In vivo transfection of enhanced green fluorescent protein in rat retinal ganglion cells mediated by ultrasound-induced microbubbles

BACKGROUND： Studies have demonstrated that ultrasound-mediated microbubble destruction significantly improves transfection efficiency of enhanced green fluorescent protein （EGFP） in in vitro cultured retinal ganglial cells （RGCs）. OBJECTIVE： To investigate the feasibility of ultrasound-mediated microbubble destruction for EGFP transfection in rat RGCs, and to compare efficiency and cell damage with traditional transfection methods. DESIGN, TIME AND SETTING： In vivo, gene engineering experiment. The study was performed at the Central Laboratory, Institute of Ultrasonic Imaging, Chongqing Medical University from March to July 2008. MATERIALS： Eukaryotic expression vector plasmid EGFP and microbubbles were prepared by the Institute of Ultrasonic Imaging, Chongqing Medical University. The microbubbles were produced at a concentration of 8.7 × 10^11/L, with a 2-4 μm diameter, and 10-hour half-life in vitro. METHODS： A total of 50 Sprague Dawley rats were randomly assigned to four groups. Normal controls （n = 5） were infused with 5 μL normal saline to the vitreous cavity; the naked plasmid group （n = 15） was infused with 5 pL EGFP plasmid to the vitreous cavity; in the plasmid with ultrasound group （n = 15）, the eyes were irradiated with low-energy ultrasound wave （0.5 W/cm^2） for a total of 60 seconds （irradiated for 5 seconds, at 10-second intervals） immediately following infusion of EGFP plasmids to the vitreous cavities. In the microbubble-ultrasound group （n = 15）, the eyes were irradiated with the same power of ultrasonic wave immediately following infusion of microbubbles containing EGFP plasmids to the vitreous cavities. MAIN OUTCOME MEASURES： After 7 days, retinal preparations and EGFP expression in RGCs were observed by fluorescence microscopy. RGC quantification in the retinal ganglion cell layer was performed. In addition, EGFP mRNA expression was semi-quantitatively determined by RT-PCR. RESULTS： The transfection efficiency of EGFP to RGCs by microbubbles with ultrasound was significantly greater than the other groups, and no obvious damage was detected in the RGCs. CONCLUSION： Under irradiation of low-frequency ultrasound waves, ultrasound-mediated microbubble destruction was effective and resulted in safe transfection of the EGFP gene to the RGCs.

Rapid Purification of Enhanced Green Fluorescent Protein from Escherichia coli

As an excellent reporter molecule, enhanced green fluorescent protein (eGFP) was widely used for gene expression and regulation and was generally expressed in Escherichia coli strain. A rapid procedure consisting of ammonium sulfate precipitation, size exclusion chromatography, and anion exchange chromatography was devel- oped for the purification of eGFP. Based on the proposed procedure, recombinant eGFP with an electrophoretic pu- rity was achieved in combination with an overall yield of 66% and a purification factor of 17.9. The fluorescent spectrometry of purified eGFP and lysate from E. coli strain expressing eGFP exhibited the same wavelength of ex- citation and emission maxima, indicating that the purification procedure did not influence the construct and fluo- rescent characteristics of desired protein. The procedure mentioned was easy to scale up for the purification of large quantities of eGFP.

Construction and Co-expression of Grass Carp Reovirus VP6 Protein and Enhanced Green Fluorescence Protein in the Insect Cells

Grass carp reovirus(GCRV),a disaster agent to aquatic animals,belongs to Genus Aquareovirus of family Reoviridea.Sequence analysis revealed GCRV genome segment 8(s8) was 1 296 bp nucleotides in length encoding an inner capsid protein VP6 of about 43kDa.To obtain in vitro non-fusion expression of a GCRV VP6 protein containing a molecular of fluorescence reporter,the recombinant baculovirus,which contained the GCRVs8 and eGFP(enhanced green fluorescence protein) genes,was constructed by using the Bac-to-Bac insect expression system.In this study,the whole GCRVs8 and eGFP genes,amplified by PCR,were constructed into a pFastBacDual vector under polyhedron(PH) and p10 promoters,respectively.The constructed dual recombinant plasmid(pFbDGCRVs8/eGFP) was transformed into DH10Bac cells to obtain recombinant Bacmid(AcGCRVs8/eGFP) by transposition.Finally,the recombinant bacluovirus(vAcGCRVs8/eGFP) was obtained from transfected Sf9 insect cells.The green fluorescence that was expressed by transfected Sf9 cells was initially observed 3 days post transfection,and gradually enhanced and extended around 5 days culture in P1(Passage1) stock.The stable high level expression of recombinant protein was observed in P2 and subsequent passage budding virus(BV) stock.Additionally,PCR amplification from P1 and amplified P2 BV stock further confirmed the validity of the dual-recombinant baculovirus.Our results provide a foundation for expression and assembly of the GCRV structural protein in vitro.

