CN111607614A - 白喉毒素调控清除免疫细胞的cd45-dtr转基因小鼠的构建方法与应用 - Google Patents
白喉毒素调控清除免疫细胞的cd45-dtr转基因小鼠的构建方法与应用 Download PDFInfo
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Abstract
本发明公开了白喉毒素调控清除免疫细胞的CD45‑DTR转基因小鼠的构建方法与应用,通过酶切线性化BAC Clone CD45‑DTR,利用电穿孔法导入小鼠胚胎干细胞BALB/C ES细胞,得到插入IRES‑DTR至CD45外显子1位点的BALB/C ES细胞,随后将此BALB/C ES细胞通过囊胚注射法构建插入IRES‑DTR至CD45外显子1位点的CD45‑DTR转基因小鼠。本发明提供一种新型可诱导的、选择性清除免疫细胞的小鼠模型的构建方法,可以广泛应用于制备人源化小鼠,研究免疫细胞功能等领域。
Description
技术领域
本发明涉及动物模型构建领域。更具体地,涉及一种白喉毒素调控清除免疫细胞的CD45-DTR转基因小鼠的构建方法。
技术背景
免疫细胞是指参与免疫应答或与免疫应答相关的细胞。包括淋巴细胞、树突状细胞、单核/巨噬细胞、粒细胞、肥大细胞等。免疫细胞可以分为多种,在人体中各种免疫细胞担任着重要的角色,其功能是不能被忽视的。为了研究免疫细胞的功能,一个重要的实验就是在体内清除免疫细胞,所以开发能够特异性清除免疫细胞的动物模型具有重要作用,白喉毒素受体-白喉毒素(DTR-DT)系统是近年来发展起来的一种用于诱导特定细胞群凋亡的系统。白喉毒素(DT)是白喉棒状杆菌产生的一种毒素,包含A和B两个亚基,而白喉毒素受体(DTR)又称人类肝素结合的表皮生长因子样生长因子(hbEGF)。当DT通过B亚基与DTR结合时,整个毒素将通过受体介导的内吞作用内化,毒素的亚单位A随后可抑制蛋白质的翻译,导致细胞凋亡而死亡。目前使用DT来诱导制备免疫细胞清除动物模型方面还存在着传代不稳定,免疫细胞清除效率低,选择性差,正常细胞容易受影响等不足。
发明内容
本发明所要解决的技术问题是为了克服现有的免疫细胞清除动物模型的不足,提供一种新型可诱导的、选择性清除免疫细胞的小鼠模型的构建方法,CD45是一种特异性表达于免疫细胞表面的信号分子,所以我们选择CD45作为驱动DTR表达的基因构建转基因小鼠,这样在免疫细胞中会表达DTR,而不表达的组织中不表达DTR,注射合适剂量的DT就可以选择性地诱导免疫细胞凋亡。
为了实现上述发明目的,本发明提供以下技术方案:
一种白喉毒素调控清除免疫细胞的CD45-DTR转基因小鼠的构建方法,包括以下步骤:
S1.酶切线性化BAC Clone CD45-DTR载体(由本公司构建,具体构建过程见实施例),利用电穿孔法导入小鼠胚胎干细胞BALB/C ES细胞,得到插入IRES-DTR至CD45外显子1位点的BALB/C ES细胞;
S2.利用插入IRES-DTR至CD45外显子1位点的BALB/C ES细胞,囊胚注射法构建插入IRES-DTR至CD45外显子1位点的CD45-DTR转基因小鼠。
进一步的,上述一种白喉毒素调控清除免疫细胞的CD45-DTR转基因小鼠的构建方法,所述步骤S1具体包括以下步骤:
(1)小鼠胚胎成纤维细胞用MEF培养基培养,经30Grayγ射线灭活处理后作为滋养层细胞。BALB/C ES细胞(以下简述为ES细胞)种在滋养层细胞上,用ES培养基培养;
(2)电穿孔法将利用PI-SceI酶切线性化的打靶载体BAC Clone CD45-DTR转入ES细胞,使BAC Clone CD45-DTR整合至基因组中;
(3)利用G418阳选,挑选得到插入BAC Clone CD45-DTR的BALB/C ES细胞克隆;
(4)提取ES细胞基因组DNA为模板,进行PCR鉴定。
进一步的,上述一种白喉毒素调控清除免疫细胞的CD45-DTR转基因小鼠的构建方法,所述步骤S1中的步骤4具体为:
提取ES细胞基因组DNA为模板,利用序列为SEQ ID NO.17的正向引物和序列为SEQID NO.18的反向引物进行PCR鉴定,插入BAC Clone CD45-DTR的ES细胞阳性克隆可见约4.1kb条带。
进一步的,上述一种白喉毒素调控清除免疫细胞的CD45-DTR转基因小鼠的构建方法,所述步骤S2具体包括以下步骤:
(1)插入BAC Clone CD45-DTRBALB/C ES细胞注射入C57BL/6J囊胚,再接种到假孕的KM母鼠子宫中,出生得到毛色为黑色和白色混杂嵌合体小鼠;
(2)提取转基因小鼠鼠尾DNA作为模板,PCR鉴定。
(3)利用白色占比最高的5只雄性转基因嵌合体小鼠F0与BALB/c雌鼠交配,得到毛色为白色的小鼠可能为DC SIGN-DTR转基因杂合小鼠。小白鼠经鼠尾PCR鉴定后得到DCSIGN-DTR转基因杂合小鼠,即为F1代小鼠,鼠尾PCR鉴定方法同步骤2。
(4)F1代小鼠与C57BL/6J母鼠杂交进行传代、培育,同时对所得的子代小鼠进行鼠尾PCR鉴定,鉴定方法同步骤2,得到F2代小鼠。按相同方法对小鼠进行传代。
进一步的,上述一种白喉毒素调控清除免疫细胞的CD45-DTR转基因小鼠的构建方法,所述步骤S2中的步骤2具体为:
提取转基因小鼠鼠尾DNA作为模板,利用序列为SEQ ID NO.19的正向引物和序列为SEQ ID NO.20的反向引物作PCR鉴定,带有IRES-DTR的阳性小鼠可见约1.2kb条带。
进一步的,上述一种白喉毒素调控清除免疫细胞的CD45-DTR转基因小鼠的构建方法在在研究免疫细胞功能方面的应用。
进一步的,上述一种白喉毒素调控清除免疫细胞的CD45-DTR转基因小鼠的构建方法在制备人源化小鼠方面的应用。
进一步的,如上述白喉毒素调控清除免疫细胞的CD45-DTR转基因小鼠的构建方法建立清除免疫细胞小鼠模型的方法,包括以下步骤:在第1、第2、第4、第7天分别腹腔注射DT(20ng/g),特异性清除小鼠免疫细胞,建立清除免疫细胞小鼠模型。
进一步的,对上述建立清除免疫细胞小鼠模型的方法的验证方案,所述验证方案包括以下步骤:获取小鼠组织,制备成单细胞悬液,用Fc blocker封闭Fc受体后,利用CD45、F4/80、CD11b流式抗体染色。
进一步的,对上述建立清除免疫细胞小鼠模型的方法的验证方案,具体包括:获取小鼠表皮,制备成单细胞悬液,用2%FCS-PBS重悬细胞,经Fc blocker封闭Fc受体后,利用anti-mouse-CD45流式抗体染色,染色完毕洗去多余抗体,单细胞悬液用流式细胞仪上机检测。
综合上述技术方案,本发明公开了一种白喉毒素调控清除免疫细胞的CD45-DTR转基因小鼠的构建方法与应用,至少有以下方面的有益效果:
(1)本发明克服了现有免疫细胞清除动物模型的不足,提供了一种能稳定传代的新型可诱导型的、白喉毒素调控清除免疫细胞的CD45-DTR转基因小鼠动物模型的制备、使用和检测方法。
(2)本发明建立了一种高效、安全靶向整合外源基因至小鼠胚胎干细胞基因组CD45外显子1位点的方法,并证实敲入的外源基因表达正常。
(3)本发明提供了筛选与鉴定阳性细胞株/克隆的方法和应用实例。
(4)本发明提供了利用转基因ES细胞生产和培育转基因小鼠的方法和应用实例。
(5)本发明构建得到的白喉毒素调控清除免疫的CD45-DTR转基因小鼠动物模型,免疫细胞清除效率达80%-90%,能够较好地清除免疫细胞,且在不用白喉毒素诱导时,免疫细胞功能不受影响,利用白喉毒素诱导清除免疫细胞时无副反应,提供了一种全新的免疫细胞清除的原理和方法,提供了一种全新的免疫细胞可诱导清除的动物模型,可以用于免疫细胞的研究以及形成人源化小鼠。
附图说明
图1:构建靶向小鼠CD45的打靶质粒流程示意图;
图2:携带抗性基因Neomycin的空载体pL451-targeted-empty的质粒结构示意图;
图3:携带外源基因IRES-DTR、抗性基因Neomycin和CD45两侧同源臂的打靶载体PL451-loxP IRES-DTR CD45 3HA 5HA质粒结构示意图;
图4:打靶载体打靶原理示意图;
