摘要
研究背景和目的
由于体型小、便于操作,而且解剖生理特性与人特别相近的特点,小型猪已逐渐成为生物医药研究的重要模式动物,应用数量逐年大幅递增。本研究中的西藏小型猪是世界上体型较小的小型猪之一,来源于青藏高原、海拔2500-4300m的农区和半农牧区,是唯一能够适应高海拔气候和以放牧为主的猪种,封闭的地理环境使西藏小型猪保存了非常纯正的品种资源。本课题组于2004年将西藏小型猪从西藏引进至广州,目前已完成风土驯化及实验动物化研究,并开展了相关动物模型、药物实验及转基因克隆等研究。从免疫学、遗传学研究发现,该品系具有独特的免疫相关指标和遗传特征,加上其独特的外形,是一种优良的实验用小型猪品种。
基因工程动物的制备研究是目前生命科学领域关键技术之一,通过转基因或者基因敲除等操作改变动物基因的遗传组成,可将动物按照人们的意愿进行各种基因改造,获得满足各种需要的基因工程动物。目前,基因工程小鼠研究的最为成熟,常用的技术方法有原核显微注射法和ES细胞法。而猪的ES细胞虽然已有分离成功的报道,但其培养体系并不稳定,同时远没有达到推广应用的水平。另外猪的受精卵数量来源有限且受精卵内脂肪滴过多也不利于原核显微操作。因此利用这些传统的技术方法均难以获得转基因及基因敲除的基因修饰猪。
随着核移植克隆猪技术的出现并不断成熟,转基因克隆技术逐渐成为制备转基因及基因敲除猪的主要方法。该方法通过在体外培养的体细胞水平进行转基因及基因敲除操作,利用体细胞核移植技术达到对动物机体水平基因的修饰,获得基因修饰克隆猪。因此为了建立西藏小型猪的转基因及基因敲除研究体系,本研究拟通过转基因及基因敲除两个方面分别进行基因修饰克隆西藏小型猪的研究。
(一)多能性基因Oct-4绿色荧光报告系统转基因克隆猪的建立
转录因子Oct-4 (POU5fl)属于POU(Pit-Oct-Unc)转录因子的家族成员,能够特异性识别DNA八聚体ATTTGCAT并与之结合,借此调控下游基因的表达。它特异性的表达于胚胎干细胞、原始生殖细胞等多能性细胞中,是一种重要的细胞多能性标记因子。
Oct-4为母性遗传的转录因子,在成熟的卵母细胞中即可检测到高水平的表达。基因组激活后,Oct-4基因随之也被激活表达。直到胚胎着床后,Oct-4基因表达逐渐停止,并仅限制在生殖细胞表达,而在已分化的体细胞中不表达。在体外培养的ES细胞中,Oct-4基因可以作为一个开关,直接调控ES细胞的全能性及自我复制能力的有无。
那么,如何监测内源性Oct-4基因的表达?常规的方法有RT-PCR、western blot、免疫荧光等。但这几种方法共同缺陷是观察细胞处理后的不可存活性,阻碍其进一步的研究。本研究拟采用Oct4-GFP的方法,利用以猪Oct-4基因的5’端完整调控区启动绿色荧光蛋白基因(EGFP)的表达,通过荧光观察实时监测猪细胞内内源性Oct-4表达。应用这种可视性的策略可直观的了解内源性-Oct-4基因的表达模式。该方法目前已广泛的应用于小鼠和人的多能性干细胞研究中,而对猪的研究较少。
(二)敲除酪氨酸酶基因建立白化西藏小型猪的研究
由于猪和人的皮肤在解剖结构和生理机能等方面非常相似,猪常用于皮肤烧伤、化妆品及药物的皮肤毒性实验和过敏反应等方面研究,我国小型猪的皮肤和毛发多为黑色或花色,而黑色或花色系猪种的皮肤明显不利于实验的观察。白色猪种多见于家猪等大型猪种,不利于实验操作。西藏小型猪由于其体型大小,皮肤的解剖生理特性与人的相似性,独特的生物学特性使其成为一个优良的实验用小型猪品种。但该小型猪全身的皮肤和毛发均为黑色,因此如何将西藏小型猪的皮肤和毛色培育成白色是小型猪实验动物化中迫切需要解决的问题之一。
哺乳动物皮肤和毛发的颜色因色素的种类和含量不同而不同。酪氨酸酶是色素合成过程的限速酶,酪氨酸酶基因的缺失或者突变将直接导致人、鼠、猫等动物的白化。
本课题拟将细胞基因敲除技术与体细胞核移植技术相结合,制备酪氨酸酶基因敲除的白化西藏小型猪。白化西藏小型猪的建立,将为药物、化妆品的毒性及过敏反应提供标准化的动物模型。与传统的育种方法相比,转基因育种技术总体效率更高,不仅大大缩短了育种时间,且所获品系的遗传背景单一,是白化实验用西藏小型猪育种的更高效策略。
研究方法
(一)多能性基因Oct-4绿色荧光报告系统转基因克隆猪的建立
1、西藏小型猪Oct-4基因启动子区域的克隆与序列鉴定
根据genebank中大白猪Oct-4基因启动子区域的序列信息,设计引物oct-pro-F2/oct-pro-R2从西藏小型猪基因组内扩增Oct-4基因启动子区域的DNA序列。PCR产物连接pMD18-T载体,进行测序、比对分析。
为了进一步对西藏小型猪Oct-4基因启动子区域进行结构分析,本文将猪、人、牛、家兔、小鼠、大鼠的Oct-4基因5’调控区域进行多重比较分析。并根据已报道的功能结构区域,对西藏小型猪的功能结构域进行预测和分析。
2、pOGN2荧光表达载体的构建及体外瞬时转染检测
应用分子克隆技术将上一步克隆并测序正确的西藏小型猪Oct-4基因启动子连接入pEGFP-N2载体的HindⅢ和KpnI之间,AseⅠ和BglⅡ酶切去除CMV载体,载体末端平端化,平端化后自连。获得目的载体命名为pOGN2。载体pOGN2中猪Oct-4基因启动子替换载体中原有的CMV启动子,含有Oct4-EGFP和Neo两个表达框,便于下一步荧光观察和药物筛选实验。