Microbubble-enhanced ultrasound exposure improves gene transfer in vascular endothelial cells

AIM: To explore the effects of ultrasound exposure combined with microbubble contrast agent (SonoVue) on the permeability of the cellular membrane and on the expression of plasmid DNA encoding enhanced green fluorescent protein (pEGFP) transfer into human umbilical vein endothelial cells (HUVECs). METHODS: HUVECs with fluorescein isothiocyanate- dextran (FD500) and HUVECs with pEGFP were exposed to continuous wave (1.9 MHz, 80.0 mW/cm2) for 5 min, with or without a SonoVue. The percentage of FD500 taken by the HUVECs and the transient expression rate of pEGFP in the HUVECs were examined by fluorescence microscopy and flow cytometry, respectively. RESULTS: The percentage of FD500-positive HUVECs in the group of ultrasound exposure combined with SonoVue was significantly higher than that of the group of ultrasound exposure alone (24.0% ± 5.5% vs 66.6% ± 4.1%, P < 0.001). Compared with the group of ultrasound exposure alone, the transfection expression rate of pEGFP in HUVECs was markedly increased with the addition of SonoVue (16.1% ± 1.9% vs 1.5% ± 0.2%, P < 0.001). No statistical significant difference was observed in the HUVECs survival rates between the ultrasound group with and without the addition of SonoVue (94.1% ± 2.3% vs 91.1% ± 4.1%).CONCLUSION: The cell membrane permeability of HUVECs and the transfection efficiency of pEGFP into HUVECs exposed to ultrasound are significantly increased after addition of an ultrasound contrast agent withoutobvious damage to the survival of HUVECs. This non- invasive gene transfer method may be a useful tool for clinical gene therapy of hepatic tumors.

Production of Transgenic Korean Native Cattle Expressing Enhanced Green Fluorescent Protein Using a FIV-Based Lentiviral Vector Injected into MII Oocytes

The potential benefits of generating and using transgenic cattle range from improvements in agriculture to the production of large quantities of pharmaceutically relevant proteins. Previous studies have attempted to produce transgenic cattle and other livestock by pronuclear injection and somatic cell nuclear transfer, but these approaches have been largely ineffective; however, a third approach, lentivirus-mediated transgenesis, has successfully produced transgenic livestock. In this study, we generated transgenic （TG） Korean native cattle using perivitelline space injection of viral vectors, which expressed enhanced green fluorescent protein （EGFP） systemically. Two different types of lentiviral vectors derived from feline immunodeficiency virus （FIV） and human immunodeficiency virus （HIV） carrying EGFP were injected into the perivitelline space of MII oocytes. EGFP expression at 8-cell stage was significantly higher in the FIV group compared to the HIV group （47.5% ± 2.2% v.s. 22.9% 4± 2.9%）. Eight-cell embryos that expressed EGFP were cultured into blastocysts and then transferred into 40 heifers. Ten heifers were successfully impregnated and delivered 10 healthy calves. All of these calves expressed EGFP as detected by in vivo imaging, PCR and Southern blotting. In addition, we established an EGFP-expressing cell line from TG calves, which was followed by nuclear transfer （NT）. Recloned 8-cell embryos also expressed EGFP, and there were no differences in the rates of fusion, cleavage and development between cells derived from TG and non-TG calves, which were subsequently used for NT. These results illustrate that FIV-based lentiviruses are useful for the production of TG cattle. Moreover, our established EGFP cell line can be used for additional studies that involve induced pluripotent stem cells.

