼠脑中诱发原发性胶质瘤，而无需各种遗传修饰的小鼠。本发明将为神经胶质瘤的发生和治疗等相关研究提供重要的研究模型。
附图说明
图1为本发明实施例Pdgfa诱发的小鼠原发胶质瘤模型表型图；
图2.为本发明实施例免疫组化和原位杂交检测该原发胶质瘤中各标记物的表达情况图；
图3.为本发明PB-CAG-MCS-IRES-EGFP质粒图谱。
具体实施方式
为了弥补以上不足，本发明提供了一种小鼠原发神经胶质瘤模型的制备方法及其应用以解决上述背景技术中的问题。
一种小鼠原发神经胶质瘤模型的制备方法，包括下列步骤：
1)PB-CAG-MCS-IRES-EGFP质粒构建，所述PB-CAG-MCS-IRES-EGFP质粒包含启动子与下游的IRES-EGFP序列，在启动子与IRES-EGFP序列之间加入多克隆位点；
2)PB-CAG-Pdgfa-HA-IRES-EGFP质粒的构建，将Pdgfa基因插入到所述PB-CAG-MCS-IRES-EGFP质粒的多克隆位点，表达携带HA标签的PDGFA蛋白与用于示踪的绿色荧光蛋白EGFP；
3)原发胶质瘤小鼠模型的制备，通过将质粒PB-CAG-Pdgfa-HA-IRES-EGFP与表达转座酶的质粒pCAGGS-piggyBac Transposase共同使用，诱导小鼠，获得原发胶质瘤小鼠模型。
所述启动子为CAG启动子或其他哺乳动物细胞可用的启动子。
所述PB-CAG-Pdgfa-HA-IRES-EGFP质粒在piggyBac转座酶存在时被整合入细胞基因组中，实现目标基因在转入细胞及其子代细胞中的稳定表达。
本发明还公开了一种小鼠原发神经胶质瘤模型的制备方法用piggyBac转座子质粒PB-CAG-MCS-IRES-EGFP，所述PB-CAG-MCS-IRES-EGFP的核苷酸序列如SEQ ID No.1所示。该质粒含有CAG启动子及其下游的IRES-EGFP序列，并在CAG启动子和IRES序列之间加入了多克隆位点，供外源基因插入。该质粒包含piggyBac转座酶介导转座发生所需的核酸序列，可实现目标基因在转入的细胞及其子代细胞中的稳定表达。
表达piggyBac转座酶的质粒pCAGGS-piggyBac Transposase，命名为pCAGGS-PBase。
通过小鼠子宫内胚胎电转技术(in ovo electroporation,IOE)，将质粒PB-CAG-Pdgfa-HA-IRES-EGFP与表达转座酶的质粒pCAGGS-piggyBac Transposase共同电转至胚胎期小鼠大脑皮层，从而诱导小鼠在出生后产生原发性胶质瘤。
该发明基于转座子基因递送系统，通过胚胎电转方法实现小鼠脑中Pdgfa持续过量表达，从而获得小鼠原发胶质瘤模型。在使用该转座子系统的技术条件下，将本发明中CAG启动子更换其他哺乳动物细胞可用的启动子，或者将胚胎电转的小鼠发育时期进行调整，比如胚胎期12天(E12)～出生后1周(P7)等，都在本发明专利的权利要求范围。
本发明还公开了一种小鼠原发神经胶质瘤模型制备方法的应用，为神经胶质瘤的发生与治疗研究提供重要的研究模型。
为了使本发明实现的技术手段、创作特征、达成目的与功效易于明白了解，下面结合具体实施例，进一步阐述本发明。
实施例：
1.PB-CAG-MCS-IRES-EGFP质粒的构建：
首先需要获取CAG-MCS-IRES-EGFP片段。该片段包含CAG启动子和IRES-EGFP两个片段。通过重叠延伸PCR的技术，可将两个DNA片段拼接在一起。其中，CAG启动子片段来自pCAGGS-mCherry质粒，IRES-EGFP片段来自pCIG2-Flag-mNuak1质粒。引物设计如下：
CAG-IRES-F:
5-gccaccatgtgtacagaactcgagtttaaatcggatccgatccgcccctctccctcccccccccctaacgttact-3
CAG-IRES-R:
5-ctcgagttctgtacacatggtggctgacatctgatgatggctagccccgggcccgcggtaccgtcgact-3
CAG-F:5-GGACTAGTtattaatagtaatcaattacggggtcattagttcatagcccat-3EGFP-R:
5-GCACGCGTCGACtgtaccgctcatctgttagtggtgcctagattacttgtagagctcgtccatgcc-3
用引物CAG-F和CAG-IRES-R做PCR，模板为质粒pCAGGS-mCherry(Addgene,#41583)，进行PCR-1#扩增，目标产物约1.7kb。用引物CAG-IRES-F和EGFP-R为引物，模板为质粒pCIG2-Flag-mNuak1(Addgene,#168507)，进行PCR-2#扩增，目标产物约1.4kb。2次PCR均为30uL体系，5个循环。酶采用TAKARA公司的PrimeSTAR GXL DNA Polymerase(#R050Q)，扩增体系参考DNA聚合酶说明书。PCR-1#,PCR-2#结束之后，以这两份PCR产物作为模板(用量为1uL/30uL体系)，以CAG-F和EGFP-R为引物，进行第三轮PCR反应，即为PCR-3#。PCR反应体系同上，但扩大为60uL，循环数设定为30，目标片段大小约3.2kb。扩增结束后，PCR-3#产物进行1％琼脂糖凝胶电泳，之后通过胶回收试剂盒纯化目标片段(50uL水洗脱产物)。该DNA片段即为CAG-MCS-IRES-EGFP。目标片段经SpeI和SalI双酶切(酶购自Thermo,#FD1254,#FD0644)，体系50uL,胶回收产物43uL,Buffer 5uL,两种内切酶各1uL，37℃孵育过夜。DNAClean-Up试剂盒回收酶切后的PCR产物至30uL去离子水，备用。
