CN112852869A - 一种构建四肢畸形小鼠模型的方法 - Google Patents
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Abstract
本发明提供了一种构建四肢畸形小鼠模型的方法，利用条件性基因打靶Cre/LoxP系统，将Hnrnpk基因两侧带有同向LoxP位点的标记小鼠，与在组织特异性Prx1的增强子控制下表达Cre重组酶的Prx1‑Cre小鼠进行杂交，筛选，获得四肢畸形小鼠模型；所述两个同向的LoxP位点分别位于Hnrnpk基因的4～7号外显子两侧，以Hnrnpk基因的4～7号外显子作为敲除区域。本发明四肢畸形的小鼠模型的构建方法简便、经济，在筛选治疗四肢畸形药物的中具有极大的前景。
Description
技术领域
本发明涉及动物模型技术领域，具体涉及一种构建四肢畸形小鼠模型的方法。
背景技术
据统计，四肢畸形是人类第二大常见的先天性畸形疾病，约占出生缺陷疾病的2％，该类疾病的病理基础来源于胚胎时期肢芽发育出现了异常改变，然而肢芽发育过程中的具体分子调控机制尚未完全阐明。肢芽发育的早期(胚胎期11.5之前)是以间充质细胞的增殖为主，而该过程发生异常则会导致肢体出现缺失、少趾等常见疾病的发生。
脊椎动物四肢骨骼起源于胚胎的侧板中胚层，胚胎肢芽(Limb bud)开始出现于人胚胎发育的26天左右和小鼠E9.5左右。肢芽细胞来自于中胚层的间质细胞，是一类多能干细胞，称为间质祖细胞(MPCs)。肢芽出现后，肢芽内的MPCs开始增殖、聚集、逐渐分化形成软骨原基，随后软骨细胞经历增殖、肥大、血管入侵及骨化形成骨，这些MPCs也被称为骨软骨祖细胞或骨骼干细胞。在此过程中，MPCs的增殖和存活是四肢骨发育的始动和核心环节。研究表明，肢体的大小由MPCs的数量，以及控制MPCs的外部信号决定，而MPCs的数量取决于调控细胞增殖与凋亡平衡的分子网络。在肢芽发育早期(小鼠E9.5-E11.5)，未分化的MPCs被外胚层覆盖，表达FGF10，诱导外胚层增厚形成顶端外胚层嵴(Apical Ectodermal Ridge，AER)，AER区反之分泌FGF8、FGF2、BMP-2、BMP-4、MSX2和MSX2，启动MPCs的增殖。在肢芽的后缘，会形成极化区(Zone of Polarizing Activity,ZPA)，该ZPA区通过分泌Sonichedgehog(Shh)和BMP4调控肢体前后轴(拇指到小指轴)的发育。Shh被证明通过刺激MPCs增殖和调节顶端外胚层脊的前后长度来控制肢芽的宽度。除了这些调控细胞增殖的分子，凋亡调控分子p19,p21和RIP5也发现在AER区及AER外间质区表达，说明这些分子在调控肢芽细胞的凋亡中扮演重要角色。细胞周期抑制因子p21缺陷小鼠已证明出现MPCs的增殖抑制。在小鼠E12.5的时候，增殖的MPCs聚集，表达BMP和Sox9等信号分子/转录因子启动MPCs的成软骨分化，形成软骨原基。此外Wnt、Notch信号分子、Hox转录调控分子等家族成员之间相互作用的复杂网络在调控MPCs的增殖及转归中也十分重要。这些调控MPCs命运的关键因子的功能缺失或调控失衡均可影响随后的软骨内骨化导致四肢发育畸形。然而调控MPCs的分子机制非常复杂，仍然有许多参与调控的蛋白未被发现和证明。
hnRNPK首次发现于hnRNP复合物，是进化上高度保守的蛋白质，HNRNPK基因位于人9q21.32。hnRNPK含有3个KH结构域，其中相邻的2个KH结构域，KH1和KH2位于氨基端，而KH3位于羧基端16；此外，hnRNPK还含有一个核定位信号、核穿梭结构域17。通过这些结构域，hnRNPK可与RNA和DNA结合发挥多种功能。研究表明hnRNPK可以作为转录因子，参与基因表达的转录调控过程；同时hnRNPK可通过KH基序与核酸结合介导mRNA稳定性、剪接和翻译，hnRNPK还可以稳定染色质的结构。通过这些功能hnRNPK参与了细胞的周期调控、增殖、凋亡和分化。hnRNPK对于胚胎形成和发育至关重要，其等位基因的完全缺失将导致小鼠胚胎死于E13.5之前，单倍体剂量不足将导致部分新生致死以及新生小鼠的发育缺陷。
然而hnRNPK的研究多集中在肿瘤方面，在骨骼发育中的作用报导较少，只有少量体外研究。研究表明，hnRNPK通过抑制转录复合物与骨钙素启动子结合，抑制骨钙素基因的转录。Fan X等发现在RANKL诱导的破骨细胞分化途径中，hnRNPK发挥着重要功能。Shen HY等研究表明，在骨髓间充质干细胞向成骨细胞分化的过程中，hnRNPK能够与LncRNA-OG相互作用并正性调控BMP信号通路促进间充质干细胞的成骨分化。临床病例研究表明，hnRNPK的错义突变会导致hnRNPK失功能，从而导致Au-Kline综合征的发生，患者表现出智力障碍、特征面部、心脏、泌尿生殖系统及远端肢体发育异常。本课题组通过前期收集的I型轴前多指家系研究首次发现hnRNPK能够结合LMBR1基因突变区及shh启动子，调控shh在肢芽的异位表达，参与I型轴前多指发生。然而hnRNPK是否调控MPCs的增殖、存活，参与四肢骨骼发育畸形的研究目前未见报导。
为了进一步研究hnRNPK在肢芽细胞中的作用及其机制，需要构建转基因小鼠对hnRNPK进行敲除，但是由于hnRNPK的全身敲除会导致小鼠在胚胎发育早期致死，无法生存至发育后期，会影响肢芽发育的观察，因此构建一类能够影响肢芽早期功能，而又不使之胚胎早期死亡的动物模型，成为本领技术人员亟待解决的问题。
发明内容
为了解决上述问题，本发明通过条件性基因敲除的方式利用Cre/loxP重组系统在小鼠Hnrnpk基因高度保守的4-7号外显子两侧插入LoxP序列，构建了Hnrnpkfl/fl小鼠，并验证发现利用Cre工具小鼠可对该基因进行高效敲除。同时利用Prx1-Cre工具小鼠特异性地在肢芽组织中敲除Hnrnpk，构建了四肢畸形的小鼠模型，小鼠表现为上肢缺失及下肢少趾的表现。
本发明通过以下技术方案实现发明目的：
一种构建四肢畸形小鼠模型的方法，利用条件性基因打靶Cre/LoxP系统，将Hnrnpk基因两侧带有同向LoxP位点的标记小鼠，与在组织特异性Prx1的增强子控制下表达Cre重组酶的Prx1-Cre小鼠进行杂交，筛选，获得四肢畸形小鼠模型；所述两个同向的LoxP位点分别位于Hnrnpk基因的4～7号外显子两侧，以Hnrnpk基因的4～7号外显子作为敲除区域。
本发明人之所以将LoxP位点分别设置于Hnrnpk基因的4～7号外显子两侧，是因为第一个外显子前面一般是启动子，放置LoxP序列有可能会破坏或改变启动子活性，造成下游基因不能正常表达。在选择要敲除的外显子的时候(在一个外显子的两侧各放一个LoxP)，该外显子的碱基数目不能是3N，否则新基因pre-RNA拼接得到的mRNA不能产生移码突变，会产生一个与野生蛋白相比少了一段中间序列的新蛋白。如果一个外显子的碱基数目是3N+1或3N+2，敲除这个外显子之后会产生移码突变，就可以达到基因敲除的目的。因此，筛选要敲除(Floxed)的外显子的时候，一般是从最上游的外显子开始筛选适合敲除的外显子。因此，本发明将LoxP位点分别设置于Hnrnpk基因的4～7号外显子两侧才能更好的敲除Hnrnpk基因，而不影响其他基因的表达。
进一步地，所述构建四肢畸形小鼠模型的方法的具体步骤如下：
(1)构建标记的小鼠：通过同源重组的方法构建Hnrnpk基因两侧带有同向LoxP位点的标记小鼠；
(2)构建Cre小鼠：构建在组织特异性Prx1的增强子作用下表达Cre重组酶的Cre小鼠；
(3)将标记小鼠与Cre小鼠杂交，筛选，获得敲除Hnrnpk基因的纯合子小鼠即为四肢畸形小鼠模型。
进一步地，所述步骤(1)构建标记的小鼠的具体方法为：根构建含有同源序列、目的序列、DTA、LoxP、Neo、Amp抗性以及多个限制性酶切位点的质粒，所述质粒的核苷酸如SEQID NO:1所示。本发明构建的质粒同时包含了同源序列、目的序列、DTA、LoxP、Neo、Amp抗性以及多个限制性酶切位点，在转化细胞的过程中，只需将该线性质粒注射如胚胎干细胞中即可，操作简单方便。
进一步地，所述Prx1的增强子序列如SEQ ID NO:2所示。本发明的Prx1只在肢芽细胞的间质细胞及颅顶成骨细胞中特异性的表达，而不能在其他组织细胞中表达其下游基因，具有一定的特异性。
本发明还提供了由本发明构建四肢畸形小鼠模型的方法制备获得的四肢畸形小鼠模型在制备或筛选治疗四肢畸形药物中的应用。
本发明还提供了提高Hnrnpk基因表达水平的制剂或药物在制备提高肢芽间质祖细胞活性或该细胞增殖或延缓该细胞凋亡的制剂或药物中的应用。
本发明的有益效果为：本发明提供一种简便、经济四肢畸形的小鼠模型。由于全部Hnrnpk基因的敲除会引起小鼠在出生后不久死亡，因而，极大的限制了其在药物筛选中的应用。同时，在构建四肢畸形的小鼠模型的过程中，还发现了Hnrnpk基因与肢芽内的间质祖细胞(MPCs)之间的关系，Hnrnpk基因能够抑制MPCs增殖、促进MPCs的凋亡以及降低该细胞的活性，从而造成胚胎发育过程中四肢畸形。
附图说明
图1为本发明构建的质粒结构示意图；
图2为质粒转入ES细胞之后的PCR筛选结果示意图；
图3为鉴定基因型时的DNA凝胶电泳图；
图4为基因敲除小鼠的敲除效率检测以及发育畸形表现示意图；
图5为Hnrnpk敲除影响小鼠肢芽MPCs的增殖凋亡示意图；
图6为Hnrnpk调控肢芽MPCs的分子信号通路富集示意图。
具体实施方式
