从伪狂犬病病毒中删除Cre/LoxP介导的报告基因

根据pEGFP-C1序列,设计并合成一对引物,通过PCR扩增出两端各含一个同向LoxP位点的GFP表达盒,克隆于转移载体pSKLR获得pSKLR-G-LoxP。通过经典同源重组方法,得到表达GFP的TK基因缺失伪狂犬病毒重组毒株S03109。该重组病毒在转染了pOG231(表达Cre重组酶)的293T细胞上连续传代,筛选得到重组病毒株S0419。荧光显微镜观察、Western blot及PCR检测结果表明,S03109感染细胞后持续表达GFP,而S0419不表达GFP。PCR证实S0419为含单个LoxP位点的TK基因缺失病毒,并且在细胞培养上遗传稳定,测序结果(GenBank登录号AY822465)表明,在Cre重组酶的作用下,两个同向LoxP序列之间的GFP表达盒被正确除去。对BALB/c小鼠的半数致死量(LD50)及免疫保护实验结果表明,S03109和S0419对BALB/c小鼠的LD50值均大于3×105PFU,免疫BALB/c小鼠,攻毒后平均存活率分别为67.5%与70%。以上结果表明,利用Cre/LoxP位点特异性重组系统,成功去除了插入重组伪狂犬病病毒基因组中的GFP报告基因。     

伪狂犬病病毒Fa株胸苷激酶基因缺失株的构建

采用外切除缺失法对已克隆于ｐＢＲ３２２中包含伪狂犬病病毒（ＰＲＶ）胸苷激酶（ＴＫ）基因的ＢａｍＨＩ－１１片段（ｐＰＢ１１）进行改建,经筛选获得４株ＴＫ基因缺失重组质粒（ｐＤＴＫ３－１．ｐＤＴＫ３－６,ｐｄＴＫ３－８和ｐＤＴＫ５－６）对其进行酶切鉴定和Ｓｏｕｔｈｅｒｎ转印杂交,结果其１１片段迁移率均发生改变,缺失的碱基数分别约１２５０,１１００,１２５０和２００ｂｐ,用ＰＲＶＦａ－ＤＮＡ或ＰＲＶＦａ     

伪狂犬病病毒上海株gE和gI基因的克隆及缺失转移载体的构建

根据已发表的伪狂犬病病毒 (PseudorabiesVirus,PRV)的 gI和 gE 基因序列 ,设计两对引物用PCR方法扩增出PRV SH gI和 gE 基因 ,将其克隆入 pUC18载体中 ,获得了缺失部分gI基因的转移载体质粒 ,命名为 pgEI。     

伪狂犬病病毒上海株gE和gI基因的克隆及缺失转移载体的构建

根据已发表的伪狂犬病病毒(Pseudorabies Virus,PRV)的gI和gE基因序列,设计两对引物用PCR方法扩增出PRV-SH gI和gE基因,将其克隆入pUC18载体中,获得了缺失部分gI基因的转移载体质粒,命名为pgEI.     

伪狂犬病基因缺失疫苗的研究进展

伪狂犬病是由疱疹病毒科伪狂犬病病毒引起的多种畜、禽及野生动物的一种急性传染病.动物感染后表现为从隐性临床症状到严重的呼吸道和神经症状综合症.该病给世界养猪业造成巨大的损失,目前预防该病主要是以疫苗接种为主.综述了当今广泛应用的伪狂犬病基因缺失疫苗的研究现状和进展.     

伪狂犬病病毒上海株(PRV-SH)gE-/gI/GFP+缺失株的构建

在构建了含伪狂犬病病毒（Pseudorabies Virus,PRV）上海株gI基因和gE基因克隆鉴定的基础上,采用酶切的方法构建了载体pgEI。然后用限制性内切酶BamHI和BstpI缺失掉gE基因5'端363bp,同时把绿色荧光蛋白（GFP）基因表达盒插入到缺失部分,并在下游引入一个多克隆位点,构建了缺失转移载体pgEIGFP。用DOTAP转染试剂盒将pgEIGFP转染了感染PRVSH的BHK21细胞,待出现80%病变后收获病毒,并以蚀斑法得到纯化的缺失了gE/gI重组病毒株。小鼠试验证实了缺失株的毒力有所下降。     

猪伪狂犬病综合诊断及分离毒株gE和TK基因遗传变异分析

2012年以来,许多使用gE基因缺失活疫苗免疫过的猪场广泛性出现伪狂犬病毒(PRV)感染,gE抗体阳性率不断升高,伪狂犬典型病例不断增加。2018年3月鲁南地区几个种猪场先后发生疑似伪狂犬病疫情,怀孕母猪流产、产死胎和木乃伊胎,仔猪出现神经症状且死亡率高。通过对病死猪及死胚剖检进行初步诊断,取病料进一步进行病理组织学诊断及病毒分离鉴定。结果显示,病死猪均可见病毒性脑炎、肝细胞变性坏死及淋巴组织坏死等病理变化,在病变的神经元、肝细胞、扁桃体隐窝上皮细胞等细胞核内见红染包涵体。对分离到的4株PRV进行了gE和TK基因的序列测定及遗传变异分析发现,4株PRV的gE和TK核苷酸序列的同源性分别为98.8%~99.3%和98.9%~99.6%,与国内流行毒株其核苷酸序列的同源性分别99.1%~99.7%和98.6%~99.8%,与匈牙利和美国等流行毒株核苷酸序列的同源性分别为97.3%~97.8%和98.8%~99.5%,表明4株分离株高度同源,与国内PRV变异株处在同一分支,而与匈牙利和美国等毒株遗传距离较远。传统疫苗对PRV变异毒株不能提供有效保护,给猪场伪狂犬病的防控和净化工作带来了新的挑战。     