之后，需要获取可被转座酶识别的质粒片段。设计引物如下：
PB-F:5-GCACGCGTCGACaatcaacctctggattacaaaatttgtg-3
PB-R:5-GGACTAGTggccttggaggccttttccccgtatccccccag-3
以引物PB-F和PB-R做PCR，模板为质粒PB-CMV-MCS-EF1α-GFP-Puro(SBI,#PB513B-1)。PCR产物片段大小约4.9kb，反应体系同前。PCR产物琼脂糖凝胶电泳后，纯化回收之后，经SpeI和SalI双酶切。之后的片段回收方法同上。将双酶切后的该片段与前述CAG-MCS-IRES-EGFP片段进行连接：3.2kb双酶切片段3uL，CAG-MCS-IRE-EGFP双酶切片段5.5uL，Ligase Buffer 1uL，T4 Ligase 0.5uL。室温连接30min，转化大肠杆菌DH5a感受态细胞。次日，挑单克隆菌落测序。测序正确的克隆可接种培养后提取质粒。即获取PB-CAG-MCS-IRES-EGFP质粒。该质粒在CAG启动子和IRES之间引入多克隆位点，包含NheI和BsrGI限制性内切酶识别位点，可供后续外源基因片段的插入(图3)。
2.PB-CAG-Pdgfa-HA-IRES-EGFP质粒的构建：
为了获取Pdgfa片段，设计引物如下：
Pdgfa-F:5-GCTCTAGAGCCACCatgaggacctgggcttgcctgctg-3
Pdgfra-R:
5-GCGCTGGTACCttatgcataatcaggcacatcgtaaggatacctcacatctgtctcctcctcccg-3
PCR方法、DNA片段回收方法、DNA片段双酶切和纯化方法同前所述。PCR所用模板为出生后2周的小鼠脑组织提取的总RNA经逆转录后的得到的cDNA，目的片段大小约为630bp。
接着，对载体PB-CAG-MCS-IRES-EGFP进行NheI、BsrGI双酶切，即质粒5ug，50uL体系，内切酶每种2.5uL，37℃孵育过夜。1％琼脂糖凝胶电泳，通过胶回收试剂盒纯化约8kb的目标片段(50uL水洗脱产物)。将该片段与前述Pdgfa-HA片段进行连接：载体片段1uL,Pdgfa-HA双酶切片段5uL，去离子水2.5uL,Ligase Buffer 1uL，T4 Ligase 0.5uL。室温连接30min，转化大肠杆菌DH5a感受态细胞。次日，挑单克隆菌落测序。测序正确的克隆可接种培养后提取质粒。即获取PB-CAG-Pdgfa-HA-IRES-EGFP质粒，该质粒在CAG启动子的驱动下，可以同时表达绿色荧光蛋白EGFP和携带HA标签的PDGFA蛋白。
3.原发胶质瘤小鼠模型的制备：
通过除内毒素的质粒提取试剂盒，从大肠杆菌中提取质粒PB-CAG-Pdgfa-HA-IRES-EGFP，溶解于无内毒素的去离子水中，使其浓度为5000ng/μl。雌性和雄性成年小鼠在傍晚放入同一个笼子，次日早上分开，至中午记为胚胎期0.5天(E0.5)。取E14.5孕鼠进行小鼠胚胎质粒电转。详细的实验操作按照之前的文献报道执行。大致流程为：孕鼠麻醉后进行腹部表面消毒，剪去腹部毛发，打开腹腔，将包含胚胎的小鼠子宫拉出，通过微量注射仪向胚胎侧脑室注射入混入终浓度0.01％FastGreen染料的质粒2-3uL，将直径6cm的圆形电极放在胚胎脑部两侧，施加45V脉冲电压(每次50ms，每次间隔950ms，连续电击5次)；将子宫小心移回小鼠子宫，缝合伤口，消毒后放在37℃保持体温直至小鼠苏醒。小鼠幼崽大概在4-5天后出生，出生后的小鼠存活至1月龄时检查胶质瘤的形成情况。对照组小鼠电转质粒PB-CAG-MCS-IRES-EGFP。
结果表明，出生一个月之后，小鼠头部出现畸形，小鼠活动能力受到严重影响，体型也比对照组小鼠更小(图1A-C)。解剖后对脑组织进行切片分析，结果表明：小鼠大脑皮层的肿瘤部位的体积已经相当于正常脑组织的大小，针对Pdgfa mRNA的原位杂交实验证实肿瘤形成区存在广泛的Pdgfa基因的高表达(图1D,E)。免疫荧光和免疫组化染色显示，在肿瘤部位的细胞高表达OLIG2、PDGFRA、SOX10等PN-GBM的标记物(图2A,B,A’,B’)。同时，在肿瘤区域，缺乏GFAP+/Glast+星形胶质细胞(图2D)，几乎没有NeuN+神经元(图2C)，但有一定数量的IBA1+/CD68+活化的小胶质细胞(图2E,D)。这些结果表明，使用该方法成功构建了小鼠原发胶质瘤模型。以上显示和描述了本发明的基本原理、主要特征及本发明的优点。本行业的技术人员应该了解，本发明不受上述实施例的限制，上述实施例和说明书中描述的只是说明本发明的原理，在不脱离本发明精神和范围的前提下，本发明还会有各种变化和改进，这些变化和改进都落入要求保护的本发明范围内。本发明要求保护范围由所附的权利要求书及其等效物界定。
