三种试剂转染293T细胞的效率及毒性比较

该文旨在比较不同的转染试剂介导两种荧光素酶报告基因转染人胚肾细胞293T的效果。采用脂质体转染试剂Lipofectamine~? 2000、非脂质体成分X-tremeGENE HP DNA和FuGENE~? HD转染试剂,选取不同配比的质粒DNA和转染试剂分别将含有绿色荧光蛋白(GFP)和红色荧光蛋白(RFP)的质粒转入293T细胞中,应用倒置荧光显微镜和流式细胞仪分析转染效率,锥虫蓝染色法检测细胞存活率。X-tremeGENE HP DNA转染试剂转染293T细胞的效率最高,不管目的基因、质粒DNA和转染试剂的配比如何,其转染效率均显著高于脂质体转染试剂(P均0.05);在相同的质粒DNA和转染试剂比例下,FuGENE~? HD试剂介导GFP转染的效率显著高于脂质体试剂Lipofectamine~? 2000(P均0.05),但二者介导RFP转染的效率无显著性差异(P均0.05);在质粒DNA和转染试剂比为1?2时,X-tremeGENE HP DNA试剂转染GFP和RFP的效率均显著高于FuGENE~? HD试剂(P均0.05),当比例递变为1?4时,二者的转染效率无显著性差异(P均0.05)。随着转染试剂使用量的增多,Lipofectamine~? 2000和FuGENE~? HD试剂的转染效率明显升高,而X-tremeGENE HP DNA试剂的转染效率则下降。与转染GFP基因相比,三种试剂的转染效率均随着目的基因RFP片段的增大而显著降低(P均0.05)。在质粒DNA和转染试剂的比例相同的情况下,三种试剂转染后的存活率无显著性差异(P均0.05),但随着转染试剂用量的增加,三种试剂转染后的细胞存活率均显著降低(P均0.05)。目的基因大小与转染效率成反比,综合考虑转染试剂用量、转染效率和细胞活性,该研究认为X-tremeGENE HP DNA转染试剂效果最好。     

TALEN质粒转染HEK-293T细胞的条件优化

本研究探讨了TALEN质粒转染HEK-293T细胞的最佳转染条件。以TALEN质粒右臂中的绿色荧光蛋白为报告基因,分别研究转染试剂、质粒DNA的量、转染试剂与质粒DNA的比例对转染效果的影响,转染24 h后荧光显微镜下统计具有代表性视野的绿色荧光细胞,计算绿色荧光细胞/总细胞数的比例得出转染率,并利用CCK-8试剂盒检测细胞存活率。实验结果表明,TALEN质粒转染293T细胞,采用Fugene HD转染试剂比Lipofectamine 2000转染试剂细胞存活率高;以6孔板为例,转染质粒DNA量为4μg时转染效率最高(49.0±8.0)%;Fugene HD与质粒DNA比例(μg/μL)为3:1时转染效率最高。本研究建立TALEN表达质粒转染HEK-293T细胞的最佳转染条件,可作为相关研究或应用提供参考。     

慢病毒载体导入种蛋内鸡胚技术研究

种蛋内鸡胚含有潜在的胚胎干细胞(BCs)或原生殖细胞(PGCs),是目前主要的转基因鸡研究方法。采用绿色荧光蛋白(GFP)基因的pLenti6/v5-DEST-GFP慢病毒表达载体,白来航鸡与伊萨鸡种蛋,结合种蛋赤道面开窗专利技术,对含这两种细胞胚的转基因技术进行了比较研究:转染白来航鸡囊胚,孵化13天时,GFP基因的PCR检出率为64.7%,孵化率极低;转染孵化72h伊萨鸡胚血液循环中PGCs,实验蛋孵化率为35.0%,在出壳后死亡的3只小鸡肝脏中,GFP基因的PCR检出率为100%,存活的4只鸡中有3只在12月龄的血液样品中,经PCR扩增出了GFP基因;转染孵化72～79h白来航鸡胚PGCs,7批次实验的平均孵化率为21.1%,能在赤道面窗口注射胚的种蛋比率,以73～77h胚龄的最高,为75.0%～92.9%,注射病毒组出壳雏鸡血液DNA中,GFP基因PCR检出率为44.4%。两种方法比较,PGCs方法在实验蛋孵化率、胚定位在赤道面窗口率等方面有较强优势。因此为种蛋内胚细胞的转基因鸡技术研究提供了系统、可操作性强的方法。     

