CN110938652A - 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 - Google Patents
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 Download PDFInfo
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Abstract
本发明涉及动物模型构建领域，具体公开了一种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，所述靶向打靶载体序列如SEQ ID NO.8所示，命名为F4/80 5HA L‑IRES‑DTR‑PGK‑EM7‑NEO‑F4/80 3HA L‑MCL‑HSV TK载体。胞生产和培育转基因小鼠的方法和应用实例。本发明构建得到的白喉毒素调控清除组织固有巨噬细胞的F4/80‑DTR转基因小鼠动物模型组织固有巨噬细胞清除效率达70％‑90％，能够较好地清除组织固有巨噬细胞，且在不用白喉毒素诱导时巨噬细胞功能不受影响，利用白喉毒素诱导清除巨噬细胞时无副反应，提供了一种全新的组织固有巨噬细胞清除的原理和方法，提供了一种全新的组织固有巨噬细胞可诱导清除的动物模型。
Description
技术领域
本发明涉及动物模型构建领域，主要是一种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。
背景技术
单核巨噬细胞系统包括骨髓中的前单核细胞，外周血中的单核细胞和组织中的巨噬细胞。近年来研究发现，大部分组织中的巨噬细胞是组织固有的，而且早在胚胎发育时期就形成了，并且在组织中自我更新和维持稳态，而与成年后外周血中单核细胞无关。不同组织中的巨噬细胞可以有组织特异性的功能，如脑中的巨噬细胞(即小胶质细胞)能够在发育过程中修剪突触，脾红髓巨噬细胞能够吞噬红细胞回收血红素，腹腔巨噬细胞通过与腹膜B细胞相互作用调节肠道免疫球蛋白IgA的产生。在癌症的发生发展中，肿瘤相关巨噬细胞也发挥了重要作用。因此，研究组织中的组织固有巨噬细胞的功能具有重要意义。
研究巨噬细胞，一个很重要的实验就是在体内清除巨噬细胞。现有的较为成熟的巨噬细胞清除体系主要有氯化钆和氯膦酸二钠脂质体法，其主要原理是巨噬细胞吞噬氯化钆或氯膦酸二钠脂质体，经溶酶体水解释放钆离子或氯膦酸根离子，并对巨噬细胞产生毒性作用和诱导凋亡。然而这些方法依赖于巨噬细胞的吞噬能力，吞噬能力较弱的巨噬细胞无法被清除，并且清除巨噬细胞的特异性不够强，一些拥有较强吞噬能力的非单核巨噬细胞，如中性粒细胞，也会吞噬氯化钆或氯膦酸二钠脂质体，从而功能受到影响，并可能产生副反应。为了研究组织中的组织固有巨噬细胞的功能，开发能够在体内特异性清除组织中组织固有巨噬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。啮齿类动物的hbEGF因为3个氨基酸的变化，对DT的亲和力比人类低103-105倍，所以给小鼠注射DT可以特异性诱导表达DTR(即人类hbEGF)的细胞群体凋亡，从而特异性清除该细胞群体，并且无明显副反应。
F4/80又被称为含表皮生长因子样黏液素样激素受体样1(EMR1)，它广泛表达于许多髓系细胞，包括单核细胞、巨噬细胞，但不包括中性粒细胞。并且敲除F4/80不影响巨噬细胞的存活和功能。最近，有研究表明组织固有巨噬细胞通常表达更高的F4/80水平，所以我们选择F4/80作为驱动DTR表达的基因构建转基因小鼠，这样在高表达F4/80的组织固有巨噬细胞中会高表达DTR，而低表达或不表达F4/80的其他细胞中低表达或不表达DTR，注射合适剂量的DT就可以选择性地诱导组织固有巨噬细胞凋亡。
发明内容
本发明的目的在于克服现有技术存在的不足，而提供一种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。
本发明的目的是通过如下技术方案来完成的。一种靶向打靶载体，所述靶向打靶载体序列如SEQ ID NO.8所示，命名为F4/80 5HA L-IRES-DTR-PGK-EM7-NEO-F4/80 3HA L-MCL-HSV TK载体。
本发明提供了一种用于构建靶向打靶载体的提取载体，所述提取载体序列如SEQID NO.7所示，命名为载体retrieval 5HA-retrieval 3HA-MCL-HSV TK。
本发明提供的一种提取载体的制备方法，所述方法包括如下步骤：
(1)以RP23-212F14 BAC DNA为模板，利用序列如SEQ ID NO.1所示的正向引物和序列如SEQ ID NO.2所示的反向引物进行PCR，扩增出序列如SEQ ID NO.3所示的基因片段F4/80 5’侧同源臂；以RP23-212F14 BAC DNA为模板，利用序列如SEQ ID NO.4所示的正向引物和序列如SEQ ID NO.5所示的反向引物进行PCR，扩增出序列如SEQ ID NO.6所示的基因片段F4/80 3’侧同源臂；
(2)将retrieval 5HA和retrieval 3HA克隆至带阴性选择标记的载体MCL-HSVTK，得到序列如SEQ ID NO.7所示的打靶载体retrieval 5HA-retrieval 3HA-MCL-HSV TK。
本发明提供了一种靶向打靶载体的制备方法，所述方法包括如下步骤：
(1)电穿孔法将质粒pRED/ET转化入BAC Clone F4/80-IRES-DTR-PGK-EM7-Neo，并用L-阿拉伯糖诱导Red/ET表达；
(2)电穿孔法将利用SpeI酶切线性化的retrieval 5HA-retrieval 3HA-MCL-HSVTK提取载体转化入上述表达RED/ET的BAC Clone F4/80-IRES-DTR-PGK-EM7-Neo，同源重组得到序列如SEQ ID NO.8所示的带有F4/80 5’同源臂(～20kb)和3’同源臂(～2kb)的打靶载体，命名为打靶载体F4/80 5HA L-IRES-DTR-PGK-EM7-NEO-F4/80 3HA L-MCL-HSV TK；
(3)挑选单克隆，利用序列如SEQ ID NO.9所示的正向引物和序列如SEQ ID NO.10所示的反向引物作菌落PCR，成功提取IRES-DTR-PGK-EM7-NEO和F4/80同源臂的载体能够得到2341bp的条带。
本发明同时提供一种靶向打靶载体在靶向整合外源基因IRES-DTR至F4/80外显子22位点，构建白喉毒素调控清除组织固有巨噬细胞的F4/80-DTR转基因小鼠的应用。
本发明提供了所述转基因小鼠的构建方法包括以下步骤：
(1)构建靶向整合外源基因IRES-DTR至F4/80外显子22位点构建小鼠胚胎干细胞株；
(1.1)电穿孔法将利用BamHI酶切线性化的打靶载体F4/80 5HA L-IRES-DTR-PGK-EM7-NEO-F4/80 3HA L-MCL-HSV TK转入ES细胞；
(1.2)利用G418阳选和ganciclovir阴选，筛选选得到靶向插入IRES-DTR-PGK-EM7-NEO至F4/80外显子22位点的Balb/c ES细胞克隆；
(1.3)提取ES细胞基因组DNA为模板，利用序列为SEQ ID NO.11的正向引物和序列为SEQ ID NO.28的反向引物进行PCR鉴定，靶向插入IRES-DTR-PGK-EM7-NEO至F4/80外显子12位点的ES细胞阳性克隆可见约2.2kb条带；
(2)靶向插入IRES-DTR至F4/80外显子22位点的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；
(3)提取转基因小鼠鼠尾DNA作为模板，利用序列为SEQ ID NO.27的正向引物和序列为SEQ ID NO.28的反向引物作PCR鉴定，带有IRES-DTR的阳性小鼠可见约2.2kb条带；
(4)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，得到毛色为白色的小鼠可能为F4/80-DTR转基因杂合小鼠。小白鼠经鼠尾PCR鉴定后得到F4/80-DTR转基因小鼠。
本发明的有益效果为：本发明克服了现有巨噬细胞清除体系存在的局限和不足，提供了一种能稳定传代的新型可诱导型的、白喉毒素调控清除组织固有巨噬细胞的F4/80-DTR转基因小鼠动物模型的制备、使用和检测方法。
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F4/80外显子22位点的方法，并证实敲入的外源基因表达正常。
本发明提供了靶向小鼠F4/80外显子22位点的打靶载体左、右同源臂DNA序列信息及构建方法。
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。
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。
本发明构建得到的白喉毒素调控清除组织固有巨噬细胞的F4/80-DTR转基因小鼠动物模型组织固有巨噬细胞清除效率达70％-90％，能够较好地清除组织固有巨噬细胞，且在不用白喉毒素诱导时巨噬细胞功能不受影响，利用白喉毒素诱导清除巨噬细胞时无副反应，提供了一种全新的组织固有巨噬细胞清除的原理和方法，提供了一种全新的组织固有巨噬细胞可诱导清除的动物模型。
附图说明
图1：外显子22位点插入IRES-DTR-PGK-EM7-Neo的BAC CloneF4/80-IRES-DTR-PGK-EM7-Neo的F4/80基因部分结构示意图，黑色片段代表外显子位点；
图2：带长同源臂的靶向F4/80基因外显子22位点的打靶载体的构建，及同源重组从F4/80BAC提取长同源臂和目的基因后进行PCR筛选与鉴定的引物(黑色箭头)示意图；
图3：靶向整合外源基因IRES-DTR至小鼠胚胎干细胞F4/80外显子22位点，及PCR筛选与鉴定阳性克隆的引物(黑色箭头)示意图；
图4：PCR筛选与鉴定靶向F4/80外显子22位点的小鼠胚胎干细胞单克隆，正确靶向的单克隆细胞出现约2.2kb条带；
图5：利用靶向插入IRES-DTR至F4/80外显子22位点的ES细胞培育F4/80-DTR转基因小鼠流程图；
图6：F4/80-DTR转基因小鼠(F1)PCR鉴定结果图；
图7：F4/80-DTR转基因小鼠腹腔注射DT特异性清除小鼠组织固有巨噬细胞，及流式细胞术检测小鼠脑中小胶质细胞敲除效率结果图。
具体实施方式
下面将结合附图对本发明做详细的介绍：
本发明所要解决的技术问题是为了克服现有的巨噬细胞清除动物模型存在的缺陷，提供一种新型可诱导的、选择性清除组织固有巨噬细胞的小鼠模型的构建方法。
本发明的目的是提供一种靶向整合外源基因IRES-DTR至F4/80外显子22位点，构建白喉毒素调控清除组织固有巨噬细胞的F4/80-DTR转基因小鼠的方法和应用。
本发明上述目的通过以下技术方案予以实现：
S1.构建带长同源臂的靶向整合外源基因IRES-DTR至F4/80外显子22位点的打靶载体，命名为打靶载体F4/80 5HA L-IRES-DTR-PGK-EM7-NEO-F4/80 3HA L-MCL-HSV TK；
S2.电穿孔法将酶切线性化的打靶载体导入小鼠胚胎干细胞Balb/c ES细胞，得到靶向插入IRES-DTR至F4/80外显子22位点的Balb/c ES细胞；
S3.利用靶向插入IRES-DTR至F4/80外显子22位点的Balb/c ES细胞，囊胚注射法构建靶向插入IRES-DTR至F4/80外显子22位点的F4/80-DTR转基因小鼠；
其中S1步骤所述构建带长同源臂的靶向整合外源基因IRES-DTR至F4/80外显子22位点的打靶载体的具体方法如下：
(1)以RP23-212F14 BAC DNA为模板，利用序列如SEQ ID NO.1所示的正向引物和序列如SEQ ID NO.2所示的反向引物进行PCR，扩增出序列如SEQ ID NO.3所示的基因片段F4/80 5’侧同源臂(retrieval5HA)；以RP23-212F14 BAC DNA为模板，利用序列如SEQ IDNO.4所示的正向引物和序列如SEQ ID NO.5所示的反向引物进行PCR，扩增出序列如SEQ IDNO.6所示的基因片段F4/80 3’侧同源臂(retrieval3HA)；
(2)将retrieval 5HA和retrieval 3HA利用酶切位点NotI、SpeI、BamHI克隆至带阴性选择标记的载体MCL-HSV TK(counter-Selection BAC Modification Kit,Genebridges)，得到序列如SEQ ID NO.7所示的提取载体retrieval 5HA-retrieval 3HA-MCL-HSV TK；
(3)电穿孔法将质粒pRED/ET转化入BAC Clone F4/80-IRES-DTR-PGK-EM7-Neo(由本实验室构建，正在申报专利，部分基因结构如附图1所示)，并用L-阿拉伯糖诱导Red/ET表达；
(4)电穿孔法将利用SpeI酶切线性化的retrieval 5HA-retrieval 3HA-MCL-HSVTK提取载体转化入上述表达RED/ET的BAC Clone F4/80-IRES-DTR-PGK-EM7-Neo，同源重组得到序列如SEQ ID NO.8所示的带有F4/80 5’同源臂(～20kb)和3’同源臂(～2kb)的打靶载体，命名为打靶载体F4/80 5HA L-IRES-DTR-PGK-EM7-NEO-F4/80 3HAL-MCL-HSV TK；
(5)挑选单克隆，利用序列如SEQ ID NO.9所示的正向引物和序列如SEQ ID NO.10所示的反向引物作菌落PCR，成功提取IRES-DTR-PGK-EM7-NEO和F4/80同源臂的载体能够得到2341bp的条带。
至此，我们得到打靶载体F4/80 5HA L-IRES-DTR-PGK-EM7-NEO-F4/80 3HAL-MCL-HSV TK。
上述打靶载体可用于靶向整合外源基因IRES-DTR至F4/80外显子22位点，如小鼠胚胎干(ES)细胞F4/80外显子22位点。
本发明还提供利用上述打靶载体靶向整合外源基因IRES-DTR至小鼠胚胎干细胞F4/80外显子22位点的方法，即步骤S2。具体步骤如下：
(1)小鼠胚胎成纤维细胞(MEFs)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BamHI酶切线性化的打靶载体F4/80 5HA L-IRES-DTR-PGK-EM7-NEO-F4/803HA L-MCL-HSV TK转入ES细胞，使IRES-DTR-PGK-EM7-NEO同源重组至F4/80外显子22位点；
(3)利用G418阳选和ganciclovir阴选，挑选得到靶向插入IRES-DTR-PGK-EM7-NEO至F4/80外显子22位点的Balb/c ES细胞克隆；
(4)提取ES细胞基因组DNA为模板，利用序列为SEQ ID NO.11的正向引物和序列为SEQ ID NO.12的反向引物进行PCR鉴定，靶向插入IRES-DTR-PGK-EM7-NEO至F4/80外显子22位点的ES细胞阳性克隆可见约2.2kb条带。
至此，我们得到靶向插入IRES-DTR至F4/80外显子22位点的Balb/c ES细胞。
上述靶向插入IRES-DTR至F4/80外显子22位点的ES细胞可用于培育F4/80-DTR转基因小鼠。
本发明还提供利用上述靶向插入IRES-DTR至F4/80外显子22位点的ES细胞培育F4/80-DTR转基因小鼠的方法，即步骤S3。具体步骤如下：
(1)靶向插入IRES-DTR至F4/80外显子22位点的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，利用序列为SEQ ID NO.11的正向引物和序列为SEQ ID NO.12的反向引物作PCR鉴定，带有IRES-DTR的阳性小鼠可见约2.2kb条带；
(3)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，得到毛色为白色的小鼠可能为F4/80-DTR转基因杂合小鼠，即F0代小鼠。小白鼠经鼠尾PCR鉴定后得到F4/80-DTR转基因杂合小鼠，即为F1代小鼠。
(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，得到F2代小鼠。按相同方法对小鼠进行传代。
至此，我们得到F4/80-DTR转基因小鼠。
上述F4/80-DTR转基因小鼠可用白喉毒素诱导，建立清除组织固有巨噬细胞的小鼠模型。
本发明还提供利用上述F4/80-DTR转基因小鼠，经白喉毒素诱导建立清除组织固有巨噬细胞的小鼠模型的方法。
白喉毒素调控清除组织固有巨噬细胞的F4/80-DTR转基因小鼠模型使用方法：因组织固有巨噬细胞高表达F4/80，故F4/80-DTR转基因小鼠的巨噬细胞也高表达DTR，白喉毒素(DT)能够结合DTR，特异性诱导巨噬细胞凋亡，从而清除巨噬细胞。优选地，在第1、第2、第4、第7天分别腹腔注射DT(20ng/g)，特异性清除小鼠组织固有巨噬细胞。
本发明还提供利用流式细胞术检测小鼠组织中巨噬细胞敲除效率的方法。
利用流式细胞术检测小鼠组织中巨噬细胞：获取小鼠组织，制备成单细胞悬液，经36％percoll密度梯度离心去除脂肪组织，经ACK lysis buffer去除红细胞，用Fc blocker封闭Fc受体后，利用CD45、MHCII流式抗体染色，其中CD45中表达，MHCII表达的细胞为巨噬细胞。