序列表
<110> 浙江欧赛思生物科技有限公司
<120> 一种小鼠原发神经胶质瘤模型的制备方法及其应用
<160> 2
<170> SIPOSequenceListing 1.0
<210> 1
<211> 8070
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 1
aaattgtaag cgttaatatt ttgttaaaat tcgcgttaaa tttttgttaa atcagctcat 60
tttttaacca ataggccgaa atcggcaaaa tcccttataa atcaaaagaa tagaccgaga 120
tagggttgag tgttgttcca gtttggaaca agagtccact attaaagaac gtggactcca 180
acgtcaaagg gcgaaaaacc gtctatcagg gcgatggccc actacgtgaa ccatcaccct 240
aatcaagttt tttggggtcg aggtgccgta aagcactaaa tcggaaccct aaagggagcc 300
cccgatttag agcttgacgg ggaaagccgg cgaacgtggc gagaaaggaa gggaagaaag 360
cgaaaggagc gggcgctagg gcgctggcaa gtgtagcggt cacgctgcgc gtaaccacca 420
cacccgccgc gcttaatgcg ccgctacagg gcgcgtccca ttcgccattc aggctgcgca 480
actgttggga agggcgatcg gtgcgggcct cttcgctatt acgccagctg gcgaaagggg 540
gatgtgctgc aaggcgatta agttgggtaa cgccagggtt ttcccagtca cgacgttgta 600
aaacgacggc cagtgagcgc gcctcgttca ttcacgtttt tgaacccgtg gaggacgggc 660
agactcgcgg tgcaaatgtg ttttacagcg tgatggagca gatgaagatg ctcgacacgc 720
tgcagaacac gcagctagat taaccctaga aagataatca tattgtgacg tacgttaaag 780
ataatcatgc gtaaaattga cgcatgtgtt ttatcggtct gtatatcgag gtttatttat 840
taatttgaat agatattaag ttttattata tttacactta catactaata ataaattcaa 900
caaacaattt atttatgttt atttatttat taaaaaaaaa caaaaactca aaatttcttc 960
tataaagtaa caaaactttt atgagggaca gccccccccc aaagccccca gggatgtaat 1020
tacgtccctc ccccgctagg gggcagcagc gagccgcccg gggctccgct ccggtccggc 1080
gctccccccg catccccgag ccggcagcgt gcggggacag cccgggcacg gggaaggtgg 1140
cacgggatcg ctttcctctg aacgcttctc gctgctcttt gagcctgcag acacctgggg 1200
ggatacgggg aaaaggcctc caaggccact agttattaat agtaatcaat tacggggtca 1260
ttagttcata gcccatatat ggagttccgc gttacataac ttacggtaaa tggcccgcct 1320
ggctgaccgc ccaacgaccc ccgcccattg acgtcaataa tgacgtatgt tcccatagta 1380
acgccaatag ggactttcca ttgacgtcaa tgggtggact atttacggta aactgcccac 1440
ttggcagtac atcaagtgta tcatatgcca agtacgcccc ctattgacgt caatgacggt 1500
aaatggcccg cctggcatta tgcccagtac atgaccttat gggactttcc tacttggcag 1560
tacatctacg tattagtcat cgctattacc atgggtcgag gtgagcccca cgttctgctt 1620
cactctcccc atctcccccc cctccccacc cccaattttg tatttattta ttttttaatt 1680