优化脂质体对肝癌细胞株HepG2细胞转染效率

摸索用Lipofectamine 2000(Lipo)转染质粒pEGFP-C1到肝癌细胞HepG2较为合适的转染条件。以HepG2细胞为研究对象,采用脂质体Lipofectamine 2000转染pEGFP-C1质粒,在24孔板先按每孔0.5μg固定pEGFP-C1质粒用量,摸索Lipo在1μL、1.5μL、2μL、2.5μL量上较为合适的用量,确定Lipo用量后,然后固定Lipo为1μL,摸索质粒用量0.5μg、1μg,确定脂质体与质粒的最佳比例。此外,对转染中培养基是否含血清,Lipofectamine 2000与pEGFP-C1质粒比例为1:0.5的基础上,扩大用量,即Lipofectamine 2000 2μL和pEGFP-C1质粒1μg,以及脂质体复合物孵育细胞时间进行优化。最后,采用荧光倒置显微镜观察细胞转染效率和方差分析及秩和检验的统计学方法进行统计分析。结果显示,pEGFP-C1质粒固定为0.5μg时,Lipo用量在1μL及1.5μL用量组转染效率最好,之后随着Lipo用量加大,转染效率下降;固定Lipo用量1μL,pEGFP-C1质粒0.5μg时转染效率最好(p0.05),比例不变,扩大Lipo和质粒用量,并不增加转染效率。转染后于3 h、6 h、8 h、12 h、24 h换成正常培养基培养,转染6 h后换液较好。此外,研究发现培养基是否含血清不影响转染效率。本研究表明在24孔板板中用Lipofectamine 2000转染HepG2细胞时较为合适的转染条件为每孔1μL Lipofectamine 2000和0.5μg质粒,脂质体与质粒的最佳比例为2:1,血清不影响转染效率,用含血清培养基转染后6 h换液培养。     

表达绿色荧光蛋白的重组鸡痘病毒的构建

[目的]旨在构建表达绿色荧光蛋白(GFP)的重组鸡痘病毒。[方法]使用overlap PCR进行表达载体质粒的构建,使用菌液PCR和测序技术进行了鉴定;使用鸡胚成纤维细胞作为重组鸡痘病毒同源重组的介质,尝试了可能影响重组鸡痘病毒构建的不同条件;最后使用PCR和Western Blot方法对重组病毒进行了鉴定。[结果]将人工合成的痘苗病毒早晚期启动子SP与绿色荧光蛋白基因一同插入到鸡痘病毒载体质粒的多克隆位点,挑取LA平板上的重组子,菌液PCR及测序验证鉴定结果为阳性;利用重组质粒转染被野生鸡痘病毒感染的鸡胚成纤维细胞制备重组病毒,利用倒置荧光显微镜观察到含有重组病毒的在激发光下呈绿色的细胞;改进重组质粒转染的条件以达到更好的重组病毒包装成效,研究得出被野生痘病毒感染的细胞进行重组质粒传染制备重组病毒的效率是用野生痘病毒感染已转染重组质粒的细胞的6倍,且感染用野生病毒滴度高时重组病毒的制备效果更好;最后,用PCR方法和Western Blot方法对重组病毒进行鉴定,鉴定结果为阳性。[结论]成功构建了表达GFP的重组鸡痘病毒。     