为了更加简洁明了的展示本发明的技术方案、目的和优点，下面结合具体实施例和附图详细说明本发明的技术方案。
实施例1构建四肢畸形小鼠模型
步骤一：构建Hnrnpkfl/fl小鼠，具体步骤如下：
A.设计Hnrnpk基因靶位点：从NCBI上获取小鼠Hnrnpk基因序列，选取高度保守的4～7号外显子作为敲除区域。构建合成含有同源序列、目的序列、DTA、LoxP、Neo、Amp抗性以及多个限制性酶切位点的质粒，所述质粒的核苷酸如SEQID NO:1所示，其结构如图1所示；
B.利用相应限制性内切酶检测质粒序列的正确性；
C.利用电转的方式将上述线性质粒注射到C57BL/6小鼠的胚胎干细胞中；
D.根据氨苄霉素抗性作用进行药筛，得到耐药克隆，再针对耐药克隆进行PCR筛选，找出序列正确的受精卵，针对两个区域进行PCR，挑选两个区域PCR结果都是正确的干细胞。再次利用特定的限制性内切酶位点对上述挑选的结果再次进行酶切，然后利用Southern blot进行分析，条带正确者即为目标克隆；
E.将上述筛选正确的胚胎干细胞通过显微注射的方法进入小鼠成纤维细胞中，诱导长成嵌合体小鼠，之后将其与野生型小鼠进行交配，后代剪取鼠尾进行PCR验证(表1)，挑选杂合子小鼠进行传代。
表1验证过程中使用到的PCR引物序列以及扩增结果：
由图3和表1可以看出，实验结果表明在小鼠Hnrnpk基因的4-7号外显子两侧成功插入Loxp序列，该小鼠会在Cre重组酶作用下会将该段基因序列敲除。
步骤二：将Prx1-Cre小鼠与Hnrnpkfl/fl小鼠进行杂交，具体步骤如下：
本实施例中的使用的Prx1-Cre小鼠购自Jackson实验室，该小鼠的Cre重组酶表达序列位于组织特异性Prx1的增强子控制之下，所述Prx1增强子的核苷酸序列如SEQ ID NO:2所示。
A.由于Prx1会在雌鼠卵巢中表达影响后代基因敲除位置，所以选用雄性Prx1-Cre小鼠。使用基因型为Hnrnpkfl/fl的雌鼠与基因型为Prx1-Cre的雄鼠杂交，再用其后代中基因型为Prx1-Cre；Hnrnpkfl/+的雄鼠与Hnrnpkfl/fl的雌鼠杂交，以产生含有基因型为Prx1-Cre；Hnrnpkfl/fl的小鼠；
B.PCR验证Prx1-Cre及Hnrnpkfl/fl的序列正确性，剪下10-100mg小鼠组织，利用1:50的Protease Plus溶液进行消化提取DNA，分别加入Prx1-Cre引物和Hnrnpk-loxp引物(表1)，之后使用3％琼脂凝胶进行DNA电泳，使用紫外灯曝光，验证基因型。
C.Prx1-Cre；Hnrnpkfl/+的雄鼠与Hnrnpkfl/fl的雌鼠合笼后，以见阴道栓为E0.5，分离E10.5胚胎前后肢芽的RNA和蛋白，qPCR及Western印记检测hnRNPK的表达，以确定Cre酶的重组效率。
步骤三：观察CKO小鼠的表型以及发育异常的始发阶段
利用大体观察、骨骼染色及组织切片染色等方法观察不同发育阶段CKO小鼠(即本发明构建的条件性基因敲除小鼠)表型的差异，具体步骤如下：
A.统计CKO小鼠出生的比例，推测其是否符合孟德尔遗传定律；
B.取E10.5，E11.5，E12.5，E16.5，E18.5胎鼠及P0的WT与CKO胎鼠/小鼠，称体重，体视显微镜观察小鼠的体型大小，观察是否出现四肢畸形及其他骨骼异常；
C.取E18.5及P0的WT与CKO的同窝胎鼠/小鼠进行全胚骨骼染色(阿利新蓝-茜素红染色)，并比较不同基因型小鼠之间的四肢骨骼组成、长度、宽度及形态的区别；
D.分离E10.5，E11.5，E12.5，E13.5，E16.5，E18.5 WT与CKO的同窝胎鼠/小鼠的肢芽/胫骨，石蜡包埋切片，进行HE、阿利新蓝/茜素红染色，观察并比较不同发育阶段CKO小鼠与同窝其它基因型小鼠之间的细胞组织分布、生长板及骨化中心差异；对E12.5肢芽进行Sox9染色及原位杂交，观察成软骨分化差异。
结果如图4a所示，通过PCR以及凝胶电泳的方法确定所取小鼠基因型，，在Cre的作用下杂合子小鼠可扩增出两条带，说明Cre酶敲除了其中一条染色体上的Hnrnpk；而纯合子小鼠由于两条染色体上的Hnrnpk基因均带有flox序列，在Cre的作用到两条染色体的Hnrnpk都被敲除，因此扩增出一条条带。如图4b、4c所示：Western结果表明，与野生型小鼠相比，CKO小鼠的Hnrnpk表达水平较低，qPCR结果表明，在Hnrnpk条带位置CKO小鼠扩增出的Hnrnpk较低，说明Cre重组酶敲除效率合格。如图4d所示，与野生小鼠相比，CKO小鼠肢芽发育过程中，肢芽畸形自E11.5之后开始出现，表现为P0天CKO小鼠出现上肢缺失，下肢严重短小，枕骨缺如，小鼠软骨内骨化过程异常，以及肢芽细胞成软骨分化减弱，未出现正常指/趾结构。如图4e、4f、4g所示，通过骨骼染色、阿辛蓝染色以及Sox9染色，显示了CKO小鼠出现上肢缺失，下肢严重短小，枕骨缺如，小鼠软骨内骨化过程异常，以及肢芽细胞成软骨分化减弱，未出现正常指/趾结构。
上述结果表明通过本发明方法表明成功的构建了四肢畸形小鼠，且在胚胎发育过程中没有出现胚胎死亡现象，可完整的观察到Hnrnp基因对于四肢发育的影响过程。
实施例2
Hnrnpk敲除影响小鼠肢芽MPCs的增殖凋亡
由实施例1的结果表明发育异常起始于E11.5，因该阶段肢芽的MPCs正处在以增殖为主的改变，因此Hnrnpk的敲除可能是影响到了体内肢芽MPCs的增殖凋亡过程，因此，为了验证这个猜测，发明人进行了如下实验，其具体步骤如下：
A.利用免疫荧光染色的方法对增殖关键分子(Ki67、PCNA)进行染色：
分离E10.5 WT与CKO的同窝胎鼠肢芽，并利用0.25％胰酶进行消化，使肢芽MPCs分散，并种植在低糖DMEM培养基中；贴壁后刮取细胞提取RNA及蛋白质，利用qPCR及Western验证Ki67和PCNA的表达量；
B.CCK8实验测量MPCs的增殖效率：
分离E10.5 WT与CKO的同窝胎鼠肢芽，并利用0.25％胰酶进行消化，待细胞贴壁之后消化，按照10000细胞/孔的密度种植在96孔板中，培养第0、1、2、3、4、5、6天后加入10％CCK8孵育3小时后进行450nm波长的光谱测量，观察细胞增殖情况；
C.利用免疫荧光染色的方法对凋亡关键分子(Caspase3-cleaved、Tunel)进行染色：
分离E10.5 WT与CKO的同窝胎鼠肢芽，并利用0.25％胰酶进行消化，使肢芽MPCs分散，并种植在低糖DMEM培养基中；贴壁后刮取细胞提取RNA及蛋白质，利用qPCR及Western验证Caspase3-cleaved和Tunel的表达量。
结果如图5所示，CKO小鼠在10.5与11.5时期，其增殖关键分子Ki67和PCNA表达量均低于野生小鼠，其凋亡关键分子Caspase3-cleaved和Tunel表达量均高于野生小鼠；在CCK8实验中，其OD值也显著低于野生小鼠。这些结果表明在肢芽发育早期，CKO小鼠就出现肢芽MPCs的增殖受限以及凋亡异常增加的现象，说明敲除Hnrnpk基因会抑制小鼠肢芽MPCs的增殖以及增加肢芽MPCs的凋亡，降低MPCs的细胞活性。
实施例3
Hnrnpk调控肢芽MPCs的分子信号通路
为了验证hnRNPK调控肢芽MPCs增殖调亡的分子机制，发明人利用RNA高通量测序结合生物信息学分析的方法，对WT与CKO同窝胎鼠肢芽MPCs表达谱差异通路进行富集，并体外进行验证，具体步骤如下：
A.提取E10.5时期的WT与CKO胎鼠三个生物学重复的肢芽MPCs的RNA，Nanodrop检测RNA的纯度(OD260/280)、浓度及核酸吸收峰是否正常，并用Agilent 2100精确检测RNA的完整性；
B.样品检测合格后，富集mRNA并进行文库构建，之后使用Qubit2.0进行初步定量，使用Agilent 2100对文库的insert size进行检测，完成文库质检；对目标下机数据量进行pooling，用IluminaHiSeq平台进行测序并进行GO分析及KEGG分析，富集表达差异通路；
C.提取E10.5时期WT与CKO小鼠肢芽细胞RNA及蛋白质；对RNA逆转录合成cDNA进行qPCR，验证Hnrnpk、Mdm2、p53、p21、Bcl2、Bax、Puma、Ptprv的表达量的改变，以Gapdh的表达量为内参；对蛋白质进行Western印记，检测蛋白hnRNPK、p53、p21、PCNA、BAX、BCL2其表达量的改变，以GAPDH的表达量为内参。
结果如图6所示，利用高通量测序及生物信息学分析，可富集出改变最为明显的是p53通路，该通路在CKO小鼠肢芽中异常激活，导致肢芽MPCs的增殖受抑制以及凋亡增加。
以上所述实施例仅表达了本发明的几种实施方式，其描述较为具体和详细，但并不能因此而理解为对发明专利范围的限制。应当指出的是，对于本领域的普通技术人员来说，在不脱离本发明构思的前提下，还可以做出若干变形和改进，这些都属于本发明的保护范围。因此，本发明专利的保护范围应以所附权利要求为准。
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<110> 中山大学附属第一医院中山大学
<120> 一种构建四肢畸形小鼠模型的方法
<130> 1.7
<160> 2
<170> PatentIn version 3.3
<210> 1
<211> 17138
<212> DNA
<213> 合成
<400> 1
cgcttacaat ttccattcgc cattcaggct gcgcaactgt tgggaagggc gatcggtgcg 60