attttgtgca gcgatggggg cggggggggg gggggcgcgc gccaggcggg gcggggcggg 1740
gcgaggggcg gggcggggcg aggcggagag gtgcggcggc agccaatcag agcggcgcgc 1800
tccgaaagtt tccttttatg gcgaggcggc ggcggcggcg gccctataaa aagcgaagcg 1860
cgcggcgggc gggagtcgct gcgttgcctt cgccccgtgc cccgctccgc gccgcctcgc 1920
gccgcccgcc ccggctctga ctgaccgcgt tactcccaca ggtgagcggg cgggacggcc 1980
cttctcctcc gggctgtaat tagcgcttgg tttaatgacg gctcgtttct tttctgtggc 2040
tgcgtgaaag ccttaaaggg ctccgggagg gccctttgtg cgggggggag cggctcgggg 2100
ggtgcgtgcg tgtgtgtgtg cgtggggagc gccgcgtgcg gcccgcgctg cccggcggct 2160
gtgagcgctg cgggcgcggc gcggggcttt gtgcgctccg cgtgtgcgcg aggggagcgc 2220
ggccgggggc ggtgccccgc ggtgcggggg ggctgcgagg ggaacaaagg ctgcgtgcgg 2280
ggtgtgtgcg tgggggggtg agcagggggt gtgggcgcgg cggtcgggct gtaacccccc 2340
cctgcacccc cctccccgag ttgctgagca cggcccggct tcgggtgcgg ggctccgtac 2400
ggggcgtggc gcggggctcg ccgtgccggg cggggggtgc cggcaggtgg gggtgccggg 2460
cggggcgggg ccgcctcggg cctgggaggg ctcgggggag gggcgcggcg gcccccggag 2520
cgccggcggc tgtcgaggcg cggcgagccg cagccattgc cttttatggt aatcgtgcga 2580
gagggcgcag ggacttcctt tgtcccaaat ctgtgcggag ccgaaatctg ggaggcgccg 2640
ccgcaccccc tctagcgggc gcggggcgaa gcggtgcggc gccggcagga aggaaatggg 2700
cggggagggc cttcgtgcgt cgccgcgccg ccgtcccctt ctccctctcc agcctcgggg 2760
ctgtccgcgg ggggacggct gccttcgggg gggacggggc agggcggggt tcggcttctg 2820
gcgtgtgacc ggcggctcta gagcctctgc taaccatgtt catgccttct tctttttcct 2880
acagctcctg ggcaacgtgc tggttattgt gctgtctcat cattttggca aagaattcct 2940
cgagatctcg agctcaagct tcgaattctg cagtcgacgg taccgcgggc ccggggctag 3000
ccatcatcag atgtcagcca ccatgtgtac agaactcgag tttaaatcgg atccgatccg 3060
cccctctccc tccccccccc ctaacgttac tggccgaagc cgcttggaat aaggccggtg 3120
tgcgtttgtc tatatgttat tttccaccat attgccgtct tttggcaatg tgagggcccg 3180
gaaacctggc cctgtcttct tgacgagcat tcctaggggt ctttcccctc tcgccaaagg 3240
aatgcaaggt ctgttgaatg tcgtgaagga agcagttcct ctggaagctt cttgaagaca 3300
aacaacgtct gtagcgaccc tttgcaggca gcggaacccc ccacctggcg acaggtgcct 3360
ctgcggccaa aagccacgtg tataagatac acctgcaaag gcggcacaac cccagtgcca 3420
cgttgtgagt tggatagttg tggaaagagt caaatggctc tcctcaagcg tattcaacaa 3480
ggggctgaag gatgcccaga aggtacccca ttgtatggga tctgatctgg ggcctcggta 3540
cacatgcttt acatgtgttt agtcgaggtt aaaaaaacgt ctaggccccc cgaaccacgg 3600