宫颈细胞系化学转染与电穿孔转染效率的比较与优化

目的 定量比较4种化学转染试剂在五株常用的宫颈(癌)细胞系中的DNA转染效率,以助于针对不同细胞系选择合适的转染试剂;同时探索电穿孔转染法在难转细胞系Ect1/E6E7、CaSki中的应用,选择最佳转染条件。方法 以pcDNA3.1-EGFP为报告载体,采用FuGENE HD、jetPRIME、Lipofectamine 2000和Lipofectamine 3000四种转染试剂分别转染常用宫颈(癌)细胞系HeLa、SiHa、CaSki、C33A和Ect1/E6E7,转染24h后用倒置荧光显微镜观察记录,并采用Cellometer K2对转染效率进行定量分析。针对化学转染法难转细胞系Ect1/E6E7、CaSki,采用电穿孔转染法进行转染,优化转染条件,并对转染效率进行定量比较。结果 jetPRIME和Lipofectamine 3000转染试剂在HeLa细胞中的转染效果很好,而对SiHa、C33A细胞转染效率相对较低,在CaSki、E6E7细胞中的转染效果很差。FuGENE HD转染试剂除了对HeLa、SiHa细胞转染效果较好之外,在其他3株细胞中的转染效果均不理想。Lipofectamine 2000在5株细胞中的转染效果也不理想。在穿孔电压170V、脉冲波长7ms、脉冲间隙10ms和驱动电压20V的条件下电穿孔转染法在Ect1/E6E7、CaSki细胞中的转染效率最佳且均高于化学转染法。结论 针对不同的宫颈(癌)细胞系选择合适的转染方式和转染试剂可达到更高的转染效率。相对电转染,化学转染细胞存活率高,电转染更适用于难转细胞的稳定转染。     

不同电转仪的电转参数、质粒用量和拓扑结构对猪胎儿成纤维细胞转染效率的影响

为获得猪胎儿成纤维细胞(porcine fetal fibroblasts, PFFs)最佳的电转染效率,本研究利用荧光激活细胞分选技术(fluorescence activated cell sorting, FACS)辅助优化NEPA 21和Nucleofector? 2b两种电转仪电转染PFFs细胞的参数,比较不同质粒用量和拓扑结构在ECM^? 830、NEPA 21和Nucleofector? 2b中的转染效率。结果显示：NEPA 21电转PFFs的最佳穿孔参数为脉冲电压200 V,脉冲长度3 ms,脉冲间隔50 ms,脉冲次数3次,脉冲电压衰减幅度10%;Nucleofector? 2b在U-023的转染参数下达到最高转染效率。ECM^? 830和Nucleofector? 2b的最适质粒用量都为10 μg,而NEPA 21为8 μg;超螺旋质粒比线性化质粒的转染效率更高,且3种仪器中Nucleofector? 2b转染效果最佳。本研究综合考虑电转仪、电转参数、质粒用量和拓扑结构的影响因素以优化PFFs的电转条件,为高效制备转基因猪及基因编辑猪的研究奠定基础。     

利用流式细胞仪分选拟南芥根尖发育早期非根毛细胞

建立了应用流式细胞仪分选植物特定类型细胞的方法。以拟南芥(Arabidopsis thaliana)Wer::GFP转基因株系为材料,用激光共聚焦显微镜鉴定GFP的表达位置,采用酶解法制备拟南芥根尖原生质体,应用流式细胞仪荧光激活细胞分选技术(FACS)分选收集GFP阳性细胞,并提取细胞的RNA。结果表明,Wer::GFP转基因株系仅在根表皮发育早期的非根毛细胞中表达GFP;利用酶解法制备的根尖原生质体数目较多;从FACS分选收集的细胞中提取的RNA质量较好,可用于研究特定类型细胞的基因表达谱。应用流式细胞仪分选拟南芥非根毛细胞的方法为研究植物特定类型细胞的基因表达谱及基因功能奠定了技术基础。     

脂质体介导法转染肿瘤细胞效率的优化

目的:研究优化影响脂质体转染效率的因素,以提高脂质体转染效率,为相关研究和应用提供参考.方法:以绿色荧光蛋白(GFP)作为报告基因,采用脂质体Lipofectamine 2000包裹pU6H1-GFP-FAK重组质粒转染Caco-2细胞,研究了细胞接种密度、DNA用量、脂质体与DNA的比例、脂质体-DNA复合物的形成时间、细胞与脂质体复合物的孵育时间、血清的有无及细胞的传代次数等因素对脂质体转染效率的影响.结果:2-5次细胞传代,2×105接种密度、4μg DNA用量、2.5:1的脂质体与DNA比例、30min脂质体-DNA复合物形成时间以及6h细胞与复合物孵育时间,转染效率最高.血清在本实验室条件下并不影响转染效率.结论:实验获得的优化条件可以明显提高脂质体对肿瘤细胞的转染效率,可作为有关研究或应用的参考.     

