CN113832107A - 一种基于同源重组技术的磷酸化突变体sufu转基因小鼠模型的构建及应用 - Google Patents
一种基于同源重组技术的磷酸化突变体sufu转基因小鼠模型的构建及应用 Download PDFInfo
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- CN113832107A CN113832107A CN202110667220.XA CN202110667220A CN113832107A CN 113832107 A CN113832107 A CN 113832107A CN 202110667220 A CN202110667220 A CN 202110667220A CN 113832107 A CN113832107 A CN 113832107A
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Abstract
本发明提供了一种基于同源重组技术的磷酸化突变体SUFU转基因小鼠模型的构建及应用,包括如下步骤:构建出含有磷酸化突变体SUFU基因的打靶载体pROSA26PA;将其线性化后转染129/S6ES细胞;利用PCR法扩增并测序筛选发生正确同源重组的ES细胞克隆;显微注射技术将ES细胞注入C57BL/6小鼠的囊胚中,与C57BL/6小鼠交配,产生出棕色和黑色小鼠;对棕色小鼠用PCR方法筛选出阳性小鼠,与Ddx4‑Cre小鼠交配,通过PCR筛选得到杂合型F1代小鼠;自交得到F2代小鼠,筛选出纯合F2代小鼠,即为磷酸化突变体SUFU转基因小鼠模型。本发明可用于研究SUFU对Hedgehog(Hh)信号通路的双向调控机制,将其引入Ptch+/‑自发髓母细胞癌小鼠模型中可进一步研究Hh亚型髓母细胞癌的发病机制,为筛选新一代抗髓母细胞癌的Hh拮抗剂提供有力工具;且该模型小鼠也可应用于研究Hh对精子发育的影响。
Description
技术领域
本发明涉及构建人源基因SUFU的特异位点磷酸化修饰突变小鼠模型的方法以及该小鼠模型在研究髓母细胞癌发生和精子发育中的应用,属于基础医学技术领域。
背景技术
Hedgehog(Hh)信号通路在昆虫到人类的各个物种间具有一定保守性,其主要作用是调节胚胎发育以及成体组织稳态。该信号功能异常会导致髓母细胞癌(Medulloblastoma,MB)的发生以及影响精子发育。
髓母细胞癌是儿童中常见的小脑肿癌,其发病率占所有儿童脑癌的20%。这种恶性肿癌起源于小脑皮层的颗粒性神经元前体细胞 (Granule Neuron Precursor Cells,GNPCs),或深层的神经干细胞 (Neural Stem Cells)。处于外颗粒层的颗粒性神经元前体细胞依赖Hh信号促进其快速分裂增生。当Hh通路活性异常增强后,就会导致GNPCS过度扩增,进而产生癌变。近年来大样本的基因表达谱研究以及肿癌全基因组测序研究从分子水平上,将髓母细胞癌分成了4种不同亚型。其中的Hh亚型,具有Hh本身和Smo基因上的激动性促癌变异,以及Ptch1和Sufu基因的抑癌变异。长期以来基于Ptch1基因敲除小鼠的自发髓母细胞癌模型为研究Hh通路信号转导机制和开发Vismodegib等Smo阻断剂类药物提供了宝贵工具,但是这一模型并不适宜用于针对通路下游基因突变所导致的髓母细胞癌癌研究。
在精子发育方面,Hh 信号通路通过直接或间接的方式调节着精原干细胞(Spermatogonial Stem Cells ,SSCs )的增殖和分化。睾丸支持细胞(Sertoli cells,SCs)自分泌的 Hh 信号分子可以促进维持自身多种SSCs 增殖的细胞生长因子的表达。由SCs 产生的Hh 信号分子将对维持正常睾丸间质细胞(leydig cells,LCs)的数量以及促使其分泌睾酮具有重要作用,睾酮的分泌将有助于 SSCs 的分化。同时,LCs 也会分泌一些抑制因子来抑制 SSCs 的分化。但是这其中具体的分子机制还不是很清楚。
作为Hh通路核心成员以及所有Gli转录因子的结合蛋白,Sufu蛋白在哺乳类动物胚胎发育中具有重要的作用。2006年,Rune Toftgard和 Stephan Teglund发表的文章显示,敲除Sufu会在E9.5导致头部和神经管的发育缺陷,同时取自Sufu-/-胎鼠的MEF细胞也表现出Hh信号通路的异常激活,表明Sufu对Hh信号通路的转导具有负向调控作用。然而,有文献报导Sufu在Hh信号转导过程中同时还具有正向的调节作用,体现在它对通路的转录激活因子Gli1蛋白有稳定作用,而且为Gli1入核及与DNA结合的功能所必须,且在小脑发育和髓母细胞癌中Gli1与Sufu都呈现高表达,后续也证实Sufu是Hh通路高度激活的必要条件。这些复杂的机制也反映在Sufu敲除小鼠的表型上,Sufu杂合缺失小鼠在P53变异的背景下自发产生髓母细胞癌,但是Sufu小脑组织特异性纯合缺失的小鼠并不产生肿癌,甚至抑制由Ptch1缺失引起的髓母细胞癌癌的发生。这些小鼠肿癌模型研究中似乎矛盾的现象与Sufu的正向与负向调控Hh信号转导的双重功能有密切关联。新近基因组学研究表明Hh通路下游Sufu 基因突变也是产生髓母细胞癌的重要因素。而在精子发育中,当 SSCs 分化时,Sufu的表达增加,抑制了 Hh 信号通路的活性,从而有益于 SSCs 的分化。
前期通过质谱分析,确立了在Sufu S342和S346位点具有磷酸化的调控机制,并进一步发现该过程是由PKA和GSK3β所介导,且该磷酸化可以影响Sufu的蛋白稳定性和核质分布,磷酸化后的Sufu更加稳定且倾向分布于细胞核。此外,这一双重磷酸化能够影响软骨细胞发育和细胞凋亡过程,具有一定生理意义。癌因此构建磷酸化突变体Sufu转基因小鼠模型,癌将为以Sufu为着眼点研究Hh影响髓母细胞癌发生和精子发育提供有力工具。
发明内容
鉴于以上所述现有技术的缺点,本发明的目的在于提供一种磷酸化突变体SUFU转基因小鼠模型构建方法,并将其引入髓母细胞癌自发小鼠模型背景中,为寻找新一代髓母细胞癌的Hh拮抗剂提供有力工具。另一方面,该模型小鼠也可应用于研究Hh对精子发育的影响。以期解决上述背景技术中提出的问题。
为实现上述目的,本发明采用了如下技术方案:
步骤一、SUFUWT/SUFUS342/346D/ SUFUS342/346A 双磷酸化突变体编码基因以及野生型SUFU编码基因转载在含有tpA转录终止序列的pBigT的载体上。
步骤二、把含有tpA转录终止序列和目的基因的pBigT载体装到包含有基因组DNARosa26序列的pRosa26PA上。
步骤三、转染SUFUWT/SUFUS342/346D/ SUFUS342/346A 双磷酸化突变体编码基因以及野生型SUFU编码基因打靶载体 :将构建好的SUFUWT/SUFUS342/346D/ SUFUS342/346A 双磷酸化突变体编码基因以及野生型SUFU编码基因打靶载体pROSA26PA,用酶切线性化,然后将线性化基因打靶载体利用电击法导入来源于129/S6小鼠的ES细胞中;然后加入G418 筛选出发生同源重组的ES细胞克隆 ;将ES细胞提取DNA,再利用PCR法扩增并测序鉴定ES克隆是敲入了SUFUWT/SUFUS342/346D/ SUFUS342/346A 双磷酸化突变体编码基因以及野生型SUFU编码基因,筛选出发生正确同源重组的ES细胞克隆。
所述步骤三中的内容均由南京大学模式动物研究所完成;
步骤四、显微注射和PCR 筛选制作磷酸化突变体SUFU转基因小鼠模型:将筛选鉴定正确的ES细胞通过显微注射技术注入C57BL/6小鼠的囊胚中,经注射的囊胚转移至假孕母鼠的子宫内着床,生出嵌合体小鼠;将嵌合体小鼠再与 C57BL/6 小鼠交配,产生出棕色和黑色后代小鼠;将其中的棕色小鼠用PCR方法1筛选出阳性小鼠,再将阳性小鼠与Ddx4-Cre小鼠交配,通过PCR方法1筛选得到杂合型 F1代小鼠;最后将杂合型F1代小鼠自交得到F2代小鼠,用PCR方法2筛选出纯合 F2 代小鼠,即为磷酸化突变体SUFU转基因小鼠模型;
其中,上述PCR方法1扩增引物序列是:
SD-left-2820-120 :TGGGGATCGACGGTATCGTA
SD-right-2820-120 :TACCGTCGACCTCGAGGG