至此，本发明提供了白喉毒素调控清除组织固有巨噬细胞的F4/80-DTR转基因小鼠制备、使用和检测方法。
实施例1：构建带长同源臂的靶向整合外源基因IRES-DTR至F4/80外显子22位点的打靶载体
从genome.ucsc.edu网站检索并下载获得小鼠F4/80基因组序列，结合小鼠F4/80(Genbank No:NM_010130.4)序列，确定各外显子和内含子位置及序列。包含整个F4/80基因的BAC Clone:RP23-212F14从Chieldren’hospotal Oakland Research Institute购买获得，BAC DNA利用
HiPure Plasmid Filter Maxiprep Kit(Invitrogen)制备备用。
PCR扩增目的基因片段F4/80 5’侧同源臂(retrieval 5HA)和F4/80 3’侧同源臂(retrieval 3HA)：以RP23-212F14 BAC DNA为模板，利用正向引物F4/80-180659-F(NotI)-ACCACCGCGGCCGCGACAGCCAAATATTGGCAT(SEQ ID NO.1)和反向引物F4/80-180900-R(SpeI)-ACCACCACTAGTTACCTGCA AACCCCCAATAA(SEQ ID NO.2)作PCR扩增F4/80 5’侧同源臂(retrieval 5HA)(SEQ ID NO.3)；以RP23-212F14 BAC DNA为模板，利用正向引物F4/80-192354-F(SpeI)-ACCACCACTAGTGCACTGAGAATGGTGCTGTGT(SEQ ID NO.4)和反向引物F4/80-192569-R(BamHI)-ACCACCGGATCCGCATAGACTCTTGATGGTGCATATT(SEQ ID NO.5)作PCR扩增F4/80 3’侧同源臂(retrieval 3HA)(SEQ ID NO.6)。下划线序列为酶切位点。
PCR反应体系如下：
PCR扩增条件如下：
将retrieval 5HA和retrieval 3HA克隆至带阴性选择标记基因的载体MCL-HSVTK：retrieval 5HA用NotI、SpeI(NEB)双酶切，retrieval 3HA用SpeI、BamHI(NEB)双酶切，带阴性选择标记基因的载体MCL-HSV TK(counter-Selection BAC Modification Kit(Genebridges,USA))用NotI、BamHI(NEB)双酶切。酶切的PCR产物经AxyPrep PCR Clean-UpKit(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retrieval 5HA和retrieval 3HA插入成功并命名为提取载体retrieval 5HA-retrieval3HA-MCL-HSV TK，载体序列如SEQ ID NO.7所示。
电穿孔法将质粒pRed/ET转化入BAC Clone F4/80-IRES-DTR-PGK-EM7-Neo：BACClone F4/80-IRES-DTR-PGK-EM7-Neo(本实验室构建，正在申报专利)是将外源基因IRES-DTR-PGK-EM7-Neo靶向插入包含整个小鼠F4/80基因的BAC CloneRP23-212F14得到，部分基因结构如附图1所示。将BAC Clone F4/80-IRES-DTR-PGK-EM7-Neo接种于含氯霉素(15μg/mL)、新霉素(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,15μg/mL新霉素)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,15μg/mL新霉素)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 BACModification 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 10μF600Ohms，将pRed/ET转化入BAC Clone F4/80-IRES-DTR-PGK-EM7-Neo。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)、新霉素(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提取载体retrieval 5HA-retrieval 3HA-Mcl-HSV TK转化入带pRED/ET的F4/80-IRES-DTR-EM7-PGK-NEO BAC Clone：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SpeI(NEB)酶切线性化的提取载体retrieval 5HA-retrieval 3HA-MCL-HSV TK，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 10μF600Ohms，将线性化的提取载体retrieval 5HA-retrieval 3HA-MCL-HSV TK转化入BACClone F4/80-IRES-DTR-EM7-PGK-NEO。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及两侧长同源臂被同源重组提取至retrieval 5HA-retrieval 3HA-MCL-HSV TK，得到带有F4/80 5’同源臂(～20kb)和3’同源臂(～2kb)的打靶载体，命名为F4/80 5HA L-IRES-DTR-PGK-EM7-NEO-F4/80 3HA L-MCL-HSV TK，序列如SEQ ID NO.8所示。取100μL大肠杆菌于含氯霉素(15μg/mL)的LB平板，涂布均匀，37℃培养过夜，pRed/ET在37℃时会丢失。
筛选和鉴定得到的含长同源臂的打靶载体F4/80 5HA L-IRES-DTR-PGK-EM7-NEO-F4/80 3HA L-MCL-HSV TK：挑选10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Kana-193782-F-GAAAATGGCCGCTTTTCTG(SEQ IDNO.9)和反向引物F480-195458-R-CACAGCACCA TTCTCAGTGC(SEQ ID NO.10)作菌落PCR，剩余菌液4℃保存备用。
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，成功提取IRES-DTR-PGK-EM7-NEO及两侧长同源臂的大肠杆菌BAC阳性克隆可见约2.3kb条带。阳性克隆用含氯霉素(15μg/mL)的LB培养基扩增，用
HiPure Plasmid Filter Maxiprep Kit(Invitrogen)制备F4/80 5HAL-IRES-DTR-PGK-EM7-NEO-F4/80 3HA L-MCL-HSV TK备用。阳性BAC克隆菌液可离心后去上清，用20％甘油的LB培养基重悬，-80℃保存。
实施例2：制备靶向插入IRES-DTR至F4/80外显子22位点的小鼠胚胎干(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、0.05mM 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。打靶载体F4/80 5HA L-IRES-DTR-PGK-EM7-NEO-F4/80 3HA L-MCL-HSV TK经NotI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。
靶向插入IRES-DTR至F4/80外显子22位点的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 G418和200μM ganciclovir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10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GHpA-2452-F-TGGGCTCTATGGCTTCTGAG(SEQ ID NO.11)和反向引物F480-15091-R-TCCCCTGATTCTATTCTTCCAC(SEQ ID NO.12)进行PCR扩增。
PCR反应体系如下：表1
PCR扩增条件为：表2
PCR产物经1％琼脂糖凝胶电泳鉴定，靶向插入IRES-DTR至F4/80外显子22位点的ES细胞阳性克隆可见约2.2kb条带，如附图4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。
实例3：繁育靶向插入IRES-DTR至F4/80外显子22位点的F4/80-DTR转基因小鼠
囊胚注射ES细胞获得F4/80-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。将靶向插入IRES-DTR至F4/80外显子22位点的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GHpA-2452-F(SEQ ID NO.11)和反向引物F480-15091-R(SEQ ID NO.12)作PCR扩增，PCR反应体系同表1，PCR扩增条件同表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2.2kb条带。一共获得13只雄性转基因嵌合体小鼠。
繁育F4/80-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F4/80-DTR转基因杂合小鼠应为白色，若为黑色与白色混杂则不带F4/80-DTR。5只雄性转基因嵌合体小鼠只有1只的后代为白色，共得到6只鼠仔，经鼠尾PCR鉴定后得到2只F4/80-DTR转基因杂合小鼠，即为F1代小鼠。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：F4/80-DTR转基因小鼠经白喉毒素诱导建立清除组织固有巨噬细胞的小鼠模型
本实施例采用腹腔注射白喉毒素(DT)，检测小鼠脑中小胶质细胞的占比来检测巨噬细胞的敲除效率，理论上小鼠全身的巨噬细胞都能被敲除。
F4/80-DTR转基因小鼠巨噬细胞清除：F4/80-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，取小鼠脑组织，用流式细胞术检测脑组织中小胶质细胞的表达情况。
流式细胞术检测脑组织中小胶质细胞的表达：获取小鼠脑组织，制备成单细胞悬液，经36％percoll(GE)密度梯度离心去除脂肪组织，经ACK lysis buffer(beyotime)去除红细胞，用2％FCS-PBS重悬细胞，经Fc blocker封闭Fc受体后，利用anti-mouse-CD45、anti-mouse-MHCII流式抗体染色，染色完毕洗去多余抗体，单细胞悬液用CytoFLEX LXFlow Cytometer上机检测。其中CD45中表达，MHCII表达的细胞为小胶质细胞。经DT诱导后小鼠小胶质细胞敲除率达70-90％，如附图7所示。
可以理解的是，对本领域技术人员来说，对本发明的技术方案及发明构思加以等同替换或改变都应属于本发明所附的权利要求的保护范围。
SEQUENCE LISTING
<110>浙江大学医学院附属第一医院
<120>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
<160>
<170>PatentIn version 3.3
<210>1
<211> 33
<212> DNA
<213>引物F4/80-180659-F(NotI)
<400>1
ACCACCGCGG CCGCGACAGC CAAATATTGG CAT
<210>2
<211> 32
<212> DNA
<213>引物F4/80-180900-R(SpeI)
<400>2
ACCACCACTA GTTACCTGCA AACCCCCAAT AA
<210>3
<211> 256
<212> DNA
<213>基因片段retrieval 5HA
<400>3
ACCACCGCGG CCGCGACAGC CAAATATTGG CATAAAAGAA GACAATAGAG GAACCAGAAT 60
AAACCAACCA GTGCTCACCT AATTTTTGAC AAAAGTATCA AATATGTATA GTAGAAAAAA 120
GGGAGCCAGT CTTATAGTTA GTTATGAGCT CAGTTTTGAA TGATGTAGCA TTCTTGGACT 180
TGCTAGAGGA TATGTTAATC CAGGGAGTAT TGATGTTTTA TAGATTATTG GGGGTTTGCA 240
GGTAACTAGT GGTGGT
<210>4
<211> 33
<212> DNA
<213>引物F4/80-192354-F(SpeI)
<400>4
ACCACCACTA GTGCACTGAG AATGGTGCTG TGT
<210> 5
<211> 37
<212> DNA
<213>引物F4/80-192569-R(BamHI)
<400>5
ACCACCGGAT CCGCATAGAC TCTTGATGGT GCATATT
<210>6
<211> 240
<212> DNA
<213>基因片段retrieval 3HA
<400>6
ACCACCACTA GTGCACTGAG AATGGTGCTG TGTAGAGTGA GTATATCTTC CCACTTCAAT 60
AATCTAATCA CAATCAAGTT GACACCTGAG ATGAACAATC AAAATCTACC CCATGACAAA 120
TTCATACTGA AAACCATCAC TTGTAATTGT AACCTTCTAC TCATCCCCAC CAAAGTCTCT 180
TAGTCATTTT ATAATGCAAA AATAATATGC ACCATCAAGA GTCTATGCGG ATCCGGTGGT 240
<210>7
<211> 5784
<212> DNA
<213>提取载体retrieval 5HA- retrieval 3HA-MCL-HSV TK
<400>7
CTCTCAAGGA TCTTACCGCT GTTGAGATCC AGTTCGATGT AACCCATTCG TGCACCCAAC 60
TTGATCTTCA GCATCTTTTA CTTTCACCAG CGTTTCTGGG TGAGCAAAAA CAGGAAGGCA 120
AAATGCCGCA AAAAAGGGAA TAAGGGCGAC ACGGAAATGT TGAATACTCA TACTCTTCCT 180
TTTTCAATAT TATTGAAGCA TTTATCAGGG TTATTGTCTC ATGAGCGGAT ACATATTTGA 240
ATGTATTTAG AAAAATAAAC AAATAGGGGT TCCGCGCACA TTTCCCCGAA AAGTGCCACC 300
TGACGCGCCC TGTAGCGGCG CATTAAGCGC GGCGGGTGTG GTGGTTACGC GCAGCGTGAC 360
CGCTACACTT GCCAGCGCCC TAGCGCCCGC TCCTTTCGCT TTCTTCCCTT CCTTTCTCGC 420
CACGTTCGCC GGCTTTCCCC GTCAAGCTCT AAATCGGGGG CTCCCTTTAG GGTTCCGATT 480
TAGTGCTTTA CGGCACCTCG ACCCCAAAAA ACTTGATTAG GGTGATGGTT CACGTAGTGG 540
GCCATCGCCC TGATAGACGG TTTTTCGCCC TTTGACGTTG GAGTCCACGT TCTTTAATAG 600
TGGACTCTTG TTCCAAACTG GAACAACACT CAACCCTATC TCGGTCTATT CTTTTGATTT 660
ATAAGGGATT TTGCCGATTT CGGCCTATTG GTTAAAAAAT GAGCTGATTT AACAAAAATT 720
TAACGCGAAT TTTAACAAAA TATTAACGCT TACAATTTCC ATTCGCCATT CAGGCTGCGC 780
AACTGTTGGG AAGGGCGATC GGTGCGGGCC TCTTCGCTAT TACGCCAGCT GGCGAAAGGG 840
GGATGTGCTG CAAGGCGATT AAGTTGGGTA ACGCCAGGGT TTTCCCAGTC ACGACGTTGT 900
AAAACGACGG CCAGTGAATT GTAATACGAC TCACTATAGG GCGAATTGGA GCTCCACCGC 960