图5:PCR筛选与鉴定靶向CD45外显子1位点的DC-SIGN BAC单克隆,正确靶向的单克隆出现约1.4kb条带;
图6:PCR筛选与鉴定插入CD45-DTR外显子1位点的小鼠胚胎干细胞单克隆,正确插入的单克隆细胞出现约1.4kb条带;
图7:利用插入CD45-DTR的ES细胞培育CD45-DTR转基因小鼠流程图;
图8:CD45-DTR转基因小鼠(F1)PCR鉴定结果图;
图9:CD45-DTR转基因小鼠腹腔注射DT特异性清除小鼠免疫细胞的流式细胞术检测敲除效率结果图。
具体实施方式
下面详细描述本发明的实施例,所述实施例的示例在附图中示出,其中自始至终相同或类似的标号表示相同或类似的元件或具有相同或类似功能的元件。下面通过参考附图描述的实施例是示例性的,旨在用于解释本发明,而不能理解为对本发明的限制。
实施例1
BAC Clone CD45-DTR载体的构建。
(1)构建靶向整合外源基因至小鼠CD45外显子1位点的打靶载体,流程如附图1所示:
从genome.ucsc.edu网站检索并下载获得小鼠CD45基因组序列,结合小鼠CD45(Genbank No:NM_001111316.2)序列,确定各外显子和内含子位置及序列。包含整个CD45基因的BAC Clone:RP23-180D23从Children’hospotal Oakland Research Institute购买获得,BAC DNA利用HiPure Plasmid Filter Maxiprep Kit(Invitrogen)制备备用。
PCR扩增得目的基因片段CD45 5’侧同源臂(CD45 5HA):以RP23-180D23 BAC DNA为模板,利用正向引物CD45-5HA-F(KpnI)-ACCACCGGTACCGTGCAAAGTATGCGTTCTTTTCTTTTAG(SEQ ID NO.1),反向引物CD45-5HA-R(SalI)-AACAACGTCGAC ATCTGGAGATCAGCTGTGCCC(SEQID NO.2)作PCR扩增5’侧同源臂5HA(SEQ ID NO.3)。下划线序列为酶切位点。
表1:PCR反应体系
表2:PCR扩增条件:
将目的基因片段CD45 5HA克隆至带阳性选择标记基因的载体pL451-targeted-empty,载体结构如附图2所示:PCR产物经琼脂糖凝胶电泳分离后,分别切出目的条带并利用AxyPrep DNA Gel Extraction Kit(Axygen)回收。带Neomycin阳性选择标记的载体pL451-targeted-empty(counter-Selection BAC Modification Kit,Genebridges)和回收后的CD45 5HA经KpnI、SalI(NEB)双酶切。酶切的PCR产物经AxyPrep PCR Clean-Up Kit(Axygen)纯化回收,酶切的载体经琼脂糖凝胶电泳分离后切出目的条带并利用AxyPrepDNA Gel Extraction Kit(Axygen)回收。利用T4 DNA ligase(NEB)将载体与目的片段连接,接着转化至大肠杆菌DH5α,挑取单个菌落培养,提取质粒DNA,经酶切初步鉴定后送商业公司测序。测序结果显示CD45 5HA插入成功并命名为中间载体pL451-CD45 5HA-PEN。
PCR扩增得目的基因片段IRES和DTR:以pLVX-EF1α-IRES-mCherry质粒(addgene)为模板,利用正向引物IRES-F(SalI)-ACCACCGTCGACGCCCCTCTCCCTCCCCCC(SEQ ID NO.4),反向引物IRES-R-CATGGTTGTGGCAAGCTTATCATCGTG(SEQ ID NO.5)作PCR扩增基因片段IRES(SEQ ID NO.6);以pIRES-proHB EGF WT质粒(addgene)为模板,利用正向引物DTR-F-CACGATGATAAGCTT GCCACAACCATGAAGCTGCTGCCGTCG(SEQ ID NO.7),反向引物DTR-R(EcoRI)-ACCACCGAATTCTTAGTGGGAATTAGTCATGCCC(SEQ ID NO.8)作PCR扩增DTR(SEQ IDNO.9)。下划线序列为酶切位点。PCR反应体系见表1,PCR扩增条件见表2。
PCR产物经琼脂糖凝胶电泳分离后,分别切出目的条带并利用AxyPrep DNA GelExtraction Kit(Axygen)回收。IRES和DTR进行Overlap PCR,合成融合基因片段IRES-DTR(SEQ ID NO.10),具体条件如表3和表4:
表3:Overlap PCR反应体系:
表4:Overlap PCR扩增条::
将目的基因片段IRES-DTR克隆至中间载体pL451-CD45 5HA-PEN:PCR产物经琼脂糖凝胶电泳分离后,分别切出目的条带并利用AxyPrep DNA Gel Extraction Kit(Axygen)回收。纯化后的IRES-DTR和中间载体pL451-DCSIGN 5HA-PEN分别经SalI、EcoRI(NEB)双酶切。酶切的PCR产物经AxyPrep PCR Clean-Up Kit(Axygen)纯化回收,酶切的质粒载体经琼脂糖凝胶电泳分离后切出目的条带并利用AxyPrep DNA Gel Extraction Kit(Axygen)回收。利用T4 DNA ligase(NEB)将载体与目的片段连接,接着转化大肠杆菌DH5α,挑取单个菌落培养,提取质粒DNA,经酶切初步鉴定后送商业公司测序。测序结果显示IRES-DTR插入成功并命名为中间载体pL451-CD45 5HA-IRES-DTR-PEN。
PCR扩增目的基因片段CD45 3’侧同源臂(CD45 3HA):以RP23-180D23 BAC DNA为模板,利用正向引物CD45-3HA-F(BamHI)-ACCACCGGATCCACCATGGGTTTGTGGCTCAAAC(SEQ IDNO.11),反向引物DC-SIGN-3HA-R(NotI)-ACCACCGCGGCCGCATCCATCACACGCAACTTTTAAAAATAG(SEQ ID NO.12)作PCR扩增3’侧同源臂CD45 3HA(SEQ IDNO.13)。下划线序列为酶切位点。PCR反应体系见表1,PCR扩增条件见表2。
将目的基因片段CD45 3HA克隆至中间载体pL451-CD45 5HA-IRES-DTR-PEN:PCR产物经琼脂糖凝胶电泳分离后,分别切出目的条带并利用AxyPrep DNA Gel Extraction Kit(Axygen)回收。纯化后的CD45 3HA和中间载体pL451-CD45 5HA-IRES-DTR-PEN分别经BamHI、(NEB)双酶切。酶切的PCR产物经AxyPrep PCR Clean-Up Kit(Axygen)纯化回收,酶切的质粒载体经琼脂糖凝胶电泳分离后切出目的条带并利用AxyPrep DNA GelExtraction Kit(Axygen)回收。利用T4 DNA ligase(NEB)将载体与目的片段连接,接着转化大肠杆菌DH5α,挑取单个菌落培养,提取质粒DNA,经酶切初步鉴定后送商业公司测序。测序结果显示CD45 3HA插入成功并命名为打靶载体PL451-loxP IRES-DTR CD45 3HA 5HA,载体结构见附图3所示,序列如SEQ ID NO.14所示。至此靶向整合外源基因至小鼠CD45外显子1位点的打靶质粒构建完成。
利用打靶载体靶向整合外源基因至小鼠CD45 BAC:
(2)电穿孔法将pRed/ET转化入包含整个CD45基因的大肠杆菌BAC Clone RP23-180D23:将包含整个CD45基因的大肠杆菌BAC Clone RP23-180D23接种于含氯霉素(15μg/mL)的LB平板,37℃培养16h。挑选1个克隆于有1mL LB培养基(含15μg/mL氯霉素)的2mL离心管(盖子上戳一个洞使能够通气),37℃200rpm培养过夜。取30μL培养过夜的大肠杆菌克隆于有1.4mL LB培养基(含15μg/mL氯霉素)的2mL离心管,37℃1000rpm,培养2-3h扩增得对数期的大肠杆菌。扩增的大肠杆菌2℃11000rpm离心30s,吸弃上清。大肠杆菌沉淀用1mL冰上预冷的ddH20清洗3遍,最后留下约30μL ddH2O重悬大肠杆菌,加入1μL pRed/ET(20ng/μL,counter-Selection BAC Modification Kit(Genebridges,USA)),轻弹混匀,冰上静置。转移大肠杆菌悬液至冰上预冷的1mm电穿孔管,利用Electroporator 2510进行电穿孔,1350V 10μF 600 Ohms,将pRed/ET转化入BAC Clone RP23-180D23。用1mL无抗生素LB培养基重悬大肠杆菌并转移至2mL离心管,30℃1000rpm培养70min使抗性基因表达。
L-阿拉伯糖诱导Red/ET表达:取100μL转入pRed/ET并诱导抗性基因表达的大肠杆菌于含四环素(3μg/mL)、氯霉素(15μg/mL)的LB平板,涂布均匀,30℃避光培养16h。挑取1个单克隆于有1mL LB培养基(含3μg/mL四环素,15μg/mL氯霉素)的2mL盖子戳洞的离心管,30℃200rpm培养过夜。取30μL大肠杆菌于1个有1mL LB培养基(含3μg/mL四环素,15μg/mL氯霉素)的2mL盖子戳洞的离心管,30℃1100rpm培养2h,直到OD600大约0.3。加入50μL L-阿拉伯糖至终浓度为0.3-0.4%,37℃200rpm培养45-60min,诱导Red/ET表达。
电穿孔法将线性化的打靶载体PL451-loxP IRES-DTR CD45 3HA 5HA转化入表达Red/ET的BAC Clone RP23-180D23:表达Red/ET的大肠杆菌进行离心,2℃11000rpm 30s,吸弃上清。大肠杆菌沉淀用1mL冰上预冷的ddH20清洗3遍,最后留下约30μL ddH2O重悬大肠杆菌,加入1-2μl(100-200ng)用NotI(NEB)酶切线性化的打靶载体PL451-loxP IRES-DTRCD45 3HA 5HA,轻弹混匀,冰上静置。转移大肠杆菌悬液至冰上预冷的1mm电穿孔管,利用Electroporator 2510进行电穿孔,1350V 10μF 600 Ohms,将线性化的打靶载体PL451-loxP IRES-DTR CD45 3HA 5HA转化入BAC Clone RP23-180D23。用1mL无抗生素LB培养基重悬大肠杆菌并转移至2mL离心管,30℃1000rpm培养70min使IRES-DTR-PGK-EM7-Neo同源重组插入CD45 BAC,并表达新霉素抗性基因。取100μL大肠杆菌于含四环素(3μg/mL)、氯霉素(15μg/mL)、新霉素(15μg/mL)的LB平板,涂布均匀,30℃培养大于20h,得到插入IRES-DTR-PGK-EM7-Neo的大肠杆菌克隆,命名为BAC clone CD45-DTR。
外源基因靶向整合至小鼠CD45外显子1位点的BAC克隆的筛选与鉴定,整合的示意图如附图4所示。
菌落PCR筛选靶向插入IRES-DTR-PGK-EM7-Neo的大肠杆菌BAC Clone:挑选XX个单克隆分别于有10μl ddH2O的1.5mL离心管,吹打混匀。取1μl菌液作为模板,利用正向引物GHpA-F-TTCTGAGGCGGAAAGAACC(SEQ ID NO.15)和反向引物CD45-R-TATTTTGAACAGAAGCAGTGTATGC(SEQ ID NO.16)作菌落PCR。剩余菌液加入含5mL LB培养基(含15μg/mL氯霉素,15μg/mL新霉素)的15mL离心管,37℃220rpm培养16h,pRed/ET在37℃时会丢失。
菌落PCR反应体系如下:
菌落PCR反应条件如下:
PCR产物经1%琼脂糖凝胶电泳鉴定,靶向插入IRES-DTR-PGK-EM7-Neo至DC-SIGN外显子7位点的大肠杆菌BAC阳性克隆可见约1kb条带,如附图5所示,
PCR鉴定正确的靶向插入外源基因的阳性BAC克隆剩余菌液离心后去上清,用20%甘油的LB培养基重悬,-80℃保存。
实施例2
插入CD45-DTR至小鼠胚胎干(ES)细胞:
小鼠胚胎成纤维细胞(MEFs)培养:MEFs(ATCC)培养和维持于MEF培养基,适时传代和冻存。MEF培养基为DMEM培养基加入10%FBS、100U/ml青链霉素、0.05 mM 2巯基乙醇、2mML-谷氨酰胺。
Balb/c ES细胞培养:MEFs细胞使用前用30Grayγ射线灭活处理作为滋养层细胞,将Balb/c ES细胞(Merck)接种至灭活的MEFs上,用ES培养基培养和维持。ES细胞长至70%丰度以1:3比例传代。ES培养基为DMEM培养基加入15%FBS、100U/ml青链霉素、1mM丙酮酸钠、0.1mM非必需氨基酸、0.05mM 2巯基乙醇、2mM L-谷氨酰胺、1μg/L白血病抑制因子(LIF)。
ES细胞电转:ES细胞经胰酶消化收集后,经PBS清洗1次,用电转缓冲液重悬(Millipore),调整细胞密度至0.5X107/ml。将实施例1中制备的BAC clone CD45-DTR载体经PI-SceI酶切线性化,将50μg线性化的打靶载体加入0.6mL上述ES细胞悬液,,并转移至Eppendorf电转仪配套电转杯中,室温静置5min。利用Electroporator 2510电转仪320V 500μF电转,取出电转杯室温放置5min。转移至含30ml培养液的50ml离心管中混匀,再均匀分配至事先准备好含滋养层细胞的3个10cm培养皿中,37℃5%CO2培养箱中培养,CD45-DTR插入目标基因的原理如附图4所示。
插入BAC clone CD45-DTR的ES细胞的筛选和鉴定:电转后的ES细胞在24小时后,换成含800μg/ml培养液,此后每天更换含药物的培养液。连续药物处理7天后,在尼康SMZ1500显微镜下用特制吸管挑取胚胎干细胞单克隆并转移至事先准备好的含滋养层细胞及培养液的48孔培养板中,每种打靶载体各挑取12个左右单克隆,于37℃5%CO2培养箱中培养。24小时后,用胰酶消化挑取的单克隆细胞并转移至新的事先准备好的含滋养层细胞及培养液的48孔培养板中,于37℃5%CO2培养箱中培养3-4天,每天换液。待单克隆细胞长至80%丰度,细胞经胰酶消化后,将细胞悬液均分转移至事先准备好的含滋养层细胞及培养液的24孔培养板中,并于37℃5%CO2培养箱中培养。待细胞丰度达70%,消化和收集ES细胞,利用QIAamp DNA Mini Kit(Qiagen)提取基因组DNA。以提取的小鼠ES细胞基因组DNA为模板,利用正向引物ES-screen-F-ATTGCATCGCATTGTCTGAG(SEQ ID NO.17)和反向引物
ES-screen-R-CGAGTGGGGACTTACAGTGG(SEQ ID NO.18)进行PCR扩增。
PCR反应体系如下:
PCR扩增条件为:
PCR产物经1%琼脂糖凝胶电泳鉴定,插入BAC clone CD45-DTR的ES细胞阳性克隆可见约1.4kb条带,如附图6所示。阳性克隆ES细胞消化后用胚胎干细胞冻存液(Millipore)重悬,-80℃冻存备用。
实施例3
繁育插入BAC clone CD45-DTR转基因小鼠,流程如附图7所示。
囊胚注射ES细胞获得CD45-DTR转基因嵌合体小鼠:选8周龄发育良好的C57BL/6J公鼠,与C57BL/6J母鼠按1:2合笼,次日清晨挑选阴栓阳性母鼠,若母鼠怀孕5天后可在子宫取得囊胚。将结扎KM公鼠与正常KM母鼠按1:2合笼,选取有阴栓且阴门肿胀、红润的母鼠,即为假孕KM母鼠。将插入CD45-DTR的ES细胞注射到C57BL/6J小鼠囊胚中,再接种到假孕的KM母鼠子宫中,出生得到嵌合体小鼠(F0),即C57来源的细胞和BALB/c来源的ES细胞共同存在于同一个体中,表现为动物毛色为黑色和白色混杂。
提取小鼠鼠尾DNA方法:小鼠鼠尾置于1.5mL离心管,加入300μL NaOH溶液(50mM),金属浴100℃30min。冷却至室温,加入30μL Tris-HCl(1M,pH6.8)混匀调节pH。10000g离心1min,上清即为含小鼠鼠尾DNA的溶液,可直接用于PCR。