ggacgtggtt ttcctttgaa aaacacgatg ataatatggc cacaaccatg gtgagcaagg 3660
gcgaggagct gttcaccggg gtggtgccca tcctggtcga gctggacggc gacgtaaacg 3720
gccacaagtt cagcgtgtcc ggcgagggcg agggcgatgc cacctacggc aagctgaccc 3780
tgaagttcat ctgcaccacc ggcaagctgc ccgtgccctg gcccaccctc gtgaccaccc 3840
tgacctacgg cgtgcagtgc ttcagccgct accccgacca catgaagcag cacgacttct 3900
tcaagtccgc catgcccgaa ggctacgtcc aggagcgcac catcttcttc aaggacgacg 3960
gcaactacaa gacccgcgcc gaggtgaagt tcgagggcga caccctggtg aaccgcatcg 4020
agctgaaggg catcgacttc aaggaggacg gcaacatcct ggggcacaag ctggagtaca 4080
actacaacag ccacaacgtc tatatcatgg ccgacaagca gaagaacggc atcaaggtga 4140
acttcaagat ccgccacaac atcgaggacg gcagcgtgca gctcgccgac cactaccagc 4200
agaacacccc catcggcgac ggccccgtgc tgctgcccga caaccactac ctgagcaccc 4260
agtccgccct gagcaaagac cccaacgaga agcgcgatca catggtcctg ctggagttcg 4320
tgaccgccgc cgggatcact ctcggcatgg acgagctcta caagtaatct aggcaccact 4380
aacagatgag cggtacagtc gacaatcaac ctctggatta caaaatttgt gaaagattga 4440
ctggtattct taactatgtt gctcctttta cgctatgtgg atacgctgct ttaatgcctt 4500
tgtatcagtt aacttgttta ttgcagctta taatggttac aaataaagca atagcatcac 4560
aaatttcaca aataaagcat ttttttcact gcattctagt tgtggtttgt ccaaactcat 4620
caatgtatct tatcatgtct ggaattgact caaatgatgt caattagtct atcagaagct 4680
atctggtctc ccttccgggg gacaagacat ccctgtttaa tatttaaaca gcagtgttcc 4740
caaactgggt tcttatatcc cttgctctgg tcaaccaggt tgcagggttt cctgtcctca 4800
caggaacgaa gtccctaaag aaacagtggc agccaggttt agccccggaa ttgactggat 4860
tcctttttta gggcccattg gtatggcttt ttccccgtat ccccccaggt gtctgcaggc 4920
tcaaagagca gcgagaagcg ttcagaggaa agcgatcccg tgccaccttc cccgtgcccg 4980
ggctgtcccc gcacgctgcc ggctcgggga tgcgggggga gcgccggacc ggagcggagc 5040
cccgggcggc tcgctgctgc cccctagcgg gggagggacg taattacatc cctgggggct 5100
ttgggggggg gctgtccctg atatctataa caagaaaata tatatataat aagttatcac 5160
gtaagtagaa catgaaataa caatataatt atcgtatgag ttaaatctta aaagtcacgt 5220
aaaagataat catgcgtcat tttgactcac gcggtcgtta tagttcaaaa tcagtgacac 5280
ttaccgcatt gacaagcacg cctcacggga gctccaagcg gcgactgaga tgtcctaaat 5340
gcacagcgac ggattcgcgc tatttagaaa gagagagcaa tatttcaaga atgcatgcgt 5400
caattttacg cagactatct ttctagggtt aatctagctg catcaggatc atatcgtcgg 5460