GGTGGCGGCC GCGACAGCCA AATATTGGCA TAAAAGAAGA CAATAGAGGA ACCAGAATAA 1020
ACCAACCAGT GCTCACCTAA TTTTTGACAA AAGTATCAAA TATGTATAGT AGAAAAAAGG 1080
GAGCCAGTCT TATAGTTAGT TATGAGCTCA GTTTTGAATG ATGTAGCATT CTTGGACTTG 1140
CTAGAGGATA TGTTAATCCA GGGAGTATTG ATGTTTTATA GATTATTGGG GGTTTGCAGG 1200
TAACTAGTGC ACTGAGAATG GTGCTGTGTA GAGTGAGTAT ATCTTCCCAC TTCAATAATC 1260
TAATCACAAT CAAGTTGACA CCTGAGATGA ACAATCAAAA TCTACCCCAT GACAAATTCA 1320
TACTGAAAAC CATCACTTGT AATTGTAACC TTCTACTCAT CCCCACCAAA GTCTCTTAGT 1380
CATTTTATAA TGCAAAAATA ATATGCACCA TCAAGAGTCT ATGCGGATCC TCTAGAGTCG 1440
AGCAGTGTGG TTTTCAAGAG GAAGCAAAAA GCCTCTCCAC CCAGGCCTGG AATGTTTCCA 1500
CCCAATGTCG AGCAGTGTGG TTTTGCAAGA GGAAGCAAAA AGCCTCTCCA CCCAGGCCTG 1560
GAATGTTTCC ACCCAATGTC GAGCAAACCC CGCCCAGCGT CTTGTCATTG GCGAATTCGA 1620
ACACGCAGAT GCAGTCGGGG CGGCGCGGTC CCAGGTCCAC TTCGCATATT AAGGTGACGC 1680
GTGTGGCCTC GAACACCGAG CGACCCTGCA GCGACCCGCT TAACAGCGTC AACAGCGTGC 1740
CGCAGATCTT GGTGGCGTGA AACTCCCGCA CCTCTTCGGC CAGCGCCTTG TAGAAGCGCG 1800
TATGGCTTCG TACCCCGGCC ATCAGCACGC GTCTGCGTTC GACCAGGCTG CGCGTTCTCG 1860
CGGCCATAGC AACCGACGTA CGGCGTTGCG CCCTCGCCGG CAGCAAGAAG CCACGGAAGT 1920
CCGCCCGGAG CAGAAAATGC CCACGCTACT GCGGGTTTAT ATAGACGGTC CCCACGGGAT 1980
GGGGAAAACC ACCACCACGC AACTGCTGGT GGCCCTGGGT TCGCGCGACG ATATCGTCTA 2040
CGTACCCGAG CCGATGACTT ACTGGCGGGT GCTGGGGGCT TCCGAGACAA TCGCGAACAT 2100
CTACACCACA CAACACCGCC TTGACCAGGG TGAGATATCG GCCGGGGACG CGGCGGTGGT 2160
AATGACAAGC GCCCAGATAA CAATGGGCAT GCCTTATGCC GTGACCGACG CCGTTCTGGC 2220
TCCTCATATC GGGGGGGAGG CTGGGAGCTC ACATGCCCCG CCCCCGGCCC TCACCCTCAT 2280
CTTCGACCGC CATCCCATCG CCGCCCTCCT GTGCTACCCG GCCGCGCGAT ACCTTATGGG 2340
CAGCATGACC CCCCAGGCCG TGCTGGCGTT CGTGGCCCTC ATCCCGCCGA CCTTGCCCGG 2400
CACAAACATC GTGTTGGGGG CCCTTCCGGA GGACAGACAC ATCGACCGCC TGGCCAAACG 2460
CCAGCGCCCC GGCGAGCGGC TTGACCTGGC TATGCTGGCC GCGATTCGCC GCGTTTACGG 2520
GCTGCTTGCC AATACGGTGC GGTATCTGCA GGGCGGCGGG TCGTGGCGGG AGGATTGGGG 2580
ACAGCTTTCG GGGACGGCCG TGCCGCCCCA GGGTGCCGAG CCCCAGAGCA ACGCGGGCCC 2640
ACGACCCCAT ATCGGGGACA CGTTATTTAC CCTGTTTCGG GCCCCCGAGT TGCTGGCCCC 2700
CAACGGCGAC CTGTACAACG TGTTTGCCTG GGCCTTGGAC GTCTTGGCCA AACGCCTCCG 2760
TCCCATGCAC GTCTTTATCC TGGATTACGA CCAATCGCCC GCCGGCTGCC GGGACGCCCT 2820
GCTGCAACTT ACCTCCGGGA TGATCCAGAC CCACGTCACC ACCCCAGGCT CCATACCGAC 2880
GATCTGCGAC CTGGCGCGCA CGTTTGCCCG GGAGATGGGG GAGGCTAACT GAAACACGGA 2940
AGGAGACAAT ACCGGAAGGA ACCCGCGCTA TGACGGCAAT AAAAAGACAG AATAAAACGC 3000
ACGGGTGTTG GGTCGTTTGT TCATAAACGC GGGGTTCGGT CCCAGGGCTG GCACTCTGTC 3060
GATACCCCAC CGAGACCCCA TTGGGGCCAA TACGCCCGCG TTTCTTCCTT TTCCCCACCC 3120
CACCCCCCAA GTTCGGGTGA AGGCCCAGGG CTCGCAGCCA ACGTCGGGGC GGCAGGCCCT 3180
GCCATAGCCA CGGGCCCCGT GGGTTAGGGA CGGGGTCCCC CATGGGGAAT GGTTTATGGT 3240
TCGTGGGGGT TATTATTTTG GGCGTTGCGT GGGGTCAGTC CACGACTGGA CTGAGCAGAC 3300
AGACCCATGG TTTTTGGATG GCCTGGGCAT GGACCGCATG TACTGGCGCG ACACGAACAC 3360
CGGGCGTCTG TGGCTGCCAA ACACCCCCGA CCCCCAAAAA CCACCGCGCG GATTTCTGGC 3420
GCCGCCGGAC GAACTAAACC TGACTACGGC ATCTCTGCCC CTTCTTCGCT GGTACGAGGA 3480
GCGCTTTTGT TTTGTATTGG TCACCACGGC CGAGTTTCCG CGGGACCCCG GCCAGATCAA 3540
GCTAGCTTAT CGATACCGTC GACCTCGAGG GGGGGCCCGG TACCAGCTTT TGTTCCCTTT 3600
AGTGAGGGTT AATTTCGAGC TTGGCGTAAT CATGGTCATA GCTGTTTCCT GTGTGAAATT 3660
GTTATCCGCT CACAATTCCA CACAACATAC GAGCCGGAAG CATAAAGTGT AAAGCCTGGG 3720
GTGCCTAATG AGTGAGCTAA CTCACATTAA TTGCGTTGCG CTCACTGCCC GCTTTCCAGT 3780
CGGGAAACCT GTCGTGCCAG CTGCATTAAT GAATCGGCCA ACGCGCGGGG AGAGGCGGTT 3840
TGCGTATTGG GCGCTCTTCC GCTTCCTCGC TCACTGACTC GCTGCGCTCG GTCGTTCGGC 3900
TGCGGCGAGC GGTATCAGCT CACTCAAAGG CGGTAATACG GTTATCCACA GAATCAGGGG 3960
ATAACGCAGG AAAGAACATG TGAGCAAAAG GCCAGCAAAA GGCCAGGAAC CGTAAAAAGG 4020
CCGCGTTGCT GGCGTTTTTC CATAGGCTCC GCCCCCCTGA CGAGCATCAC AAAAATCGAC 4080
GCTCAAGTCA GAGGTGGCGA AACCCGACAG GACTATAAAG ATACCAGGCG TTTCCCCCTG 4140
GAAGCTCCCT CGTGCGCTCT CCTGTTCCGA CCCTGCCGCT TACCGGATAC CTGTCCGCCT 4200
TTCTCCCTTC GGGAAGCGTG GCGCTTTCTC ATAGCTCACG CTGTAGGTAT CTCAGTTCGG 4260
TGTAGGTCGT TCGCTCCAAG CTGGGCTGTG TGCACGAACC CCCCGTTCAG CCCGACCGCT 4320
GCGCCTTATC CGGTAACTAT CGTCTTGAGT CCAACCCGGT AAGACACGAC TTATCGCCAC 4380
TGGCAGCAGC CACTGGTAAC AGGATTAGCA GAGCGAGGTA TGTAGGCGGT GCTACAGAGT 4440
TCTTGAAGTG GTGGCCTAAC TACGGCTACA CTAGAAGAAC AGTATTTGGT ATCTGCGCTC 4500
TGCTGAAGCC AGTTACCTTC GGAAAAAGAG TTGGTAGCTC TTGATCCGGC AAACAAACCA 4560
CCGCTGGTAG CGGTGGTTTT TTTGTTTGCA AGCAGCAGAT TACGCGCAGA AAAAAAGGAT 4620
CTCAAGAAGA TCCTTTGATC TTTTCTACGG GGTCTGACGC TCAGTGGAAC GAAAACTCAC 4680
GTTAAGGGAT TTTGGTCATG AGATTATCAA AAAGGATCTT CACCTAGATC CTTTTAAATT 4740
AAAAATGAAG TTTTAAATCA ATCTAAAGTA TATATGAGTA AACTTGGTCT GACAGTTACC 4800
AATGCTTAAT CAGTGAGGCA CCTATCTCAG CGATCTGTCT ATTTCGTTCA TCCATAGTTG 4860
CCTGACTCCC CGTCGTGTAG ATAACTACGA TACGGGAGGG CTTACCATCT GGCCCCAGTG 4920
CTGCAATGAT ACCGCGAGAC CCACGCTCAC CGGCTCCAGA TTTATCAGCA ATAAACCAGC 4980
CAGCCGGAAG GGCCGAGCGC AGAAGTGGTC CTGCAACTTT ATCCGCCTCC ATCCAGTCTA 5040
TTAATTGTTG CCGGGAAGCT AGAGTAAGTA GTTCGCCAGT TAATAGTTTG CGCAACGTTG 5100
TTGCCATTGC TACAGGCATC GTGGTGTCAC GCTCGTCGTT TGGTATGGCT TCATTCAGCT 5160
CCGGTTCCCA ACGATCAAGG CGAGTTACAT GATCCCCCAT GTTGTGCAAA AAAGCGGTTA 5220
GCTCCTTCGG TCCTCCGATC GTTGTCAGAA GTAAGTTGGC CGCAGTGTTA TCACTCATGG 5280
TTATGGCAGC ACTGCATAAT TCTCTTACTG TCATGCCATC CGTAAGATGC TTTTCTGTGA 5340
CTGGTGAGTA CTCAACCAAG TCATTCTGAG AATAGTGTAT GCGGCGACCG AGTTGCTCTT 5400
GCCCGGCGTC AATACGGGAT AATACCGCGC CACATAGCAG AACTTTAAAA GTGCTCATCA 5460
TTGGAAAACG TTCTTCGGGG CGAAAACTCT CAAGGATCTT ACCGCTGTTG AGATCCAGTT 5520
CGATGTAACC CACTCGTGCA CCCAACTGAT CTTCAGCATC TTTTACTTTC ACCAGCGTTT 5580
CTGGGTGAGC AAAAACAGGA AGGCAAAATG CCGCAAAAAA GGGAATAAGG GCGACACGGA 5640
AATGTTGAAT ACTCATACTC TTCCTTTTTC AATATTATTG AAGCATTTAT CAGGGTTATT 5700
GTCTCATGAG CGGATACATA TTTGAATGTA TTTAGAAAAA TAAACAAATA GGGGTTCCGC 5760
GCACATTTCC CCGAAAAGTG CCAC
<210>8
<211>30420
<212> DNA
<213>打靶载体F4/80 5HA-IRES-DTR-PGK-EM7-NEO-F4/80 3HA-MCL-HSV TK
<400>8
CTCTCAAGGA TCTTACCGCT GTTGAGATCC AGTTCGATGT AACCCATTCG TGCACCCAAC 60
TTGATCTTCA GCATCTTTTA CTTTCACCAG CGTTTCTGGG TGAGCAAAAA CAGGAAGGCA 120
AAATGCCGCA AAAAAGGGAA TAAGGGCGAC ACGGAAATGT TGAATACTCA TACTCTTCCT 180
TTTTCAATAT TATTGAAGCA TTTATCAGGG TTATTGTCTC ATGAGCGGAT ACATATTTGA 240
ATGTATTTAG AAAAATAAAC AAATAGGGGT TCCGCGCACA TTTCCCCGAA AAGTGCCACC 300
TGACGCGCCC TGTAGCGGCG CATTAAGCGC GGCGGGTGTG GTGGTTACGC GCAGCGTGAC 360
CGCTACACTT GCCAGCGCCC TAGCGCCCGC TCCTTTCGCT TTCTTCCCTT CCTTTCTCGC 420
CACGTTCGCC GGCTTTCCCC GTCAAGCTCT AAATCGGGGG CTCCCTTTAG GGTTCCGATT 480
TAGTGCTTTA CGGCACCTCG ACCCCAAAAA ACTTGATTAG GGTGATGGTT CACGTAGTGG 540
GCCATCGCCC TGATAGACGG TTTTTCGCCC TTTGACGTTG GAGTCCACGT TCTTTAATAG 600
TGGACTCTTG TTCCAAACTG GAACAACACT CAACCCTATC TCGGTCTATT CTTTTGATTT 660
ATAAGGGATT TTGCCGATTT CGGCCTATTG GTTAAAAAAT GAGCTGATTT AACAAAAATT 720
TAACGCGAAT TTTAACAAAA TATTAACGCT TACAATTTCC ATTCGCCATT CAGGCTGCGC 780
AACTGTTGGG AAGGGCGATC GGTGCGGGCC TCTTCGCTAT TACGCCAGCT GGCGAAAGGG 840
GGATGTGCTG CAAGGCGATT AAGTTGGGTA ACGCCAGGGT TTTCCCAGTC ACGACGTTGT 900
AAAACGACGG CCAGTGAATT GTAATACGAC TCACTATAGG GCGAATTGGA GCTCCACCGC 960
GGTGGCGGCC GCGACAGCCA AATATTGGCA TAAAAGAAGA CAATAGAGGA ACCAGAATAA 1020
ACCAACCAGT GCTCACCTAA TTTTTGACAA AAGTATCAAA TATGTATAGT AGAAAAAAGG 1080
GAGCCAGTCT TATAGTTAGT TATGAGCTCA GTTTTGAATG ATGTAGCATT CTTGGACTTG 1140
CTAGAGGATA TGTTAATCCA GGGAGTATTG ATGTTTTATA GATTATTGGG GGTTTGCAGG 1200
TATGTTATTG TGTCTTAAGA GTGCTCTATA GTAAACAATA TAGATTTTTA TCATCTTAGC 1260
TCTGGAGACC TGAAACCTAA TATGATGGAT CAAGCTCTCC CAATAGCACT AAGGATGGAT 1320
CTACTCTGTG ATTTTCTTCT CTCTTTTTTC CTTTTCTCTT TCTTACTGCT CTTTCCTTTA 1380
ACTGTCTATA ATCATGATCT CTTGCTCCTG CCCCTTTTCT TTTTTTTCCC CATTTTTATT 1440