鼠尾PCR鉴定转基因嵌合体小鼠:以转基因小鼠鼠尾DNA作为模板,利用正向引物CD45 DTR-screen-F-GGTGGTGCTGAAGCTCTTTC
(SEQ ID NO.19)和反向引物CD45-DTR-screen-R-CCCATGACACCTCTCTCCAT
(SEQ ID NO.20)作PCR扩增,PCR反应体系同实施例2,PCR扩增条件同实施例2。PCR产物经1%琼脂糖凝胶电泳鉴定,插入IRES-DTR的阳性小鼠可见约0.4kb条带,如附图8所示。
繁育CD45-DTR转基因小鼠:白色占比越高的嵌合体小鼠,ES细胞发育成生殖细胞的可能性越大,利用白色占比最高的5只雄性转基因嵌合体小鼠与BALB/c雌鼠交配,得到的CD45-DTR转基因杂合小鼠应为白色。小白鼠经鼠尾PCR鉴定后得到CD45-DTR转基因杂合小鼠,即为F1代小鼠。鼠尾PCR鉴定方法同上。F1代小鼠与C57BL/6J母鼠杂交进行传代,培育。培养方法按照常规小鼠饲养模式进行。
实施例4
CD45-DTR转基因小鼠经白喉毒素诱导建立清除免疫细胞的小鼠模型。
本实施例采用腹腔注射白喉毒素(DT),检测小鼠淋脾脏中免疫细胞的占比来检测免疫的敲除效率。
CD45-DTR转基因小鼠免疫细胞清除:CD45-DTR转基因小鼠在第1、第2、第4、第7天分别腹腔注射DT(20ng/g),小鼠生长状态无明显变化。对照组小鼠在第1、第4天用4.5Grayγ射线照射4小时。第8天用脱颈椎法处死小鼠,取小鼠表皮,用流式细胞术检测淋巴结中免疫细胞情况。
流式细胞术检测脾脏中免疫细胞表达:获取小鼠表皮,制备成单细胞悬液,用2%FCS-PBS重悬细胞,经Fc blocker封闭Fc受体后,利用anti-mouse-CD45流式抗体染色,染色完毕洗去多余抗体,单细胞悬液用CytoFLEX LX Flow Cytometer上机检测。其中CD45代表不同类型免疫细胞。经DT诱导后小鼠免疫细胞敲除如附图9所示,率约达80-90%,证明本发明所述的方案能够很好的清除免疫细胞。
以上所述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,这些改进也应视为本发明的保护范围。
SEQUENCE LISTING
<110> 乾元康安(苏州)生物科技有限公司
<120> 白喉毒素调控清除免疫细胞的CD45-DTR转基因小鼠的构建方法与应用
<130> 2020
<160> 20
<170> PatentIn version 3.3
<210> 1
<211> 40
<212> DNA
<213> 人工
<223> 引物CD45-5HA-F(KpnI)
<400> 1
accaccggta ccgtgcaaag tatgcgttct tttcttttag 40
<210> 2
<211> 33
<212> DNA
<213> 人工
<223> 引物CD45 -5HA-R(SalI)
<400> 2
aacaacgtcg acatctggag atcagctgtg ccc 33
<210> 3
<211> 151
<212> DNA
<213> 人工
<223> 基因片段DCSIGN 5HA
<400> 3
accaccggta ccgtgcaaag tatgcgttct tttcttttag ttttgactta gctttacaga 60
gacaaacttc aagagagata accattattt tgcctttcag ggagacccta tttcttaggg 120
gcacagctga tctccagatg tcgacgttgt t 151
<210> 4
<211> 30
<212> DNA
<213> 人工
<223> 引物IRES-F(SalI)
<400> 4
accaccgtcg acgcccctct ccctcccccc 30
<210> 5
<211> 27
<212> DNA
<213> 人工
<223> 引物IRES-R
<400> 5
catggttgtg gcaagcttat catcgtg 27
<210> 6
<211> 602
<212> DNA
<213> 人工
<223> 基因片段IRES
<400> 6
accaccgtcg agcccctctc cctccccccc ccctaacgtt actggccgaa gccgcttgga 60
ataaggccgg tgtgcgtttg tctatatgtt attttccacc atattgccgt cttttggcaa 120
tgtgagggcc cggaaacctg gccctgtctt cttgacgagc attcctaggg gtctttcccc 180
tctcgccaaa ggaatgcaag gtctgttgaa tgtcgtgaag gaagcagttc ctctggaagc 240
ttcttgaaga caaacaacgt ctgtagcgac cctttgcagg cagcggaacc ccccacctgg 300
cgacaggtgc ctctgcggcc aaaagccacg tgtataagat acacctgcaa aggcggcaca 360
accccagtgc cacgttgtga gttggatagt tgtggaaaga gtcaaatggc tctcctcaag 420
cgtattcaac aaggggctga aggatgccca gaaggtaccc cattgtatgg gatctgatct 480
ggggcctcgg tgcacatgct ttacatgtgt ttagtcgagg ttaaaaaacg tctaggcccc 540
ccgaaccacg gggacgtggt tttcctttga aaaacacgat gataagcttg ccacaaccat 600
ga 602
<210> 7
<211> 42
<212> DNA
<213> 人工
<223> 引物DTR-F
<400> 7
cacgatgata agcttgccac aaccatgaag ctgctgccgt cg 42
<210> 8
<211> 34
<212> DNA
<213> 人工
<223> 引物DTR-R(EcoRI)
<400> 8
accaccgaat tcttagtggg aattagtcat gccc 34
<210> 9
<211> 663
<212> DNA
<213> 人工
<223> 基因片段DTR
<400> 9
cacgatgata agcttgccac aaccatgaag ctgctgccgt cggtggtgct gaagctcttt 60
ctggctgcag ttctctcggc actggtgact ggcgagagcc tggagcggct tcggagaggg 120
ctagctgctg gaaccagcaa cccggaccct cccactgtat ccacggacca gctgctaccc 180
ctaggaggcg gccgggaccg gaaagtccgt gacttgcaag aggcagatct ggaccttttg 240
agagtcactt tatcctccaa gccacaagca ctggccacac caaacaagga ggagcacggg 300
aaaagaaaga agaaaggcaa ggggctaggg aagaagaggg acccatgtct tcggaaatac 360
aaggacttct gcatccatgg agaatgcaaa tatgtgaagg agctccgggc tccctcctgc 420
atctgccacc cgggttacca tggagagagg tgtcatgggc tgagcctccc agtggaaaat 480
cgcttatata cctatgacca cacaaccatc ctggccgtgg tggctgtggt gctgtcatct 540
gtctgtctgc tggtcatcgt ggggcttctc atgtttaggt accataggag aggaggttat 600