其中,上述 PCR 方法2扩增引物序列是:
Rosa26-F8731:5’-CTTGCTCTCCCAAAGTCGCT-3’
Rosa26-R:5’-GCGGGAGAAATGGATATGAAGTA-3’
pBigT-R:5’-CGGCCTCGACTCTACGATAC-3’
上述磷酸化突变体SUFU转基因小鼠模型的制备方法中,所述质粒的图谱如图1所示,基因序列见序列表。
本发明成功构建出磷酸化突变体SUFU打靶载体pROSA26PA,并实现了磷酸化突变体SUFU转基因小鼠的制备。本发明的突出效果是:
(1) 利用基因打靶技术获得了磷酸化突变体SUFU基因敲入小鼠。
(2) 利用本发明方法制成的磷酸化突变体SUFU基因敲入小鼠能够了解磷酸化SUFU的生理学意义。
(3) 利用本发明方法制成的磷酸化突变体SUFU基因敲入小鼠是进一步研究SUFU调控Shh双向功能的理想模型,特别是将此模型小鼠引入Ptch+/-自发髓母细胞癌小鼠背景后,可以更好地研究SUFU(磷酸化SUFU)对髓母细胞癌的正向调控作用。
附图说明
图1 基因打靶载体pROSA26PA构建图示。
图2 磷酸化突变体SUFU转基因打靶载体构建图示。
图3 PCR方法1扩增验证携带有Neo和SUFU基因点突变小鼠的阳性筛选结果图示。
图4 PCR 方法2扩增验证SUFU磷酸化点突变模型小鼠的筛选结果图示。
图5 提取鼠尾RNA并反转成cDNA,检测Rosa26S342D/S346D变异体敲入小鼠中SUFUWT/SUFUS342/346D/ SUFUS342/346A 双磷酸化突变体编码基因以及野生型SUFU编码基因的敲入和表达情况。
图6 磷酸化突变体SUFU转基因小鼠多趾表型分析。
图7 磷酸化突变体SUFU转基因小鼠中高表达量的SUFU导致Hh信号通路的过度激活。
图8 磷酸化突变体SUFU转基因小鼠引入Ptch+/-自发髓母细胞癌肿癌小鼠模型配繁方案。
图9 磷酸化突变体SUFU转基因小鼠引入Ptch+/-自发髓母细胞癌肿癌小鼠后,髓母细胞癌的发生率癌有所提升。
图10磷酸化突变体SUFU转基因小鼠生育率低下表型分析结果图示。
图11 磷酸化突变体SUFU转基因小鼠成熟精子的向前运动力、活力、数量以及畸形率分析。
具体实施方式
下面将结合本发明的附图,对本发明的技术方案进行清楚、完整地描述,显然,所描述的实施例仅仅是本发明的一部分实施例,而不是全部的实施例,基于本发明中的实施例,本领域普通技术人员在没有做出创造性劳动前提下所获得的所有其他实施例,都属于本发明的保护范围。
小鼠的饲养和繁育:Rosa26SufuWT或Rosa26SufuS342/S346D或Rosa26SufuS342/S346A品系的小鼠在南京医科大学实验动物中心进行构建。
Ddx4-Cre小鼠购于南京医科大学实验动物中心。
Ptch+/-小鼠精子购自南京大学模式动物研究所,于南京医科大学实验动物中心复苏。
实验小鼠饲养于南京医科大学SPF级实验动物中心,由该中心的饲养员和保种员进行不同品系小鼠之间的配繁。饲养条件:全价饲料喂养,自由进食饮水,小鼠成年后进行雌雄分笼,剪指(趾)标记,剪尾提取基因组DNA做基因型鉴定。
实施例1
将SUFUWT/SUFUS342/346D/ SUFUS342/346A 双磷酸化突变体编码基因以及野生型SUFU编码基因转载在含有tpA转录终止序列的pBigT载体上,然后把含有tpA转录终止序列和目的基因的pBigT载体装到包含有基因组DNA Rosa26序列的pRosa26PA上,通过同源重组,将pBigT序列整合到小鼠的基因组上。
在pBigT上游的Rosa26序列上设计了一条正向引物Rosa26-F8731(5’-CTTGCTCTCCCAAAGTCGCT-3’);
在pBigT上设计了一条反向引物pBigT-R(5’-CGGCCTCGACTCTACGATAC-3’);
在pBigT下游的Rosa26序列上设计了另一条反向引物Rosa26-R(5’-GCGGGAGAAATGGATATGAAGTA-3’)。
借助这两对引物,就可以鉴定是否插入了含有目的基因的片段。鉴定判断标准如下:
实施例2
分离原代的小鼠胚胎成纤维细胞:取妊娠14.5d的孕鼠,断颈处死,按常规撕开子宫和胎膜取出胚胎,置于无菌的1×PBS中。
用无菌镊子去除尾部、四肢、头和内脏(一切鲜红的组织都应去除干净),只留躯干背部组织,用无菌1×PBS清洗血迹。
用无菌手术刀切割躯干背部组织,尽可能切成1-3mm的碎块。
将同一胚胎的碎块移至同一个15ml的离心管里,静置至组织块沉入管底,小心吸弃上清。
向离心管里加入1-2ml 0.25%胰蛋白酶-EDTA,用滴管反复吹打使之和组织碎块混合均匀。
将其放置于细胞培养箱中静置20分钟,再用滴管反复吹打使组织碎块被充分消化成单个细胞。
加入10ml含10%胎牛血清的DMEM培养液终止胰酶的消化作用,用移液管吹打混匀细胞后转移至10cm的培养皿中,至37℃,5%CO2的细胞培养箱中培养至12小时后,观察细胞的贴壁状态,若是70%-80%贴壁,则可吸弃培养液,用1×PBS清洗2遍,然后换上10ml新鲜含10%胎牛血清的DMEM培养液继续培养。
实施例3
对原代MEF细胞加入ShhN配基或是Smoothen的激动剂SAG刺激,模拟MEF细胞中Hedgehog信号通路激活状态:
先将原代MEF细胞种于10cm的P100培养皿,待细胞密度达到90%的时候,弃去之前的培养液,用1×PBS清洗细胞2遍,准备进行加配基或药物刺激,整个刺激过程均在无血清无双抗的DMEM环境下进行。
具体用量如下:无血清无双抗DMEM : ShhN=9 : 1;无血清无双抗DMEM : DMSO=1000 : 1;无血清无双抗DMEM : SAG=2000 : 1。
刺激24小时后,便可以收取细胞的总RNA和总蛋白进行后续实验。
实施例4
E17.5胎鼠全身骨染色;
取妊娠17.5d的孕鼠,断颈处死,按常规撕开子宫和胎膜取出胚胎,置于装有无菌的1×PBS的六孔板中,并编号,同时将胚外膜取出,放入装有1ml冰1×PBS的1.5mlEP管中,之后需提取基因组DNA,进行基因型鉴定。
注意胚胎的完整性。
实验前,将水浴锅升温至70℃。然后用眼科镊头部轻轻镊挂住胚胎的脖颈部,缓慢地将胚胎浸入水浴锅中烫20-30sec,这将有助于后面的剥皮。此过程中,胎鼠刚进入锅中时,躯干和尾部会伸展开来,然后大约3-5sec后,可见尾部、躯干依次开始回缩,可根据此掌控烫胎鼠的火候。
用手术刀或利刃将胚鼠从颅骨顶端先沿着背部向尾部浅浅地划开一条口子,再沿着腹部一侧到尾部浅浅地划开一道口子,接着用显微镊轻轻地沿着开口小心地将皮肤剥离,一定要特别注意四肢皮肤的剥离,若是无法剥离干净,可以放弃,因为后续的消化染色步骤,会慢慢将四肢上的皮肤组织消化干净。
剥完皮后,用显微镊从脐带连接处扎出一个小洞,然后将该小洞扩张,将腹部,胸腔中的器官组织全部剥离出来,注意一定要保证胚胎的完整性,特别不能损伤到四肢、尾部以及肋骨。
将处理好的胎鼠放入95%乙醇,4℃,脱水固定1-3天。
配制Alcian Blue和Alizarin Red染液:Alcian Blue染液(0.15mg/ml,总体积为100mL):
Alcian Blue 8GX(Sigma A3157) 称取15mg;
95%乙醇量取80mL;冰乙酸量取20ml。
配制Alizarin Red染液(0.05mg/ml,总体积为100mL):Alizarin Red(SigmaA5533)称取5mg;2%KOH量取100ml。
将胚鼠浸泡在Alcian Blue染液中,以六孔板为例,每孔大约需要加入10ml染液,避光染色24小时。
染色24小时后,将1ml枪头剪开,然后缓慢轻柔地将染液吸尽,缓缓地沿着皿侧壁加入95%乙醇轻柔荡洗两遍,每遍30sec-1分钟,然后将其浸泡在95%乙醇中避光静置24小时。
此过程需要重复一遍。
小心吸走95%乙醇浸泡液,仍然缓缓地加入Alizarin Red染液,避光染色1-3小时。此步染色时间是关键,由于染色液中有高浓度的KOH,染色过久,对胎鼠的组织腐蚀消化过度,最后的标本容易破碎,无法拍摄完整图像。
所以,此步染色的时候,1个小时需要观察下,标本的完整度,是否有消化过度的现象,一旦发现标本开始出现消化过度的现象,立即轻柔吸弃染液,开始后续的清洗工作。
一切以保证标本完整为基本原则。标本的完整度可以以四肢的完整度作为判断标准。