gtcttttttc cggctcagtc atcgcccaag ctggcgctat ctgggcatcg gggaggaaga 5520
agcccgtgcc ttttcccgcg aggttgaagc ggcatggaaa gagtttgccg aggatgactg 5580
ctgctgcatt gacgttgagc gaaaacgcac gtttaccatg atgattcggg aaggtgtggc 5640
catgcacgcc tttaacggtg aactgttcgt tcaggccacc tgggatacca gttcgtcgcg 5700
gcttttccgg acacagttcc ggatggtcag cccgaagcgc atcagcaacc cgaacaatac 5760
cggcgacagc cggaactgcc gtgccggtgt gcagattaat gacagcggtg cggcgctggg 5820
atattacgtc agcgaggacg ggtatcctgg ctggatgccg cagaaatgga catggatacc 5880
ccgtgagtta cccggcgggc gcgcttggcg taatcatggt catagctgtt tcctgtgtga 5940
aattgttatc cgctcacaat tccacacaac atacgagccg gaagcataaa gtgtaaagcc 6000
tggggtgcct aatgagtgag ctaactcaca ttaattgcgt tgcgctcact gcccgctttc 6060
cagtcgggaa acctgtcgtg ccagctgcat taatgaatcg gccaacgcgc ggggagaggc 6120
ggtttgcgta ttgggcgctc ttccgcttcc tcgctcactg actcgctgcg ctcggtcgtt 6180
cggctgcggc gagcggtatc agctcactca aaggcggtaa tacggttatc cacagaatca 6240
ggggataacg caggaaagaa catgtgagca aaaggccagc aaaaggccag gaaccgtaaa 6300
aaggccgcgt tgctggcgtt tttccatagg ctccgccccc ctgacgagca tcacaaaaat 6360
cgacgctcaa gtcagaggtg gcgaaacccg acaggactat aaagatacca ggcgtttccc 6420
cctggaagct ccctcgtgcg ctctcctgtt ccgaccctgc cgcttaccgg atacctgtcc 6480
gcctttctcc cttcgggaag cgtggcgctt tctcatagct cacgctgtag gtatctcagt 6540
tcggtgtagg tcgttcgctc caagctgggc tgtgtgcacg aaccccccgt tcagcccgac 6600
cgctgcgcct tatccggtaa ctatcgtctt gagtccaacc cggtaagaca cgacttatcg 6660
ccactggcag cagccactgg taacaggatt agcagagcga ggtatgtagg cggtgctaca 6720
gagttcttga agtggtggcc taactacggc tacactagaa ggacagtatt tggtatctgc 6780
gctctgctga agccagttac cttcggaaaa agagttggta gctcttgatc cggcaaacaa 6840
accaccgctg gtagcggtgg tttttttgtt tgcaagcagc agattacgcg cagaaaaaaa 6900
ggatctcaag aagatccttt gatcttttct acggggtctg acgctcagtg gaacgaaaac 6960
tcacgttaag ggattttggt catgagatta tcaaaaagga tcttcaccta gatcctttta 7020
aattaaaaat gaagttttaa atcaatctaa agtatatatg agtaaacttg gtctgacagt 7080
taccaatgct taatcagtga ggcacctatc tcagcgatct gtctatttcg ttcatccata 7140
gttgcctgac tccccgtcgt gtagataact acgatacggg agggcttacc atctggcccc 7200
agtgctgcaa tgataccgcg agacccacgc tcaccggctc cagatttatc agcaataaac 7260
cagccagccg gaagggccga gcgcagaagt ggtcctgcaa ctttatccgc ctccatccag 7320