gatgtggaaa atgaagagaa agtgaagttg ggcatgacta attcccacta agaattcggt 660
ggt 663
<210> 10
<211> 1238
<212> DNA
<213> 人工
<223> 基因片段IRES-DTR
<400> 10
accaccgtcg acgcccctct ccctcccccc cccctaacgt tactggccga agccgcttgg 60
aataaggccg gtgtgcgttt gtctatatgt tattttccac catattgccg tcttttggca 120
atgtgagggc ccggaaacct ggccctgtct tcttgacgag cattcctagg ggtctttccc 180
ctctcgccaa aggaatgcaa ggtctgttga atgtcgtgaa ggaagcagtt cctctggaag 240
cttcttgaag acaaacaacg tctgtagcga ccctttgcag gcagcggaac cccccacctg 300
gcgacaggtg cctctgcggc caaaagccac gtgtataaga tacacctgca aaggcggcac 360
aaccccagtg ccacgttgtg agttggatag ttgtggaaag agtcaaatgg ctctcctcaa 420
gcgtattcaa caaggggctg aaggatgccc agaaggtacc ccattgtatg ggatctgatc 480
tggggcctcg gtgcacatgc tttacatgtg tttagtcgag gttaaaaaac gtctaggccc 540
cccgaaccac ggggacgtgg ttttcctttg aaaaacacga tgataagctt gccacaacca 600
tgaagctgct gccgtcggtg gtgctgaagc tctttctggc tgcagttctc tcggcactgg 660
tgactggcga gagcctggag cggcttcgga gagggctagc tgctggaacc agcaacccgg 720
accctcccac tgtatccacg gaccagctgc tacccctagg aggcggccgg gaccggaaag 780
tccgtgactt gcaagaggca gatctggacc ttttgagagt cactttatcc tccaagccac 840
aagcactggc cacaccaaac aaggaggagc acgggaaaag aaagaagaaa ggcaaggggc 900
tagggaagaa gagggaccca tgtcttcgga aatacaagga cttctgcatc catggagaat 960
gcaaatatgt gaaggagctc cgggctccct cctgcatctg ccacccgggt taccatggag 1020
agaggtgtca tgggctgagc ctcccagtgg aaaatcgctt atatacctat gaccacacaa 1080
ccatcctggc cgtggtggct gtggtgctgt catctgtctg tctgctggtc atcgtggggc 1140
ttctcatgtt taggtaccat aggagaggag gttatgatgt ggaaaatgaa gagaaagtga 1200
agttgggcat gactaattcc cactaagaat tcggtggt 1238
<210> 11
<211> 34
<212> DNA
<213> 人工
<223> 引物CD45-3HA-F(BamHI)
<400> 11
accaccggat ccaccatggg tttgtggctc aaac 34
<210> 12
<211> 42
<212> DNA
<213> 人工
<223> 引物CD45-3HA-R(NotI)
<400> 12
accaccgcgg ccgcatccat cacacgcaac ttttaaaaat ag 42
<210> 13
<211> 196
<212> DNA
<213> 人工
<223> 基因片段CD45 3HA
<400> 13
accaccggat ccaccatggg tttgtggctc aaacttctgg cctttggatt tgcccttctg 60
gacacagaag tctttgtcac aggtaagcac acccatattc atttttactt ccttgtattt 120
tgttgatttt gtggcaaagg ctaggttgaa atggctattt ttaaaagttg cgtgtgatgg 180
atgcggccgc gttgtt 196
<210> 14
<211> 6307
<212> DNA
<213> 人工
<223> 打靶载体PL451-loxP IRES-DTR CD45 3HA 5HA
<400> 14
ctaaattgta agcgttaata ttttgttaaa attcgcgtta aatttttgtt aaatcagctc 60
attttttaac caataggccg aaatcggcaa aatcccttat aaatcaaaag aatagaccga 120
gatagggttg agtgttgttc cagtttggaa caagagtcca ctattaaaga acgtggactc 180
caacgtcaaa gggcgaaaaa ccgtctatca gggcgatggc ccactacgtg aaccatcacc 240
ctaatcaagt tttttggggt cgaggtgccg taaagcacta aatcggaacc ctaaagggag 300
cccccgattt agagcttgac ggggaaagcc ggcgaacgtg gcgagaaagg aagggaagaa 360
agcgaaagga gcgggcgcta gggcgctggc aagtgtagcg gtcacgctgc gcgtaaccac 420
cacacccgcc gcgcttaatg cgccgctaca gggcgcgtcc cattcgccat tcaggctgcg 480
caactgttgg gaagggcgat cggtgcgggc ctcttcgcta ttacgccagc tggcgaaagg 540
gggatgtgct gcaaggcgat taagttgggt aacgccaggg ttttcccagt cacgacgttg 600
taaaacgacg gccagtgaat tgtaatacga ctcactatag ggcgaattgg gatccgtgca 660
aagtatgcgt tcttttcttt tagttttgac ttagctttac agagacaaac ttcaagagag 720
ataaccatta ttttgccttt cagggagacc ctatttctta ggggcacagc tgatctccag 780
atgtcgacgc ccctctccct cccccccccc taacgttact ggccgaagcc gcttggaata 840
aggccggtgt gcgtttgtct atatgttatt ttccaccata ttgccgtctt ttggcaatgt 900
gagggcccgg aaacctggcc ctgtcttctt gacgagcatt cctaggggtc tttcccctct 960
cgccaaagga atgcaaggtc tgttgaatgt cgtgaaggaa gcagttcctc tggaagcttc 1020
ttgaagacaa acaacgtctg tagcgaccct ttgcaggcag cggaaccccc cacctggcga 1080