为了验证载体pOGN2中Oct-4基因启动子的有效性和特异性,研究将质粒pOGN2经HindⅢ线性化并经乙醇纯化后,转染猪IPS细胞(已知有Oct-4基因表达)。同时转染pEGFP-N2(CMV-EGFP)。观察两个实验组转染后IPS细胞和饲养层细胞绿色荧光表达情况。
3、pOGN2转基因猪胚胎成纤维细胞的建立
在西藏小型猪怀孕30-35d时,无菌剖腹手术取出胎儿。利用胶原酶消化方法体外分离培养猪胚胎成纤维细胞。细胞药物筛选前,进行G418药物毒性试验。设置G418浓度分别为0μg/ml,200μg/ml,400μg/ml,600μg/ml,800μg/ml, 1000μg/ml,1200μg/ml。根据细胞死亡情况确定G418筛选浓度。
HindⅢ酶切线性化质粒pOGN2,线性化后质粒经脂质体lipo2000转染西藏小型猪胚胎成纤维细胞。转染后1：25传代培养,48小时后加G418药物进行筛选。7-10天后出现细胞克隆,使用克隆环挑取克隆,转移至48孔板中培养。待细胞长满后进一步传代扩大培养,并取部分细胞裂解基因组DNA,应用PCR进行转基因鉴定。将鉴定为阳性的转基因细胞克隆液氮冻存放置。
4、pOGN2转基因核移植胚胎及pOGN2转基因猪的建立
猪卵母细胞的体外成熟培养：屠宰场取卵巢,置38℃左右的含青霉素和硫酸链霉素的生理盐水中,运回实验室。用配有14号针头的注射器抽吸3-8mm的卵泡。于成熟培养液中,38.5℃成熟培养42小时。卵母细胞成熟培养后,透明质酸酶消化去除卵丘,体视显微镜下挑选有第一极体的卵细胞,作为体细胞核移植的受体细胞。
体细胞核移植：挑取具有第一极体的成熟卵细胞,采用盲吸法吸取第一极体及其附近10-20%可能含有卵母细胞核的细胞质。挑选准备好的转基因体细胞,从去核切口放入卵周隙内。将重组卵转入电融合槽中,使供体细胞和受体卵细胞膜接触面与电极平行,用BSL融合仪施加2个直流电脉冲(1.2kV/cm,60μs)诱导融合并激活,转入矿物油覆盖的胚胎培养液PZM-3中,于38.5℃、5%二氧化碳、95%空气的气体环境,饱和湿度条件下培养0.5h-lh后取出,在体视显微镜下判定是否融合。
转基因核移植胚胎的体外培养及绿色荧光表达的观察：核移植胚胎置38.5℃、5%C02培养5-7天后,观察胚胎发育情况。荧光倒置显微镜下观察绿色荧光的表达情况。同时将表达绿色荧光的胚胎,进行Oct-4蛋白的免疫荧光染色,检测内源性Oct-4基因的表达情况。
核移植胚胎的移植：将核移植胚胎体外培养表达绿色荧光的体细胞克隆,继续体细胞核移植,并将核移植胚胎移植受体母猪的输卵管内。
5、利用pOGN2转基因猪胚胎成纤维细胞进行猪IPS诱导重编程研究
将人的pMXs-Oct4,pMXs-Sox2,pMXs-Klf4,pMXs-c-Myc同时转染HEK293T,包装逆转录病毒。将四因子病毒感染pOGN2转基因猪胚胎成纤维细胞,48小时后,消化离心细胞,重新铺细胞至100mm培养皿中,1×104／皿。更换IPS诱导培养液。培养17-22天,镜下观察可出现IPS样的细胞克隆。荧光显微镜下观察IPS样细胞克隆是否表达绿色荧光,并挑取绿色荧光表达的细胞克隆,传代培养,进行IPS细胞的鉴定。
(二)敲除酪氨酸酶基因建立白化西藏小型猪研究
1、构建TYR基因基因打靶载体
(1)克隆TYR基因的未知基因组序列。通过比对人、牛、犬、鼠等已知的TYR序列,设计兼并引物,进行嵌套PCR克隆获得西藏小型猪TYR未知基因组序列。
(2)构建TYR基因正负筛选敲除载体,根据克隆所得的TYR基因组序列
设计同源重组的左臂和右臂,利用分子克隆技术分别克隆入打靶载体pSSC-9载体中,获得TYR正负筛选基因敲除载体pSSC-tyrKO。
(3)TYR特异性锌指的设计、合成及体外筛选鉴定,TYR修复质粒的制备。参考小鼠TYR锌指的序列及锌指open库筛选结果,设计针对西藏小型猪TYR第一外显子的锌指一对。同时将该对锌指蛋白分别连接两组不同的核酸酶切酶(DD/RR,KK/EL)。设计引物进行overlap法合成锌指并测序。同时构建相应的修复质粒pSSC-tyrZFNKO。
制备锌指体外细胞鉴定的转基因细胞模型,将含有锌指靶序列的GFP表达框转入293T细胞,获得稳定整合的转基因293T细胞系。将合成的锌指及修复质粒共同转染该转基因293T细胞系,根据细胞的绿色荧光表达情况来鉴定锌指是否有效。
将分别连接核酸酶DD/RR、KK/EL的TYR锌指转染转基因293T细胞系后,进行流式分析,检测锌指的修复效率.
2、打靶质粒转染猪胚胎成纤维细胞,阳性基因敲除猪胚胎成纤维细胞的筛选和鉴定。
(1)正负筛选基因打靶
将TYR正负筛选基因敲除载体pSSC-tyrKO进行酶切线性化,纯化后进行转染。采用lipo2000进行转染,转染48h后加入正筛选药物G418和负筛选药物GANC进行双筛。筛选12-15天后,挑取抗性克隆,进行传代培养。
取部分细胞,进行体外裂解,取裂解产物为模板,设计嵌套引物进行PCR鉴定。PCR鉴定阳性的克隆连接T载体,进一步测序鉴定。
(2)锌指介导基因打靶
待转染锌指及修复质粒的准备。无内毒素提取锌指质粒,修复质粒酶切线性化。将锌指及线性化的修复质粒共同转染猪胚胎成纤维细胞,转染后48小时加正筛选药物G418和负筛选药物GANC进行双筛。筛选12-15天后,挑取抗性克隆,进行传代培养。
取部分细胞,进行体外裂解,取裂解产物为模板,设计引物进行PCR鉴定。PCR鉴定阳性的克隆连接T载体,进一步测序鉴定。