伪狂犬病病毒鄂A株包膜糖蛋白gD基因的克隆与表达

克隆了伪狂犬病病毒鄂A株编码包膜糖蛋白gD的基因并进行了序列测定 ,与国外报道的Rice株相比 ,其核苷酸序列具有 98%的同源性 ,推导氨基酸序列同源性为 97%。将此基因克隆于具有全期启动子盒的杆状病毒转移载体pSX35A中 ,构建成重组转移质粒pSX35A gD ,与致死缺失型线性化苜蓿丫纹夜蛾核型多角体病毒 (AcMNPV OCC- )基因组DNA一起共转染粉纹夜蛾Hi5细胞 ,经同源重组 ,获得含gD基因的重组病毒AcMNPV OCC+ gD。重组病毒经空斑纯化后感染Hi5细胞进行表达分析 ,细胞裂解物的SDS PAGE及Western Blot ting均显示分子量约 47kD的gD蛋白得到了特异性表达 ,其表达量占细胞总蛋白的 6 2 % ,表达的gD蛋白具有免疫原性。     

利用Cre/LoxP系统删除转基因山羊体内的选择标记基因

在制备转基因家畜过程中的一个关键步骤是使用选择标记基因 (Selectable marker genes,SMGs) 将转基因整合细胞从大量的正常细胞中筛选出来,这导致了SMGs整合入家畜的基因组内持续传递给后代。SMGs已被证明能够显著影响基因组内整合位点处的基因调控,也增加了对转基因动物安全评价的复杂性。为了确定转基因山羊制备过程中SMGs的删除时机和删除方法,在体细胞克隆前后两个时段内,利用Cre/loxP系统删除SMGs的可行性,同时比较了蛋白转导和质粒共转染两种Cre导入方式的删除效率。结果表明：尽管在首次对山羊成纤维细胞进行遗传修饰后即可进行SMGs删除,但两次遗传修饰导致细胞严重老化,无法用于后续的体细胞克隆羊制备。在转基因山羊的成体细胞中删除SMGs不存在上述问题,成功率高,缺点是试验周期长、耗资增大。Cre表达质粒瞬时转染能够删除SMGs,但有超过30%的无SMGs细胞克隆中整合有质粒序列。TAT-CRE蛋白质转导方法可以避免引入的新外源基因,SMGs删除率达到43.9%~72.8%,是一种较佳的SMGs删除手段。     

骨骼肌特异性敲除TβRⅡ小鼠模型的建立

目的:建立骨骼肌特异性敲除转化生长因子受体Ⅱ(TβRⅡ)小鼠模型,为进一步研究TβRⅡ在骨骼肌发育和分化中的作用奠定基础。方法:首先将TβRⅡflox/flox转基因小鼠与上游携带肌酸激酶(MCK)启动子的MCK-Cre转基因小鼠进行杂交,培育繁殖出TβRⅡflox/wt/MCK-Cre(+)双转基因小鼠。然后利用TβRⅡflox/wt/MCK-Cre(+)双转基因小鼠与TβRⅡflox/flox转基因小鼠进行杂交,繁殖培育出在骨骼肌内特异敲除TβRⅡ基因的TβRⅡflox/flox/MCK-Cre(+)小鼠。结果:利用Cre/loxP技术世界上首次成功繁殖培育出有活力的且发育正常的TβRⅡ基因敲除小鼠。     

含有融合标记基因和LoxP/FRT位点的植物表达载体构建及应用

本研究利用化学合成法合成了包含pCAMBIA0390左右边界T-DNA和LoxP/FRT (LF)位点的DNA片段Ⅰ,利用SacⅡ和SphⅠ酶切位点,去除了pCAMBIA0390左右边界T-DNA和多克隆位点之间的序列,然后连接DNA片段Ⅰ和载体片段,构建了植物表达载体pGM323-LF-enTP.随后,再合成含有适合在单子叶植物中表达的由玉米Ubi-1启动子驱动的融合标记基因(Bar::gus)和水稻actin-1启动子驱动的Bt抗虫蛋白基因(Cry1Ab)表达元件的DNA片段Ⅳ,在pGM323-LF-enTP的基础上,利用SalⅠ和PstⅠ位点构建了同时含有LF位点、Bar::gus以及Cry1Ab基因表达元件的表达载体pGM626-LF-ABt.利用含有pGM626-LF-ABt的农杆菌遗传转化烟草和玉米,以草丁膦作为抗性筛选剂,非转化细胞得到了有效抑制,快速获得了转基因植株,利用GUS组织化学检测和RT-PCR分析了转基因植株中标记基因的表达,结果表明pGM626-LF-ABt可以用于农杆菌介导的单、双子叶植物遗传转化.本研究为培育安全抗虫转基因植物奠定了基础.     