tctattaatt gttgccggga agctagagta agtagttcgc cagttaatag tttgcgcaac 7380
gttgttgcca ttgctacagg catcgtggtg tcacgctcgt cgtttggtat ggcttcattc 7440
agctccggtt cccaacgatc aaggcgagtt acatgatccc ccatgttgtg caaaaaagcg 7500
gttagctcct tcggtcctcc gatcgttgtc agaagtaagt tggccgcagt gttatcactc 7560
atggttatgg cagcactgca taattctctt actgtcatgc catccgtaag atgcttttct 7620
gtgactggtg agtactcaac caagtcattc tgagaatagt gtatgcggcg accgagttgc 7680
tcttgcccgg cgtcaatacg ggataatacc gcgccacata gcagaacttt aaaagtgctc 7740
atcattggaa aacgttcttc ggggcgaaaa ctctcaagga tcttaccgct gttgagatcc 7800
agttcgatgt aacccactcg tgcacccaac tgatcttcag catcttttac tttcaccagc 7860
gtttctgggt gagcaaaaac aggaaggcaa aatgccgcaa aaaagggaat aagggcgaca 7920
cggaaatgtt gaatactcat actcttcctt tttcaatatt attgaagcat ttatcagggt 7980
tattgtctca tgagcggata catatttgaa tgtatttaga aaaataaaca aataggggtt 8040
ccgcgcacat ttccccgaaa agtgccacct 8070
<210> 2
<211> 615
<212> DNA
<213> Mus musculus
<400> 2
atgaggacct gggcttgcct gctgctcctc ggctgcggat acctcgccca tgccctggcc 60
gaggaagccg agataccccg ggagttgatc gagcggctgg ctcgaagtca gatccacagc 120
atccgggacc tccagcgact cttggagata gactccgtag gggctgagga tgccttggag 180
acaagtctga gagcccatgg gtcccatgcc attaaccatg tgcccgagaa gcggcctgtg 240
cccattcgca ggaagagaag tattgaggaa gccattcctg cagtttgcaa gaccaggacg 300
gtcatttacg agatacctcg gagccaggtg gaccccacat cggccaactt cctgatctgg 360
cccccatgtg tggaggtgaa gcgctgcact ggctgttgta acaccagcag cgtcaagtgc 420
cagccttcac gggtccacca ccgcagtgtc aaggtggcca aagtggagta tgtcaggaag 480
aagccaaaat tgaaagaggt ccaggtgagg ttagaggaac acctggagtg tgcatgtgcg 540
acctccaacc tgaacccaga ccatcgggag gaggagacag atgtgaggta tccttacgat 600
gtgcctgatt atgca 615
Claims (2)
1.一种小鼠原发神经胶质瘤模型的制备方法，其特征在于，包括下列步骤：
1）PB-CAG-MCS-IRES-EGFP质粒构建，所述PB-CAG-MCS-IRES-EGFP质粒包含启动子与下游的IRES-EGFP序列，在启动子与IRES-EGFP序列之间加入多克隆位点，所述PB-CAG-MCS-IRES-EGFP的核苷酸序列如SEQ ID No.1所示；
2）PB-CAG-Pdgfa-HA-IRES-EGFP质粒的构建，将Pdgfa基因插入到所述PB-CAG-MCS-IRES-EGFP质粒的多克隆位点，表达携带HA标签的PDGFA蛋白与用于示踪的绿色荧光蛋白EGFP；
3）原发胶质瘤小鼠模型的制备，通过将质粒PB-CAG-Pdgfa-HA-IRES-EGFP与表达转座酶的质粒pCAGGS-piggyBac Transposase共同使用，诱导小鼠，获得原发胶质瘤小鼠模型；所述PB-CAG-Pdgfa-HA-IRES-EGFP质粒在piggyBac转座酶存在时被整合入细胞基因组中，实现目标基因在转入细胞及其子代细胞中的稳定表达。
2.一种如权利要求1所述的小鼠原发神经胶质瘤模型制备方法的应用，其特征在于：为神经胶质瘤的发生与治疗研究提供研究模型。
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