3、以TYR基因敲除的细胞作为核供体,经体细胞克隆技术制备TYR基因敲除的西藏小型猪(TYR+/-)。
研究结果
(一)多能性基因Oct-4绿色荧光报告系统转基因克隆猪的建立
1、扩增获得的西藏小型猪Oct-4基因启动子序列与genebank中大白猪进行比对发现,序列同源性为97%。证明已成功的扩增出西藏小型猪Oct-4基因启动子DNA。与人、牛、家兔、小鼠、大鼠序列进行多重比较时发现,猪的Oct-4基因启动子在4个序列保守区域(CR1-4)与其他物种高度保守。其中人与猪的序列相似度最高。核心启动子位于CRl内,远端增强子位于CR4内,在功能区域位点不同的物种间序列高度保守。近端增强子PE-1B位于CR2保守区域内,而-PE-1A位于CR2的外侧,不同的物种间序列差异较大。证明与PE-1A相关的调控方式为种间特异性的。
2、构建的载体pOGN2经测序证实载体构建成功。测序正确的载体线性化后转染猪IPS细胞,同时以pEGFP-N2为对照。转染后荧光显微镜观察发现,对照组pEGFP-N2转染猪IPS细胞后,IPS细胞核饲养层细胞均有细胞表达绿色荧光。而pOGN2(Oct4-EGFP)转染组,仅在IPS细胞表达绿色荧光,在饲养层细胞未见任何绿色荧光细胞出现。表明pOGN2载体中Oct-4启动子的特异性,仅在Oct-4表达的多能性细胞中表达。同时也说明Oct-4启动子能有效地启动GFP的表达。证实了pOGN2载体中Oct-4启动子的有效性和特异性启动表达功能。
3、线性化质粒pOGN2转染猪胚胎成纤维细胞后,经G418药物筛选后获得的克隆进行PCR鉴定,发现有10个克隆鉴定为阳性转基因克隆。其中8个克隆细胞生长良好。
4、将生长良好的8个转基因胚胎成纤维克隆进行体细胞核移植重编程,进行胚胎体外培养和观察。结果发现8个转基因胚胎成纤维细胞克隆所获得重构胚胎中,其中#3、#4、#8克隆表达强绿色荧光。#1、#2克隆荧光表达较弱。而其他几个克隆没有观察到绿色荧光的表达。将表达绿色荧光的重构胚进行Oct-4蛋白的免疫荧光染色后发现,内源性Oct-4的表达与绿色荧光的表达一致。将#3、#4、#8克隆重新进行核移植,并将核移植胚胎移植发情的受体母猪。共移植13头受体母猪,目前已有3头怀孕,待分娩。
5、取抗性细胞克隆,复苏后细胞扩大培养,取部分细胞进行IPS诱导研究。经过17-22天的诱导培养后,镜下观察,出现少量绿色荧光表达的IPS样细胞克隆,同时也有许多表达绿色荧光的单个细胞存在。大多数细胞克隆没有检测到绿色荧光的表达。
(二)敲除酪氨酸酶基因建立白化西藏小型猪的研究
1、正负筛选基因打靶
(1)成功克隆西藏小型猪TYR基因的部分未知基因组序列。经过兼并引物嵌套PCR方法成功克隆出TYR基因的部分未知序列,序列经比对分析证明为正确的序列。
(2)取第二外显子为靶序列,以左侧第一内含子4000bp及右侧1200bp序列为同源臂,构建正负筛选打靶载体。所获得载体pSSC-tyrKO经酶切和测序鉴定均为正确。
(3)将该载体转染猪胚胎成纤维细胞后,经正负筛选共获得抗性克隆166个。经PCR鉴定,其中有一个细胞克隆为TYR基因敲除克隆。
(4)以该细胞克隆为核供体,进行体细胞核移植。获重组胚胎400枚,共移植两个受体母猪。其中有一头36天解剖发现有着床点但被吸收,另一头返情没有受孕。
2、锌指介导基因打靶
(1)overlap法合成锌指tyrZFN-131 F/tyrZFN-131R,测序获得序列正确的克隆。分别连入载体pST1374-DD/RR及pST1374-KK/EL。经酶切及测序证明载体构建正确。
(2)锌指和修复质粒共同转染转基因293T细胞系,3天后观察到GFP阳性的细胞出现,表明锌指能特异性切割靶序列,证明锌指的有效性。
流式分析结果表明,tyrKK/EL的修复效率最高,可达0.29%。而tyrDD/RR稍低,为0.23%。只转染修复质粒的细胞组肉眼观察未见绿色荧光细胞的出现。
结论
(一)多能性基因Oct-4绿色荧光报告系统转基因克隆猪的建立
1、扩增获得西藏小型猪Oct-4基因5’上游启动子区域
2、成功构建含有Oct4-EGFP和NEO两个表达框的载体pOGN2,并通过猪IPS细胞转染验证载体构建成功。
3、获得整合载体pOGN2的西藏小型猪转基因胚胎成纤维细胞。
4、通过核移植重编程和IPS重编程研究结果表明,本研究已成功的建立了多能性基因Oct-4表达GFP报告系统。可广泛的应用于猪IPS诱导重编程体系的建立和完善。
(二)敲除酪氨酸酶基因建立白化西藏小型猪的研究
1、成功克隆TYR部分未知基因序列并构建TYR正负筛选基因打靶载体。转染细胞后,经正负筛选获得一个阳性基因敲除细胞克隆。
2、本实验设计并合成了西藏小型猪TYR基因的锌指一对,并在293T细胞验证该锌指有效。
3、设计构建了锌指鉴定的293T细胞模型,用于锌指合成后的鉴定筛选。
本研究的创新之处
1、本项目利用我国独特的小型猪品系-西藏小型猪,将体细胞基因修饰和克隆技术相结合,培育具有我国自主知识产权的基因修饰克隆西藏小型猪。
2、本研究首次将猪Oct-4基因5’上游完整的调控区域启动绿色荧光的表达,利用该报告系统,可直观、方便地根据绿色荧光的表达监测猪细胞内多能性基因Oct-4的表达情况。
3、根据酪氨酸酶基因结构和功能特点,采用基因敲除克隆猪技术制备白化西藏小型猪。为进行化妆品、药品的皮肤毒性实验及过敏反应等研究提供标准化的白化实验小型猪。此外,也为人类白化病发病机制和基因治疗研究提供大型实验动物模型。
