绵羊胎儿成纤维细胞体外培养及转基因研究

目的用增强型绿色荧光蛋白(EGFP)基因转染体外培养绵羊胎儿成纤维细胞,探讨绿色荧光蛋白对绵羊胎儿成纤维细胞生物学特性的影响.方法体外分离培养绵羊胎儿成纤维细胞,经脂质体介导EGFP基因转染第一代成纤维细胞,G418筛选10～12*!d,挑选转基因单克隆细胞,传代培养,进行细胞形态观察、生长曲线以及染色体核型分析,并进行了培养细胞性别鉴定.结果整合有EGFP基因的绵羊胎儿成纤维细胞生物学行为与未转染外源基因的细胞无明显差别,根据荧光强度可直接反应外源基因的表达量.结论 EGFP基因作为体内报告基因可用于转基因细胞的研究,并将整合有EGFP基因的转基因细胞为克隆动物提供核供体奠定了基础.     

重组人gdnf乳腺特异表达载体转染牛胎儿成纤维细胞的研究

为了制备重组人GDNF牛乳腺生物反应器,采用组织块贴壁法分离培养雌性牛胎儿成纤维细胞,连续继代培养75d,进行形态观察和染色体分析,在此基础上,转染带有新霉素抗性和红色荧光蛋白双重筛选标记的重组人gdnf乳腺特异表达载体pNR-GDNF,G418筛选阳性抗性克隆,进行PCR法鉴定。结果表明,分离培养的牛胎儿成纤维细胞具有正常的形态、分裂增殖特性和染色体数目;目的基因已整合到转基因细胞的染色体上。     

利用体细胞核移植技术制作人胰岛素原转基因牛 （英文）

通过体细胞核移植技术制作了人胰岛素原转基因牛。在CMV启动子指导下以内部核糖体进入位点序列(IRES)连接的新霉素抗性基因和绿色荧光蛋白基因组成了双重标记基因的筛选系统,用于转基因细胞的富集以及细胞和植入前胚胎的筛选。转基因通过电穿孔的方法(900V/cm,5ms)转入体外培养的牛胎儿成纤维细胞,基因转染细胞在添加G418 (800μg/mL)的培养基中培养10天以富集转基因细胞。选择表达绿色荧光蛋白的转基因细胞作为核供体进行体细胞核移植,重构胚经体外培养至囊胚阶段,选择表达绿色荧光蛋白的囊胚进行胚胎移植。为比较基因转染以及供体细胞所处周期对转基因细胞核移植胚胎发育的影响,用作核移植供体的转基因细胞或非转基因细胞先饥饿培养2—4天(0.5 ?S) ,然后恢复培养(10?S) 10 h使细胞同步化于G1期,以正常培养的细胞作为对照进行核移植。结果表明,转基因细胞作为核供体得到的核移植胚胎的体外囊胚发育率低于以非转基因细胞为核供体的对照组(23.2% VS 35.2 %,P<0.05) ;转基因细胞周期同步化处理与否对其克隆胚囊胚发育率无显著影响(23.2% VS 18.9 %,P>0.05)。胚胎移植后2个月直肠检查发现7头受体牛(每头移植2—4枚胚胎)中有一头妊娠,并最终发育足月产下一头小牛。聚合酶链反应(PCR)检测和DNA测序分析表明其为转人胰岛素原基因的转基因克隆牛。     

利用启动子缺陷型打靶载体敲除牛胎儿成纤维细胞中的Prnp

利用启动子捕获对中靶细胞进行富集是提高体细胞基因打靶效率常用的策略之一.敲除动物的Prnp可使其具有抵抗Prion病感染的能力.采用启动子捕获策略,构建了牛Prnp启动子缺陷型打靶载体BoPrneo,线性化后,再通过电穿孔转染牛胎儿成纤维细胞BFF,用250 μg/mLG418进行药物筛选,共得到99个药物抗性细胞克隆.对细胞克隆进行PCR、测序及Southern blotting鉴定,结果表明,其中的4个细胞克隆为中靶细胞,说明牛胎儿成纤维细胞中的Prnp一条等位基因被成功敲除.本研究为牛Prnp的敲除提供了一种简单、安全、有效的方法.     