caggtgcctc tgcggccaaa agccacgtgt ataagataca cctgcaaagg cggcacaacc 1140
ccagtgccac gttgtgagtt ggatagttgt ggaaagagtc aaatggctct cctcaagcgt 1200
attcaacaag gggctgaagg atgcccagaa ggtaccccat tgtatgggat ctgatctggg 1260
gcctcggtgc acatgcttta catgtgttta gtcgaggtta aaaaacgtct aggccccccg 1320
aaccacgggg acgtggtttt cctttgaaaa acacgatgat aatatggcca caaccatgaa 1380
gctgctgccg tcggtggtgc tgaagctctt tctggctgca gttctctcgg cactggtgac 1440
tggcgagagc ctggagcggc ttcggagagg gctagctgct ggaaccagca acccggaccc 1500
tcccactgta tccacggacc agctgctacc cctaggaggc ggccgggacc ggaaagtccg 1560
tgacttgcaa gaggcagatc tggacctttt gagagtcact ttatcctcca agccacaagc 1620
actggccaca ccaaacaagg aggagcacgg gaaaagaaag aagaaaggca aggggctagg 1680
gaagaagagg gacccatgtc ttcggaaata caaggacttc tgcatccatg gagaatgcaa 1740
atatgtgaag gagctccggg ctccctcctg catctgccac ccgggttacc atggagagag 1800
gtgtcatggg ctgagcctcc cagtggaaaa tcgcttatat acctatgacc acacaaccat 1860
cctggccgtg gtggctgtgg tgctgtcatc tgtctgtctg ctggtcatcg tggggcttct 1920
catgtttagg taccatagga gaggaggtta tgatgtggaa aatgaagaga aagtgaagtt 1980
gggcatgact aattcccact aaacgcgtac cgggcccccc ctcgaggtcg acggtatcga 2040
taagcttgat atcgaattcc gaagttccta ttctctagaa agtataggaa cttcaggtct 2100
gaagaggagt ttacgtccag ccaagctagc ttggctgcag gtcgtcgaaa ttctaccggg 2160
taggggaggc gcttttccca aggcagtctg gagcatgcgc tttagcagcc ccgctgggca 2220
cttggcgcta cacaagtggc ctctggcctc gcacacattc cacatccacc ggtaggcgcc 2280
aaccggctcc gttctttggt ggccccttcg cgccaccttc tactcctccc ctagtcagga 2340
agttcccccc cgccccgcag ctcgcgtcgt gcaggacgtg acaaatggaa gtagcacgtc 2400
tcactagtct cgtgcagatg gacagcaccg ctgagcaatg gaagcgggta ggcctttggg 2460
gcagcggcca atagcagctt tgctccttcg ctttctgggc tcagaggctg ggaaggggtg 2520
ggtccggggg cgggctcagg ggcgggctca ggggcggggc gggcgcccga aggtcctccg 2580
gaggcccggc attctgcacg cttcaaaagc gcacgtctgc cgcgctgttc tcctcttcct 2640
catctccggg cctttcgacc tgcagcctgt tgacaattaa tcatcggcat agtatatcgg 2700
catagtataa tacgacaagg tgaggaacta aaccatggga tcggccattg aacaagatgg 2760
attgcacgca ggttctccgg ccgcttgggt ggagaggcta ttcggctatg actgggcaca 2820
acagacaatc ggctgctctg atgccgccgt gttccggctg tcagcgcagg ggcgcccggt 2880
tctttttgtc aagaccgacc tgtccggtgc cctgaatgaa ctgcaggacg aggcagcgcg 2940
gctatcgtgg ctggccacga cgggcgttcc ttgcgcagct gtgctcgacg ttgtcactga 3000
agcgggaagg gactggctgc tattgggcga agtgccgggg caggatctcc tgtcatctca 3060
ccttgctcct gccgagaaag tatccatcat ggctgatgca atgcggcggc tgcatacgct 3120
tgatccggct acctgcccat tcgaccacca agcgaaacat cgcatcgagc gagcacgtac 3180
tcggatggaa gccggtcttg tcgatcagga tgatctggac gaagagcatc aggggctcgc 3240
gccagccgaa ctgttcgcca ggctcaaggc gcgcatgccc gacggcgagg atctcgtcgt 3300
gacccatggc gatgcctgct tgccgaatat catggtggaa aatggccgct tttctggatt 3360
catcgactgt ggccggctgg gtgtggcgga ccgctatcag gacatagcgt tggctacccg 3420
tgatattgct gaagagcttg gcggcgaatg ggctgaccgc ttcctcgtgc tttacggtat 3480
cgccgctccc gattcgcagc gcatcgcctt ctatcgcctt cttgacgagt tcttctgagg 3540
ggatcaattc tctagagctc gctgatcagc ctcgactgtg ccttctagtt gccagccatc 3600
tgttgtttgc ccctcccccg tgccttcctt gaccctggaa ggtgccactc ccactgtcct 3660
ttcctaataa aatgaggaaa ttgcatcgca ttgtctgagt aggtgtcatt ctattctggg 3720
gggtggggtg gggcaggaca gcaaggggga ggattgggaa gacaatagca ggcatgctgg 3780
ggatgcggtg ggctctatgg cttctgaggc ggaaagaacc agctggggct cgactagagc 3840
ttgcggaacc cttcgaagtt cctattctct agaaagtata ggaacttcat cagtcaggta 3900
cataatggat ctaccatggg tttgtggctc aaacttctgg cctttggatt tgcccttctg 3960
gacacagaag tctttgtcac aggtaagcac acccatattc atttttactt ccttgtattt 4020