将经过Alizarin Red染色的标本用1%KOH轻柔浸泡式清洗一定时间,以每30分钟观察下标本的完整度,来判定染色的时间。
切记不可清洗过久;清洗过后,请立即拍照,如有需要,可将样本经过含20%,40%,60%,80%甘油的1%KOH浸泡,每次时间根据标本完整度来调整,梯度浸泡后,标本便可以4℃低温封存。
实施例5
Ptch1+/-品系小鼠从南京大学模式动物研究所引进,该小鼠精子冻于南京医科大学实验动物中心。
我们将其复苏,与Sufu+/+;Rosa26+/SD配繁(其中的”SD”表示SUFUS342/346D双磷酸化突变体SUFU编码基因),将子代基因型为Ptch1+/-;Sufu+/+;Rosa26+/SD雌雄鼠配繁,预计将会得到Ptch1+/+;Sufu+/+,Ptch1+/+;Sufu+/+;Rosa26+/SD,Ptch1+/-;Sufu+/+,Ptch1+/-;Sufu+/+;Rosa26+/SD,然后再将Ptch1+/+;Sufu+/+;Rosa26+/SD和Ptch1+/-;Sufu+/+;Rosa26+/SD雌雄鼠配繁,便可以得到最终的目的小鼠Ptch1+/-(作为对照)和Ptch1+/-;Sufu+/+;Rosa26SD/SD。
因为Ptch1突变基因组DNA上插入了lacZ基因,所涉及的一对引物可以特异性识别lacZ基因(lacZ-F:5’-GACACCAGACCAACTGGTAATGGTAGCGAC-3’;lacZ-R:5’-GCATTGAGCTGGGTAATAAGCGTTGGCAAT-3’),扩增出800bp的片段出来。
鉴定判断标准如下:
实施例6
小鼠小脑组织的提取、固定、脱水、包埋以及HE染色:根据实验室之前的研究,小鼠出现如下表型特征:精神状态差、脑水肿、步履蹒跚、平衡失调、后肢瘫痪等一些典型的神经病学症状,将会被判定为出现髓母细胞癌的疑似小鼠。
小鼠小脑组织的提取:发现具有上文所述表型特征的小鼠,用3.6%水合氯醛进行腹腔注射后,待小鼠无力行动后,将小鼠四肢固定,使其腹部向上。剪开小鼠腹腔,剪开肠系膜上的血管,使微量血液流出,继续剪开小鼠胸腔,暴露心脏,然后用静脉输液针在左心室上扎一个小口。用装有1×PBS的10mL注射器连接静脉输液针,将1×PBS从小鼠左心室泵入体循环,将体内血液排空,待肝脏变白后,将1×PBS换成10mL 4%多聚甲醛(PFA)。灌流后约5分钟,剪开小鼠头部皮肤,露出颅骨,小心用剪刀沿矢状缝剪开颅骨,小心不要损坏肿癌。分离脑组织,放到50ml 4%多聚甲醛中4度过夜,第二天对完整的脑进行拍照记录,再将脑组织的大脑切去一部分,然后沿矢状缝将小脑分成两部分,固定12小时。
小鼠小脑组织的脱水和石蜡包埋:
①将固定后的脑组织从4%多聚甲醛中取出,用1×PBS清洗3次,每次3分钟,然后按下述步骤进行梯度脱水:50%乙醇30分钟,70%乙醇30分钟、85%乙醇30分钟2次、90%乙醇30分钟、100%乙醇30分钟、100%乙醇 :100%正丁醇=1:1 15分钟、100%正丁醇1小时 3次。
②石蜡包埋机中的石蜡提前一天融化,将已经透明处理的脑组织在65℃石蜡中浸泡3次,每次一小时,将脑组织放入包埋模具中央进行包埋,将模具放在冷台上至石蜡完全凝固后将包裹组织模具平移至-20℃的冷冻台上冷冻30分钟,然后就可以将石蜡包埋的组织从模具中分离。
③切片厚度5µm。将切好的切片放到42℃的蒸馏水上,借水的张力和温度使石蜡切片展开。用防脱载玻片将切片捞出,室温晾干后,放于55℃烘箱中烤片6-8小时,增加组织在载玻片上的粘附性。
HE染色:
①脱蜡:用二甲苯去除切片上的石蜡,将切片浸在二甲苯中10分钟,换成新的二甲苯重复一次。
②在二甲苯:无水乙醇 1:1 的试剂中静置1分钟,然后依次放入100%、95%、85%、75%和50%乙醇中,每个梯度放入浸泡2分钟,使组织水化。
③在ddH2O中浸泡1分钟,再放入苏木素染液中染色,染色时间依染色效果而定。苏木素染色后切片颜色偏紫,用流动自来水冲洗蓝化。
④1% 盐酸酒精(1%HCl in 70%乙醇)分化,即快速提拉1~2次后迅速放入蒸馏水中,再次浸洗蓝化5~10分钟,镜下观察至满意为止。
⑤放在伊红染液中染色,染色时间依染液效果而定;
⑥95%乙醇浸泡2次,每次1分钟,100%乙醇浸泡2次,每次2分钟,二甲苯浸泡2次,每次2分钟。
⑦用中性树脂封片。
实施例7
小鼠睾丸组织的提取、固定、脱水、包埋以及HE染色,免疫荧光染色:前期我们通过配繁发现两个月的雄性Sufu+/+; Rosa26SD / SD基因型小鼠生育率低下。为了进一步研究生育率低下的表型,我们首先观测了Sufu在WT小鼠不同脏器中的蛋白表达情况,发现Sufu主要在睾丸中表达,且Sufu+/+; Rosa26SD/SD小鼠睾丸中表达的Sufu确实比同窝的Sufu+/+小鼠要多,再次验证了Sufu+/+; Rosa26SD/SD敲入小鼠模型的成功构建。为了探究为何Sufu+/+;Rosa26SD / SD基因型小鼠生育率低下,我们对Sufu+/+; Rosa26SD/SD和Sufu+/+小鼠进行取材发现Sufu+/+; Rosa26SD/SD小鼠睾丸变小,睾丸于体重比有一定统计学差异。通过对Sufu+/+和Sufu+/+; Rosa26SD/SD小鼠睾丸HE染色,我们发现Sufu+/+; Rosa26SD/SD小鼠睾丸中有很多异常的曲细精管,PLZF免疫荧光染色对Sufu+/+和Sufu+/+; Rosa26SD/SD小鼠的精原干细胞进行了标记并且统计分析,发现相比于对照,Sufu+/+; Rosa26SD/SD小鼠的精原干细胞数量都明显变少,且Sufu+/+; Rosa26SD/SD小鼠中Sufu与PLZF有很好的共定位,且主要定位于细胞质中。
小鼠睾丸组织的脱水和石蜡包埋:
①:将生育力低下的小鼠进行安乐死,将小鼠四肢固定,使其腹部向上,剪开小鼠腹部,在小鼠左右下腹找出睾丸,用镊子提起附睾处,用剪刀剪下睾丸,将其放入MDF固定液固定24小时,24小时后将睾丸取出对称切两半分别放入MDF固定液再固定24小时,然后按下述步骤进行梯度脱水:70%乙醇12h,80%乙醇1小时2次,90%乙醇1小时2次,100%酒精1小时,二甲苯:酒精=1:1 45分钟,二甲苯45分钟。
②石蜡包埋机中的石蜡提前一天融化,将已经透明处理的睾丸组织在65℃石蜡中浸泡2次,每次45分钟,将睾丸组织放入包埋模具中央进行包埋,将模具放在冷台上至石蜡完全凝固后将包裹组织模具平移至-20℃的冷冻台上冷冻30分钟,然后就可以将石蜡包埋的组织从模具中分离。
③切片厚度5µm。将切好的切片放到42℃的蒸馏水上,借水的张力和温度使石蜡切片展开。用防脱载玻片将切片捞出,室温晾干后,放于55℃烘箱中烤片6-8小时,增加组织在载玻片上的粘附性。
HE染色:
①脱蜡:用二甲苯去除切片上的石蜡,将切片浸在37℃二甲苯中15分钟,换成新的二甲苯重复一次。
②依次放入100%、100%、90%、80%、70%乙醇中,每个梯度放入浸泡2分钟,使组织水化。
③在ddH2O中浸泡10分钟,再放入苏木素染液中染色3分钟,用流动自来水冲洗10分钟蓝化。
④在1%盐酸快速震荡一次,用流水自来水冲洗10分钟
⑤放在伊红染液中染色5分钟
⑥70%乙醇震荡4次,80%乙醇震荡4次。90%乙醇震荡4次,100%乙醇震荡4次,二甲苯浸泡2次,每次15分钟。
⑦用中性树脂封片。
免疫荧光染色:
① 脱蜡:用二甲苯去除切片上的石蜡,将切片浸在37℃二甲苯中15分钟,换成新的二甲苯重复一次。
② 依次放入100%、100%、90%、80%、70%乙醇中,每个梯度放入浸泡2分钟,使组织水化。
③ 在PBS中浸泡10分钟,用柠檬酸钠高温修复3分钟,低火7分钟,待冷却后,用0.1%TritonX-100通透15分钟,3次。
④ 用组化笔花圈,5%BSA常温封闭2小时。
⑤ 用5%BSA作为一抗稀释液,抗体比例1:100,孵育一抗,4℃过夜。
⑥ PBST清洗切片15分钟,3次。
⑦ 用5%BSA作为二抗稀释液,荧光二抗比例1:50,常温避光孵育2小时。
⑧ PBST清洗切片15分钟,3次。
⑨ 滴DAPI封片。
实施例8