Background and objective
Since the anatomical and physiological similarites to humans and the relative ease with which they can be handled with in experiment, the minipigs are considered improtant model animals in biomedical research. The Tibet-minipig in this study coming from the farming and farming&herding region in attitude 2500-4300m of Qing-zang plateau, is the only one that can accommodate the high elevation climate. The closed geographical environment makes it keep the genuine breed. The Tibet-minipigs were introduced from Tibet to Guangzhou for laboratory animal research in 2004. Until now, we have finished acclimatization and experimental animalization. We also carried out lots of studies about animal model, drug experiment and transgenic cloning. The research results about immunology and genetics showed that this minipig presented unique characteristics and was an excellent experimental miniature pig.
Preparation and research about genetic engineering animal is the one of key technologis in life science field. We can obtain different genetically modified animals by use of transgenic and gene knockout technology. Now the establishment of genetically modified mice with pronuclear microinjection and ES methods is the most mature and common technology. However porcine ES cells are not availabe in many labs, yet successful isolation has been reported recently. Pronuclear microinjection is also not suitable for pig because of hard to get fertilized eggs and excess fat drop. Therefore, it is difficult to achieve genetically modified pigs with the traditional methods.
Since the advent of nuclear transfer technology, we can produce genetically modified pigs from gene modified somatic cells. In order to establish the research platform of gene modified Tibet minipig, here we report producing transgenic and gene knockout Tibet minipig.
I. Establishment of porcine Oct-4 promoter-driven EGFP reporter system for evaluation of Oct-4 status in porcine pluripotenct cells
Oct-4, a member of Class V of the POU (Pit-Oct-Unc) transcription factor family, plays a crucial role in establishment and maintenace of transcriptional regulation during early embryo development and cell differentiation. The Oct-4 protein contains a POU domain that binds to the octamer sequence ATGCAAAT located in the promoter or enhancer regions of its target genes, to regulate the expression of target genes.