tgttgatttt gtggcaaagg ctaggttgaa atggctattt ttaaaagttg cgtgtgatgg 4080
atgcggccgc caccgcggtg gagctccagc ttttgttccc tttagtgagg gttaatttcg 4140
agcttggcgt aatcatggtc atagctgttt cctgtgtgaa attgttatcc gctcacaatt 4200
ccacacaaca tacgagccgg aagcataaag tgtaaagcct ggggtgccta atgagtgagc 4260
taactcacat taattgcgtt gcgctcactg cccgctttcc agtcgggaaa cctgtcgtgc 4320
cagctgcatt aatgaatcgg ccaacgcgcg gggagaggcg gtttgcgtat tgggcgctct 4380
tccgcttcct cgctcactga ctcgctgcgc tcggtcgttc ggctgcggcg agcggtatca 4440
gctcactcaa aggcggtaat acggttatcc acagaatcag gggataacgc aggaaagaac 4500
atgtgagcaa aaggccagca aaaggccagg aaccgtaaaa aggccgcgtt gctggcgttt 4560
ttccataggc tccgcccccc tgacgagcat cacaaaaatc gacgctcaag tcagaggtgg 4620
cgaaacccga caggactata aagataccag gcgtttcccc ctggaagctc cctcgtgcgc 4680
tctcctgttc cgaccctgcc gcttaccgga tacctgtccg cctttctccc ttcgggaagc 4740
gtggcgcttt ctcatagctc acgctgtagg tatctcagtt cggtgtaggt cgttcgctcc 4800
aagctgggct gtgtgcacga accccccgtt cagcccgacc gctgcgcctt atccggtaac 4860
tatcgtcttg agtccaaccc ggtaagacac gacttatcgc cactggcagc agccactggt 4920
aacaggatta gcagagcgag gtatgtaggc ggtgctacag agttcttgaa gtggtggcct 4980
aactacggct acactagaag aacagtattt ggtatctgcg ctctgctgaa gccagttacc 5040
ttcggaaaaa gagttggtag ctcttgatcc ggcaaacaaa ccaccgctgg tagcggtggt 5100
ttttttgttt gcaagcagca gattacgcgc agaaaaaaag gatctcaaga agatcctttg 5160
atcttttcta cggggtctga cgctcagtgg aacgaaaact cacgttaagg gattttggtc 5220
atgagattat caaaaaggat cttcacctag atccttttaa attaaaaatg aagttttaaa 5280
tcaatctaaa gtatatatga gtaaacttgg tctgacagtt accaatgctt aatcagtgag 5340
gcacctatct cagcgatctg tctatttcgt tcatccatag ttgcctgact ccccgtcgtg 5400
tagataacta cgatacggga gggcttacca tctggcccca gtgctgcaat gataccgcga 5460
gacccacgct caccggctcc agatttatca gcaataaacc agccagccgg aagggccgag 5520
cgcagaagtg gtcctgcaac tttatccgcc tccatccagt ctattaattg ttgccgggaa 5580
gctagagtaa gtagttcgcc agttaatagt ttgcgcaacg ttgttgccat tgctacaggc 5640
atcgtggtgt cacgctcgtc gtttggtatg gcttcattca gctccggttc ccaacgatca 5700
aggcgagtta catgatcccc catgttgtgc aaaaaagcgg ttagctcctt cggtcctccg 5760
atcgttgtca gaagtaagtt ggccgcagtg ttatcactca tggttatggc agcactgcat 5820
aattctctta ctgtcatgcc atccgtaaga tgcttttctg tgactggtga gtactcaacc 5880
aagtcattct gagaatagtg tatgcggcga ccgagttgct cttgcccggc gtcaatacgg 5940
gataataccg cgccacatag cagaacttta aaagtgctca tcattggaaa acgttcttcg 6000
gggcgaaaac tctcaaggat cttaccgctg ttgagatcca gttcgatgta acccactcgt 6060
gcacccaact gatcttcagc atcttttact ttcaccagcg tttctgggtg agcaaaaaca 6120
ggaaggcaaa atgccgcaaa aaagggaata agggcgacac ggaaatgttg aatactcata 6180
ctcttccttt ttcaatatta ttgaagcatt tatcagggtt attgtctcat gagcggatac 6240
atatttgaat gtatttagaa aaataaacaa ataggggttc cgcgcacatt tccccgaaaa 6300
gtgccac 6307
<210> 15
<211> 25
<212> DNA
<213> 人工
<223> 引物GHpA-F
<400> 15
tattttgaac agaagcagtg tatgc 25
<210> 16
<211> 25
<212> DNA
<213> 人工
<223> 引物DCSIGN-R
<400> 16
tattttgaac agaagcagtg tatgc 25
<210> 17
<211> 20
<212> DNA
<213> 人工
<223> 引物ES-screen-F
<400> 17
attgcatcgc attgtctgag 20
<210> 18
<211> 20
<212> DNA
<213> 人工
<223> 引物ES-screen-R
<400> 18
cgagtgggga cttacagtgg 20
<210> 19
<211> 20
<212> DNA
<213> 人工
<223> 引物CD45 DTR-screen-F
<400> 19
ggtggtgctg aagctctttc 20
<210> 20
<211> 20
<212> DNA
<213> 人工
<223> 引物CD45-DTR-screen-R
<400> 20
cccatgacac ctctctccat 20
Claims (10)
1.一种白喉毒素调控清除免疫细胞的CD45-DTR转基因小鼠的构建方法,包括以下步骤:
S1. 酶切线性化BAC Clone CD45-DTR,利用电穿孔法导入小鼠胚胎干细胞BALB/C ES细胞,得到插入IRES-DTR至CD45外显子1位点的BALB/C ES细胞;
S2. 利用插入IRES-DTR至CD45外显子1位点的BALB/C ES细胞,囊胚注射法构建插入IRES-DTR至CD45外显子1位点的CD45-DTR转基因小鼠。