ggcctcttcg ctattacgcc agctggcgaa agggggatgt gctgcaaggc gattaagttg 120
ggtaacgcca gggttttccc agtcacgacg ttgtaaaacg acggccagtg aattgtaata 180
cgactcacta tagggcgaat tggagctcca ccgcccgggc tggttctttc cgcctcagaa 240
gccatagagc ccaccgcatc cccagcatgc ctgctattgt cttcccaatc ctcccccttg 300
ctgtcctgcc ccaccccacc ccccagaata gaatgacacc tactcagaca atgcgatgca 360
atttcctcat tttattagga aaggacagtg ggagtggcac cttccagggt caaggaaggc 420
acgggggagg ggcaaacaac agatggctgg caactagaag gcacagtcga ggctgatcag 480
cgagctctag gatctgcatt ccaccactgc tcccattcat cagttccata ggttggaatc 540
taaaatacac aaacaattag aatcagtagt ttaacacatt atacacttaa aaattttata 600
tttaccttag agctttaaat ctctgtaggt agtttgtcca attatgtcac accacagaag 660
taaggttcct tcacaaagag atcgcctgac acgatttcct gcacaggctt gagccatata 720
ctcatacatc gcatcttggc cacgttttcc acgggtttca aaattaatct caagttctac 780
gcttaacgct ttcgcctgtt cccagttatt aatatattca acgctagaac tcccctcagc 840
gaagggaagg ctgagcacta cacgcgaagc accatcaccg aaccttttga taaactcttc 900
cgttccgact tgctccatca acggttcagt gagacttaaa cctaactctt tcttaatagt 960
ttcggcatta tccactttta gtgcgagaac cttcgtcagt cctggatacg tcactttgac 1020
cacgcctcca gcttttccag agagcgggtt ttcattatct acagagtatc ccgcagcgtc 1080
gtatttattg tcggtactat aaaacccttt ccaatcatcg tcataatttc cttgtgtacc 1140
agattttggc ttttgtatac ctttttgaat ggaatctaca taaccaggtt tagtcccgtg 1200
gtacgaagaa aagttttcca tcacaaaaga tttagaagaa tcaacaacat catcaggatc 1260
catggcacgc gcttctacaa ggcgctggcc gaagaggtgc gggagtttca cgccaccaag 1320
atctgcggca cgctgttgac gctgttaagc gggtcgctgc agggtcgctc ggtgttcgag 1380
gccacacgcg tcaccttaat atgcgaagtg gacctcggac cgcgccgccc cgactgcatc 1440
tgcgtgttcg aattcgccaa tgacaagacg ctgggcgggg tttgctcgac attgggtgga 1500
aacattccag gcctgggtgg agaggctttt tgcttcctct tgcaaaacca cactgctcga 1560
cattgggtgg aaacattcca ggcctgggtg gagaggcttt ttgcttcctc ttgaaaacca 1620
cactgctcga tttgttagca gcctcgaatc aacccgggcg atcctaggcg atgagatcta 1680
gctgtcgcga ttaattaata cgcttgcctt taatccctgc tttgggagga cgaggcaggt 1740
ggatctcttg agagaccctc cgggtcaaaa accaccatat gaaaaatctt gagccaggag 1800
tgggggcaca cgcttttaat gccagcgttt gagaggcagc ggcaggagat ctacaggatc 1860
ccacgaaagc cccagccaca gtctgggcta cttctgcact agtcagttgc tgtcaggtaa 1920
tcagaaagga tgcgagaggc tcgaccccct ccgctccacc agtgtaacat cctcggagcg 1980
gagcgggacg ggcgggtcgc gctggctgga gcggggtcgg acgccgtagt cggctgtccg 2040
gggtggcgtt cggggcggcc gtggcccgcc ggggaacgtt gcccgggttc ccctcccccc 2100
accgccgcca tttccccgag cgtgcccctg cggttggcgt gccggcgcta ggtggcttga 2160
gggacccggg aaggagaggc ctgtgggttt gcgacgcggg cgccccgtgt ggagggagcg 2220
ccgggccgtc cggtggactc gcgggtgtgg tgtgtgtgtg tgcgccccgg ccgccctttg 2280
tccctccacg gcgtgggcct ccctcctcac cccacctccg gggtgggggg tgctgcgcgg 2340
cggggccgat ggggcggccg cggcggcttt gtctcgggtg gtgggggagg tgcgggggac 2400
gggcgctcgc gagcccgcgt gctgggtggg ggaggggggt ctcgcgcgac tccgcctagt 2460
cgcacgtagt cgccattacg cgggccgcca tttcctgtcg gatccgggtg aggagaggct 2520
cgccgctcgg cgacggtcgc tggccgctgc ttccaggccc ggcccgtggg cgggaggcga 2580
gcgcgcgcgg tcggtctcgc tcggcggacc tgttaacacc agcgcgctgt gggaacagcg 2640
gccgcgggac gagagggctg gctgggcctg tttgtgtgcg tagacccggc tgatttgcct 2700
gaatccattt acccccgcgg ccgggggttt agcggagtgg ccggccgatc tctgcattta 2760
aagtggtccc cagagggatc gtcatttgct gccggcgttt tggaatagga gaaggcatga 2820
gggtgatgcc tcgactttaa gatcctgtgt tggtccttgg ctttggtagg actacgagtt 2880
aactggacca gttgtgatgg tttggttgca gtcttggaga gttgggttag aggtaaaatt 2940
gttgggaaat agcctttttt acgttgccta aaataatggt gttggtgatt aaagattccg 3000
tggctttgct gaataatggt ggtcgtttcc atctggttct ttagatttct gtgtctgaaa 3060
gttcaatgta ccttgtattt gccgctaagt tttttaaagt tggcagattt cattgattag 3120
tgttgtcagt cacgaccaaa ggcggggaaa aaattactac tagccctact ataacactag 3180
ttttgtgata agacgtggtc ttttcaagaa ttagtctcgt gaacaaaatt atatgtcgac 3240
tagtttttcc gttgtaatga aatgaagcaa gttgctgagg acggaggtaa ccggcgcgcc 3300
ataacttcgt atagcataca ttatacgaag ttatgtttaa acattactgg tcccccacac 3360
cccttgcggg gggggggggc gcaaggctct ggtggtggag gtggttaggg tgtgcttcat 3420
tgagacaatc tagtctaaaa gaagtccatt atgcctgtct ctcctcaatt atagttttta 3480