Oct-4 is a maternally inherited transcription factor and can be detected in matured oocyte. Oct-4 gene begins to be expressed after the genome activate. Until embryo implantation, its expression may stop gradually and only restricted in germ cells but not in differentiated somatic cells. Thus Oct-4 is a gatekeeper in regulateing the pluripotency of ES cells and in the beginnings of mammalian development.
Then, how can monitor the in vivo Oct-4 expression? The common methods used include RT-PCR, westent blot, immunofluorescence assay and so on. But all the methods have the defective of cell death after treatment and hinder further research. In this study we utilized the method of Oct4-GFP report system, GFP driven by the porcine Oct-4 promoter. The endogenous Oct-4 expressing status can be detected and monitored by GFP fluorescence observation. This visualized method has been applied in research of mouse and human pluripotent stem cells, but not in pig.
II. Establishment of TYR knockout albinism Tibet minipig by nuclear transfer
Pig is usually used for research about skin burn, skin toxic experiment and allergic reaction of cosmetics and drugs because of the skin anatomical and physiological similarites to human. However, the skin color of most Chinese minipig is black or mixed colour, leading to unsuitable for research observation. Therefore the production of albinism Tibet minipig is key question for experimental animalization.
The skin and hair color of mammalian depend on the type and content of pigment. Tyrosinase is the rate-limiting enzyme in the process of the pigment synthesis.TYR deletion and mutation would result in albinism in human, mouse, cat and so on. This study intend to produce TYR knockout albinism Tibet minipig with combination of somatic cell gene knockout and nuclear transfer. The albinism Tibet minipig will act as standard animal model for toxicity and anaphylactic response of cosmetics and drugs. Compared with traditional breeding method, it is more effective by use of breeding with transgenic technology. This method not only saves the breeding time but also produces strain with uniform genetic backgrounds.