2.根据权利要求1所述的一种白喉毒素调控清除免疫细胞的CD45-DTR转基因小鼠的构建方法,其特征在于,所述步骤S1具体包括以下步骤:
(1) 小鼠胚胎成纤维细胞用MEF培养基培养,经30Grayγ射线灭活处理后作为滋养层细胞,BALB/C ES细胞(以下简述为ES细胞)种在滋养层细胞上,用ES培养基培养;
(2) 电穿孔法将利用PI-SceI酶切线性化的打靶载体BAC Clone CD45-DTR转入ES细胞,使BAC Clone CD45-DTR整合至基因组中;
(3) 利用G418阳选,挑选得到插入BAC Clone CD45-DTR的BALB/C ES细胞克隆;
(4) 提取ES细胞基因组DNA为模板,进行PCR鉴定。
3.根据权利要求2所述的一种白喉毒素调控清除免疫细胞的CD45-DTR转基因小鼠的构建方法,其特征在于,所述步骤4具体为:
提取ES细胞基因组DNA为模板,利用序列为SEQ ID NO.17的正向引物和序列为SEQ IDNO.18的反向引物进行PCR鉴定,插入BAC Clone CD45-DTR的ES细胞阳性克隆可见约4.1kb条带。
4.根据权利要求1所述的一种白喉毒素调控清除免疫细胞的CD45-DTR转基因小鼠的构建方法,其特征在于,所述步骤S2具体包括以下步骤:
(1) 插入BAC Clone CD45-DTRBALB/C ES细胞注射入C57BL/6J囊胚,再接种到假孕的KM母鼠子宫中,出生得到毛色为黑色和白色混杂嵌合体小鼠;
(2) 提取转基因小鼠鼠尾DNA作为模板,PCR鉴定;
(3) 利用白色占比最高的5只雄性转基因嵌合体小鼠F0与BALB/c雌鼠交配,得到毛色为白色的小鼠可能为DC SIGN-DTR转基因杂合小鼠,小白鼠经鼠尾PCR鉴定后得到DC SIGN-DTR转基因杂合小鼠,即为F1代小鼠,鼠尾PCR鉴定方法同步骤2;
(4) F1代小鼠与C57BL/6J母鼠杂交进行传代、培育,同时对所得的子代小鼠进行鼠尾PCR 鉴定,鉴定方法同步骤2,得到F2代小鼠,按相同方法对小鼠进行传代。
5.根据权利要求4所述的一种白喉毒素调控清除免疫细胞的CD45-DTR转基因小鼠的构建方法,其特征在于,所述步骤2具体为:
提取转基因小鼠鼠尾DNA作为模板,利用序列为SEQ ID NO.19的正向引物和序列为SEQID NO.20的反向引物作PCR鉴定,带有IRES-DTR的阳性小鼠可见约1.2kb条带。
6.如权利要求1-5任一项所述的白喉毒素调控清除免疫细胞的CD45-DTR转基因小鼠的构建方法在研究免疫细胞功能方面的应用。
7.如权利要求1-5任一项所述的白喉毒素调控清除免疫细胞的CD45-DTR转基因小鼠的构建方法在制备人源化小鼠方面的应用。
8.如权利要求1-5任一项所述的白喉毒素调控清除免疫细胞的CD45-DTR转基因小鼠的构建方法建立清除免疫细胞小鼠模型的方法,包括以下步骤:在第1、第2、第4、第7天分别腹腔注射DT(20ng/g),特异性清除小鼠免疫细胞,建立清除免疫细胞小鼠模型。
9.一种对权利要求8所述的建立清除免疫细胞小鼠模型的方法的验证方案,其特征在于,所述验证方案包括以下步骤:获取小鼠组织,制备成单细胞悬液,用Fc blocker 封闭Fc受体后,利用CD45、F4/80、CD11b流式抗体染色。
10.根据权利要求9所述的建立清除免疫细胞小鼠模型的方法的验证方案,其特征在于,具体包括:获取小鼠表皮,制备成单细胞悬液,用2%FCS-PBS重悬细胞,经Fc blocker封闭Fc受体后,利用anti-mouse-CD45流式抗体染色,染色完毕洗去多余抗体,单细胞悬液用流式细胞仪上机检测。
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Cited By (3)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN110938652A (zh) * | 2019-11-07 | 2020-03-31 | 浙江大学医学院附属第一医院 | 打靶载体及构建白喉毒素调控清除巨噬细胞的f4/80-dtr转基因小鼠的方法和应用 |
CN113604473A (zh) * | 2021-10-09 | 2021-11-05 | 广东南模生物科技有限公司 | 一种可诱导自然杀伤细胞缺陷小鼠模型的构建方法及应用 |
CN114214346A (zh) * | 2021-11-04 | 2022-03-22 | 中国人民解放军海军军医大学第三附属医院 | 靶向肝前体细胞的质粒系统及应用 |
Citations (2)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN110938652A (zh) * | 2019-11-07 | 2020-03-31 | 浙江大学医学院附属第一医院 | 打靶载体及构建白喉毒素调控清除巨噬细胞的f4/80-dtr转基因小鼠的方法和应用 |
CN111088280A (zh) * | 2019-12-31 | 2020-05-01 | 浙江大学医学院附属第一医院 | 打靶载体及白喉毒素调控清除单核细胞衍生树突状细胞的转基因小鼠的构建方法和应用 |
-
2020
- 2020-05-22 CN CN202010438512.1A patent/CN111607614A/zh active Pending
Patent Citations (2)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN110938652A (zh) * | 2019-11-07 | 2020-03-31 | 浙江大学医学院附属第一医院 | 打靶载体及构建白喉毒素调控清除巨噬细胞的f4/80-dtr转基因小鼠的方法和应用 |
CN111088280A (zh) * | 2019-12-31 | 2020-05-01 | 浙江大学医学院附属第一医院 | 打靶载体及白喉毒素调控清除单核细胞衍生树突状细胞的转基因小鼠的构建方法和应用 |
Non-Patent Citations (2)
Title |
---|
MANICKAM ARUN KUMAR: "Development of a new mouse host for (syngeneic, allogeneic and xenogeneic) hematopoietic cell transplantations without lethal irradiation as preconditioning" * |
OVERHOLT等: "Identification of a murine CD45-F4/80lo HSC-derived marrow endosteal cell associated with donor stem cell engraftment" * |
Cited By (4)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN110938652A (zh) * | 2019-11-07 | 2020-03-31 | 浙江大学医学院附属第一医院 | 打靶载体及构建白喉毒素调控清除巨噬细胞的f4/80-dtr转基因小鼠的方法和应用 |
CN113604473A (zh) * | 2021-10-09 | 2021-11-05 | 广东南模生物科技有限公司 | 一种可诱导自然杀伤细胞缺陷小鼠模型的构建方法及应用 |
CN114214346A (zh) * | 2021-11-04 | 2022-03-22 | 中国人民解放军海军军医大学第三附属医院 | 靶向肝前体细胞的质粒系统及应用 |
CN114214346B (zh) * | 2021-11-04 | 2024-06-11 | 中国人民解放军海军军医大学第三附属医院 | 靶向肝前体细胞的质粒系统及应用 |
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