aaaacagttc acttttagga atattttagt ttcctcgaat cagaccttgt ttttatatac 3540
tactgtagca ctaggatgtc acttgtgtgt gctggaagca gaatcctttt tttttttttt 3600
ttttttagct tctggaagtt tattatttta tttcaaagca aacagattag agtgggtgga 3660
tttgcttcca ttttctgata attctaagcc aagaaaaatg taatggagct agctaccgaa 3720
agcaaggtat aagctcttta aaatttggag gttggttagt agttaaatat aaatggatgc 3780
tttttcactt tgaactctgc tttggctctg gaaacctttc agaattttat aacacgttga 3840
ataaaaggtt tttaaagggt tcatttgttt tgagacccct cacaggtgag gcgggccttg 3900
agctcttgat cctctgtctt tgattactgg gattccgagt gtgcgctatc acatttagtt 3960
gtggttctta atgataagca gatttcagaa ctgccggtag tgctgaaatt ttaatggtga 4020
aagccctatt accagggtcc accacagatc agctagagag atgagtggag taactgtaga 4080
cacaggtttt aagtgttggt tgcatcttat ttccccagga aactagttta cttaagtcta 4140
ccccatcact tatttcaagt cacaatataa aagtaggtac tgtttagatt ctgtttgttt 4200
ttttacaaga gggcacttgt aatctgtgct ttctaggaat tttatattca tgcctggtgt 4260
tctgaatact ccaaagacca taaaactatt catcttgatt tttttctttc tcaagatttt 4320
cagtgctgtg cacaaaattt ttttaacatt tttgtcagaa gttattccta aaaaagaatg 4380
tatagcctgg agttaaaaca gtgaatacta ttggaaattt tcaaggagta aatggcaaga 4440
atattaaggc tctccgtaca gacgtaagta ttaaactact gaacaagcaa gcacattcat 4500
tgtatgtttc tagaatttta aaggttcaca tcttagaaat cactgactag ctacaaacca 4560
tttgtaataa ttaatggtta tgtaaatgac ttcttttttt ttaaaaccct gattaaggtg 4620
gaacataatt aaatcttttc atattttaat aaaacccttc aaattaattc atttcaaata 4680
agattaaaac atcttaatta ggaatggaga caaagtgtaa aaacccttaa ttgtataatt 4740
cctcttgtat gtgtaagagg gatttagtca tttctgaaag ccattgaata tggctgataa 4800
aaactttatt tccttttttt ttcttttaaa tcatctgttc cacagtttgc aacatggagc 4860
tcatgctgac tgatttctaa atatgggtaa tgaatagatt aatcttggca atcagaactg 4920
tttgcctagg atttgagtct tcataattta tagtgggttg actcttgtga ttagacacat 4980
taaatgttgg cagcaccaga agtgtgtgtg tatttctaga actgattaca ccaaaattta 5040
tttttttccc tcatactatc tacatttctt aatgtttatc ttggtaaaat tgaggcaaat 5100
aactttgagt acctaacagt atttcatttc tcttggtaac agatctttgt gtttctctta 5160
ataaacttta gatttcgttg taatttttat aaatctgctt ataagatgtg ttttatacct 5220
tatgaaacca aatcagtctt aatatttggg ggtttggtgt gggaattttt gttttgtctt 5280
tgaacctctt tcctcttaat ccagatgttc tgtataatta tctgggttat ttccccattt 5340
tcctcaattt cagtaaactt ttgagtcatg acaggttttg tgtctaccct cacgagccag 5400
cctgctactg aaacattaaa aaacaaaaca aaacctagaa ccatcattta aggtccttaa 5460
atgttaagaa gccacggtca ggtatagaga agtccattat tgtgttagtc ttcagaacaa 5520
cttaagcttg tttcattgag agtatgagta tagtttgtat taaattaaaa tttattgctt 5580
ttcccccttt tccctctcct tgtttttttg gccatatatc tccgttgccc atgtactgga 5640
tcgacttacc cctgcgttgc ccaccatgac gttggcccat ccgcccctga acgcccatgt 5700
gaaaaccctg gttggctatg cccaaaaatg tgctcgttgc caaacgggta caatacttga 5760
caacttctga ttgaatgccc atgcccaatg ccaacatcca cgaacgccaa tgaccaatgc 5820
agtacaatgc cagtgtttca gtcccagaca gcagtggccc cgagcggtat gtcccaacag 5880
tttatgcctc actgcctgat tagaaagaga gaaatgtatt ttattacctg ccttttatat 5940
aattaaatag tatcccctta gacggtgtat tgtttttcaa gtttctgtct tattttcccc 6000
attaagtcct tttgtcactg aacttttacc gtgagttgcc cacccaaacg atccactaat 6060
tttatttcga tggaaggagc acagcttcaa gtgttgcata tttgctgcat tgtaaatcaa 6120
actgttaccc aaatagtctc tcgctctgtc tttgtggccc accttgcttc ccaacattgt 6180
ctgttcataa ttttttctga tgaaacgcta actggttcac tttctttcac ttctcatttg 6240
tactgtcttt gttttgtctt gcatttgtat ttgttttctt gcagagccca ttaagtagtc 6300
taatttctaa ctggtattca taaccttact ccagatacag tgttcaccac ttgcatattc 6360
ccccaccgcg gcacttaagt agttaacttt aaataatgcc aattatttaa agttaatagg 6420
cgatcgcacc atatgaatct cgaggttatg tacctgactg atgaagttcc tatactttct 6480
agagaatagg aacttcgaag ggttccgcaa gctctagtcg agccccagct ggttctttcc 6540
gcctcagaag ccatagagcc caccgcatcc ccagcatgcc tgctattgtc ttcccaatcc 6600
tcccccttgc tgtcctgccc caccccaccc cccagaatag aatgacacct actcagacaa 6660
tgcgatgcaa tttcctcatt ttattaggaa aggacagtgg gagtggcacc ttccagggtc 6720
aaggaaggca cgggggaggg gcaaacaaca gatggctggc aactagaagg cacagtcgag 6780
gctgatcagc gagctctaga gaattgatcc cctcagaaga actcgtcaag aaggcgatag 6840
aaggcgatgc gctgcgaatc gggagcggcg ataccgtaaa gcacgaggaa gcggtcagcc 6900