The labeling of C57BL/6j derived embryonic stem cells with enhanced green fluorescent protein

Objective To labele MESPU35, a embryonic stem (ES) cell line derived from C57BL/6j mouse, with enhanced green fluorescent protein (EGFP) for further application.Methods The EGFP gene was controlled by the hybrid CA promoter/enhancer (CMV enhancer/ chicken beta-actin promoter/ beta-actin intron) to construct the vector of the transgene, pCA-EGFP. The vector was transfected into MESPU35 by electroporation.Results We generated EGFP expressing ES cells demonstrating normal properties. The green fluorescence of EGFP expressing cells was maintained in propagation of the ES cells for more than 30 passages as well as in differentiated cells. Cultured in suspension, the 'green' ES cells aggregated, and formed embryoid bodies maintaining the green fluorescence at varying developmental stages. The 'green' embryoid bodies could expand and differentiate into various types of cells, exhibiting ubiquitous green fluorescence. Conclusions The hybrid CA promoter/enhancer used to control the EGFP expressing ES cells, resulted in more intense and ubiquitous activity. The EGFP transfected cells yield bright green fluorescence, which can be visualized in real time and in situ. In addition, the ES cells, MESPU35, are derived from C57BL/6j mice, which are the most widely used in oncology, physiology and genetics. Compared to 129 substrains, C57BL/6j mice avoid a number of potential problems apparent in the other strains.

不同途径注射质粒微泡对超声微泡破碎技术介导EGFP基因在兔骨缺损处转染的影响

目的探讨超声微泡破碎技术介导增强型绿色荧光蛋白(enhanced green fluorescent protein,EGFP)基因在兔骨缺损处转染时,不同途径注射质粒微泡混悬液对转染效率及局部组织的影响。方法3月龄新西兰大白兔10只,制备右尺骨骨缺损模型,按照随机数字表法分为静脉组和断端间组(n=5)。静脉组和断端间组造模后第10天分别经耳缘静脉或骨缺损断端间向兔体内注射携带EGFP基因的质粒微泡混悬液(0.3 ml/kg)。在超声频率1 MHz,超声强度1.0 W/cm^(2),占空比20%条件下,对两组骨缺损部位超声辐照1 min,进行EGFP基因转染。在基因转染后1周时处死兔,于骨缺损处获取标本制作切片,荧光染色观察各组EGFP表达情况。采用病理图像分析软件分析计算平均光密度。HE染色观察断端间软组织病理特点。结果静脉组和断端间组均观察到绿色荧光蛋白表达。断端间组平均光密度高于静脉组,差异有统计学意义(0.0345±0.0028 vs 0.0004±0.0001,P<0.05)。表达绿色荧光蛋白的细胞主要为骨骼肌细胞,各组未见细胞坏死征象。结论超声微泡破碎技术介导EGFP基因在兔骨缺损处转染时,其效率受不同途径注射质粒微泡混悬液的影响,骨缺损断端间直接注射优于静脉注射。

黑曲霉尿苷/尿嘧啶营养缺陷型转化系统的构建及应用

黑曲霉(Aspergillus niger)是一种重要的工业发酵菌株,它具有强大的蛋白分泌表达能力。为了提高黑曲霉遗传操作效率及优化重组菌株的筛选策略,构建以尿苷/尿嘧啶营养缺陷型为筛选标记的转化系统。利用CRISPR/Cas9技术实现pyrG基因的敲除,在含有尿嘧啶核苷和5-氟乳清酸(5-FOA)的抗性培养基中筛选表型正确的转化子。经基因组PCR验证,黑曲霉营养缺陷型菌株可稳定遗传。利用该转化系统可成功实现增强型绿色荧光蛋白在黑曲霉中的表达。通过结合增强型绿色荧光蛋白和流式细胞仪建立了黑曲霉转化子的高通量筛选模型。

巴西固氮螺菌Yu62的EGFP标记及其在小麦体内的定殖研究

以质粒pEGFP-C1为模板,采用PCR方法特异性扩增增强型绿色荧光蛋白(EGFP)基因全长序列,将其与原核表达载体pVK-100连接,构建成重组载体pVK-EGFP。利用电转化法将重组载体导入巴西固氮螺菌Yu62中,得到EGFP标记菌株。用EGFP标记菌接种小麦‘小偃107’种子,室内限菌条件下培养10 d后,用荧光显微镜观测标记菌在小麦体内的定殖规律并观察接菌植株的田间生长状况。结果显示,巴西固氮螺菌Yu62能定殖于小麦根毛区、茎组织的细胞间隙等部位,而且接菌小麦‘小偃107’植株在根系发育、株高、分蘖数等方面比对照有较明显的优势。研究表明,巴西固氮螺菌Yu62能够定殖于小麦根茎内,并具有促进植物生长的作用。