AGGATTTAGC TCATTTACAT TTCCAATGCT ATACCAAAAG CCCCCATACC CATCCACCCC 1500
CACTCCCCTA CCCACCCACT CCCCCTTTTT GGCCCTGGCG TTCCCCTGTA CTGGGGCATA 1560
TAAAGTTTGC AAGTCCAATG GGCCTCTCTT TGCAGTGATG GCCGACTAGG CCATCTTTTG 1620
ATACATATGC AGCTAGAGAC AAGAGCTCCG GGGTACTGCT TAGTTCATAT TGTTGTTCCA 1680
CCTATAGGGT TGCAGTTCCC TTTAGTTCCT TGGGTGCTTT CTCTAGTTCC TCCATTGGGG 1740
GCCCTGTGGT CCATTCAATA GCTGACTGTG AGCATCCACT TCTGTGTTTG CTAGGCCCTG 1800
GCATAGTCTC ACAAGAGACA GCTATATCTG GGTCCTTTCA GCAAAATCTT GCTAGTGTAT 1860
GCAATGGTGT CAGCGTTTGG AAGCTGATCA TGGGATGGAT CTCCCCTGCC CCTTTTCTTA 1920
AGTAAGATCC AATGCTTAGA CCAAAAAATT ACTTGTTTTC TTTTCTTTGC CAGGATTTTT 1980
GATCACTACT TCATCTACAA TGCAATCCTA GATTTATGTG GTGGTCTTCT TCCTTTATAT2040
GGAACCAAAA CCATCCCTTT TATTTAAAGA AAGATCATTA TCCCAGGGAT TCAGACACCT 2100
TCAGGAGTGT ACAACATGTA GTCTGTGGGC TGCATGTGCT CCGGGATATC TATGAGTGAA 2160
GCATAATAAA ATTATGGGTG ACAACTTTGC ATTACAATGT CAGTTGGTCA GACATCCTTG 2220
ATGATTTTCA TATTATGGCT CTATAATTAT GTGTATTTGT GAATCTGTTG TTTTTTATGA 2280
TATCAAAAAA TTAGTTGTGA ATCTGTCTTT TGACAGTTGG TAGTGACTAG TTTTGAGAAT 2340
GATATTCCTT AAAGCCAACA TTGACTCTGA CATACTTCTT TCTGTTCATT ATCATTGTCC 2400
CTCCCCTTCA TGCATTCCTC AAAAGTTCTC TATTCAATGA TGACACCTTT ATAAGTCCAT 2460
GGTTTCTCTC ATTGTCCTCA AGATTATTTT CTTAGTTAGG GTTACTATTG TTGGATGAAA 2520
CATCACCACC AAAAGCATCT TAAGGGGAGA AAGGATTTAT TTCACTCACA ATTCTATATA 2580
AGATTTTATT ATCAAAATCA GTGAGGGTAA GAACTCAAGC AGATGTAGAG GCCATGGAAG 2640
AGTGTTCTTC TCTGGCTTGC TCTTCATGGC TTGTTTAGTC TGCTTTCTTA TGGCACCCAG 2700
GACCACTTGG CCAGTGGTGG CCCCACCCAC CTTGAGCCAG GCCCTCACCC ATCAATCACT 2760
AAATAAGAAA ACACTCTAAA GGCTTGTCTA CAGCCTGATC TTATGGAGGC ATTTTCTCAG 2820
GAGGCTCCCT TCTCTCAGAT GACTCTAGCT TGTGTCAAGT TGACACAAAA CCAGCCAGTA 2880
CAATGTGTGT CATCTCTTAA ACCTGACTGG CTGCTGGTGG TATGGCCTTT CCTGGGGAAA 2940
TGTGAATAAA TTTCTACTTA CCTCAGATAC AGCACCAAAG TAATGATTCT ACTACAGTTC 3000
AGTTTGGCAG ATCAATGAAC TTACTGGGAT TATACACAGG AGCATGGGTT GGTGTAACCT 3060
TGAGCATAGA TGGTTTGAAT GCATCTGCAT CATTAAAAAG CTGGGGGCAT GAATGTTTCA 3120
GGAATAATCA GCTGAGGGAC ATGACAAGCT GGGGTACAAT TCATGAAAGG GTTCCCAGAC 3180
ATCCTTTCAT GGATCAGGCA TTTAACAGGT CAGAGAATCT CCCTTTCCAG GAGTCATTAC 3240
TATTTCTATA ACCTACAGAG AGGCCTTGCA AATCTTACAA GTTTAAGGAG ATTCCTGAGA 3300
CTTGTGAGTT GTTTCCTGAG TCTTGACTGT AGGACAGATT GTTTTAGCTT GGAGATTGTT 3360
ACACAAGCAT TATTCTTCTT CACCTAGCAC TGTTACATCT TCTAGTGTCC CTCTTAGGGA 3420
TTGCCTGCAT GCTTACTTGT CTAGCTGTAT CAAAGGATTT TCTTCTCCCA CACAATTCCA 3480
GCCCTGTCAT GGAGGGTTGT TTTGGAAAGC AGAGAATGTG ATTAAGATAA TTAGCACAAT 3540
GTCTGACATT GACTGTTCCT CAATACATAA TAGGTTTGAG AAATAATGGG GATAAAAATA 3600
ATAATGAACA AATACCACCA GGAGAGAGCT GGTCTCCCAG GAGTTCAGAC AAGCCTATAA 3660
GTGCAGGTAA GACCACCATT GATGCTCAGA GGTACCCACC TGGAACCCTC AGGACACAGG 3720
ATGTAAGGAG CAGCCTAGAC AGGAGCCTTC TTGTTTTCGT CTGCATCCAG AGCTGACCCT 3780
GTGCCACAGA GCTACATACA TAAATAACTC TAGGAGAGTG CTGGTCTCCT AGGAGAACAA 3840
ACAAACCTGT GAATGCAGGT AAGACTACCA CATCTCTTCA AATTCCTGGC CCAAGAAGGA 3900
TGGTCCTAGA GCCATCAGGA CACATGAACT AAGGATCAGC TGGGGACAGT ATCCTTCTGG 3960
TTTCTGTCTG CACCCCAGAT CTGACCCTAT ACCACAATTC TCCATACATA AATTCCTCCT 4020
GGAGAGAATC AGTCCAAGGA GTACTGACAT ATAGGCTTGC AGGAGGGTCA AGCCACAGTC 4080
AGAGACAGCA AGGCCAGCTA ACACCAGAGA TAAAAGATGG CAAAAGGCAA GGGCAAGAAC 4140
ATAAGCAACA GAAGCCAAGT CTATGTGGCA TCATCAAAGC TCAGTCCTCC CACCACAGCA 4200
AGCCCTGGTT TCCCCAACAC ACGAGAAAAG CAAGACTATG ATTTAAAGTC ACATCTCATG 4260
ATGGTAATAG AGGACTTTCA GAAGGACATA AATAACTCCC TTAAAGAAAT ACGAGAGAAC 4320
ACAGATAAAC AGGTAGAAGA TCTTAAAGAG AAAACACAAA AATTCCTTAA GGAATTACAG 4380
GAAAACACAA CCAAACAGGT GAAGGAACTG AACAAAACCA TCCAGAATCT AAAAATGGAA 4440
ATAGAAACAA TAAAGAAATC ACAAAGGGAG ACAACCCTGG AGATAGAAAC CCTAGGAAAG 4500
AGATCAGGAG TTATAGATGC AAGCACCACC AACATTGACA CAACAGCTAA AGAAAATGCA 4560
AAATTCAAAA AGCTCCTAAT CCAAAACATC CAGGAAGTCC AGGACACAAA GAGAAGACCA 4620
AGCCTAAGGA TAATAGGTAT GGAAGAGAGT GAAGATTCCC AGCACAAAGG GCTAATAAAT 4680
ATCTTCAGCA AAATTATAGA AGAAAACTTC CCTAACTTAA AGAAAGTGAT GCCCATGAAC 4740
ATACAAGAAG AATAGAGCAC TCCAAATAGA TTGAACCATA AAAAATTCCT CCTATCAAAT 4800
AATAGTCAAA CCACCAAATG CACAAAAAAG AAAGAATATT AAAAGCGGTA AGGGAAAAAG 4860
GCCAAGTAAC ATATAAACTC AGACCTATCA GAATTACACC AGATTTCTCA CCAGAGACTA 4920
TGAAAGCTAG AAGATCCTGG GTAGATGTCA TACAGACCCT AAGAGAACAC AAATGCCAGC 4980
ACAGACTACT ATACCCAGCA AAACTCTCAA TACTGTACAT GGAGAAAACA AGATATTCCA 5040
TGACAAAACC AATTTTATAT AATATTTTTA CACAAATCCA GTCCTACAAA GGATAATAGA 5100
TGGAAAACAC CAGCACAAGA AGGGGAACTA TACCCTAGAA ATGACAGAAA GTAATCTTTT 5160
TCAACAAACC CAAAAGGAGA TAACCACACA AACATAAAAT AACAGGAAGC AATGATCACT 5220
ACTCCTTAAT ATATCGTAAC ATCAATGGAT TTAATTCCCC AATGAAAAGA GAAAGACTAG 5280
AAGACTGGAT ATGTAAATAG GACACAGCAT TTTGTTGCAT ATAAGAAAAG AATCTCAGTG 5340
TTAAAGACAA ACAATACCTC AGAGTAAAAG ACCTGGAAAA CAATTTTCCA AGCAATGGTC 5400
TCAAAAAACA AGCTAGAATA GCCATTATAA TAGCGGATAA AATCGACTTT CAACCAAAAG 5460
TCATCAAAAA AGATAAGGAA GGACAGTTCA CACACATCAA AGAAAAAATC TACCAAGAAC 5520
TCTCAATTCT GAACATCTAT GCTCCAAATG CAAGGGCACC CACATTTGTA AAAGAAACTT 5580
TACTAAATCT CAAAGCACAC ATTGCACCTC ACACACTAAT AGTGGGAGAC TTCAACATGC 5640
CACTCTTATC AATGGATAGA TCATGGAAAC ACAAACTAAA CAGAGAAGCT AACATTATTT 5700
ATGAACCAAA TGGATTTAAC AGATATCTAT AGAACATTTC ATTCTAAATC AAAAGAATAT 5760
ACCTTCTTCT CAGCACCTCA TGGTACCTTT CCAAAATTGA CCGTATAATC TGTCACAAAA 5820
CAGACCTCAA CAGATATAGG AAGATTGAAA TAATCCCATG CATCCTATCA AATTACTACA 5880
GATCAAGGCT GGTCTTCAGT AGCAATAAAA ACACAGAAAT CCCACATACA TATCGAAGCT 5940
GAACAACACT CTACTCAATG ATAATTTGGT CAAGGAAGAA ATAGAGAAAG AAATCAAAGA 6000
CTTTTTAGAA TTTAATGAAA ATGAAGGTAC AACTTACCCA AACTTATAGG ACACAATGAA 6060
AGCAGTGCTA AGAGGAAAAC TCATAGCTCT GAGTGCCTCC AAAAAGAAAG TGGGGAGAGC 6120
ATACACTAGC AGCTTGACAG CACACCTGAA AGCTTTAGAA CAAAAAGAAG AAAATACAAC 6180
CAAGAGGAGT AGACAGGAGG AAATAATTGA ACTCAGGGCC AAAATCAACC AAGAAGAAAC 6240
AAAAAGAACT ACACAAAGAA TCAACAAAAC CAGGAGTTGG TTCTTTGAGA AAATCAACAA 6300
AGTCAATAAA CCCTTAGCCA GAGTAACCAG AGGGCACAGA GACAGTATCC AAATTAATAA 6360
AATCAGAAAT GAAAAGGGAG ACATAACAAT GGAAACTGAG GAAATTTTAA AAAATCATCA 6420
GATCCTACTA CAAAAGCCTA TACTCAACCA AATTGGAAAA TGTGGATGAA ATGGACAATT 6480
TTCTAGACAG ATACCAGGTG CCAAAGTTAA ATCAGGATCA TCTAAACCAT CTAGAGTCCC 6540
ACAACCCCTA AAGAAATAGA AGCAGTCATT AAAAGTCTCC CATAATCAGC ATCCAAATGC 6600
TGACACCATT GCATACACTA GCAAGATTTT GCTGAAAGGA CCCAGATATA GCTCTCTCTT 6660
GTGAGACTAT GCCGGGGCCT AGCAAACACA GAAGTGGAAG ATCACAGTCA GCTATTGGAT 6720
GGGTCACACG GCCCCCAATG GAGGAGCTAG AGAAATTGCC CAAGGAGCTA ATGGGAACTG 6780
CAACCCTATA GGTAGAACAA CAATATGAAC TAACCAGTAC CCCAGAGCTC TTGTCTCTAG 6840
CTGCATATGT ATCAAAAGAT GGCCTAGTTG GCCATCACTG CAAAGAGAGG CCCATTAGAC 6900
ACGCAAACTT TATATGCCCC AGTACAGGGG AACGCCAGGG CCAAAAAGGG GGAGTGGGTG 6960
GGTAGGGAAT TGGGGGGGTG GGTATGGGGG ACCTTTGGGA TAGCATTGAA AATGTAAACG 7020
AGGAAAATAC CTAATAAAAA AAATAAAAAA ACCAAAAAAC CAAAAACCAA ACCAAACAAA 7080
CAAAAAAGTC TCCCAACCAA AAAAAAAAAA AAGCCCAGGA CCAGATGAGT TTAGTGCAGA 7140
GTTCTATCAG ACCTTTAAGG AAGACCTTCA AACTATTCCA CAAAAGAGAA ACAGAAAGAA 7200
CACTACCCAA TTACTCTGAT ACAAAAAGCA CACAAAGACC CAACAAAGAA AGAGAACTTC 7260
AGACCAATTT CATTTATGAA TATTGATGGG AAAATGCTCA ATTAAATTCT TGCCAACTGA 7320
ATTCAAGAAC ACATCAAAAT GATCACTCAC CACAATCAAG TAGAGTTCAT CTCAGGGTTG 7380
CCAGGATATT TCAATAGAAA GAAATCCATC AATGTAAACT ACTACAAAAA AAAAAAAAAA 7440
CCTCAAAGAA AAAAACCACA TGATAATTTC ATTGGATGCT GAAAAAACAT TTGACAAAAT 7500
TCAACATCCC TTCATGTTAA AAGTCTTGGA AAGATCAGGA ATTCAAGGCC TATACCTAAA 7560
CATAGTAAAA GCAACATACA GCAAACCAGT TGCCACCATC AAAATAAATG GAGAGAACCT 7620
TGAAGCAATC CCACTAAAAT CAGGGACTAG ACAATGCTAT CCACTGTTTC CCTTTTGAAG 7680
GTCTAGCTAG AGCAATTAGA CAACAAAAGT AGGTCAAAGG TATACAAACT GCCAAGAATT 7740
AAGTCAAAAT ATCACTATTT GCAGATAGTA TACTTAAGTG ACCCCAAAAA TTCCACCAGA 7800