小鼠成熟精子的向前运动力,活力,数量以及畸形率统计:为了解Sufu+/+;Rosa26SD/SD组中精子的运动能力,我们使用了计算机辅助的精子自动分析系统(CASA)分析了附睾尾中成熟精子的运动状态,结果显示与对照组相比,Sufu+/+;Rosa26SD/SD精子数量在2月龄时有一定减少,总体呈现成熟精子少弱畸的表型。而6月龄时,无论是精子数量、活力还是前向运动力与Sufu+/+小鼠相比都有明显差异,并且这种差异比2个月的小鼠更加明显。
小鼠casa仪实验:
① 小鼠进行安乐死,将小鼠四肢固定,使其腹部向上,剪开小鼠腹部,在小鼠左右下腹找出睾丸,用镊子提起睾丸处,用镊子剪下附睾,顺着附睾尾十字剪开,放入37℃水浴锅中HTF溶液中15分钟,HTF溶液提前预热。
② 取10uL HTF溶液上机。
精子涂片HE染色:
①取材:取出小鼠附睾,在尾部十字剪口,放入HTF溶液中15分钟。
②涂片:滴取适当体积悬液涂片,自然晾干,4%PFA 20-40分钟,PBS洗片5分钟/次,每次5分钟。
③染色:依次放入100%、100%、90%、80%、70%乙醇中,每个梯度放入浸泡2分钟,在ddH2O中浸泡10分钟,再放入苏木素染液中染色10分钟,用流动自来水冲洗10分钟蓝化。在1%盐酸快速震荡一次,用流水自来水冲洗10分钟,放在伊红染液中染色15分钟,70%乙醇震荡2分钟,80%乙醇震荡2分钟,90%乙醇震荡2分钟,100%乙醇震荡2分钟,二甲苯浸泡2次,每次15分钟。
以上显示和描述了本发明的基本原理、主要特征和本发明的优点。本行业的技术人员应该了解,本发明不受上述实施例的限制,上述实施例和说明书中描述的仅为发明的优选例,并不用来限制本发明,在不脱离本发明新型精神和范围的前提下,本发明还会有各种变化和改进,这些变化和改进都落入要求保护的本发明范围内。本发明要求保护范围由所附的权利要求书及其等效物界定。
序列表
<110> 南京医科大学
<120> 一种基于同源重组技术的磷酸化突变体SUFU转基因小鼠模型的构建及应用
<160> 4
<170> SIPOSequenceListing 1.0
<210> 2
<211> 8946
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 2
cttaattaag ggatctgtag ggcgcagtag tccagggttt ccttgatgat gtcatactta 60
tcctgtccct tttttttcca cagctcgcgg ttgaggacaa actcttcgcg gtctttccag 120
tggggatcga cggtatcgta gagtcgaggc cgctctagaa ctagtggatc cggaaccctt 180
aatataactt cgtataatgt atgctatacg aagttattag gtccctcgac ctgcaggaat 240
tctaccgggt aggggaggcg cttttcccaa ggcagtctgg agcatgcgct ttagcagccc 300
cgctggcact tggcgctaca caagtggcct ctggcctcgc acacattcca catccaccgg 360
tagcgccaac cggctccgtt ctttggtggc cccttcgcgc caccttctac tcctccccta 420
gtcaggaagt tcccccccgc cccgcagctc gcgtcgtgca ggacgtgaca aatggaagta 480
gcacgtctca ctagtctcgt gcagatggac agcaccgctg agcaatggaa gcgggtaggc 540
ctttggggca gcggccaata gcagctttgc tccttcgctt tctgggctca gaggctggga 600
aggggtgggt ccgggggcgg gctcaggggc gggctcaggg gcggggcggg cgcgaaggtc 660
ctcccgaggc ccggcattct cgcacgcttc aaaagcgcac gtctgccgcg ctgttctcct 720
cttcctcatc tccgggcctt tcgacctgca gccaatatgg gatcggccat tgaacaagat 780
ggattgcacg caggttctcc ggccgcttgg gtggagaggc tattcggcta tgactgggca 840
caacagacaa tcggctgctc tgatgccgcc gtgttccggc tgtcagcgca ggggcgcccg 900
gttctttttg tcaagaccga cctgtccggt gccctgaatg aactgcagga cgaggcagcg 960
cggctatcgt ggctggccac gacgggcgtt ccttgcgcag ctgtgctcga cgttgtcact 1020
gaagcgggaa gggactggct gctattgggc gaagtgccgg ggcaggatct cctgtcatct 1080
caccttgctc ctgccgagaa agtatccatc atggctgatg caatgcggcg gctgcatacg 1140
cttgatccgg ctacctgccc attcgaccac caagcgaaac atcgcatcga gcgagcacgt 1200
actcggatgg aagccggtct tgtcgatcag gatgatctgg acgaagagca tcaggggctc 1260
gcgccagccg aactgttcgc caggctcaag gcgcgcatgc ccgacggcga tgatctcgtc 1320
gtgacccatg gcgatgcctg cttgccgaat atcatggtgg aaaatggccg cttttctgga 1380
ttcatcgact gtggccggct gggtgtggcg gaccgctatc aggacatagc gttggctacc 1440
cgtgatattg ctgaagagct tggcggcgaa tgggctgacc gcttcctcgt gctttacggt 1500
atcgccgctc ccgattcgca gcgcatcgcc ttctatcgcc ttcttgacga gttcttctga 1560
ggggatccgc tgtaagtctg cagaaattga tgatctatta aacaataaag atgtccacta 1620
aaatggaagt ttttcctgtc atactttgtt aagaagggtg agaacagagt acctacattt 1680
tgaatggaag gattggagct acgggggtgg gggtggggtg ggattagata aatgcctgct 1740
ctttactgaa ggctctttac tattgcttta tgataatgtt tcatagttgg atatcataat 1800
ttaaacaagc aaaaccaaat taagggccag ctcattcctc ccactcatga tctatagatc 1860
tatagatctc tcgtgggatc attgtttttc tcttgattcc cactttgtgg ttctaagtac 1920
tgtggtttcc aaatgtgtca gtttcatagc ctgaagaacg agatcagcag cctctgttcc 1980
acatacactt cattctcagt attgttttgc caagttctaa ttccatcaga agcttgcaga 2040
tctgcgactc tagaggatct gcgactctag aggatcataa tcagccatac cacatttgta 2100
gaggttttac ttgctttaaa aaacctccca cacctccccc tgaacctgaa acataaaatg 2160
aatgcaattg ttgttgttaa cttgtttatt gcagcttata atggttacaa ataaagcaat 2220
agcatcacaa atttcacaaa taaagcattt ttttcactgc attctagttg tggtttgtcc 2280