构建LEF1基因毛囊特异表达载体并稳定转染胎儿成纤维细胞

旨在构建内蒙古白绒山羊(Capra hircus)淋巴样增强因子-1(Lymphoid enhancer factor,LEF1)基因真核表达载体并转染胎儿成纤维细胞,获得稳定表达红色荧光蛋白及毛囊特异性表达LEF1的转基因细胞克隆。以pCDsRed2载体为基本骨架将LEF1基因亚克隆到KAP6-1启动子下游,连接红色荧光蛋白表达元件,构建LEF1基因毛囊特异表达载体pCDsRed-KL。外源表达载体以lipofectamineTM2000介导转染胎儿成纤维细胞,通过G418筛选获得稳定转染的细胞克隆。PCR鉴定外源基因在细胞基因组中的整合。测序显示构建的表达载体pCDsRed-KL序列中,LEF1基因正确连接在KAP6-1启动子下游,顺序连接CMV启动子和红色荧光蛋白基因,载体构建正确。脂质体介导的稳定转染效率约为14.0%,经G418筛选得到高效表达红色荧光蛋白转基因细胞克隆。PCR检测显示外源KAP6-1启动子和LEF1基因整合到胎儿成纤维细胞基因组中。     

利用体细胞核移植技术制作人胰岛素原转基因牛

通过体细胞核移植技术制作了人胰岛素原转基因牛。在CMV启动子指导下以内部核糖体进入位点序列（IRES）连接的新霉素抗性基因和绿色荧光蛋白基因组成了双重标记基因的筛选系统，用于转基因细胞的富集以及细胞和植入前胚胎的筛选。转基因通过电穿孔的方法（900 V/cm, 5 ms）转入体外培养的牛胎儿成纤维细胞，基因转染细胞在添加G418(800 μg/mL) 的培养基中培养10天以富集转基因细胞。选择表达绿色荧光蛋白的转基因细胞作为核供体进行体细胞核移植，重构胚经体外培养至囊胚阶段，选择表达绿色荧光蛋白的囊胚进行胚胎移植。为比较基因转染以及供体细胞所处周期对转基因细胞核移植胚胎发育的影响，用作核移植供体的转基因细胞或非转基因细胞先饥饿培养2—4天(0.5% FBS)，然后恢复培养(10% FBS)10?h使细胞同步化于G1期，以正常培养的细胞作为对照进行核移植。 结果表明，转基因细胞作为核供体得到的核移植胚胎的体外囊胚发育率低于以非转基因细胞为核供体的对照组(23.2% VS 35.2%, P<0.05)；转基因细胞周期同步化处理与否对其克隆胚囊胚发育率无显著影响（23.2% VS 18.9%, P>0.05）。胚胎移植后2个月直肠检查发现7头受体牛（每头移植2—4枚胚胎）中有一头妊娠，并最终发育足月产下一头小牛。聚合酶链反应（PCR）检测和DNA测序分析表明其为转人胰岛素原基因的转基因克隆牛。     

以不同类型的转基因细胞为核供体生产牛的转基因克隆胚胎

利用所构建的含新霉素抗性(Neo^r)基因和绿色荧光蛋白(GFP)基因的双标记选择载体, 通过电穿孔的方法, 分别转染了牛胎儿成纤维细胞、胎儿输卵管上皮细胞、胎儿卵巢上皮细胞、颗粒细胞, 经过800 μg/mL的G418筛选14 d后, 均获得了阳性细胞株. 分别以未转基因牛颗粒细胞和4种细胞系的转基因细胞为核供体, 进行了牛的体细胞核移植. 结果表明: (ⅰ) 转基因与未转基因牛颗粒细胞的重组胚的囊胚发育率(44.6% vs 42.8%)、移植妊娠率(19% vs 25%)差异不显著(P＞0.05); (ⅱ) 比较4种类型转基因细胞的重组胚的囊胚发育率, 发现胎儿输卵管上皮细胞(49.1%)和颗粒细胞(44.6%)最高, 牛胎儿成纤维细胞(37.2%)次之, 胎儿卵巢上皮细胞的重组胚囊胚发育率(22.5%)最低, 三者之间差异显著(P＜0.05). 以上结果显示, 供体细胞的转基因与否对牛克隆胚胎的体外和体内早期发育影响不明显; 通过体细胞核移植技术, 牛胎儿输卵管上皮细胞和颗粒细胞可以有效地生产牛转基因囊胚, 并且绿色荧光蛋白作为一种无毒性作用的筛选标记, 可用于转基因胚胎的筛选.     