GAGCTCCTAA AGCTGATAAA CAACTTCAGC AAGGTGGTCG GATATAACAT TAACTCAAAC 7860
AAATCAGTAG CCTTCCTGTC CTCAAAGGAT TAACAAGCTG AGAAAGAAAT GAGGGAAACT 7920
AAACCCTTCA CAATAGTTAC AAACAATATA TAATATCTTT GTGTGACTCT AACCAAACAG 7980
ATGAAAGATC TGTATGACAA GAACTTCAAG TCTCTGAAGA AAGAAATCAA AGAAGACCTC 8040
AGATGATGGA AAGATCTCCC ATGCTCATGG ATTGGCAGGA TTAATATTGT AAAACTGGCC 8100
ATCTTGTCTA GTGCAATCCC AGTCAACATT TCAATTCATA ACTCTTCATA GTGTTAGAAA 8160
GAGCAATTCT CAAATTCATC TGGAATAATA ATAATAAAAA ACCCAGGGTA GTAAAAACTA 8220
TTCTCAACAA TAAAAGAACT TCTGGGGGAA TCAGCATCCT GGACCTCAAG GTATACTACA 8280
GAGTAATAGT GATAAAAACT GCATGGTATT GGTACAGTGA CAGGTAGATC TATGGAATGG 8340
AATTAAAGAT CCTGAAATGA ACCCACACAC CTACAGTCAC TTGATCTTTG ACAAAGGAGC 8400
TAAAACCATC CAGTGGAAAA AAGATAGCAT TTTCAACAAA TGGTGCTGAT TCAACTGGAG 8460
GTCAACATGT AGCAGAATGC AAATTGATCC ATTATTATCT CCCTGTACAA ACCTCTAGTC 8520
CAAGTGGATC AAGGTCCTTC ATATAAAACC AGATACACTG AATCTAATAG AAGAGAAAGT 8580
GGGGAAACCT CTCAAACACA TGGGTACAGG GAAAAATTCC TGAACAGAAC ACCAATGGCT 8640
TATGATCTAA GATCAAGAAT TGACAAATGA GACCTCATAA AATTGCAAAG CTTCTATAAG 8700
TCAAAGGGCA CTGTCAATAG GACAAAACTG GAACCAACAC ATTGGTAAAA GATCTTTACT 8760
AATTGTACAT ATGATAGAGA GCTACTATCC AATATATACA AAAAACTTAA GAACGTAGAC 8820
TCCAGACATC CAAATATCCC TATGAATAAA TGGGGAACAG AGCTAAACAA AGAATTCTCA 8880
ACTGAGGAAA GATGAAAAGC TCAGAAACAC CTAAAGAAAT GTTCAACATC CTTGGTCATC 8940
GGGGAAATGC AAATCAAAAC AACCCTGAGA TTCCACCTCA CACCAGTCAG AATGGCTAAG 9000
ATCAAAAACT CAGGTGACAG CAGATGCTAG CGAGGATGTG GAGAAAGAGG AACCCCCCTC 9060
CATTGCTGGT GGGATTGCAA GCTGTTATAA CCACTCTGGT AATAAGTCTT GTGGATCCTC 9120
AGAAAATTGG GCATAGTACT ACCCAAGGAT CCATCTATAC CACTTCTGGG CATATACCCA 9180
GAAGATGTTC CAACATGTAA TAAGGACACA TGCTCCACCA TGTTCATAGA AGCCATATGA 9240
CTAATAGCCA GAAGCCATAG TTAACCTAGA TGTCCCTCAA CAAATGAATG GATACAGAAA 9300
ATGTGGTACA TTTACACAGT GGAGTACTAC TCAGCCATTA AAAACGACTT CATGAAATTT 9360
GTAGGCAAAT GGATGAAACT TGAGAATATC ATCTTGAGTG AGATAACTCA GTCATAAAGG 9420
AACAAACATG GTATGCAATC ACTGATAAGT GGATATTAGC CCAAAAGTTC AGAATACCCA 9480
AGATTCCATT CACAGACCAT ATGAAAATTA AGAAGAAGGA AGACCAAAAT GTAGATTCTT 9540
CAGTGCTTTT TAGAAGGGTG AACAAAATAC TCAGAGGAGC AAATAAGGAG ATAAAGTGTG 9600
AAGCAGAGCC CGAAGGAAAG GCCATCCAGA GAGTGCCCCA CCTGGGGATC CATCCAATAT 9660
ACAGCCACCA ATCCTGGATG TTACTGTGGA TGCCAGGAAG TGCTTGCTGA CAGGAGCCTG 9720
ATATAGGTGA CTCCTGAGAG GTTATGCCAG AGCCTGACAA ATACAGAGGA GGAAGCTTGC 9780
AGTCAACTAT TGGATTGAGC TTGGGGGTCC CCAATTGAGG AGATGGAGAA AGGACTGAGG 9840
GTGTTTGTGG CCCCATGGAG GGAGCAACAA TGTCAATAGG CCAGATCCCT GCCCAGGCAC 9900
TCCTGGGGAC TGGACCACCA ACCAAAGAAT ACACATGGAG TCACCTATGG TCCTGGCCAC 9960
ATATGTTGTA GAAGATGGAC TTGTTGGACA TCAGTGGGAG GAGAGGCCCT CGGTCCTGAG 10020
GGTGTTCAGT GCCCCAGTGT AGGGGAATGC TGGGGCTGTA GAATTGGAGT TGGTGGGTGG 10080
GGGAGAACCC TCATAGAGTC AGGTGGAGGG AGGATGCAAT AGGGGGTTTC CTAAGGGGAG 10140
ACCTGGAAAG GGAAAAATAT TTGAAATATA TATAAAGAAA ATACCCAAGG GGGGAAAAAG 10200
AAAGAAAAAA AAGTAATGAA ATGTGTAGCA GAGGATGACC TAGTTGGCCA TCAATGGGAA 10260
AAGAGGCCCT TGGTCTTGTG AAGGTTCTAT GCCCCAGTAT AGGGGAATGC CAGGGCCGGA 10320
AGCAGGAGTG GGTGGGTTAG TGAGCAGGGG ACGGGGGAGA GGACAGGGGA TTTTTGGAGG 10380
GGAAATTAGG AAAGGGCATA CCTTTTGAAA TGTAAATGAA GAAAATATGT AATAAAAAAT 10440
AAATTTAAAA CAACAGAAAC AAACCAAAAA ACCAAAAAAC CAAAAACCAA AACAAGAATA 10500
ACAAAAAAGA ATAAACTCAT AGACTTAGTT TTGCTCTATG TGCAGACCCT GGGAATCTTT 10560
TTGTGTGGTT TTATAGTCCT GTATATAACA ATAGAAGCAT TTTAACTTCT GCCTTAATAT 10620
TTCTATGTAA ATAAATAGAA TCATTTCCAA AAAAAAATTA AAATATTTTT ATATTTTACA 10680
TATCTTTTCT GTGAGGTGGG ATTCAATTCC AACCAGGAAG TAATTGTTTA CTTCCAAAAA 10740
TATTCATGCC ACTATTGCAC TAGGAGGCAT AGCTTGCCTG GCAGGTTAGT ATTTTATTAA 10800
TTTTACACAG GGTCCAGAGA GGTTAGACTG TTGATGGTCA TTCTCTCCTG TCCAGCACAG 10860
AGGCTTCCAG AACTATGAAT GTTAGCCAGC ATGGAGGAAG TTGAAATGTT TATTTCACTT 10920
ACTTCCAAAT TTTCAGCACT ATTTTTCCTC CTGAAAATCT TAGTTGCCAG GAGAAATAAT 10980
ACCCTTCCAC CATACATTGG CTTTCCACTG GGGCAATTTT GCCTTTCAGG TGACTGATGC 11040
GAAAAATTTT TTTTGTTGTT GTTGTTCTCA TAACAAGGAA GGAGGGTAAC ACTGTCATCT 11100
AATGCACAGT GTCCAAGAAG TTGTTCCACA AACTACCAGA AGAAAGCACT GTTTGTCCCA 11160
CAATAGAGGT GATAGGACCT CAGATGTCAA TATTAGCAAC ATGGAAAACC TAGGTTCTTG 11220
GTTTACATGA ATCCGATTGT CTTAATTTCC TTCCCTGTTG CTGTCATAAA GTGCACTGTT 11280
GATTTCTCCC ATTCATAGCA CTCCTGGGTT GTCTGTTATT CTTTGTGTAG AGCTGAAGCC 11340
TCATGGTCTT TCCTTTTTCC ACTTTGACAT GTCCATTGCT ACTGTTTTTG TCCAGCCTAT 11400
GTTTCTTCAG TCATGTTGAG ACTTCCTGTG TGTAACTACT GATGTTGTTA GGAAATATAA 11460
TCTCACAGCA AAATCCCTCA TAGTCCAGCT CCTAGAATAT TTTTGTTCCC TCTTCTACAA 11520
ATTTCCTCAA ACCTTAAGTA TAGGAATTAT ATTCTCAAGA AAGAGCATAT CAATTGGTTA 11580
TCCAGTAACA AGAGGTCATC CCTGAAAACA CACATACATG TAATATTATA TAGACTAAGC 11640
AGGTTGCATT TAGGAATATA TATGTATATA ATAACAATGA AAAAAGAGGC CATAGATATC 11700
AAAGAAAGCA AGGAGGGGTA TATTGGAGGG TTTGGAAGAA GGACAAGGAA GGGGAATATG 11760
AGTATAATTA TATTCTAATC TCTCCCTCCC TCTCAGTATC CTGACCAAAG CAATGTAAGG 11820
GAGAAAGCTT ATGTGGCTCA CAGTTCCCAG GTATATCCAG TTGTTGCAAC TGGTAATAAG 11880
GACACATGCT CCACTATGTT CATAGCAGCC TTATTTATAA TAGCCAGAAG CCAAAAAGAA 11940
CCCAGATGTC CCTCAACAGA GGAATGGATA CAGAAAATGT GGTACATTTA CACAATGAAG 12000
TACTACTCAG CTATTAAAAA GAATGAATTT ATGAAATTCC CAGGCAAATG GATGGACCTG 12060
GAGGGTATCA TCCTGAGTGA GGTAACCCAA TCACAAAAGA ACTCAAATGG TATGTACTCA 12120
CTGATAAGTG GATATTAACC CAGAAACTTA GAATACCCAA GTTACAAGTT GCAAAACACA 12180
TGAATCTCAA GAAGAACGAA GACCAAAGTG TGGCCACTTT GCCCCTCCTT AGAATTGGGT 12240
ACAAAACACC CATGGAAGGA GTTACAAAGT TTGGAGCTGA GATGAAAGAA TGGACCACCT 12300
AGAGACTGAC GCACCTGGGG ATCCATCCCA TAATCAGCCT CCAAATGCAG ACACCATCGC 12360
ATATGCCAGC AAGATTTTGC TGAAAGGACC CTGATATAGC TGTCTCTTTT GAGGCTATGC 12420
AGGTGCCTGG CAAACACAGA AGTGGATGCT CACAGTCAGC TATTGGATGG ATCACAGGGC 12480
CCCCAATGGA GGAGCTAGAA AAAGTACCAA AGGAGCTAAA AGGGTCTGCA ATCCTGTAGG 12540
TGGAACAACG ATATGAACTA ACCAGTACCC CCAGAGCTCG TGTCTCTAGC TGCATATGTA 12600
GTAGAAGATG GCCTAGTTGG CCATCATTCG GAAGAGAGGC CCCTTGGTTT TGCAAGCTTT 12660
ATATGCCTCA GTACATGGGA ACACCAGGGC CAAGAAGTGG GAGTGGGTGG GTAGGGAAGC 12720
AGGGCGGGGG GGGGGGGGAG GGTATAGGCG ACTTTTGGGT AGCATTTGAA ATGTAAATGA 12780
AGAAAATACC TAAGTAAAAA AAAAGCATAA AAAAAGAGAA GATAAGTCTA AAAGAGCTCA 12840
AAGCAGTCCA TCATATTACA TTCAAGAGCA ATGATCAATG AATTTGTGCA CTGTGCTCAG 12900
CTCACTGTCC CACTCTCTTA AGACTCTGTT TAGGGTATGG TACTACCCAC AATGGCTGAG 12960
TCTTTCTACT AATCAAGATA ATCAAGATAA TCCCCCACAT GAATACTCAT AGACTAACCT 13020
TAAATTAGAC AGTCCCTTAC TGGTGTTCCT GGAGGCTTGC CTTCTAGGTG TTGACAATTA 13080
ATACCAATCA TCACACCACT TTGAGAAGGT TGAGGGAACA CCTTTGAAGA GAAGGAACCA 13140
TCTGTTCCGT GTCACAGTCT ATGGCATCAA CATTGGTTTC AGTTCTAACT GAAATGATGG 13200
GAAACAGCTG GCAGAAATAA AAATATTTAC ATGCAAACAA CAAAGAACAA TTTGTTGCAA 13260
GGTAGCTACA GTATAATAGC AGAGGTATGC ATTTTCATCA CTTACAATGT ATACTCAGAA 13320
GCAGGCAGGG AATGTCAGTG ATTAAGAAAT GGGCTTTGAA TGCTGTGGCA TGAATTAGCA 13380
GGTTGGGAAA CTACAAGTGG GGTGTCTTAT GGGGGCCATC AGTGGTGATG CTGGCTTTCT 13440
GCTGTCCATC CTAAATTGGA AGAAGAGGCA TCAGCTGGGT AACTGTTGGC TATGTTTCAA 13500
ATCCAGGCTA TTCAGACCTG ACAAATCCAG GATTGCAACC AGGCATCCTA CATCCAGGAT 13560
AGGTTACTAG AGGCCATATT TTAATGTGTA GATCGTCATG GTCAGTCTAT GTATCTTGTC 13620
TACACACCCT CTGCTTATCC TTACAGATAA ACTCTGTCCT CCTTGCCTGG ACACTGTGGG 13680
TTCTGAGGCA GAAGCTCTGC AGTGTCAGCT CAGAAGTCTC TAAACTCAAG GACACGAGGT 13740
ACAGTCTCTT TCTCACCTTA ACTCATTCCT TTCCTTCTTC TGACAAAGCT TTACCACTCT 13800
CTCAGCTTTC TCTATCATGT TCATTTCTCT ATCATGTTCA CATGCAGGTG TGTGCATGCC 13860
ATGATACACA TGCAGAGGTC AGAGGAATCC TAGGGTATCG GTTTCTACTC TCCTGTCTCC 13920
TCCTCCCATC TCACCTCAGG AGCACTGGTA TTACAGATGT TCATGTTATA CGCACATGGT 13980
TTATGTGGGT TCTCAGGACT TAAACTCAGG TCTTCACATT TGTGCATTAA GCAATTCAAC 14040