aaactcatca atgtatctta tcatgtctgg atctgcgact ctagaggatc ataatcagcc 2340
ataccacatt tgtagaggtt ttacttgctt taaaaaacct cccacacctc cccctgaacc 2400
tgaaacataa aatgaatgca attgttgttg ttaacttgtt tattgcagct tataatggtt 2460
acaaataaag caatagcatc acaaatttca caaataaagc atttttttca ctgcattcta 2520
gttgtggttt gtccaaactc atcaatgtat cttatcatgt ctggatctgc gactctagag 2580
gatcataatc agccatacca catttgtaga ggttttactt gctttaaaaa acctcccaca 2640
cctccccctg aacctgaaac ataaaatgaa tgcaattgtt gttgttaact tgtttattgc 2700
agcttataat ggttacaaat aaagcaatag catcacaaat ttcacaaata aagcattttt 2760
ttcactgcat tctagttgtg gtttgtccaa actcatcaat gtatcttatc atgtctggat 2820
ccccatcaag ctgatccgga acccttaata taacttcgta taatgtatgc tatacgaagt 2880
tattaggtcc ctcgacctgc agcccaagct agcttatcga taccgtcgac ctcgaggggc 2940
ccccgcgggc ggccgcgagc tcgctgatca gcctcgactg tgccttctag ttgccagcca 3000
tctgttgttt gcccctcccc cgtgccttcc ttgaccctgg aaggtgccac tcccactgtc 3060
ctttcctaat aaaatgagga aattgcatcg cattgtctga gtaggtgtca ttctattctg 3120
gggggtgggg tggggcagga cagcaagggg gaggattggg aagacaatag caggcatgct 3180
ggggatgcgg tgggctctat ggcttctgag gcggaaagaa ccagctgggg ctcgatcctc 3240
tagttggcgc gccaacggta ccagatgggc gggagtcttc tgggcaggct taaaggctaa 3300
cctggtgtgt gggcgttgtc ctgcagggga attgaacagg tgtaaaattg gagggacaag 3360
acttcccaca gattttcggt tttgtcggga agttttttaa taggggcaaa taggaaaatg 3420
gaggatagga gtcatctggg gtttatgcag caaaactaca ggtatattgc ttgtatccgc 3480
ctcggagatt tccatgagga gataaagaca tgtcacccga gtttatactc tcctgcttag 3540
atcctactac agtatgaaat acagtgtngc gaggtagact atgtaagcag atttaatcat 3600
tttaaagagc ccagtacttc atatccattt ctcccgctcc ttctgcagcc ttatcaaaag 3660
gtatttagaa cactcatttt agccccattt tcatttatta tactggctta tccaacccct 3720
agacagagca ttggcatttt ccctttcctg atcttagaag tctgatgact catgaaacca 3780
gacagattag ttacatacac cacaaatcga ggctgtagct ggggcctcaa cactgcagtt 3840
cttttataac tccttagtac actttttgtt gatcctttgc cttgatcctt aattttcagt 3900
gtctatcacc tctcccgtca ggtggtgttc cacatttggg cctattctca gtccagggag 3960
ttttacaaca atagatgtat tgagaatcca acctaaagct taactttcca ctcccatgaa 4020
tgcctctctc ctttttctcc attataactg agctatnacc attaatggtt tcaggtggat 4080
gtctcctccc ccaatatacc tgatgtatct acatattgcc aggctgatat tttaagacat 4140
naaaggtata tttcattatt gagccacatg gtattgatta ctgctactaa aattttgtca 4200
ttgtacacat ctgtaaaagg tggttccttt tggaatgcaa agttcaggtg tttgttgtct 4260
ttcctgacct aaggtcttgt gagcttgtat tttttctatt taagcagtgc tttctcttgg 4320
actggcttga ctcatggcat tctacacgtt attgctggtc taaatgtgat tttgccaagc 4380
ttcttcagga cctataattt tgcttgactt gtagccaaac acaagtaaaa tgattaagca 4440
acaaatgtat ttgtgaagct tggtttttag gttgttgtgt tgtgtgtgct tgtgctctat 4500
aataatacta tccaggggct ggagaggtgg ctcggagttc aagagcacag actgctcttc 4560
cagaagtcct gagttcaatt cccagcaacc acatggtggc tcacaaccat ctgtaatggg 4620
atctgatgcc ctcttctggt gtgtctgaag accacaagtg tattcacatt aaataaataa 4680
tcctccttct tcttcttttt ttttttttaa agagaatnct gtctccagta gaattactga 4740
agtaatgaaa tactttgtgt ttgttccaat atggnagcca ataatcaaat actcttnagc 4800
actggaaatg taccaaggaa ctattttatt taagtgnact gtggacagag gagccataac 4860
tgcagacttg tgggatacag aagaccaatg cagacttaat gtcttttctc ttacactaag 4920
caataaagaa ataaaaattg aacttctagt atcctatttg ttaaactgct agctttacta 4980
acttttgtgc ttcatctata caaagctgaa agctaagtct gcagccatta ctaaacatga 5040
aagcaagtaa tgataatttt ggatttcaaa aatgtagggc cagagtttag ccagccagtg 5100
gtggtgcttg cctttatgcc ttaatcccag cactctggag gcagagacag gcagatctct 5160
gagtttgagc ccagcctggt ctacacatca agttctatct aggatagcca ggaatacaca 5220
cagaaaccct gttggggagg ggggctctga gatttcataa aattataatt gaagcattcc 5280