cattcgccgc caagctcttc agcaatatca cgggtagcca acgctatgtc ctgatagcgg 6960
tccgccacac ccagccggcc acagtcgatg aatccagaaa agcggccatt ttccaccatg 7020
atattcggca agcaggcatc gccatgggtc acgacgagat catcgccgtc gggcatgcgc 7080
gccttgagcc tggcgaacag ttcggctggc gcgagcccct gatgctcttc gtccagatca 7140
tcctgatcga caagaccggc ttccatccga gtacgtgctc gctcgatgcg atgtttcgct 7200
tggtggtcga atgggcaggt agccggatca agcgtatgca gccgccgcat tgcatcagcc 7260
atgatggata ctttctcggc aggagcaagg tgagatgaca ggagatcctg ccccggcact 7320
tcgcccaata gcagccagtc ccttcccgct tcagtgacaa cgtcgagcac agctgcgcaa 7380
ggaacgcccg tcgtggccag ccacgatagc cgcgctgcct cgtcctgcag ttcattcagg 7440
gcaccggaca ggtcggtctt gacaaaaaga accgggcgcc cctgcgctga cagccggaac 7500
acggcggcat cagagcagcc gattgtctgt tgtgcccagt catagccgaa tagcctctcc 7560
acccaagcgg ccggagaacc tgcgtgcaat ccatcttgtt caatggccga tcccatggtt 7620
tagttcctca ccttgtcgta ttatactatg ccgatatact atgccgatga ttaattgtca 7680
acaggctgca ggtcgaaagg cccggagatg aggaagagga gaacagcgcg gcagacgtgc 7740
gcttttgaag cgtgcagaat gccgggcctc cggaggacct tcgggcgccc gccccgcccc 7800
tgagcccgcc cctgagcccg cccccggacc caccccttcc cagcctctga gcccagaaag 7860
cgaaggagca aagctgctat tggccgctgc cccaaaggcc tacccgcttc cattgctcag 7920
cggtgctgtc catctgcacg agactagtga gacgtgctac ttccatttgt cacgtcctgc 7980
acgacgcgag ctgcggggcg ggggggaact tcctgactag gggaggagta gaaggtggcg 8040
cgaaggggcc accaaagaac ggagccggtt ggcgcctacc ggtggatgtg gaatgtgtgc 8100
gaggccagag gccacttgtg tagcgccaag tgcccagcgg ggctgctaaa gcgcatgctc 8160
cagactgcct tgggaaaagc gcctccccta cccggtagaa tttcgacgac ctgcagccaa 8220
agcgctagag ccagacatga taagatacat tgatgagttt ggacaaacca caactagaat 8280
gcagtgaaaa aaatgcttta tttgtgaaat ttgtgatgct attgctttat ttgtaaccat 8340
tataagctgc aataaacaag tttagctatc catcagtcga gaattggctc ccacggacac 8400
tccgcccctt ctgagatacc gcatcagtgt ttcaggcttc ttccatgtcc cttcctgcat 8460
aatctcgacc atagacacct gtttctcggc catgtctatt gcggcaccca ccctggcgga 8520
atggccggtc caggtcctat acctcccttt attgggggtg gcgtcccgtt tgttcagcag 8580
cacccaggca tcactgaaaa tcttctccat ggcaggggcg gtcaggggcg tggtagtaat 8640
gcgggctttg tttgaccggt gtataggtgg gaagagcaca gcgtctggat gttctctaag 8700
cccactgacg tccagccagt cattgagcac tgctgtggtt cgccgggaga gcactttgtc 8760
aaggcccgca gctgtggtga ttgtcttagt gtgactgatg tgcagggtca ctgtgtctcc 8820
tgtctgatcc aagtccccaa ccctgatgcg tgatatttca gacattctca tgagggtgtt 8880
ataggctacg aacaggaaag ccctgttgcg caggtcgacc agccgctcgc tcctgctgag 8940
cagcacatcc agcagcttca ggtcgtccca gcgcaggggg attgcctgtc ctgtcctctc 9000
acccttttcg gttgcggctt ctcttctaat tcttctcatg gcaagactca ctgacttgtc 9060
gtcggacaag ggtggcagtc cgcagtggga cagaagcata ttaagcatag catagtgctt 9120
gtcgatggta gtggaggcca ggtctgcatc gtgcaactga agaaagtatt cgcgtgccat 9180
ttcaggacta attggaaacc atgcaagctg ccgagcatgg caccatctgg cccagctatg 9240
aaagaccaac ctcaaatccc tcaaagtgtt aggagcgtac gccccctggt cattcataaa 9300
cctcatgaag ttttcagcgg cctcctgata ctctttgccg atgtttcgca ggaatccacc 9360
agatgaacca ctaataatca gctcagaaac cttcctcttc ttcttaggca tggccgcagg 9420
aaagcagagc cctgaagctc ccatcaccgg ccaataagag ccaagcctgc agtgtgacct 9480
catagagcaa tgtgccagcc agcctgaccc caagggccct caggcttggg cacactgtct 9540
ctaggaccct gagagaaaga catacccatt tctgcttagg gccctgagga tgagcccagg 9600
ggtggcttgg cactgaagca aaggacactg gggctcagct ggcagcaaag tgaccaggat 9660
gctgaggctt tgacccagaa gccagaggcc agaggccagg acttctcttg gtcccagtcc 9720
accctcactc agagctttac caatgccctc tggatagttg tcgggtaacg gtggacgcca 9780
ctgattctct ggccagccta ggacttcgcc attccgctga ttctgctctt ccagccactg 9840
gctgaccggt tggaagtact ccagcagtgc cttggcatcc agggcatctg agcctaccag 9900
gtccttcagt acctcctgcc agggcctgga gcagccagcc tgcaacacct gcctgccaag 9960
cagagtgacc actgtgggca caggggacac agggtggggc ccacaacagc accattgtcc 10020
acttgtccct cactagtaaa agaactctag ggttgcgggg ggtgggggag gtctctgtga 10080
ggctggtaag ggatatttgc ctggcccatg gagctagctt ggctggacgt aaactcctct 10140
tcagacctga agttcctata ctttctagag aataggaact tcggaattcg atatcaagct 10200
aagcttgatt aactttaaat aatgccaatt atttaaagtt agtagcgtcg cacgtgaaat 10260
cgatataact tcgtatagca tacattatac gaagttatgg taccaattac ccaagagaag 10320