超声微泡造影剂介导EGFP质粒转染大鼠视网膜的实验研究

目的探讨超声破坏微泡介导EGFP质粒转染大鼠视网膜的效率及可行性,为实现外源基因高效、定向的转移目的奠定基础。方法将30只Long-evans大鼠分为6组,第1组仅以0.5W/cm2的超声波辐照大鼠眼球,第2组于尾静脉输入适当剂量的微泡造影剂,并立即以相同能量的超声波辐照大鼠眼球,第3组于尾静脉输入质粒,第4组于尾静脉输入质粒,并以超声辐照大鼠眼球,第5组于尾静脉输入质粒与微泡,第6组尾静脉输入质粒、微泡,并用超声辐照眼球。转染2周后,在激光共聚焦显微镜下观察EGFP表达情况。结果超声微泡介导的EGFP质粒对大鼠视网膜的转染效率,明显高于其他实验组。一定能量和时间的超声波辐照,及适当浓度的微泡,对大鼠视网膜脉络膜无明显损伤。结论利用低频率和一定能量的超声击碎携带EGFP质粒的超声微泡造影剂,能够有效地提高EGFP质粒在大鼠视网膜的转染效率。

家蚕精子介导报告基因gfp和egfp转移技术的研究

探讨家蚕精子介导基因转移技术能否有效地将外源基因导入并整合到家蚕基因组中,使之稳定遗传.分别进行了含gfp报告基因的pG350载体和含egfp报告基因的pMD-Fib-IE-EGFP与pEGFP-N3载体的精子介导基因转移实验.结果表明,导入线性pG350载体,在14个G0代实验蛾区的DNA样品中,有5个PCR检测为阳性,阳性率为35.7%;对G1代的PCR与Southern blot检测的结果证明,导入的gfp基因存在于G1代的家蚕基因组中.导入不同浓度或构型的pMD-Fib-IE-EGFP与pEGFP-N3载体时,对G0代的PCR检测结果表明,导入线性pMD-Fib-IE-EGFP组的PCR阳性率为61.2%,而导入环状质粒组的PCR阳性率为57.1%;导入环状pEGFP-N3组的PCR阳性率为46.2%;对部分PCR阳性蛾区进行Southern blot检测结果表明,导入的egfp基因整合在G0代家蚕基因组中.本研究的结果表明,家蚕精子介导基因转移能够将外源基因有效地导入、整合于家蚕基因组,并可遗传至G1代.

绿色荧光蛋白标记的表达载体pHis-EGFP的构建

为对重组蛋白的表达进行直观检测并简化蛋白纯化的步骤,构建了能在大肠杆菌中表达融合蛋白的通用表达载体pHis-EGFP。该载体含有源自表达载体pET-32a的T7启动子、终止子和源自质粒pUC18的ColE1复制子与绿色荧光蛋白报告基因。应用该载体成功地表达并纯化了酵母GGDP(geranylgeranyldiphosphate,GGDP)合酶融合蛋白,结果表明所构建的载体是一个实用的表达载体,并建立了离子交换层析和亲和层析两步纯化融合蛋白的方法。

结核杆菌Hsp65与EGFP融合基因的构建及DC疫苗的制备

目的: 构建结核杆菌H37Rv株Hsp65与增强型绿色荧光蛋白 (EGFP)的融合基因pEGHsp65, 并以其转染小鼠的树突状细胞 (DC), 制备抗结核的DC疫苗。方法: 采用PCR技术, 从培养的结核杆菌H37Rv株中抽提Hsp65基因,克隆到含有EGFP基因的质粒pEGFP- C1中, 构建pEGHsp65融合基因。以其转染Hela细胞, 在共聚焦激光扫描荧光显微镜下观测不同时间荧光表达的强弱, 并用RT- PCR检测Hsp65mRNA的表达。以pEGHsp65融合基因转染小鼠骨髓细胞经GM- CSF和IL -4诱导分化的DC, 用MTT比色法检测DC疫苗刺激未致敏脾细胞的增殖。结果: 用EcoRⅠ和BglⅡ双酶切鉴定证实, H37Rv株Hsp65DNA已插入重组表达载体pEG -FP- C1中。将融合基因转染入Hela细胞, 48h转染率最高;用RT- PCR在mRNA水平上可检测到Hsp65mRNA的表达。用MTT比色法检测表明, 融合基因转染的DC能激活并引起未致敏的脾细胞增殖。结论: 成功地构建pEGHsp65融合基因和以其制备的DC疫苗, 为进一步观察其治疗结核病的效应奠定了基础。

稳定表达egfp基因细胞的构建与克隆

将增强的绿色荧光蛋白 ( enhanced green fluorescent protein,EGFP)基因插在 HCMV( hum ancytom egolovirus)启动子下游 ,构建了表达质粒 p CA13 - e G,用脂质体 L ipofectin介导分别转染 He L a细胞、Vero细胞 ,仅通过细胞传代 ,就获得了能稳定高效表达 EGFP的绿色细胞 .比较发现 ,质粒 p CA13 - e G转染后 ,产生能高效表达 EGFP的 He L a细胞其比率高于 Vero细胞 ;EGFP高效表达对 Vero细胞的毒性大于对 He L a细胞的毒性 .本研究表明 ,绿色细胞轮廓清晰 ,由于其特有的性质 ,在用于细胞的形态观察、细胞分裂等研究时会有所作为 .