ctaatgagcc actatggatg tggctaaatc cgtctacctt tctgatgaga tttgggtatt 5340
attttttctg tctctgctgt tggttgggtc ttttgacact gtgggctttc ttaaagcctc 5400
cttccctgcc atgtggtctc ttgtttgcta ctaacttccc atggcttaaa tggcatggct 5460
ttttgccttc taagggcagc tgctgagntt tgcagcctga tttccagggt ggggttggga 5520
aatctttcaa acactaaaat tgtcctttaa ttttttttta aaaaatgggt tatataataa 5580
acctcataaa atagttatga ggagtgaggt ggactaatat taatgagtcc ctcccctata 5640
aaagagctat taaggctttt tgtcttatac taactttttt tttaaatgtg gtatctttag 5700
aaccaagggt cttagagttt tagtatacag aaactgttgc atcgcttaat cagattttct 5760
agtttcaaat ccagagaatc caaattcttc acagccaaag tcaaattaag aatttctgac 5820
tttaatgtta tttgctactg tgaatataaa atgatagctt ttcctgaggc agggtctcac 5880
tatgtatctc tgcctgatct gcaacaagat atgtagacta aagttctgcc tgcttttgtc 5940
tcctgaatac taaggttaaa atgtagtaat acttttggaa cttgcaggtc agattctttt 6000
ataggggaca cactaaggga gcttgggtga tagttggtaa atgtgtttaa gtgatgaaaa 6060
cttgaattat tatcaccgca acctactttt taaaaaaaaa agccaggcct gttagagcat 6120
gctaagggat ccctaggact tgctgagcac acaagagtag tacttggcag gctcctggtg 6180
agagcatatt tcaaaaaaca aggcagacaa ccaagaaact acagtaaggt tacctgtctt 6240
taaccatctg catatacaca gggatattaa aatattccaa ataatatttc attcaagttt 6300
tcccccatca aattgggaca tggatttctc cggtgaatag gcagagttgg aaactaaaca 6360
aatgttggtt ttgtgatttg tgaaattgtt ttcaagtgat agttaaagcc catgagatac 6420
agaacaaagc tgctatttcg aggtctcttg gttatactca gaagcacttc tttgggtttc 6480
cctgcactat cctgatcatg tgctaggcct nccttaggct gattgttgtt caaataactt 6540
aagtttcctg tcaggtgatg tcatatgatt tcatatatca aggcaaaaca tgttatatat 6600
gttaaacatt tgnacttaat gtgaaagtta ggtctttgtg ggttttgatt ttaatttcaa 6660
aacctgagct aaataagtca ttttacatgt cttacatttg gtgaattgta tattgtggtt 6720
tgcaggcaag actctctgac ctagtaaccc tcctatagag cactttgctg ggtcacaagt 6780
ctaggagtca agcatttcac cttgaagttg agacgttttg ttagtgtata ctagttatat 6840
gttggaggac atgtttatcc agaagatatt caggactatt tttgactggg ctaaggaatt 6900
gattctgatt agcactgtta gtgagcattg agtggccttt aggcttgaat tggagtcact 6960
tgtatatctc aaataatgct ggcctttttt naaaagccct tgttctttat caccctgttt 7020
tctacataat ttttgttcaa agaaatactt gtttggatct ccttttgaca acaatagcat 7080
gttttcaagc catatttttt ttcctttttt tttttttttt tggtttttcg agacagggtt 7140
tctctgtata gccctggctg tcctggaact cactttgtag accaggctgg cctcgaactc 7200
agaaatccgc ctgcctctgc ctcctgagtg ccgggattaa aggcgtgcac caccacgcct 7260
ggctaagttg gatattttgt atataactat aaccaatact aactccactg ggtggatttt 7320
taattcagtc agtagtctta agtggtcttt attggccctt attaaaatct actgttcact 7380
ctaacagagg ctgttggact agtggnacta agcaacttcc tacggatata ctagcagata 7440
agggtcaggg atagaaacta gtctagcgtt ttgtatacct accagcttat actaccttgt 7500
tctgatagaa atatttagga catctagctt atcgatccgt cgacggtatc gataagcttg 7560
atatcgaatt ctaccgggta ggggaggcgc ttttccaagg cagtctgagc atgcggtttt 7620
cccagtcacg acgcggcgcc aggggcacgc gggacacgcc ccctcccgcc gcgccattgg 7680
cctctccgcc caccgcccca cacttattgg ccggtgcgcc gccaatcagc ggaggctgcc 7740
ggggcnncgc ctaaagaaga ggctgtgctt tggggctccg gctcctcaga gagcctcggc 7800
taggtagggg atcgggactc tggcgggagg gcggcttggt gcgtttgcgg ggatgggcgg 7860
ccgcggcagg ccctccgagc gtggtggagc cgttctgtga gacagccggg tacgagtcgt 7920
gacgctggaa ggggcaagcg ggtggtgggc aggaatgcgg tccgccctgc agcaaccgga 7980
gggggaggga gaagggagcg gaaaagtctc caccggacgc ggccatggct cggggggggg 8040
ggggcagcgg aggagcgctt ccggccgacg tctcgtcgct gattggcttc ttttcctccc 8100
gccgtgtgtg aaaacacaaa tggcgtgttt tggttggcgt aaggcgcctg tcagttaacg 8160
gcagccggag tgcgcagccg ccggcagcct cgctctgccc actgggtggg gcgggaggta 8220
ggtggggtga ggcgagctgg acgtgcgggc gcggtcggcc tctggcgggg cgggggaggg 8280