细胞培养液pH对慢病毒包装及感染效率的影响

为解决实验室中包装慢病毒转染效率低、感染效率低的问题,从培养液p H角度对病毒包装及感染进行优化。首先构建慢病毒载体cop GFP质粒,采用磷酸钙法和Lipofectamine2000法,转染不同p H培养液培养的293T细胞。在荧光显微镜下比较转染效率,发现磷酸钙法p H(6.5~7.5)转染效率高,培养液p H(6~8)对Lipofectamine2000法转染几乎无影响。使用标准p H(7.2~7.4)培养液包装慢病毒,收取病毒上清液后调节至不同p H,加或不加Polybrene感染293T细胞,发现p H在8~9附近感染效率高,调节慢病毒p H至弱碱性与Polybrene同时使用有助于提高感染效率。     

Improvement of transfection efficiency in cultured chicken primordial germ cells by percoll density gradient centrifugation

Chicken primordial germ cells (PGCs) differentiate into germ cells in gonads. Because PGCs can be cloned and cultured maintaining germline competency, they are a good means of modifying the chicken genome, but the efficiency of plasmid transfection into PGCs is very low. In this study, I attempted to improve the efficiency of PGC transfection. Cultured PGCs were purified by Percoll density gradient centrifugation, and were then transfected with plasmid DNA. For transient transfection, the transfection efficiency increased more than 7-fold by the Percoll method. The efficiency of stable transfection of PGCs also increased significantly. The stable transfectants that were isolated by this method accumulated in the developing gonads after microinjection into bloodstream of chick embryos, indicating that gene transfection by Percoll purification did not alter the function of PGCs in vivo.     

Improvement of Transfection Efficiency in Cultured Chicken Primordial Germ Cells by Percoll Density Gradient Centrifugation

Chicken primordial germ cells (PGCs) differentiate into germ cells in gonads. Because PGCs can be cloned and cultured maintaining germline competency, they are a good means of modifing the chicken genome, but the efficiency of plasmid transfection into PGCs is very low. In this study, I attempted to improve the efficiency of PGC transfection. Cultured PGCs were purified by Percoll density gradient centrifugation, and were then transfected with plasmid DNA. For transient transfection, the transfection efficiency increased more than 7-fold by the Percoll method. The efficiency of stable transfection of PGCs also increased significantly. The stable transfectants that were isolated by this method accumulated in the developing gonads after microinjection into bloodstream of chick embryos, indicating that gene transfection by Percoll purification did not alter the function of PGCs in vivo.     

Transfer of blood containing primordial germ cells between chicken eggs development of embryonic reproductive tract

The present study was carried out to investigate development of recipient chicken embryonic reproductive tracts which are transferred chicken primordial germ cells (PGCs). It is thought that differentiation of PGCs is affected by the gonadal somatic cells. When female PGCs are transferred to male embryos, it is possible that they differentiate to W-spermatogonia. However, the relationship development between PGCs and gonads has not been investigated. At stage 12–15 of incubation of fertilized eggs, donor PGCs, which were taken from the blood vessels of donor embryos, were injected into the blood vessels of recipient embryos. The gonads were removed from embryos that died after 16 days of incubation and from newly hatched chickens and organs were examined for morphological and histological features. The survival rate of the treated embryos was 13.6% for homo-sexual transfer of PGCs (male PGCs to male embryo or female PGCs to female embryo) and 28.9% for hetero-sexual transfer PGCs (male PGCs to female embryo or female PGCs to male embryo) when determined at 15 days of incubation. The gonads of embryos arising from homo-sexual transfer appeared to develop normally. In contrast, embryos derived from hetero-sexual transfer of PGCs had abnormal gonads as assessed by histological observation. These results suggest that hetero-sexual transfer of PGCs may influence gonadal development early-stage embryos.     

A study of the efficiency of different methods to transfer a reporter gene to chicken embryonic cells

The methods of transfection of a plasmid with a reporter gene involving DNA injection into chicken embryonic cells were studied. The parameters of the efficient transfection of chicken blastodermal cells with a foreign gene have been determined (20–24 and up to 40% in culture and embryos, respectively). A high efficiency of transfection of primordial germ cells isolated from the gonads has been obtained after DNA injection into the dorsal aorta of 2.5-day-old chicken embryos.     