CATTGAGCTA CCTGCCAATC TGATCACTTT TTGAACATTC ATTGCAGAGA CATGATGAGG 14100
CTTGCATAGA ATAGAAGAGT CCAGACATCA ACTTCCCACT CCATCGATGT CCTTTATGAC 14160
CTGTATGACA TTATGTTGCT TATAGGACCT CTACAAAGCT CACTTTCTTT ATTTGTAGAA 14220
AGGGGATATT AGTAGGGTTC ACTTTAGGTA GCTAATAATG TATTGGTATA ATTTCTGGGA 14280
ACACTGAGCC TTTCATGGTT CACAAATATT GATACACTCA GTGTGAGCAC CCACACACAA 14340
ACATGGACAC ACACGCAAAA GGCACATACA CATAGACATA TGTACATATA CATGCACACA 14400
TATAAACATG GATATAAATA TGCATGGGCA CATATTCACA CACACAAACA GAAACATATA 14460
TGCACATGCA CACATGTGCT CATGGACATA AGCGTACATG GACATGTATG CACACACATG 14520
AACTCACATA CATACCTACA AATGCACACA TACATATTGA CACATACAAA CACAGATATA 14580
TGCACACATA CATGCACACA ATACATACAC ATGCCCACCC CATATGCTCA AGTACACACA 14640
CATGCAAACA CAACATGAAC ATGCACATAC ACATTCACAC TCATGTACAT ATCTAACCAC 14700
TCACATGTTC ACACACAATA TACATCCATA CATGAAATCA TAAATACACA TCCATTCACA 14760
AAAATGCAGA TAAGCACACA TATACATGCA CACATACATT CACATCACAC ATGTACACAT 14820
ACACCATACT CATATGCATG CACAAAAGAC AAATATGCAC ATACAACACA AAAACATATA 14880
AGAAGGCTCA TAATGTTTTA AAATGTTTGT CTATTTTGAG ACAGATTTCC CTCCTGTAGG 14940
ACAAGCAGGT ATGAAATCCA TGATTCTTCA GTGCTGTGAT TATTCAGTGT GCCATATGGC 15000
CTGGCTTAGA ATCTCTTTCT GTGGCTGGGG AGTCATACAG GACAGTGTGA TGCATAGAGA 15060
GGGTGAGGGT CTCATAAATG TCATAAAATT AGTGATTGAA TAAATCATTC ACTAACTGGC 15120
TCCAAGCCTG AACATTGATT GTCATTACTC ACTTAATTCT TTTCTTCTTC TTTCTTCTTC 15180
TTTCTTCTTC TTCTTCTTCT TCTTCTTCTT CTTCTTCTTC TTCTTCTTCT TCTTCTTCTT 15240
CTTCTTCTTC TTCTTCTTCT TCTTCTTCTT CCTTCTTCTT CTTCTTCTTC TTCTTCTTCT 15300
TCTTCTTCTT CTTCTTCTTC TTCTTCTTCT TCTTCTTCCT TCTTCTTCTT CTTCTTCTCC 15360
TCCTCCTCCT CCTCCTCCTC CTCCTCCTCC TCCTCCTCCT CCTCCTCCTC CTCCTCCTTC 15420
TCTCCTTCTA CTTCTCCTTC CCGTCCTCCT CCTCCTTCTT CCTCCTCCTC CCACTCCTCC 15480
TCCTTCTTCT TTCTTTTCCC TCTCCCTCTC TCTCTCCCTC TCCTCTTCCT CATACTTCTC 15540
ATACTCCTCC TTCTCCATCA TTATAACAAA TGAATATTAT TATTAATTAT TTTAAAAATA 15600
GCCTTACATT GCCTATATGT TTCTGCCTTT CTTTTCCTTT CTCTTAGTTT ATTTTTGATA 15660
CCCAAATAAA TTTAACCATG CAACAGCTTG CCTTTGTCTG GATTTTTATT TATTTATTAT 15720
GAAGATTATA GGATTTATAC ATATTCTTTC AGCTACATCT GCCTCACTCC TTATTATGGT 15780
AATATAATAT TCCACTGAAT GGCCATGCTA CATTTTCTTT ATCCTACGTC TATGTTTTTA 15840
TTCTATAGTA TGTCCACTAA CTGGTTTTAA CCAAATCTGA TCTAGTCTAT ATTTATCTAT 15900
ACTTATTCTA TTCATTCATA TTTTTATGAA TATTAATTGT CTTCCATAGG AAGGGGGAGA 15960
GAGAATGATT GCTTTGCCTG CATACTGTTT CTTTTCATGT ATGTGTCTAT TTCATTTTTA 16020
AGATAATAAA ATGACTAAGA ACTATATAGT AGTGATGGTT GCATGATGTG TGAGTGTACT 16080
TAAAGCCACC GAATCACATT TTTTTTGTAC TTAACGTGAT GAATTTGCCT CAGTGAAAGG 16140
ATCTCTCAGC ATTTTATAAC CATGGGTTTA CCACTTACAC AAGATCTAAG TGTCTGTTCA 16200
TGAGCCTTTT CAGCCATTCA TTGCTAAACC AAAAATTTAT TCGACACACA CATCCTCACA 16260
TCTCTGAATA CATGTGTATT TGTGGACACC AGCATTTTAA TTGATTATAG GACTTGAGAC 16320
AGCAAATTTT TCTTTGAGAT TTTGGGTTTA TTTTTATTTT TACAAGTTTA AAATTTGTTT 16380
TTGAAATACA TAACTTAAAA TTTGCTATGT CAGACACTTC CAAGTATACA AGAGTATTGG 16440
TGTTGTAGTA AACAAGAGCA GTGTCCAAGA GACAGCAGTT TTTCTCTGTC TTTATCATCC 16500
ATGTGATGTC ATCTTCAGAC AGTGCCAGGT CTCCATGGAG ACCATGCCAG CTCTCAAGAC 16560
AATTCAATTT TGTAAACATT ATCTTCTAGG TTGCTGACCT TCAAGGCCAT TGCCCAGATT 16620
TTCATCTTGG GCTGCTCCTG GGTGCTGGGC ATTTTCCAGA TTGGCCCCTT GGCAAGCATC 16680
ATGGCATACC TGTTCACCAT TATCAACAGT CTGCAGGGGG CTTTCATCTT CCTCATTCAC 16740
TGTCTGCTCA ACCGTCAGGT ATGCAGATGT TGCCTACTTG CATCAGCTTC TCTGAATAAA 16800
ACTGCCTACT GCATGTGCTT GTAGCAGACC AACTCCCATA TCTAGGACTG GTCTGTGTGT 16860
GTGTGCTTTG TATGTTTTAT GCCTTTGTGA GTATATGTGT TTTTTTTGTG TTATGAATTA 16920
TATGTGTGGA TTCATACCTG TGCTCTTGGC TAGACACAAA CATTGGAAAT ATTTCTCTGT 16980
CATGTTCTCC CATATCTCTA GTATTTTATT TAAATAAAAT TGTTGTGCAT ATGTGTGTAA 17040
TCTCTTTGTG GAGCAGTTTG ACTGAGAAAC AGTCAAATTG TGCATCCCCC ACGTCTCCTC 17100
CTGGGCCTTC TGCATGTCTA TAGTCTGTGG TCTGTAGCCT ATTTGGCTAG CATGTGGTGT 17160
TTGTTAGATC TGTCTGAATT GCCTGTGTGT GTTATCTATC TGTTGTGCCA CTGTCTAATA 17220
AAGGTATTAC CCTATGTTTA TTGAATAGCA CAGAAAACAC AGCATAGAAA AGGTACAAGA 17280
AATTATTTTC AGAAAAATTT GACAGAGTTT TACCAGGGAA GAAATCACTT TACTAGTCAT 17340
AGCTGACCCA GAAGAACACC CTAAATTGCT GCAGATCTTA TCAATCTGTT TCCACATGTT 17400
GCTTTCTACA GTTATGAGAC ATGTGTTTTG TTTCAGGTTG CCTTCAACTT GTTTGTTGCT 17460
CTCAGCAAAT GACCATCCCC TCCCACACCC CACATACATT GTGCTGGGCC AGGGATATAG 17520
GAGGGCATAT AATTAAATGG CTTAGATTAA AAAAAACTGA TAACCTGAAT AATTGAAGGA 17580
ATAGTAAGAT CGATTACATT GTTCTCTTAA AAAGAGGTTA GACTAGACAC ACTGAGCACA 17640
TGCAGGATAG AAAGTTGAAC ACATTAATCT TAGATTATTT CAAGGTATAT TATTAACTTG 17700
GTAGCAATGT TTAGAAAAAG TTCAGTGTTT TTGAAGGCAG ATGGTATTTT CAGAAGAATG 17760
TATTACCTTT TCTATGCACA TGAGTATAGT GCATGTAGAG GCTAGAAGAG AGTGCTGGAT 17820
CGCCTAGAGC TGAAGTTGTG GGCGTTTGTG AGCCTCCTCC TGACATGTCT TTAGAACTGA 17880
GGTTGTGTCC TTCACATGAG TAAGGCAGAC TCTTAAGGCA CCAACTCTTC TCTCTATCTC 17940
TTCCATTCTG TATTTTGAGA GAGGATCTTT CACTGAGCTT GAAGTATAAA TTGATCTATA 18000
CTGATAGGCC ATCAAGTCTC AGAAAGTGTT CTCCTACGTT CACCTTCCTA GAATTGGGAT 18060
TTCTGTTGTG TGCAGCCATG TTGGTACCCA CCAAACCCAC CATCAAATTT TCCTTATTCT 18120
TGAGAATTTA GTACATACAC ACAATGAAAT ATGATCATAC CTACTCCCAT TCCTCTCCAC 18180
CAGATTCTAC ATAGTCCTCT TAACATGGTA CCCAACTAAT GTTATGTTTC ACGATTTAAA 18240
AACCGTACTA AGTCTAGTCA GTATTTTCCA TATGTGATTG AATGTGGAGC TATCCATTGG 18300
AGCATATCCC CAGAAATGAA TAATTCTTTC TCTGTCAGAA ACTATTAACT TCCAATAATT 18360
CCTCAGTAAG GGGTGAAGAC TAGACGTCTT CTAACCAATA TATGCTAGAA TTTGGTTGGC 18420
TTGATCTTGT ACAGGTACCA ACAGCTACCA TGAGTTCATG AGTTTGATGA CATGTCATGT 18480
CCAGGAGGAA GCACTTCACA GTATCCCTTA TCTCTTCTAG CTCTTACATT TTTTTCTATC 18540
TTTTTTTTTC TATGATGTTC CTTGACCCTT TGTATGTTGG TTGAAGTAGG TCTTGTTTAG 18600
GGCTAAGCAC CATACTGATT TTTTAAAAAC GTGGGTGCTG GAGATCCAAA CTTAAGAACT 18660
CATATTATAT AGCAAACTTA GTGACTGGGC CATTTCCAAG CCTGGTTTAC TTTGCTTTCA 18720
TTTGTCTCCC CCTCTAGGTA CGGGATGAAT ATAAGAAACT CCTCACCAGG AAGACTGACC 18780
TTAGCTCCCA TTCCCAGACT TCTGGGATTT TACTGTCATC TATGCCATCC ACTTCCAAGA 18840
TGGTAAGGGA CTATAAATTT CCTACTGGTA CTAATTAGAA GGGGCATATA AGCTCAACAA 18900
AACCGAAAAA TACCTTCTGT CAGTACTGCT GACTGCTAAG TGTGCTCACT GTGGGGATTG 18960
TAGGATGAGG ATATTGGGGC CAGGATCTCA GGGTTTGCAC CCTACATTTA ACACTTTTTA 19020
AACTGGGCAA CCTAAATAGG GTGACATCCA CAGACGCAAA GAGAATACAC ATGCTAAGAA 19080
TTTTATCCTC GTCAATGATC AATGAGGATC TCAGAGAGTT GTTATTACTG GTTCAAAGAA 19140
TATGACAATT TATATCCCCA AGTTCTGTGA CCAGATTAGT TGTTAAGTCA CAGGTGAGTG 19200
GATTAGGCTA AGAAGGAGTC TTAGCTACTT TTTTTGTTGC CAGGACCCAA CACCTAACAA 19260
AAGTAACATA AGAAGGAAGG GTTTATTTCG ACACAGTTCA ATGGTAGAGT CCATTATGGT 19320
GAAGAGATCA GCATCAGGAG CATGGGGCAA CTGGTCACAT GGCATCCACA ATCAGGAAGC 19380
AGCAAATGAG TGCTGATGCT TGGCTCACTT TCTCCTTTTT ATTTGATCTT GTCTCCTAGC 19440
TATTGGAATG ATACTACAAC AAAAAGAGTG GGTCTTCACA TTTCAGTTAG CATAATGTAG 19500
ATGATCACTC GCAGGCATGA CCAGAGGCTT CTTTTTTTTT CCATTTTTAT TAGGTATTTA 19560
GCTCATTTAC ATTTCCAATG CTATACCAAA AGTCCCCCAT ACCCACTCCC CCCACTCCCC 19620
TACCCCCCCA CTCCCCCTTT TTGGCCCTGG CGTTCCTCTG TACTGGGGCA TATAAAGTTT 19680
GCAAGTCCAA TGGGCCTCTC TTTGCAGTGA TGGCTGACTA GGCCATCTTT TGATACATTC 19740
CAGAATCTTT CAAGTTGACA GTAAATATTA ACCATGAGAG ATGAGGTTTT ATTTTTCCAT 19800
AAACTAAAGA AAATGAAATA AAATTTCAGA AACATGGTTA GAACTTGAAA TCATTGTGTT 19860
AAGTGAAATA ACCCAGACAC AGAAGTTTAA ATATCACATA TTGTCTGTCA TATGTAGAAT 19920
TCATGTTGGT AAGTGACATG AAATAGAAGT GTAGCCATTT GGGAGGGGAA AAAGGATCAA 19980
TAAGAATAGA AAGAATTGAA TAGAGGATAA CATGATAAAT ATTATCAATG TACATGATGT 20040
ATGTGCATAA AACCACTACA GAGAAACACA TCATTTTGTT CAATATACTA GTAAATAAAA 20100
AACAAATAGC ATATACATGT TTATATATAT TCTGTGATTA TTATATATCA CATAATGTAC 20160
ATATAATATT TATTTAATTT AGTCAATGAT TTTTGTATCA AATTTTACTT TAACATAACT 20220
TCATGCTGTG ACCTCACTAT TCACTTAGTG CATACCAAAT ACTTGGTCAA CTGGGTCTGT 20280