gagggagggt cagcgaaagt agctcgcgcg cgagcggccg cccaccctcc ccttcctctg 8340
ggggagtcgt tttacccgcc gccggccggg cctcgtcgtc tgattggctc tcggggccca 8400
gaaaactggc ccttgccatt ggctcgtgtt cgtgcaagtt gagtccatcc gccggccagc 8460
gggggcggcg aggaggcgct cccaggttcc ggccctcccc tcggccccgc gccgcagagt 8520
ctggccgcgc gcccctgcgc aacgtggcag gaagcgcgcg ctgggggcgg ggacgggcag 8580
tagggctgag cggctgcggg gcgggtgcaa gcacgtttcc gacttgagtt gcctcaagag 8640
gggcgtgctg agccagacct ccatcgcgca ctccggggag tggagggaag gagcgagggc 8700
tcagttgggc tgttttggag gcaggaagca cttgctctcc caaagtcgct ctgagttgtt 8760
atcagtaagg gagctgcagt ggagtaggcg gggagaaggc cgcacccttc tccggagggg 8820
ggaggggagt gttgcaatac ctttctggga gttctctgct gcctcctggc ttctgaggac 8880
cgccctgggc ctgggagaat cccttccccc tcttccctcg tgatctgcaa ctccagtctt 8940
tctaga 8946
<210> 2
<211> 1455
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 2
atggcggagc tgcggcctag cggcgccccc ggccccaccg cgcccccggc ccctggcccg 60
actgcccccc cggccttcgc ttcgctcttt cccccgggac tgcacgccat ctacggagag 120
tgccgccgcc tttaccctga ccagccgaac ccgctccagg ttaccgctat cgtcaagtac 180
tggttgggtg gcccagaccc cttggactat gttagcatgt acaggaatgt ggggagccct 240
tctgctaaca tccccgagca ctggcactac atcagcttcg gcctgagtga tctctatggt 300
gacaacagag tccatgagtt tacaggaaca gatggaccta gtggttttgg ctttgagttg 360
acctttcgtc tgaagagaga aactggggag tctgccccac caacatggcc cgcagagtta 420
atgcagggct tggcacgata cgtgttccag tcagagaaca ccttctgcag tggggaccat 480
gtgtcctggc acagcccttt ggataacagt gagtcaagaa ttcagcacat gctgctgaca 540
gaggacccac agatgcagcc cgtgcagaca ccctttgggg tagttacctt cctccagatc 600
gttggtgtct gcactgaaga gctacactca gcccagcagt ggaacgggca gggcatcctg 660
gagctgctgc ggacagtgcc tattgctggc ggcccctggc tgataactga catgcggagg 720
ggagagacca tatttgagat cgatccacac ctgcaagaga gagttgacaa aggcatcgag 780
acagatggct ccaacctgag tggtgtcagt gccaagtgtg cctgggatga cctgagccgg 840
ccccccgagg atgacgagga cagccggagc atctgcatcg gcacacagcc ccggcgactc 900
tctggcaaag acacagagca gatccgggag accctgagga gaggactcga gatcaacagc 960
aaacctgtcc ttccaccaat caaccctcag cggcagaatg gcctcgccca cgaccgggcc 1020
ccgagccgca aagacagcct ggaaagtgac agctccacgg ccatcattcc ccatgagctg 1080
attcgcacgc ggcagcttga gagcgtacat ctgaaattca accaggagtc cggagccctc 1140
attcctctct gcctaagggg caggctcctg catggacggc actttacata taaaagtatc 1200
acaggtgaca tggccatcac gtttgtctcc acgggagtgg aaggcgcctt tgccactgag 1260
gagcatcctt acgcggctca tggaccctgg ttacaaattc tgttgaccga agagtttgta 1320
gagaaaatgt tggaggattt agaagatttg acttctccag aggaattcaa acttcccaaa 1380
gagtacagct ggcctgaaaa gaagctgaag gtctccatcc tgcctgacgt ggtgttcgac 1440
agtccgctac actag 1455
<210> 3
<211> 1455
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 3
atggcggagc tgcggcctag cggcgccccc ggccccaccg cgcccccggc ccctggcccg 60
actgcccccc cggccttcgc ttcgctcttt cccccgggac tgcacgccat ctacggagag 120
tgccgccgcc tttaccctga ccagccgaac ccgctccagg ttaccgctat cgtcaagtac 180
tggttgggtg gcccagaccc cttggactat gttagcatgt acaggaatgt ggggagccct 240
tctgctaaca tccccgagca ctggcactac atcagcttcg gcctgagtga tctctatggt 300
gacaacagag tccatgagtt tacaggaaca gatggaccta gtggttttgg ctttgagttg 360
acctttcgtc tgaagagaga aactggggag tctgccccac caacatggcc cgcagagtta 420
atgcagggct tggcacgata cgtgttccag tcagagaaca ccttctgcag tggggaccat 480
gtgtcctggc acagcccttt ggataacagt gagtcaagaa ttcagcacat gctgctgaca 540
gaggacccac agatgcagcc cgtgcagaca ccctttgggg tagttacctt cctccagatc 600
gttggtgtct gcactgaaga gctacactca gcccagcagt ggaacgggca gggcatcctg 660
gagctgctgc ggacagtgcc tattgctggc ggcccctggc tgataactga catgcggagg 720
ggagagacca tatttgagat cgatccacac ctgcaagaga gagttgacaa aggcatcgag 780