A new method for producing transgenic birds via direct in vivo transfection of primordial germ cells

Traditional methods of avian transgenesis involve complex manipulations involving either retroviral infection of blastoderms or the ex vivo manipulation of primordial germ cells (PGCs) followed by injection of the cells back into a recipient embryo. Unlike in mammalian systems, avian embryonic PGCs undergo a migration through the vasculature on their path to the gonad where they become the sperm or ova producing cells. In a development which simplifies the procedure of creating transgenic chickens we have shown that PGCs are directly transfectable in vivo using commonly available transfection reagents. We used Lipofectamine 2000 complexed with Tol2 transposon and transposase plasmids to stably transform PGCs in vivo generating transgenic offspring that express a reporter gene carried in the transposon. The process has been shown to be highly effective and as robust as the other methods used to create germ-line transgenic chickens while substantially reducing time, infrastructure and reagents required. The method described here defines a simple direct approach for transgenic chicken production, allowing researchers without extensive PGC culturing facilities or skills with retroviruses to produce transgenic chickens for wide-ranging applications in research, biotechnology and agriculture.     

影响鸡原始生殖细胞分离克隆因素的研究（简报）

具有多向分化潜能的胚胎干细胞有两种来源：一是来自于早期胚胎内细胞团的胚胎干细胞(Em．bryonic Stem Cells,ESCs),另一种是来自于胚胎生殖腺原始生殖细胞(Primordial Germ Cells,PGCs)的胚胎生殖细胞(Embryonic Germ Cells,EGCs)。     

Improved Transfection Efficiency of Chicken Gonadal Primordial Germ Cells for the Production of Transgenic Poultry

Electroporation is a common method of DNA transfection for many types of eukaryotic cells, but has not been attempted in avian primordial germ cells (PGCs). DNA uptake in chicken primordial germ cells (PGCs) was tested using electroporation with and without dimethyl sulfoxide (DMSO). Gonadal tissue and chicken embryonic fibroblasts (CEFs) were isolated from 6-day-old embryos (stage 29), transfected with pCMV carrying the bacterial lacZ gene, and cultured for 24 h. Gonadal primordial germ cells (gPGCs) were purified from culture using a Ficoll gradient. The addition of DMSO significantly increased the transfection efficiency of gPGCs but had no effect on chicken embryonic fibroblasts. Electroporation of gPGCs resulted in an 80% transfection efficiency, compared with about 17% observed with liposomes. Approximately 200 transfected gPGCs were injected into 2.5-day-old (stage 17) recipient embryos and the eggs were incubated for an additional 3.5 days, 7.5 days or to ...     

PRODUCTION OF GERMLINE CHIMERIC CHICKENS BY TRANSFER OF CULTURED PRIMORDIAL GERM CELLS

Primordial germ cells (PGCs) from stage 27 (5.5-day-old) Korean native ogol chicken embryonic germinal ridges were cultured in vitro for 5 days. As in in vivo culture, these cultured PGCs were expected to have already passed beyond the migration stage. Approximately 200 of these PGCs were transferred into 2.5-day-old white leghorn embryonic blood stream, and then the recipient embryos were incubated until hatching. The rate of hatching was 58.8% in the manipulated eggs. Six out of 60 recipients were identified as germline chimeric chickens by their feather colour. The frequency of germline transmission of donor PGCs was 1.3–3.1% regardless of sex. The stage 27 PGCs will be very useful for collecting large numbers of PGCs, handling of exogenous DNA transfection during culture, and for the production of desired transgenic chickens.     

Optimizing Transfection of Primary Human Umbilical Vein Endothelial Cells Using Commercially Available Chemical Transfection Reagents

Primary cells, such as HUVEC, are notoriously difficult to transfect and are susceptible to the toxic effects of transfection reagents. A transfection reagent with a high transfection efficiency and low cytotoxicity was sought to retain sufficient viability of transfected HUVEC for subsequent assays. Nine chemical transfection reagents, currently commercially available, were compared for their ability to transfect HUVEC in vitro. A plasmid expressing the enhanced GFP (EGFP) was used for transfection, followed by flow cytometry of transfected HUVEC to determine the proportion of EGFP-expressing cells as a measure of transfection efficiency. Lipofectamine 2000 and Lipofectamine LTX (Invitrogen, Carlsbad, CA, USA) gave the highest transfection efficiencies of the reagents tested. Lipofectamine LTX was identified as the optimal transfection reagent as a result of its higher transfection efficiency at shorter periods of time following transfection when cytotoxicity was limited, allowing sufficient yield of transfected HUVEC for use in subsequent assays.