gtgtgccagt attaccataa cagcgagggt gtccttaact tggtggcggg gaacatgaga 10380
attcaactct gcttcaattc tggtcgatta ttgccactta atctgagccc ttccagcagc 10440
ccattctctc tgtctctctc aaagtctcct aataatgtta tgtctgtatt gggcaaaaaa 10500
taaaatgcat gtgtaaaatt cataaatagg ctaccttaaa tggggactat taagtaatta 10560
tgatgtactt tttacttctt ttgttctaaa attctgagtt tatggtatta tgatttgata 10620
tactgatttg gctgacaaag cataaactca ctgagttttt tcatcagttt ttccgctgat 10680
caatagaaaa aggcttaaaa atatgatata tggatggtct tacaactcac tgttgaccct 10740
gctccatgct gcagtgggaa ttaactacat ttgcaaagtg ctttgcagtc atttttatta 10800
tagtgttatt tgaatgttaa taattttgtg gtaacagcga catgctaaat ggctgcagtg 10860
tgggtatggt gttgcaggac ctgaaaataa aatagttttt taaggtggtg cagattgtcc 10920
tttttgggcc tggctattga ggataataag cacataactg gtaaagttat tgagataata 10980
attcaggttg cttactgcaa cagtgcatta tgttgtgctt aagcataagc tgatacagtg 11040
ctgggaaaat acttgcaaga gagtaaaatt gtgtatttcc ccctctacag taccaacatt 11100
ataaaggaag tgactttgat tgcgagttga gactgttgat tcatcagagt ctggcaggag 11160
gaataattgg tgttaaaggt gctaaaatca aagaacttcg agaagtaaga gattattttg 11220
ttctgttttg ttccttctgg attcaatagt ttttcatgga gttggttttg tgggttactt 11280
cctcagatcc ctcatcacta tttagttggg tcttaaagta acaaatctaa gttttgttaa 11340
attctcacca tttctaggtc ctgtgtagtt gaccacattc tgtattgaga gtaattgggt 11400
tcaaatatct gaacttaggg agccttgatt ttatttgatg tgctaacaac ttctaatggg 11460
ttttctcttt tgtaaatgtt atagaacact cagacaacaa tcaagctttt ccaggagtgc 11520
tgccctcact ctactgacag agttgttctt attggaggaa aacctgatag ggttgtagaa 11580
tgcatcaaga tcatccttga ccttatatct gaggtatctt tgggatattt ttatttcatt 11640
cagtttcatc taactaccag aaataagaca gtattcacac cattaatttt gacatctccc 11700
tgcaagtttt ttattgcctc ctttaatata gtgcctttgg tccttactca aaggtccctg 11760
tagtgccatg ttacccactt cttacagaga tatctggggt aatgatcctt ctgtctctga 11820
cccacatcca cctccccgcc ccccggtcag tatgttagaa atcctttcca tcattgcttc 11880
attgtgtccc tgcctctgtt accttttgag ttaataatgg ttgagatggg ttaattgata 11940
ggaccagagc tatttttctt caggctttaa ggtgctgtgt ttctgttttt agtctcccat 12000
caaaggacgt gcacaacctt atgatcccaa cttttatgat gagacctatg attatggtgg 12060
ttttacaatg atgtttgatg accgccgagg acgacctgtg ggattcccca tgaggggaag 12120
aggtggtttt gacagaatgc ctcctggtcg gggtgggcgt cccatgcctc cttctagaag 12180
agattatgat gatatgagcc ctcgtcgagg acctccacca ccaccacctg gtcgaggtgg 12240
ccggggtggc agcagagccc ggaatctgcc tcttcctcct ccaccaccac ccagaggggg 12300
gtaagttttt atccaaacat taacattaat aaaagaggct gcacttagca gtgtgtgtgt 12360
gtgggggggt gattttaatg tgaccatgta aacattgcaa catgtgcttg cagagatcta 12420
atggcttatg acagaagagg aaggcctgga gaccgctatg atggcatggt aagaattttg 12480
atttataagg gcggcatgtg tgttttcaga gaccttataa ccaaacattt gtgtgagact 12540
gtagtactaa gagctttttt aaaattgaaa ttattttcac agccctctaa aagtttggta 12600
tttatgtatg taatacaggt tgggttcagt gctgatgaaa cttgggattc tgcaattgac 12660
acatggagcc catcagaatg gcaaatggct tatgaaccac aggttgagta tcaataatgt 12720
taataatgta catattggta atttaccttg actgtccata ataattggtc tcccctccct 12780
acccctgtaa tttcttgatt cagtttattt cctggataaa ctgggtatgg gtggtatggg 12840
aggactttgc ttttctaagc tctgtgatat tttagaaata ttaagtaatt ttttttcttt 12900
tgtttttagg gtggttctgg atatggtaag tttcttcttt ttgaaatcaa acattatttc 12960
aatcttattt ttatgtgaac ttgctgctca ttatttaaat cagactattc ttatgcaggg 13020
ggccgtggct catatggtga tcttggcgga cctattatca ctacacaagt aactattccc 13080
aaagatgtaa gtatttttaa tttagagtat ttttaattcg gggaatttac ttacttgtat 13140
tggttgctgc ttcttggaca aagacttcca gtttaggatt gagataatgt ttaagaactt 13200
aatggagggg gtattttgaa ccttagatca aatgatactt agtgacaaaa agaagacagt 13260
ttggggttct taaaatacac atatgtaaaa tttatttcag gaatttaaga ctgacgttct 13320
aaataaatta ctataaagtt attacatttt gtcattgata aaagggagta aaatttcttt 13380
caagtcatca taaacactta caatcttttc agttggctgg atctattatt ggcaaaggtg 13440
gtcagcggat taaacaaatt cgtcatgaat ctggagcatc aatcaaaatt gatgaacctt 13500
tagaaggatc tgaagatcgg atcattacca ttacaggaac acaggaccag atacagaacg 13560
cacagtattt gctgcagaac aggtcagttg acccttagct ttatgttagt gtacacatac 13620
taaaaccttt aaaaagcttt tcttcccaat tgattcttct tttagaaacc atggtgtttc 13680