acagatggct ccaacctgag tggtgtcagt gccaagtgtg cctgggatga cctgagccgg 840
ccccccgagg atgacgagga cagccggagc atctgcatcg gcacacagcc ccggcgactc 900
tctggcaaag acacagagca gatccgggag accctgagga gaggactcga gatcaacagc 960
aaacctgtcc ttccaccaat caaccctcag cggcagaatg gcctcgccca cgaccgggcc 1020
ccggaccgca aagacgacct ggaaagtgac agctccacgg ccatcattcc ccatgagctg 1080
attcgcacgc ggcagcttga gagcgtacat ctgaaattca accaggagtc cggagccctc 1140
attcctctct gcctaagggg caggctcctg catggacggc actttacata taaaagtatc 1200
acaggtgaca tggccatcac gtttgtctcc acgggagtgg aaggcgcctt tgccactgag 1260
gagcatcctt acgcggctca tggaccctgg ttacaaattc tgttgaccga agagtttgta 1320
gagaaaatgt tggaggattt agaagatttg acttctccag aggaattcaa acttcccaaa 1380
gagtacagct ggcctgaaaa gaagctgaag gtctccatcc tgcctgacgt ggtgttcgac 1440
agtccgctac actag 1455
<210> 5
<211> 1214
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 5
atggcggagc tgcggcctag cggcgccccc ggccccaccg cgcccccggc ccctggcccg 60
actgcccccc cggccttcgc ttcgctcttt cccccgggac tgcacgccat ctacggagag 120
tgccgccgcc tttaccctga ccagccgaac ccgctccagg ttaccgctat cgtcaagtac 180
tggttgggtg gcccagaccc cttggactat gttagcatgt acaggaatgt ggggagccct 240
tctgctaaca tccccgagca ctggcactac atcagcttcg gcctgagtga tctctatggt 300
gacaacagag tccatgagtt tacaggaaca gatggaccta gtggttttgg ctttgagttg 360
acctttcgtc tgaagagaga aactggggag tctgccccac caacatggcc cgcagagtta 420
atgcagggct tggcacgata cgtgttccag tcagagaaca ccttctgcag tggggaccat 480
gtgtcctggc acagcccttt ggataacagt gagtcaagaa ttcagcacat gctgctgaca 540
gaggacccac agatgcagcc cgtgcagaca ccctttgggg tagttacctt cctccagatc 600
cgttggtgtc tgcactgaag agctacactc agcccagcag tggaacgggc agggcatcct 660
ggagctgctg cggacagtgc ctattgctgg cggcccctgg ctgataactg acatgcggag 720
gggagagacc atatttgaga tcgatccaca cctgcaagag agagttgaca aaggcatcga 780
gacagatggc tccaacctga gtggtgtcag tgccaagtgt gcctgggatg acctgagccg 840
gccccccgag gatgacgagg acagccggag catctgcatc ggcacacagc cccggcgact 900
ctctggcaaa gacacagagc agatccggga gaccctgagg agaggactcg agatcaacag 960
caaacctgtc cttccaccaa tcaaccctca gcggcagaat ggcctcgccc acgaccgggc 1020
cccggcccgc aaagacgcct ggaccctggt tacaaattct gttgaccgaa gagtttgtag 1080
agaaaatgtt ggaggattta gaagatttga cttctccaga ggaattcaaa cttcccaaag 1140
agtacagctg gcctgaaaag aagctgaagg tctccatcct gcctgacgtg gtgttcgaca 1200
gtccgctaca ctag 1214
Claims (4)
1.一种基于同源重组技术的磷酸化突变体SUFU转基因小鼠模型的构建及应用,其特征在于,包括如下步骤:
步骤1:SUFUWT/SUFUS342/346D/ SUFUS342/346A 双磷酸化突变体编码基因以及野生型SUFU编码基因转载在含有tpA转录终止序列的pBigT的载体上;
步骤2:把含有tpA转录终止序列和目的基因的pBigT载体装到包含有基因组DNARosa26序列的pRosa26PA上;
步骤3:转染SUFUWT/SUFUS342/346D/ SUFUS342/346A 双磷酸化突变体编码基因以及野生型SUFU编码基因打靶载体:
将构建好的SUFUWT/SUFUS342/346D/ SUFUS342/346A 双磷酸化突变体编码基因以及野生型SUFU编码基因打靶载体pROSA26PA,用酶切线性化,然后将线性化基因打靶载体利用电击法导入来源于129/S6小鼠的ES细胞中;然后加入G418 筛选出发生同源重组的ES细胞克隆。
2.将ES细胞提取DNA,再利用PCR法扩增并测序鉴定ES克隆是敲入了SUFUWT/SUFUS342 /346D/ SUFUS342/346A 双磷酸化突变体编码基因以及野生型SUFU编码基因,筛选出发生正确同源重组的ES细胞克隆;
步骤4:显微注射和PCR 筛选制作磷酸化突变体SUFU转基因小鼠模型:
将筛选鉴定正确的ES细胞通过显微注射技术注入C57BL/6小鼠的囊胚中,经注射的囊胚转移至假孕母鼠的子宫内着床,生出嵌合体小鼠;
将嵌合体小鼠再与 C57BL/6 小鼠交配,产生出棕色和黑色后代小鼠;
将其中的棕色小鼠用PCR方法1筛选出阳性小鼠,再将阳性小鼠与Ddx4-Cre小鼠交配,通过PCR筛选得到杂合型 F1代小鼠;
最后将杂合型F1代小鼠自交得到F2代小鼠,用筛选F1代小鼠相同的PCR方法2筛选出纯合 F2 代小鼠,即为磷酸化突变体SUFU转基因小鼠模型。
3.根据权利要求1所述的基于同源重组技术的磷酸化突变体SUFU转基因小鼠模型及应用,其特征在于:所述步骤4中,所述 PCR方法1扩增引物序列是:
SD-left-2820-120 :TGGGGATCGACGGTATCGTA
SD-right-2820-120 :TACCGTCGACCTCGAGGG。
4.根据权利要求1所述的基于同源重组技术的磷酸化突变体SUFU转基因小鼠模型及应用,其特征在于:所述步骤4中,所述PCR方法2扩增引物序列是:
Rosa26-F8731:5’-CTTGCTCTCCCAAAGTCGCT-3’;
Rosa26-R:5’-GCGGGAGAAATGGATATGAAGTA-3’;
pBigT-R:5’-CGGCCTCGACTCTACGATAC-3’。
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