Methods
Ⅰ. Establishment of porcine Oct-4 promoter-driven EGFP reporter system for evaluation of Oct-4 status in porcine pluripotency cells
1.Isolation and identification of porcine Oct-4 promoter region
For cloning porcine Oct-4 promoter, two primers were designed, namely oct-pro-F2/oct-pro-R2..Porcine Oct4 promoter DNA amplified was subcloned to pMD18T vector to create pT-Oct-pro for sequencing. DNA sequecne was identified by DNA blast.
In order to further analyze the structure of porcine Oct-4 promoter, we made multi-alignment between the corresponding region of human, bovine, rabbit, mouse, rat. The functional domain of porcine Oct-4 promoter region can be predicted and analyzed according to the alignment results.
2. Construction of pOGN2 vector and identification by transient transfection in vitro
The porcine Oct-4 promoter was isolated by digestion of pT-Oct-pro with HindⅢand KpnⅠand inserted upstream of EGFP cDNA in pEGFP-N2 (BD Biosciences Clontech) to produce pCOGN2. Then CMV promoter in pCOGN2 was removed by disgestiong with AseⅠand BglⅡ. The DNA fragment with 3'-and 5'-protruding ends produced by AseⅠand BglⅡdigestion was converted to that with blunt ends and was self-ligated with DNA blunting kit from Takara to create the vector pOGN2 for this research. The vector pOGN2 contained two expression cassette of Oct4-EGFP and Neo, which facilitate drug screening and fluoresence observation.
Expression assay of porcine Oct-4 promoter in pOGN2 was carried out to identify the efficiency and specification of the promoter. HindⅢlineared pOGN2 or lineared pEGFP-N2 were transfected into porcine IPS cells in which Oct-4 was expressed. After transfection, IPS cells and feeder cells were obsearved under fluorescence microscope.
3.Establishment of Oct4-GFP transgenic PFF cell clones
Porcine fetal fibroblasts (PFFs) were isolated from 35d Tibet minipig fetus. G418 toxicity trails was performed before drug screecing. The concentration gradients were as follows: Oμg/ml,200μg/ml,400μg/ml,600μg/ml,800μg/ml,1000μg/ml,1200μg/ml,1400μg/ml. On the basis of cell death results, the optimized concentration was found out. For transfection, HindⅢ-lineared pOGN2 and lipo2000 (invitrogen) was mixed together. Then the transfection was performed according to the manufacturer's protocol. The day after transfection, cells were passaged to 100-mm dishes by the ratio of 1:25.At 2 days of culture, selective medium with 1000μg/ml G418 were added and cells were cultured for additional 7-10 days. G418 resistant cell colonies were selected by the cloning ring (sigma) and propagated in a 48-well culture plate.4-6 days later, wells in which cells growed confluently were trypsinized (60μl trypsin,0.5% EDTA) and seeded in a 24-well plate for frozen soon after. Parts of the cells were used for PCR screening. Cells were resuspended in lysis buffer and 1μl lysate was used in a 20μl PCR reaction. The positive cell clones were cryopreserved in liquid nitrogen for further use.
4. Production of SCNT embryos with Oct4-GFP transgenic PFFs and establishment of Oct4-GFP transgenic pig
In vitro muturation of porcine oocytes:Immature oocytes are derived from ovaries obtained from the slaughterhouse and subsequently matured in vitro. Porcine ovaries are collected from an abattoir and transported to the laboratory in a thermos filled with saline maintained at 30-35℃within 4 h after collection. Follicular fluid from 3-6-mm antral follicles is aspirated by using an 18-gauge needle attached to a 10-mL disposable syringe. The oocytes are matured for 42-44 h at 39℃,5% CO2 in air. Oocytes with an intact plasma membrane, round shape and visible first polar body are selected and kept in manipulation medium until use.
Nuclear transfer:Enucleation was accomplished by aspirating the first polar body and the metaphase II plate in a small amount of surrounding cytoplasm. Then injection of donor cells into the perivitelline space of enucleated oocytes was carried out. Electrical fusion and activation were achieved by direct current of 1.2kV/cm,60μs. After activation, embryos are transferred to 500 mL embryo culture medium in a four-well dish and covered with light mineral oil.
In vitro culture of embryos and fluorescence observation:Oct4-GFP transgenic SCNT embryos were observed under fluorescence microscope after 5-7 days culture. Oct-4 protein was detected in GFP positive embryos with immunofluorescence.