CTTAATTTCA GTTCCTTTGA CTATACCTTG ACAAAAGCAA CTTAGTAGAA AAAGAGTTTA 20340
TTTTATCTCA AAGTTACAGA AAAACAATTC ATCATCAGGG AAGTCACTAC AGTAAGATAT 20400
TGACAGAATT GCCACATTAC ATTCACACTC AGCAGCAGAG AGCAACAAAT TAGAGCATGT 20460
ATGCAGTGCT CAGCACATTG CCCATTCTTA CACAGTTCAG AATCATATTT CTAGGAATTG 20520
GTGCCACCCA TGGTGGCTGG GTATTTCTAT ATCAGTCAGT ATAAAAGATA AATCTATTGG 20580
TCAGTCTGGT CTGATAATCC CAAACTGAGA CTCTCAGGTG ATTCTAGATT GTGTCTAGTT 20640
GACAGTTACA ATTAATCATT ACGTTTGTTA TATTCTTTGT TGCTGAACCC CTGATAAATG 20700
GACCATGATC CAGAAAATCA CCTCGCCCAA AGATGTTCAC ACAAGTAACT AGTTAAATTC 20760
AGTGAATCAC AAAGTAGGGC AATTGTGTTT AGCATTTCAA TGTTTATGCT TATAGCCTGA 20820
TTCTATATTG ATATCCATTT ACATGTGAGA CAGTTGTCCC CACAGATAGG ACTGGATAAT 20880
TTTTAGATCA ATTTTGCTTC TAACTGAGGT CATTCCCTTT TGTCTTTTTC AGGGTTAACA 20940
TCCTTTCTTG CTTTTAAATA TATTATGGGT CGACGCCCCT CTCCCTCCCC CCCCCCTAAC 20000
GTTACTGGCC GAAGCCGCTT GGAATAAGGC CGGTGTGCGT TTGTCTATAT GTTATTTTCC 20060
ACCATATTGC CGTCTTTTGG CAATGTGAGG GCCCGGAAAC CTGGCCCTGT CTTCTTGACG 20120
AGCATTCCTA GGGGTCTTTC CCCTCTCGCC AAAGGAATGC AAGGTCTGTT GAATGTCGTG 20180
AAGGAAGCAG TTCCTCTGGA AGCTTCTTGA AGACAAACAA CGTCTGTAGC GACCCTTTGC 20240
AGGCAGCGGA ACCCCCCACC TGGCGACAGG TGCCTCTGCG GCCAAAAGCC ACGTGTATAA 20300
GATACACCTG CAAAGGCGGC ACAACCCCAG TGCCACGTTG TGAGTTGGAT AGTTGTGGAA 20360
AGAGTCAAAT GGCTCTCCTC AAGCGTATTC AACAAGGGGC TGAAGGATGC CCAGAAGGTA 20420
CCCCATTGTA TGGGATCTGA TCTGGGGCCT CGGTGCACAT GCTTTACATG TGTTTAGTCG 20480
AGGTTAAAAA ACGTCTAGGC CCCCCGAACC ACGGGGACGT GGTTTTCCTT TGAAAAACAC 20540
GATGATAATA TGGCCACAAC CATGAAGCTG CTGCCGTCGG TGGTGCTGAA GCTCTTTCTG 20600
GCTGCAGTTC TCTCGGCACT GGTGACTGGC GAGAGCCTGG AGCGGCTTCG GAGAGGGCTA 20660
GCTGCTGGAA CCAGCAACCC GGACCCTCCC ACTGTATCCA CGGACCAGCT GCTACCCCTA 20720
GGAGGCGGCC GGGACCGGAA AGTCCGTGAC TTGCAAGAGG CAGATCTGGA CCTTTTGAGA 20780
GTCACTTTAT CCTCCAAGCC ACAAGCACTG GCCACACCAA ACAAGGAGGA GCACGGGAAA 20840
AGAAAGAAGA AAGGCAAGGG GCTAGGGAAG AAGAGGGACC CATGTCTTCG GAAATACAAG 20900
GACTTCTGCA TCCATGGAGA ATGCAAATAT GTGAAGGAGC TCCGGGCTCC CTCCTGCATC 20960
TGCCACCCGG GTTACCATGG AGAGAGGTGT CATGGGCTGA GCCTCCCAGT GGAAAATCGC 22020
TTATATACCT ATGACCACAC AACCATCCTG GCCGTGGTGG CTGTGGTGCT GTCATCTGTC 22080
TGTCTGCTGG TCATCGTGGG GCTTCTCATG TTTAGGTACC ATAGGAGAGG AGGTTATGAT 22140
GTGGAAAATG AAGAGAAAGT GAAGTTGGGC ATGACTAATT CCCACTAAAC GCGTACCGGG 22200
CCCCCCCTCG AGGTCGACGG TATCGATAAG CTTGATATCG AATTCCGAAG TTCCTATTCT 22260
CTAGAAAGTA TAGGAACTTC AGGTCTGAAG AGGAGTTTAC GTCCAGCCAA GCTAGCTTGG 22320
CTGCAGGTCG TCGAAATTCT ACCGGGTAGG GGAGGCGCTT TTCCCAAGGC AGTCTGGAGC 22380
ATGCGCTTTA GCAGCCCCGC TGGGCACTTG GCGCTACACA AGTGGCCTCT GGCCTCGCAC 22440
ACATTCCACA TCCACCGGTA GGCGCCAACC GGCTCCGTTC TTTGGTGGCC CCTTCGCGCC 22500
ACCTTCTACT CCTCCCCTAG TCAGGAAGTT CCCCCCCGCC CCGCAGCTCG CGTCGTGCAG 22560
GACGTGACAA ATGGAAGTAG CACGTCTCAC TAGTCTCGTG CAGATGGACA GCACCGCTGA 22620
GCAATGGAAG CGGGTAGGCC TTTGGGGCAG CGGCCAATAG CAGCTTTGCT CCTTCGCTTT 22680
CTGGGCTCAG AGGCTGGGAA GGGGTGGGTC CGGGGGCGGG CTCAGGGGCG GGCTCAGGGG 22740
CGGGGCGGGC GCCCGAAGGT CCTCCGGAGG CCCGGCATTC TGCACGCTTC AAAAGCGCAC 22800
GTCTGCCGCG CTGTTCTCCT CTTCCTCATC TCCGGGCCTT TCGACCTGCA GCCTGTTGAC 22860
AATTAATCAT CGGCATAGTA TATCGGCATA GTATAATACG ACAAGGTGAG GAACTAAACC 22920
ATGGGATCGG CCATTGAACA AGATGGATTG CACGCAGGTT CTCCGGCCGC TTGGGTGGAG 22980
AGGCTATTCG GCTATGACTG GGCACAACAG ACAATCGGCT GCTCTGATGC CGCCGTGTTC 23040
CGGCTGTCAG CGCAGGGGCG CCCGGTTCTT TTTGTCAAGA CCGACCTGTC CGGTGCCCTG 23100
AATGAACTGC AGGACGAGGC AGCGCGGCTA TCGTGGCTGG CCACGACGGG CGTTCCTTGC 23160
GCAGCTGTGC TCGACGTTGT CACTGAAGCG GGAAGGGACT GGCTGCTATT GGGCGAAGTG 23220
CCGGGGCAGG ATCTCCTGTC ATCTCACCTT GCTCCTGCCG AGAAAGTATC CATCATGGCT 23280
GATGCAATGC GGCGGCTGCA TACGCTTGAT CCGGCTACCT GCCCATTCGA CCACCAAGCG 23340
AAACATCGCA TCGAGCGAGC ACGTACTCGG ATGGAAGCCG GTCTTGTCGA TCAGGATGAT 23400
CTGGACGAAG AGCATCAGGG GCTCGCGCCA GCCGAACTGT TCGCCAGGCT CAAGGCGCGC 23460
ATGCCCGACG GCGAGGATCT CGTCGTGACC CATGGCGATG CCTGCTTGCC GAATATCATG 23520
GTGGAAAATG GCCGCTTTTC TGGATTCATC GACTGTGGCC GGCTGGGTGT GGCGGACCGC 23580
TATCAGGACA TAGCGTTGGC TACCCGTGAT ATTGCTGAAG AGCTTGGCGG CGAATGGGCT 23640
GACCGCTTCC TCGTGCTTTA CGGTATCGCC GCTCCCGATT CGCAGCGCAT CGCCTTCTAT 23700
CGCCTTCTTG ACGAGTTCTT CTGAGGGGAT CAATTCTCTA GAGCTCGCTG ATCAGCCTCG 23760
ACTGTGCCTT CTAGTTGCCA GCCATCTGTT GTTTGCCCCT CCCCCGTGCC TTCCTTGACC 23820
CTGGAAGGTG CCACTCCCAC TGTCCTTTCC TAATAAAATG AGGAAATTGC ATCGCATTGT 23880
CTGAGTAGGT GTCATTCTAT TCTGGGGGGT GGGGTGGGGC AGGACAGCAA GGGGGAGGAT 23940
TGGGAAGACA ATAGCAGGCA TGCTGGGGAT GCGGTGGGCT CTATGGCTTC TGAGGCGGAA 24000
AGAACCAGCT GGGGCTCGAC TAGAGCTTGC GGAACCCTTC GAAGTTCCTA TTCTCTAGAA 24060
AGTATAGGAA CTTCATCAGT CAGGTACATA ATGGATCTAA CAATGTCTGA AGATTGTAAG 24120
TGCTTTAAGG ATCATACTTT TAATAACTAT CTAGCTTATG GAAATTTAGG GTATCATGAG 24180
TTGATGGCAG TTTTGTTTTT AGCTCAAAAC ATGGATAGAT GGAACCAAAC TCCAGGAGTT 24240
TCATGCACCA AGGAGAAACA CAGGATATCT ACCCAAATAA GTTTGTCTTT TGTGCCTTAC 24300
AACTATGAAG CTCCACATGT TTTGAAAAGA GCACGTCCTA TTTCAACGGT ACAAGAGATT 24360
ACCATTTTTG TGTTTCCTAT GCACTTTCTT GTGGTGTTCC AGAAATTCTC ATTTAATACC 24420
CATCAAGCAG TAAACACAGA CTAACTCTTA ATAAATTATT TGTTCTCTCT CTGAATGACT 24480
TATTTATCTT TTATTTATTC ATTTACAATT TTAGACATAT ATGCATTGGT TATATCCACC 24540
CCAATTTTCC CACTTCCCGG AAAAAACATC TTCACATTCT ATTTCTCTTG TAAATCAGTG 24600
GGCCCACTTA TTGCTTCTGA TTACTGGGAT GTGAACTGAT TTTGTGAATG AATGTTGCAG 24660
CTTGATCATG TGCAGAAATG ATAGCTACAG TGAGTACAAG GACATGGGTC TATGCCATGT 24720
CCAGAAGACA TCATGGCATC TCTCTCCTCC CCACCTCCAG CTCATACATT CTACATTCTC 24780
TTTCATAATG TTCCCTGAAT TGGGAGTTCC ATATAATTGT CTTACTTAGT ACCAAGCATT 24840
AACTAATCAT TTATTTTCAG TCCTTGAAAA GTTATGAGTC TTTGGATCTT TGTAATTTAA 24900
AAGAAATGAC CAGTAAAACC AAAATGGGAA TACCAAGCAA GCAACATCAC ACAAAGGGTG 24960
CAGCAAATGT ACAAACACCA AAACCCACAT ATATTGTGTT TTTTATTAAT CACTCCATTT 25020
GTTTACATCT CAAATGACAT CCTACTCCCT GGTTACCCCT CCACACTCCC CCCCACTCCA 25080
CATCTGCCCT CTCACCCCTA TTCTTTGCCT CTATGAGGTT GCTCACCCAC CCTCTGCTGC 25140
CCCACAACTC CAGCTTCCCC TTACACTGGG GCATCAAGCC ACCACAGGAC CAAGGCATCA 25200
AGGTCATCCT CTGAGATATA TATATATATC TATCTAGCTT GAATGCCTCC CTGTACACTT 25260
CTTGATTGGG GGCCTAGTCC CTGGGAGCAC TGGGTGGTCT GGCCAGCCGA CATTGTTTTT 25320
CCTATGGGGT TGCAATCCCA ATCTGCTCCT CCAGGCCGTC CACAAGCTCC CCCACTGGGG 25380
TCATAGGTTC AGTCTATCAT CATCAAGGCA AGAGCATATC AATGTCCAGA CAGGCATGGT 25440
ATAGGAGGAG CTGAGAGTTC TATACCTGCA TATGAAGGCT GCTAGGAGAA GACTGACTCC 25500
CAAGAAGCTA GGAAAAGGGT ATTTAAGCCC ACACCATAGT GACATACCTA CTCCAAAAGG 25560
CTAGATCCCC TGATAGTGCC ATTTCTTAGG CTAAGCATAT ACAAACTATC AAATTCCACT 25620
CCCTGGACCC ATAGGCTTTT ACTTTCTTTC TGTCTTTTTT TTTTTTAAAG AAGGAAACAA 25680
ACTTATTATG GCTCATAAAT CAAGGTAGAA TCCATCATAG TAGATTAGAA GCAAATAAGA 25740
GGAGATTGAG ACAACAGATA CATTGAATTC TCAGTCAGGA AACAAGAAGA GATGAATATG 25800
TATGCTTATC TTAGTTGTTC CTTTTTGTAT AGTCTAGGAT CCAAGCACTG AGAATGGTGC 25860
TGTGTAGAGT GAGTATATCT TCCCACTTCA ATAATCTAAT CACAATCAAG TTGACACCTG 25920
AGATGAACAA TCAAAATCTA CCCCATGACA AATTCATACT GAAAACCATC ACTTGTAATT 25980
GTAACCTTCT ACTCATCCCC ACCAAAGTCT CTTAGTCATT TTATAATGCA AAAATAATAT 26040
GCACCATCAA GAGTCTATGC GGATCCTCTA GAGTCGAGCA GTGTGGTTTT CAAGAGGAAG 26100
CAAAAAGCCT CTCCACCCAG GCCTGGAATG TTTCCACCCA ATGTCGAGCA GTGTGGTTTT 26160
GCAAGAGGAA GCAAAAAGCC TCTCCACCCA GGCCTGGAAT GTTTCCACCC AATGTCGAGC 26220
AAACCCCGCC CAGCGTCTTG TCATTGGCGA ATTCGAACAC GCAGATGCAG TCGGGGCGGC 26280
GCGGTCCCAG GTCCACTTCG CATATTAAGG TGACGCGTGT GGCCTCGAAC ACCGAGCGAC 26340
CCTGCAGCGA CCCGCTTAAC AGCGTCAACA GCGTGCCGCA GATCTTGGTG GCGTGAAACT 26400
CCCGCACCTC TTCGGCCAGC GCCTTGTAGA AGCGCGTATG GCTTCGTACC CCGGCCATCA 26460
GCACGCGTCT GCGTTCGACC AGGCTGCGCG TTCTCGCGGC CATAGCAACC GACGTACGGC 26520