Maternal inheritance of Cre activity in a Sox2Cre deleter strain

The Sox2 gene is expressed in several undifferentiated cell types. In an earlier study we described a Sox2Cre transgene that mediates epiblast-specific Cre-mediated modification of gene activity in the embryo. Here we report that this transgene is active in the female germline. Consequently, all offspring that arise from female mice heterozygous for the Sox2Cre transgene have demonstrable Cre activity irrespective of whether they inherit the transgene itself. Maternal inheritance of Cre activity allows the efficient modification of gene activity for functional analysis.     

一种可切除包装信号的重组1型单纯疱疹病毒的产生

A new kind of recombinant herpes simplex virus type 1 (HSV 1) was constructed. This recombinant, named HSV1 LaL, contained an unique packaging signal (“a" sequence) flanked by two loxP sites in parallel orientation, named LaL, while the original packaging signals of HSV 1 were deleted. Based on a set of cosmids containing the entire HSV 1 genome except the “a" sequence, the LaL was inserted into HSV 1 UL44 gene on one of the cosmids, cos56, generating cos56/LaL. By co transfecting cos56/LaL with the other cosmids, HSV1 LaL was generated in the cells by recombination. By introducing cos56/LaL or HSV1 LaL respectively into E.coli or BHK cells that expressed Cre recombinase, LaLs on both of them were excised by Cre, which was proved by PCR detection. To study the potential use as helper virus in packaging amplicon vector, HSV1 LaL was compared with a control virus HSV1 lacZ that contained a lacZ gene in the UL44 gene. The titer of amplicon virus generated from HSV1 LaL infected BHK/Cre cells was basically the same as that from HSV1 lacZ infected cells, however,the former contained about 10 fold less helper virus than the later, while HSV1 LaL showed the same replication rate as HSV1 lacZ on standard cells, like BHK 21.     

Generation of a floxed allele of the mouse BMP type II receptor gene

Bone morphogenetic proteins (BMPs) regulate a wide range of cellular functions that contribute to embryonic development from mesoderm formation to organogenesis. BMP type II receptor (BMPR-II) transduces BMP signals by forming heteromeric complexes with and phosphorylating BMP type I receptors. Heterozygous germline mutations of BMPR-II gene have been identified in patients with familial and sporadic primary pulmonary hypertension, indicating that BMPR-II may contribute to the maintenance of normal pulmonary vascular structure and function. Since embryos homozygous for a null BMPR-II allele died during gastrulation, precluding further studies of BMPR-II function in organ formation and in adult tissues, we generated mice carrying a conditional mutant BMPR-II allele in which exons 4 and 5 were flanked by loxP sequences. We anticipate that studies of mice carrying a floxed BMPR-II allele and a Cre transgene (under the control of a tissue-specific promoter) will enable characterization of the role of BMPR-II in specific cell types during development and in the pathogenesis of cardiovascular diseases.     

Establishment of Cre/LoxP recombination system in transgenic rats

The rat has offered an important animal model in biomedical research including surgical procedure. However, advanced genetic manipulation has progressed less far in the rat than in the mouse. Here we report the Cre/LoxP transgenic rat system, demonstrating conditional chromosomal translocation both in the fertilization and adult stage, spatio-temporal gene controlling by catheter-based adenoviral gene transfer, and muscular fusion events in the limb transplant. Taking advantage of the larger body size of the rat than the mouse, this rat system provides a potential value to evaluate biomedical and therapeutic significance for gene therapy and regenerative medicine.     

Melanocyte homeostasis in vivo tolerates Rb1 loss in a developmentally independent fashion

There has been uncertainty regarding the precise role that the pocket protein Rb1 plays in murine melanocyte homeostasis. It has been reported that the TAT-Cre mediated loss of exon 19 from a floxed Rb1 allele causes melanocyte apoptosis in vivo and in vitro. This is at variance with other findings showing, either directly or indirectly, that Rb1 loss in melanocytes has no noticeable effect in vivo, but in vitro leads to a semi-transformed phenotype. In this study, we show that Rb1-null melanocytes lacking exon 19 do not undergo apoptosis and survive both in vitro and in vivo, irrespective of the developmental stage at which Cre-mediated ablation of the exon occurs. Further, Rb1 loss has no serious long-term ramifications on melanocyte homeostasis in vivo, with Rb1-null melanocytes being detected in the skin after numerous hair cycles, inferring that the melanocyte stem cell population carrying the Cre-mediated deletion is maintained. Consequently, whilst Rb1 loss in the melanocyte is able to alter cellular behaviour in vitro, it appears inconsequential with respect to melanocyte homeostasis in the mouse skin.