表达红色荧光及转胸腺素β4基因绵羊胎儿成纤维细胞系的制备

旨在构建胸腺素β4(thymosin beta4,Tβ4)基因真核表达载体并转染绵羊胎儿成纤维细胞,获得稳定表达胸腺素β4及红色荧光蛋白的转基因细胞克隆。将克隆载体pMD19TT中的胸腺素β4基因亚克隆到表达载体pIRES2-DsRed2的多克隆位点,构建表达载体pIRES2-DsRed2-Tβ4,脂质体介导转染绵羊胎儿成纤维细胞,G418筛选获得稳定转染的细胞克隆。RT-PCR检测Tβ4基因在宿主细胞中的转录。测序结果显示,构建的表达载体pIRES2-DsRed2-Tβ4序列中,Tβ4基因正确连接在CMV启动子下游,顺序连接IRES2序列和红色荧光蛋白基因,载体构建正确。脂质体介导的稳定转染效率约为15%,经G418筛选得到转基因细胞克隆并高效表达红色荧光蛋白。RT-PCR检测显示外源Tβ4基因在绵羊胎儿成纤维细胞中得到转录。成功构建具有红色荧光蛋白和新霉素抗性双选择标记的胸腺素β4基因真核表达载体并稳定转染绵羊胎儿成纤维细胞,筛选得到的超表达胸腺素β4绵羊胎儿成纤维细胞系为下一步通过核移植和克隆技术获得转基因绵羊提供了条件。     

牛胎儿成纤维细胞的分离与体外培养

分别用胶原酶Ⅲ和胰蛋白酶成功地分离了牛胎儿成纤维细胞,并对其进行了体外培养,同时探讨了两种酶消化分离牛胎儿组织的时间对牛胎儿成纤维细胞原代培养的影响。用组织块直接培养也成功得到了牛胎儿成纤维细胞,并探讨了不同组织块大小对牛胎儿成纤维细胞原代培养的影响。通过绘制生长曲线,可以研究牛胎儿成纤维细胞增殖规律;通过制备牛胎儿成纤维细胞的细胞染色体发现,经过传代(12代)、冷冻保存、解冻,牛胎儿成纤维细胞染色体数目不变。     

无标记转Fat-1基因真核表达载体的构建及转基因绵羊细胞系的建立

为了建立无筛选标记基因的转Fat-1基因绵羊细胞系,本研究将PCR克隆得到的Fat-1基因,合成的attB、Loxp序列并克隆入pN1-EGFP框架载体,得到可删除筛选标记基因的pEGFP-N1-Fat-1真核表达载体。体外转录合成phiC31整合酶mRNA并与线性化的pEGFP-N1-Fat-1载体共转染绵羊胎儿皮肤成纤维细胞,G418筛选得到表达绿色荧光的单克隆,再利用pET-28a-His-NLS-TAT-Cre质粒诱导Cre重组蛋白表达,将纯化后的Cre穿膜肽转导表达绿色荧光的单克隆细胞,将荧光淬灭的细胞系扩繁,提取基因组DNA,进行PCR及测序鉴定,得到无标记转Fat-1基因绵羊胎儿皮肤成纤维细胞系,为生产无筛选标记基因的转基因绵羊奠定基础。     

A Unique Protease Cleavage Site Predicted in the Spike Protein of the Novel Pneumonia Coronavirus (2019-nCoV) Potentially Related to Viral Transmissibility

正Dear Editor,In December 2019, a novel human coronavirus caused an epidemic of severe pneumonia(Coronavirus Disease 2019,COVID-19) in Wuhan, Hubei, China(Wu et al. 2020; Zhu et al. 2020). So far, this virus has spread to all areas of China and even to other countries. The epidemic has caused 67,102 confirmed infections with 1526 fatal cases     

Curcuminoids as inhibitors of thioredoxin reductase: A receptor based pharmacophore study with distance mapping of the active site

Curcumin is the yellow pigment of turmeric that interacts irreversibly forming an adduct with thioredoxin reductase (TrxR), an enzyme responsible for redox control of cell and defence against oxidative stress. Docking at both the active sites of TrxR was performed to compare the potency of three naturally occurring curcuminoids, namely curcumin, demethoxy curcumin and bis-demethoxy curcumin. Results show that active sites of TrxR occur at the junction of E and F chains. Volume and area of both cavities is predicted. It has been concluded by distance mapping of the most active conformations that Se atom of catalytic residue SeCYS498, is at a distance of 3.56 from C13 of demethoxy curcumin at the E chain active site, whereas C13 carbon atom forms adduct with Se atom of SeCys 498. We report that at least one methoxy group in curcuminoids is necessary for interation with catalytic residues of thioredoxin. Pharmacophore of both active sites of the TrxR receptor for curcumin and demethoxy curcumin molecules has been drawn and proposed for design and synthesis of most probable potent antiproliferative synthetic drugs.     