GTTGCGCCCT CGCCGGCAGC AAGAAGCCAC GGAAGTCCGC CCGGAGCAGA AAATGCCCAC 26580
GCTACTGCGG GTTTATATAG ACGGTCCCCA CGGGATGGGG AAAACCACCA CCACGCAACT 26640
GCTGGTGGCC CTGGGTTCGC GCGACGATAT CGTCTACGTA CCCGAGCCGA TGACTTACTG 26700
GCGGGTGCTG GGGGCTTCCG AGACAATCGC GAACATCTAC ACCACACAAC ACCGCCTTGA 26760
CCAGGGTGAG ATATCGGCCG GGGACGCGGC GGTGGTAATG ACAAGCGCCC AGATAACAAT 26820
GGGCATGCCT TATGCCGTGA CCGACGCCGT TCTGGCTCCT CATATCGGGG GGGAGGCTGG 26880
GAGCTCACAT GCCCCGCCCC CGGCCCTCAC CCTCATCTTC GACCGCCATC CCATCGCCGC 26940
CCTCCTGTGC TACCCGGCCG CGCGATACCT TATGGGCAGC ATGACCCCCC AGGCCGTGCT 27000
GGCGTTCGTG GCCCTCATCC CGCCGACCTT GCCCGGCACA AACATCGTGT TGGGGGCCCT 27060
TCCGGAGGAC AGACACATCG ACCGCCTGGC CAAACGCCAG CGCCCCGGCG AGCGGCTTGA 27120
CCTGGCTATG CTGGCCGCGA TTCGCCGCGT TTACGGGCTG CTTGCCAATA CGGTGCGGTA 27180
TCTGCAGGGC GGCGGGTCGT GGCGGGAGGA TTGGGGACAG CTTTCGGGGA CGGCCGTGCC 27240
GCCCCAGGGT GCCGAGCCCC AGAGCAACGC GGGCCCACGA CCCCATATCG GGGACACGTT 27300
ATTTACCCTG TTTCGGGCCC CCGAGTTGCT GGCCCCCAAC GGCGACCTGT ACAACGTGTT 27360
TGCCTGGGCC TTGGACGTCT TGGCCAAACG CCTCCGTCCC ATGCACGTCT TTATCCTGGA 27420
TTACGACCAA TCGCCCGCCG GCTGCCGGGA CGCCCTGCTG CAACTTACCT CCGGGATGAT 27480
CCAGACCCAC GTCACCACCC CAGGCTCCAT ACCGACGATC TGCGACCTGG CGCGCACGTT 27540
TGCCCGGGAG ATGGGGGAGG CTAACTGAAA CACGGAAGGA GACAATACCG GAAGGAACCC 27600
GCGCTATGAC GGCAATAAAA AGACAGAATA AAACGCACGG GTGTTGGGTC GTTTGTTCAT 27660
AAACGCGGGG TTCGGTCCCA GGGCTGGCAC TCTGTCGATA CCCCACCGAG ACCCCATTGG 27720
GGCCAATACG CCCGCGTTTC TTCCTTTTCC CCACCCCACC CCCCAAGTTC GGGTGAAGGC 27780
CCAGGGCTCG CAGCCAACGT CGGGGCGGCA GGCCCTGCCA TAGCCACGGG CCCCGTGGGT 27840
TAGGGACGGG GTCCCCCATG GGGAATGGTT TATGGTTCGT GGGGGTTATT ATTTTGGGCG 27900
TTGCGTGGGG TCAGTCCACG ACTGGACTGA GCAGACAGAC CCATGGTTTT TGGATGGCCT 27960
GGGCATGGAC CGCATGTACT GGCGCGACAC GAACACCGGG CGTCTGTGGC TGCCAAACAC 28020
CCCCGACCCC CAAAAACCAC CGCGCGGATT TCTGGCGCCG CCGGACGAAC TAAACCTGAC 28080
TACGGCATCT CTGCCCCTTC TTCGCTGGTA CGAGGAGCGC TTTTGTTTTG TATTGGTCAC 28140
CACGGCCGAG TTTCCGCGGG ACCCCGGCCA GATCAAGCTA GCTTATCGAT ACCGTCGACC 28200
TCGAGGGGGG GCCCGGTACC AGCTTTTGTT CCCTTTAGTG AGGGTTAATT TCGAGCTTGG 28260
CGTAATCATG GTCATAGCTG TTTCCTGTGT GAAATTGTTA TCCGCTCACA ATTCCACACA 28320
ACATACGAGC CGGAAGCATA AAGTGTAAAG CCTGGGGTGC CTAATGAGTG AGCTAACTCA 28380
CATTAATTGC GTTGCGCTCA CTGCCCGCTT TCCAGTCGGG AAACCTGTCG TGCCAGCTGC 28440
ATTAATGAAT CGGCCAACGC GCGGGGAGAG GCGGTTTGCG TATTGGGCGC TCTTCCGCTT 28500
CCTCGCTCAC TGACTCGCTG CGCTCGGTCG TTCGGCTGCG GCGAGCGGTA TCAGCTCACT 28560
CAAAGGCGGT AATACGGTTA TCCACAGAAT CAGGGGATAA CGCAGGAAAG AACATGTGAG 28620
CAAAAGGCCA GCAAAAGGCC AGGAACCGTA AAAAGGCCGC GTTGCTGGCG TTTTTCCATA 28680
GGCTCCGCCC CCCTGACGAG CATCACAAAA ATCGACGCTC AAGTCAGAGG TGGCGAAACC 28740
CGACAGGACT ATAAAGATAC CAGGCGTTTC CCCCTGGAAG CTCCCTCGTG CGCTCTCCTG 28800
TTCCGACCCT GCCGCTTACC GGATACCTGT CCGCCTTTCT CCCTTCGGGA AGCGTGGCGC 28860
TTTCTCATAG CTCACGCTGT AGGTATCTCA GTTCGGTGTA GGTCGTTCGC TCCAAGCTGG 28920
GCTGTGTGCA CGAACCCCCC GTTCAGCCCG ACCGCTGCGC CTTATCCGGT AACTATCGTC 28980
TTGAGTCCAA CCCGGTAAGA CACGACTTAT CGCCACTGGC AGCAGCCACT GGTAACAGGA 29040
TTAGCAGAGC GAGGTATGTA GGCGGTGCTA CAGAGTTCTT GAAGTGGTGG CCTAACTACG 29100
GCTACACTAG AAGAACAGTA TTTGGTATCT GCGCTCTGCT GAAGCCAGTT ACCTTCGGAA 29160
AAAGAGTTGG TAGCTCTTGA TCCGGCAAAC AAACCACCGC TGGTAGCGGT GGTTTTTTTG 29220
TTTGCAAGCA GCAGATTACG CGCAGAAAAA AAGGATCTCA AGAAGATCCT TTGATCTTTT 29280
CTACGGGGTC TGACGCTCAG TGGAACGAAA ACTCACGTTA AGGGATTTTG GTCATGAGAT 29340
TATCAAAAAG GATCTTCACC TAGATCCTTT TAAATTAAAA ATGAAGTTTT AAATCAATCT 29400
AAAGTATATA TGAGTAAACT TGGTCTGACA GTTACCAATG CTTAATCAGT GAGGCACCTA 29460
TCTCAGCGAT CTGTCTATTT CGTTCATCCA TAGTTGCCTG ACTCCCCGTC GTGTAGATAA 29520
CTACGATACG GGAGGGCTTA CCATCTGGCC CCAGTGCTGC AATGATACCG CGAGACCCAC 29580
GCTCACCGGC TCCAGATTTA TCAGCAATAA ACCAGCCAGC CGGAAGGGCC GAGCGCAGAA 29640
GTGGTCCTGC AACTTTATCC GCCTCCATCC AGTCTATTAA TTGTTGCCGG GAAGCTAGAG 29700
TAAGTAGTTC GCCAGTTAAT AGTTTGCGCA ACGTTGTTGC CATTGCTACA GGCATCGTGG 29760
TGTCACGCTC GTCGTTTGGT ATGGCTTCAT TCAGCTCCGG TTCCCAACGA TCAAGGCGAG 29820
TTACATGATC CCCCATGTTG TGCAAAAAAG CGGTTAGCTC CTTCGGTCCT CCGATCGTTG 29880
TCAGAAGTAA GTTGGCCGCA GTGTTATCAC TCATGGTTAT GGCAGCACTG CATAATTCTC 29940
TTACTGTCAT GCCATCCGTA AGATGCTTTT CTGTGACTGG TGAGTACTCA ACCAAGTCAT30000
TCTGAGAATA GTGTATGCGG CGACCGAGTT GCTCTTGCCC GGCGTCAATA CGGGATAATA 30060
CCGCGCCACA TAGCAGAACT TTAAAAGTGC TCATCATTGG AAAACGTTCT TCGGGGCGAA 1 30120
AACTCTCAAG GATCTTACCG CTGTTGAGAT CCAGTTCGAT GTAACCCACT CGTGCACCCA 30180
ACTGATCTTC AGCATCTTTT ACTTTCACCA GCGTTTCTGG GTGAGCAAAA ACAGGAAGGC 30240
AAAATGCCGC AAAAAAGGGA ATAAGGGCGA CACGGAAATG TTGAATACTC ATACTCTTCC 30300
TTTTTCAATA TTATTGAAGC ATTTATCAGG GTTATTGTCT CATGAGCGGA TACATATTTG 30360
AATGTATTTA GAAAAATAAA CAAATAGGGG TTCCGCGCAC ATTTCCCCGA AAAGTGCCAC 30420
< 210>9
< 211>19
< 212> DNA
< 213>引物Kana-193782-F
<400>9
GAAAATGGCC GCTTTTCTG
< 210>10
< 211> 20
< 212> DNA
< 213>引物F480-195458-R
<400>10
CACAGCACCA TTCTCAGTGC
< 210>11
< 211> 20
< 212> DNA
< 213>引物GHpA-2452-F
<400>11
TGGGCTCTAT GGCTTCTGAG
< 210>12
< 211> 22
< 212> DNA
< 213>引物F480-15091-R
<400>12
TCCCCTGATT CTATTCTTCC AC
Claims (6)
1.一种打靶载体，其特征在于：所述靶向打靶载体序列如SEQ ID NO.8所示，命名为F4/80 5HA L-IRES-DTR-PGK-EM7-NEO-F4/80 3HA L-MCL-HSV TK载体。
2.一种用于构建如权利要求1所述打靶载体的提取载体，其特征在于：所述提取载体序列如SEQ ID NO.7所示，命名为载体retrieval 5HA-retrieval 3HA-MCL-HSV TK。
3.一种提取载体的制备方法，其特征在于，所述方法包括如下步骤：
(1)以RP23-212F14 BAC DNA为模板，利用序列如SEQ ID NO.1所示的正向引物和序列如SEQ ID NO.2所示的反向引物进行PCR，扩增出序列如SEQ ID NO.3所示的基因片段F4/805’侧同源臂；以RP23-212F14 BAC DNA为模板，利用序列如SEQ ID NO.4所示的正向引物和序列如SEQ ID NO.5所示的反向引物进行PCR，扩增出序列如SEQ ID NO.6所示的基因片段F4/80 3’侧同源臂；
(2)将retrieval 5HA和retrieval 3HA克隆至带阴性选择标记的载体MCL-HSV TK，得到序列如SEQ ID NO.7所示的打靶载体retrieval 5HA-retrieval 3HA-MCL-HSV TK。
4.一种打靶载体的制备方法，其特征在于：所述方法包括如下步骤：
(1)电穿孔法将质粒pRED/ET转化入BAC Clone F4/80-IRES-DTR-PGK-EM7-Neo，并用L-阿拉伯糖诱导Red/ET表达；
(2)电穿孔法将利用SpeI酶切线性化的retrieval 5HA-retrieval 3HA-MCL-HSV TK提取载体转化入上述表达RED/ET的BAC Clone F4/80-IRES-DTR-PGK-EM7-Neo，同源重组得到序列如SEQ ID NO.8所示的带有F4/80 5’同源臂(～20kb)和3’同源臂(～2kb)的打靶载体，命名为打靶载体F4/80 5HA L-IRES-DTR-PGK-EM7-NEO-F4/80 3HA L-MCL-HSV TK；
(3)挑选单克隆，利用序列如SEQ ID NO.9所示的正向引物和序列如SEQ ID NO.10所示的反向引物作菌落PCR，成功提取IRES-DTR-PGK-EM7-NEO和F4/80同源臂的载体能够得到2341bp的条带。
5.一种打靶载体在靶向整合外源基因IRES-DTR至F4/80外显子22位点，构建白喉毒素调控清除巨噬细胞的F4/80-DTR转基因小鼠的应用。
6.根据权利要求5所述的构建白喉毒素调控清除巨噬细胞的F4/80-DTR转基因小鼠的应用，其特征在于，所述转基因小鼠的构建方法包括以下步骤：
(1)构建靶向整合外源基因IRES-DTR至F4/80外显子22位点构建小鼠胚胎干细胞株；
(1.1)电穿孔法将利用BamHI酶切线性化的打靶载体F4/80 5HA L-IRES-DTR-PGK-EM7-NEO-F4/80 3HA L-MCL-HSV TK转入ES细胞；
(1.2)利用G418阳选和ganciclovir阴选，筛选选得到靶向插入IRES-DTR-PGK-EM7-NEO至F4/80外显子22位点的Balb/c ES细胞克隆；
(1.3)提取ES细胞基因组DNA为模板，利用序列为SEQ ID NO.11的正向引物和序列为SEQ ID NO.28的反向引物进行PCR鉴定，靶向插入IRES-DTR-PGK-EM7-NEO至F4/80外显子12位点的ES细胞阳性克隆可见约2.2kb条带；
(2)靶向插入IRES-DTR至F4/80外显子22位点的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；
(3)提取转基因小鼠鼠尾DNA作为模板，利用序列为SEQ ID NO.27的正向引物和序列为SEQ ID NO.28的反向引物作PCR鉴定，带有IRES-DTR的阳性小鼠可见约2.2kb条带；
(4)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，得到毛色为白色的小鼠可能为F4/80-DTR转基因杂合小鼠。小白鼠经鼠尾PCR鉴定后得到F4/80-DTR转基因小鼠。
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| CN111607614A (zh) * | 2020-05-22 | 2020-09-01 | 乾元康安(苏州)生物科技有限公司 | 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 |
| CN111909959A (zh) * | 2020-07-17 | 2020-11-10 | 中国人民解放军陆军军医大学第二附属医院 | 一种条件性Yap1基因敲入小鼠的构建方法和应用 |
| CN111909959B (zh) * | 2020-07-17 | 2021-09-07 | 中国人民解放军陆军军医大学第二附属医院 | 一种条件性Yap1基因敲入小鼠的构建方法和应用 |
| CN114645042A (zh) * | 2020-12-18 | 2022-06-21 | 中国食品药品检定研究院 | 一种构建具有表达荧光素酶和红色荧光蛋白的重组巨噬细胞的转基因小鼠的方法 |
| CN113439711A (zh) * | 2021-07-01 | 2021-09-28 | 中国医科大学 | 一种改造的巨噬细胞及其应用 |
| 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 |
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