cgtcacttgg ggaatctaac ttctaaatat tctaattaat agctctgcct taattttctt 13740
ctgttttctt actaaaatga agacattcaa tactaatctt gctggaagaa gccttaacca 13800
agcaaacttc tcatttctct ggtgaaaact gctgccaaaa ccaccacttg taataattta 13860
tacagagcct gtagaaaatg tagaagagac attggatgtt ggcctagttc tgtgtggaag 13920
actagtgatt ttgttgtttt tagataacta aaacgacaac aaatcacagt ctgccatatg 13980
gcacaggcca cctctacagg acagataatg atggtgctgt caaacgcagc atttcacacc 14040
ttcctcatgt tcctttgtct ttcctaccaa gtattaacag cttccagttt cattttctta 14100
accctgcact actactgtct attaacattt tcttgagttt tacattgctt taatttatag 14160
ttgctattgc tctttaaaag ttcttctaat tgtaatggac atgtttatca ttttaattta 14220
tcattgaaat tgtcttaatg ttgattaaga tgttttgaac aaaaattcta attcgtactt 14280
tttttttctt ttcttttata gtgtgaagca gtatgcagat gttgaaggat tctaatgcaa 14340
gatatttttt cttttttata gtgtgaagca gtattctgga aagtttttct aagactagtg 14400
aagaactgaa ggagtcctgc atcttttttt ttttatctgc ttctgtttaa aaagccaaca 14460
ttcctctgct tcataggtgc ggccgcgtac cagcttttgt tccctttagt gagggttaat 14520
ttcgagcttg gcgtaatcat ggtcatagct gtttcctgtg tgaaattgtt atccgctcac 14580
aattccacac aacatacgag ccggaagcat aaagtgtaaa gcctggggtg cctaatgagt 14640
gagctaactc acattaattg cgttgcgctc actgcccgct ttccagtcgg gaaacctgtc 14700
gtgccagctg cattaatgaa tcggccaacg cgcggggaga ggcggtttgc gtattgggcg 14760
ctcttccgct tcctcgctca ctgactcgct gcgctcggtc gttcggctgc ggcgagcggt 14820
atcagctcac tcaaaggcgg taatacggtt atccacagaa tcaggggata acgcaggaaa 14880
gaacatgtga gcaaaaggcc agcaaaaggc caggaaccgt aaaaaggccg cgttgctggc 14940
gtttttccat aggctccgcc cccctgacga gcatcacaaa aatcgacgct caagtcagag 15000
gtggcgaaac ccgacaggac tataaagata ccaggcgttt ccccctggaa gctccctcgt 15060
gcgctctcct gttccgaccc tgccgcttac cggatacctg tccgcctttc tcccttcggg 15120
aagcgtggcg ctttctcata gctcacgctg taggtatctc agttcggtgt aggtcgttcg 15180
ctccaagctg ggctgtgtgc acgaaccccc cgttcagccc gaccgctgcg ccttatccgg 15240
taactatcgt cttgagtcca acccggtaag acacgactta tcgccactgg cagcagccac 15300
tggtaacagg attagcagag cgaggtatgt aggcggtgct acagagttct tgaagtggtg 15360
gcctaactac ggctacacta gaagaacagt atttggtatc tgcgctctgc tgaagccagt 15420
taccttcgga aaaagagttg gtagctcttg atccggcaaa caaaccaccg ctggtagcgg 15480
tggttttttt gtttgcaagc agcagattac gcgcagaaaa aaaggatctc aagaagatcc 15540
tttgatcttt tctacggggt ctgacgctca gtggaacgaa aactcacgtt aagggatttt 15600
ggtcatgaga ttatcaaaaa ggatcttcac ctagatcctt ttaaattaaa aatgaagttt 15660
taaatcaatc taaagtatat atgagtaaac ttggtctgac agttaccaat gcttaatcag 15720
tgaggcacct atctcagcga tctgtctatt tcgttcatcc atagttgcct gactccccgt 15780
cgtgtagata actacgatac gggagggctt accatctggc cccagtgctg caatgatacc 15840
gcgagaccca cgctcaccgg ctccagattt atcagcaata aaccagccag ccggaagggc 15900
cgagcgcaga agtggtcctg caactttatc cgcctccatc cagtctatta attgttgccg 15960
ggaagctaga gtaagtagtt cgccagttaa tagtttgcgc aacgttgttg ccattgctac 16020
aggcatcgtg gtgtcacgct cgtcgtttgg tatggcttca ttcagctccg gttcccaacg 16080
atcaaggcga gttacatgat cccccatgtt gtgcaaaaaa gcggttagct ccttcggtcc 16140
tccgatcgtt gtcagaagta agttggccgc agtgttatca ctcatggtta tggcagcact 16200
gcataattct cttactgtca tgccatccgt aagatgcttt tctgtgactg gtgagtactc 16260
aaccaagtca ttctgagaat agtgtatgcg gcgaccgagt tgctcttgcc cggcgtcaat 16320
acgggataat accgcgccac atagcagaac tttaaaagtg ctcatcattg gaaaacgttc 16380
ttcggggcga aaactctcaa ggatcttacc gctgttgaga tccagttcga tgtaacccac 16440
tcgtgcaccc aactgatctt cagcatcttt tactttcacc agcgtttctg ggtgagcaaa 16500
aacaggaagg caaaatgccg caaaaaaggg aataagggcg acacggaaat gttgaatact 16560
catactcttc ctttttcaat attattgaag catttatcag ggttattgtc tcatgagcgg 16620
atacatattt gaatgtattt agaaaaataa acaaataggg gttccgcgca catttccccg 16680
aaaagtgcca cctgacgcgc cctgtagcgg cgcattaagc gcggcgggtg tggtggttac 16740
gcgcagcgtg accgctacac ttgccagcgc cctagcgccc gctcctttcg ctttcttccc 16800
ttcctttctc gccacgttcg ccggctttcc ccgtcaagct ctaaatcggg ggctcccttt 16860
agggttccga tttagtgctt tacggcacct cgaccccaaa aaacttgatt agggtgatgg 16920
ttcacgtagt gggccatcgc cctgatagac ggtttttcgc cctttgacgt tggagtccac 16980
gttctttaat agtggactct tgttccaaac tggaacaaca ctcaacccta tctcggtcta 17040