Embryo transfer:Since the nuclear transfer embryos are generally of a low quality, a large number(>100) one-cell stage embryos cultured 18-22 h with good shape (intact membrane) are surgically transferred into an oviduct of the surrogate.
5.Porcine iPS cells induction with pOGN2 transgenic PFFs
Retroviral vectors encoding EGFP or human Oct-4, Sox2, Klf4 and c-Myc were produced using HEK293T cells.4×104 PFF cells per p6 well-dishes were infected with 2.0ml each of unconcentrated retroviral vector in the presence of 8 mg/ml Polybrene. Two days after infection, the cells were trypsinized, and 1×104 cells were transferred onto feeders in a 10cm dish in dFBS medium. Emerging ES-like colonies were picked from 17-22 d and observed under fluorescence microscope. The GFP positive IPS clones were passaged for identification and further research.
Ⅱ. Establishment of TYR knockout albinism Tibet minipig by nuclear transfer
1.Construction of TYR gene knockout vector
(1)Cloning of unknown TYR genomic DNA. According to the DNA multi-alignment among human, bovine and dog, two primers pairs were designed for each homologous arm. Nested PCR was performed to clone TYR gene as two homologous recombination (HR) arms.
(2) Construction of TYR PNS gene knockout vector. Two homologous arms were disigned and cloned into the vector pSSC-9 that contains Neor and two HSV-tk genes as positve and negative screening genes, respectively. The knockout vector was designated as pSSC-tyrko.
(3) Design and synthesis of TYR ZFN and identification in vitro, construction of donor plasmid. With the reference of mouse TYR ZFN sequence and the results of OPEN-ZiFit screening, one pair of ZFN specific for TYR was designed. Recognition DNA were synthesized with overlap PCR. The recognition sequence were ligated to different types of endonuclease (DD/RR, KK/EL).
After constructing mutant EGFP vector encoding TYR ZFN binding sites, then we transfected the vector into 293T cells to generate 293T cell lines with an integarted mutant EGFP. For ZFN-mediated gene targeting experiments, TYR ZFN vector and GFP donor template plasmid were cotransfected into transgenic 293T cell line. With effective gene targeting and HR, the cell became GFP positive, as detected by flow cytometry.
GFP positive cells were examined and compared between DD/RR、KK/EL by microscopy of confirm GFP expression and FCAS.
2.Disruption of TYR gene in PFFs
(1)Positive and negative TYR gene knockout strategy
For DNA transfection, appromate 1×107 cells were electroporated with 30μg Sfi I-linearized gene targeting vector DNA at 450v and 350 uf using a gene pulser II electroporator (Bio-Rad). Transfected cells were then split into twelve 100mm plates and were selected with 1000μg/ml G418 (geneticin, Invitrogen) 48h after transfection,2μM gancyclovir (GANC) 96h after transfection. Selection was carried out for-12 days. Then G418-GANC resistant colonies were selected by the cloning ring (sigma) and transferred to 48-well culture plates for expansion and analysis
(2) ZFN mediated TYR gene knockout strategy
TYR ZFN and TYR donor template plasmid were cotransfected into PFFs with lipo2000.
Transfected cells were then split into 100mm plates by ratio of 1:25 and were selected with 1000μg/ml G418 (geneticin, Invitrogen) 48h after transfection,2μM gancyclovir (GANC) 96h after transfection. Selection was carried out for 12-15 days. Then G418-GANC resistant colonies were selected by the cloning ring (sigma) and transferred to 48-well culture plates for expansion and analysis.
3.Production of TYR gene knockout pig by nuclear transfer
Results
Ⅰ. Establishment of porcine Oct-4 promoter-driven EGFP reporter system for evaluation of Oct-4 status in porcine pluripotency cells
1.Here we succeed in isolating 3.2kb DNA spanning 5'-flanking Oct-4 gene promoter region of Tibet minipig. The sequence of the promoter region is 97% homology to NCBI data (NW_001886435). By alignment with Oct-4 promoter of human, bovine, rabbit, mouse, rat, four highly conserved promoter regions (CR1-4)were identified within the porcine region. The highest degree of similarity on sequence level was found among the conserved domains between rabbit and human. The core promoter and distant enhancer located in CR-1 and CR-4 respectively, showing highly conserved at different domain. Proximal enhancer PE-1B located within CR2 and PE-1A outside CR2. PE-1A from different organism differed greatly,indicating that PE-1 related regulation is species specific.