Comparative morphology of the carpel in the Liliaceae: Hewardieae, Petrosavieae, and Tricyrteae

The young pistils in the melanthioid tribes, Hewardieae, Petrosavieae and Tricyrteae, are uniformly tricarpellate and syncarpous. They lack raphide idioblasts. All are multiovulate, with bitegmic ovules. The Petrosavieae are marked by the presence of septal glands and incomplete syncarpy. Tepals and stamens adhere to the ovary in the Hewardieae and the Petrosavieae but not in the Tricyrteae. Two vascular bundles occur in the stamens of the Hewartlieae and Tricyrtis latifolia. Ventral bundles in the upper part of the ovary of the Hewardieae are continuous with compound septal bundles and placental bundles in the lower part. Putative ventral bundles occur in the alternate position in the Tricyrteae and putative placental bundles in the opposite. position in the Petrosavieae. The dichtomously branched stigma in each carpel of the Tricyrteae is supplied by a bifurcated dorsal bundle.     

Enterovirus 71 3C proteolytically processes the histone H3 N-terminal tail during infection

Highlights
1. The N-terminal tail of histone H3 is specifically cleaved during EV71 infection.
2. Viral protease 3C is identified as a protease responsible for proteolytically processing the N-terminal H3 tail.
3. Our finding reveals a new epigenetic regulatory mechanism for Enterovirus 71 in virus-host interactions.     

Detection of EBV and HHV6 in the Brain Tissue of Patients with Rasmussen’s Encephalitis

Rasmussen’s encephalitis (RE) is a rare pediatric neurological disorder, and the exact etiology is not clear. Viral infection may be involved in the pathogenesis of RE, but conflicting results have reported. In this study, we evaluated the expression of both Epstein-Barr virus (EBV) and human herpes virus (HHV) 6 antigens in brain sections from 30 patients with RE and 16 control individuals by immunohistochemistry. In the RE group, EBV and HHV6 antigens were detected in 56.7% (17/30) and 50% (15/30) of individuals, respectively. In contrast, no detectable EBV and HHV6 antigen expression was found in brain tissues of the control group. The co-expression of EBV and HHV6 was detected in 20.0% (6/30) of individuals. In particular, a 4-year-old boy had a typical clinical course, including a medical history of viral encephalitis, intractable epilepsy, and hemispheric atrophy. The co-expression of EBV and HHV6 was detected in neurons and astrocytes in the brain tissue, accompanied by a high frequency of CD8⁺ T cells. Our results suggest that EBV and HHV6 infection and the activation of CD8⁺ T cells are involved in the pathogenesis of RE.     

Perinatal Vertical Transmission of Chikungunya Virus in Ruili,a Town on the Border between China and Myanmar

正Dear Editor,Chikungunya virus (CHIKV), an arbovirus in the family of Togaviridae, genus Alphavirus, is transmitted by the A.aegyptii or A. albopictus mosquito, and causes disease in humans characterized by fever, rash, and arthralgia (Silva and Dermody 2017; Suhrbier 2019). It was first reported in 1953 in Tanzania, and caused only a few outbreaks and sporadic cases in Africa and Asia in last century. However, in the epidemic in 2004, CHIKV acquired mutations that conferred enhanced transmission by the A. albopictus mosquito(Schuffenecker et al. 2006). Since then, it has successively caused outbreaks in Africa, the Indian Ocean, South East Asia, the South America, and Europe (Zeller et al. 2016).     

Development of a Novel Reverse Transcription Loop-Mediated Isothermal Amplification Method for Rapid Detection of SARS-CoV-2

In conclusion, the novel visual RT-LAMP assay is a simple, rapid, and sensitive approach for detection of SARS-CoV-2, and it is ready for application in primary care and community hospitals or health care centers, and even patients' own houses in response to the current SARS-CoV-2 epidemic because the assay does not require sophisticated equipment and skilled personnel. Furthermore, it is also ready to be used in fields for screening samples from wild animals and environments to facilitate the identification of potential intermediate hosts that mediate the cross-species transmission of SARS-CoV-2 from bats to humans.