ttcttttgat ttataaggga ttttgccgat ttcggcctat tggttaaaaa atgagctgat 17100
ttaacaaaaa tttaacgcga attttaacaa aatattaa 17138
<210> 2
<211> 2321
<212> DNA
<213> 合成
<400> 2
tctagaacaa tgggggaggg gaagtcagca agtggcagta gttggtttgc ctctgacctt 60
taggaaaata ccagacattt gattttctat atatgctaga tgctatattt gttattatct 120
aaacaaattt gctacacaga tgttagaaat gtatctaaat gttcccatag aaaccattaa 180
tacccaggtt gatactgtag caattggttg ataggaacat gggtaaccag ctgcggattt 240
tcaaaatgtg cctgcttcct ggagaaaagg cggaatgcaa ggaaaggcac ttaatgatat 300
ttgggggatg gtctctgatc tcttcccttg acagtcttct ggagtatcaa ccatactggc 360
tgcaaacaga aacaaggata cttcttggag gcgatatcta gggtgctaga agtcctgatc 420
ttttactcag gatttggtca caaatggcaa ttctcacgtc tgaaagaaaa aggaatccat 480
ctgtgtaagt aaagatggag agcatgagcc tccagtctga gaagatatgg gagagactga 540
tgctgtctgg ccttaggaag agagaaaggg tgtctccttt cccctaaggg agagagagtt 600
ccagagacac cggtggaaat atgcacttta acctgaccat cgagtcgctt ccactccagg 660
cactttaggc tctaggagtc tcaccgttgg aatctccgaa agcacctcag gatactcaag 720
tccccactct gtctggccca cccacgggca agtccccagc gcaagaacca gttcttctgg 780
ttagatgccc tccggcagac aaaccttcgc aaaccttcgc ggtcagagcc tgagctgccg 840
agcaggggcg accagagcac tttggcgggg ctgggggctg ccggtcgggg ggcacggcac 900
gagtggctgg agcgaggcga ggggtggaaa aaggaggggg cggcgggtgg actaatgatg 960
cctttgatgg catcaaatgt tcttttccca gatcagggct ggaagtctgg gctaactatg 1020
gccgtttgga gcacagaaac catttataca cactgggacc ctccttcttt tcaattcaat 1080
ctcatagcta caggagaagg ggaaatctag caatcagggg agcgagggga aacgtatgta 1140
tgtgtgtgtt taaagaacat aaaacgccac aaataagcac atactatttt acttactggc 1200
catatagtac tcattcctaa tgagactatt aaagctgtta ccagattcca acaggtcatg 1260
aaaacaccgt ccagcaccgc tctgccctct gtcaagcctg gtcatagatt cgaccttcct 1320
cagtctctgt gtttgccagg gcctcaccga actggataca gattagcaca gagtccaccc 1380
cacccccagc tcctcctgct ccccccaact tcttcagccc cagtgttcct gtctgctctg 1440
atctggggca gcctcggcta cgagcaagaa attagaacat tggcaaaggt aggggaatta 1500
tcaggacgga ctttcttctg gtttaatgaa tatgtaaatt ttctactgtc tgaagcctgg 1560
ggatttctgg tggcagcgaa agtctcgaat ccattcgaag ctgaagactt ctggatctgc 1620
ccaagccgtc tttagatcgt agagagcctc ccgttattat cgctcatgtt attttattac 1680
attatttaat aggctgtctc tacagagcca aacgagcttt aataggcttg ctcttttttg 1740
ccgttcagtc tcagactctc cctgcgctct ccgccaaaac aaagctgcag gaaaagttac 1800
tggggggcgg tgcagccgca gatttgactt gttatagatg tgcatctttt ccctgttcct 1860
ttttcttttt tcttttccta acaagtttgc cctcagctgt tatcaaaaca tgcaaatgag 1920
atattccaag aggatctcaa acaaatccgg aggagttaat gctgaaacaa aataatcagc 1980
cccgttttaa agtgactgcg ggtttcgggc acttttctct ctgtgatact actgcaaggg 2040
aatgataatt agacctttat tttaaaaatc caaaggggct ctctccttag cttccctccc 2100
ttttccctcc ctccctcctt cctccctccc tcctcctctc ttgctccctc cctctccctt 2160
tctctctggc tctgatgttg gcaaaggggt tttcttaatc agactgtttt ttcggtccga 2220
ggaaaaggag gtaggagatt gtgatggaga aagggggtct gtaaaacgtc aggcgccgca 2280
caaaggcttt gccacgtaat tacaggctcc tattaagtgg a 2321
Claims (6)
1.一种构建四肢畸形小鼠模型的方法，利用条件性基因打靶Cre/LoxP系统，其特征在于，将Hnrnpk基因两侧带有同向LoxP位点的标记小鼠，与在组织特异性Prx1的增强子控制下表达Cre重组酶的Prx1-Cre小鼠进行杂交，筛选，获得四肢畸形小鼠模型；所述两个同向的LoxP位点分别位于Hnrnpk基因的4～7号外显子两侧，以Hnrnpk基因的4～7号外显子作为敲除区域。
2.如权利要求1所述的构建四肢畸形小鼠模型的方法，其特征在于，其构建步骤如下：
(1)构建标记的小鼠：通过同源重组的方法构建Hnrnpk基因两侧带有同向LoxP位点的标记小鼠；
(2)构建Cre小鼠：构建在组织特异性Prx1的增强子作用下表达Cre重组酶的Cre小鼠；
(3)将标记小鼠与Cre小鼠杂交，筛选，获得敲除Hnrnpk基因的纯合子小鼠即为四肢畸形小鼠模型。
3.如权利要求2所述的构建四肢畸形小鼠模型的方法，其特征在于，所述步骤(1)构建标记的小鼠的具体方法为：根构建含有同源序列、目的序列、DTA、LoxP、Neo、Amp抗性以及多个限制性酶切位点的质粒，所述质粒的核苷酸如SEQ ID NO:1所示。
4.如权利要求2所述的构建四肢畸形小鼠模型的方法，其特征在于，所述Prx1的增强子序列如SEQ ID NO:2所示。
5.由权利要求1或2所述的构建四肢畸形小鼠模型的方法制备获得的四肢畸形小鼠模型在制备或筛选治疗四肢畸形药物中的应用。
6.提高Hnrnpk基因表达水平的制剂或药物在制备提高肢芽间质祖细胞活性或该细胞增殖或延缓该细胞凋亡的制剂或药物中的应用。
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| CA2658351A1 (en) * | 2009-03-04 | 2010-09-04 | Institut De Recherches Cliniques De Montreal | Method for targeted cell ablation |
| JP2020089332A (ja) * | 2018-12-07 | 2020-06-11 | 国立大学法人北海道大学 | 非ヒト遺伝子改変動物及びその作製方法 |
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| 吴伟: "hnRNPK基因脂肪组织特异性敲除小鼠模型的建立及其对脂肪发育的影响", 《道客巴巴》 * |
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