2. Vector pOGN2 was constructed successfully by identification with enzyme digestion and DNA sequencing. After transfection into porcine IPS cells, GFP only can be observed in IPS cell but not in feeder cell, while both IPS and feeder cell expressed GFP in contrast group transfected with pEGFP-N2. The transfection results showed that the pOGN2 merely expressed in Oct-4 active cells.
3.PFFs were transfected with lineared pOGN2 and screened with G418. After identification with PCR,10 positive cell clones were achieved and 8 of them growed well.
4. SCNT was carried out with the 8 pOGN2 transgenic PFFs. After observed at days 5-7, embryo with clone #3、#4、#8 expressed high GFP fluorescence, embryo with clone#1、#2 showed weak GFP, while no GFP fluorescence were detected in other clones. Oct-4 protein also can be detected in GFP positive embryos. Another SCNT was performed with cell clone#3、#4、#8.The SCNT embryos were transferred into an oviduct of the surrogate.
5. To further verify Oct4-EGFP reporter system for monitering the endogenous Oct-4 gene, we initially transfected pig fetal fibroblasts with Oct4-EGFP and then reprogrammed Oct4-EGFP transgenic PFFs into iPS cell colonies by infection with retroviral vectors encoding the four factors (Oct-4, Sox2, Klf4, c-Myc). EGFP expression iPS-like cell colonies were detected by day 20. And many signle cells also expressed GFP but not formed colony. Nevertheless most colonies were EGFP-negative.
ⅡEstablishment of TYR knockout albinism Tibet minipig by nuclear transfer
1.PNS mediated gene knockout
(1)After two rounds of PCR, the unknown TYR DNA was cloned successfully. Sequence blast and analysis proved the cloned DNA was correct.
(2) The targeted vector was constructed from 6kb TYR genomic DNA fragment spanned part of intron 1 (4.5kb) as a long homology arm and part of intron 2 as a short homology arm. PNS gene knockout vecotr pSSC-tyrKO was achieved successfully.
(3) We obtained 166 clones surviving from G418 and GANC screening. A large portion of these clones were transgenic clones though the addition of GANC as negative screening drug. Only one clone of these was considered as TYR gene knockout clones after PCR test.
(4) 400 SCNT embryos were produced with the gene knockout cell clone and transferred into 2 receptor gilt. One was pregnant and the other returned to estrus. When performing laparotomy at 36 days of pregency, several implantation sites were found and yet no fetus discovered for absorption.
2. ZFN mediated TYR gene knockout
(1)We designed and engineered one pair of 3-finger ZFN tyrZFN-131F/tyrZFN-131R that targeted specific porcine TYR gene by overlap PCR method. pST1374-DD/RR and pST1374-KK/EL was constructed successfully.
(2) Gene targeting and recombination was initiated by co-transfection of the expression plasmids for the pair of ZFNs and the EGFP donor template plasmid.3 days after transfection, the cells beame GFP positive,which was detected under fluorescence microscope.
The number of EGFP positive cell in cells transfected with tyrDD/RR (0.23%) appeared to be lower than transfected with tyrKK/EL (0.29%). No GFP positive cells were observed post-transfection with GFP donor template plasmid only.
Conclusions
I. Establishment of porcine Oct-4 promoter-driven EGFP reporter system for evaluation of Oct-4 status in porcine pluripotency cells
1.cloning of 5'upstream promoter region of Tibet minipig Oct-4 gene
2. Successful construction of pOGN2 containing Oct4-EGFP and NEO expression cassette and identification by IPS transfection.
3. Establishment of pOGN2 transgenic PFFs cell lines
4. SCNT embryos with pOGN2 transgenic PFFs cells expressed GFP, indicating that we have developed Oct4-GFP monitoring system. This system can be applied in porcine pluripotent research such as IPS and ES.
II. Establishment of TYR knockout albinism Tibet minipig by nuclear transfer
1.Cloning unknown TYR genomic DNA and construction of TYR PNS gene knockout vector. One gene knokcout cell clone was achieved by this method.
2.Designing and engineering one pair of ZFN targeted TYR gene. This ZFN mediated gene correction of the mutant EGFP locus in 293T cells correctly.
3.Designing the mutant EGFP transgenic 293T cell model to access the efficiency of ZFN.
Innovation points
1.This study intent to develop gene modified cloning Tibet minipig with Chinese autonomous intellectual property since the Tibet minipig was the unique minipig breed of our country.
2. It was the first time to monitor porcine endogenous Oct-4 expression with Oct4-EGFP system, in which the GFP was driven by the whole 5'upstream regulation region.
3.It was first time to establish albinism Tibet minipig by nuclear transfer technology. This albinism animal model provied perfect big animal model for human albinism disease research.
引文
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