一种新的双元表达质粒pCMV-Myc-IRES-EGFP的构建及其表达

为了研究基因的特征、理化特性及其功能机制,通常需要构建多个真核表达载体,涉及到多次的引物设计、酶切、连接和鉴定等繁琐的亚克隆过程。构建携带易于多种实验研究的多用或通用载体是基因工程载体的发展方向。为此,利用pIRES载体为骨架质粒,在A和B多克隆位点上分别插入c-Myc标签蛋白序列和增强型绿色荧光蛋白(EGFP)序列,从而构建了一个包含c-Myc标签蛋白序列并携带有核糖体结合位点(IRES)介导的增强型绿色荧光蛋白的真核表达载体pCMV-Myc-IRES-EGFP。通过荧光检测和免疫印迹实验证实该载体能在哺乳细胞中表达。该载体可用于监测细胞的转染效率、分选稳定表达的阳性细胞群体、体外转录和翻译、检测或纯化目的蛋白以及捕获相关作用蛋白等多种实验研究,为基因功能研究提供了便利。

含增强型绿色荧光蛋白(EGFP)基因的家蚕同源重组质粒载体的构建

以含家蚕丝心蛋白重链基因同源片段的质粒pG35 0为出发质粒 ,插入增强型绿色荧光蛋白 (EGFP)基因 ,构建成以EGFP为报告基因的同源重组质粒载体 pMD Fib IE EGFP ,通过PCR及酶切鉴定表明EGFP以正确的方式插入到原始质粒中 ,通过脂质体介导法转染胃癌细胞能发出很强的绿色荧光 ,表明该质粒能够在真核细胞中表达 。

重组腺病毒载体Ad5-hTRX-EGFP的构建及其表达

本研究旨在构建并制备人硫氧还蛋白(hTRX)和增强型绿色荧光蛋白(EGFP)基因腺病毒载体Ad-hTRX-EGFP,感染HEK293细胞,为基因治疗提供实验基础。设计含有NotⅠ和EcoRⅤ酶切位点的引物,PCR扩增hTRX,将扩增产物连接到带有EGFP标记的pDC316-mCMV穿梭质粒上,构建重组穿梭质粒pDC316-hTRX-EGFP,利用Lipofectamine2000脂质体的方法将AdMax腺病毒包装系统的骨架质粒pBHG lox_E1,3Cre和穿梭质粒pDC316-hTRX-EGFP共转染入HEK293细胞,进行同源重组,得到腺病毒重组质粒pAd-hTRX-EGFP,并在其中包装扩增病毒,用氯化铯高速梯度离心、纯化病毒,测定病毒颗粒数及滴度。采用PCR方法对重组腺病毒进行鉴定,用流式细胞仪测定感染HEK293细胞的效率,Western blot方法验证细胞表达hTRX蛋白。结果显示,重组腺病毒质粒经PCR和NotⅠ、EcoRⅤ酶切鉴定,证实含有hTRX基因,测序结果和设计片段的序列一致。重组腺病毒载体构建成功,病毒滴度达5.558×1010pfu/ml。病毒成功感染HEK293细胞,MOI=100时,感染效率达92.25%。经Western blot方法验证表明,感染后的HEK293细胞高表达hTRX蛋白。结论:应用细胞内同源重组方法成功构建了含hTRX基因的重组腺病毒载体,制备获得高滴度的病毒,能高效感染HEK293细胞并表达目的蛋白,为后续研究奠定了基础。

脂质体介导pIDO-EGFP转染原代培养软骨细胞的初步研究

目的:检测脂质体介导pIDO-EGFP转染原代培养的C57小鼠关节软骨细胞的瞬时表达及转染效率,建立原代培养的小鼠关节软骨细胞转染方法。方法:大肠杆菌中扩增pIDO-EGFP质粒,在最优化条件下通过lipofectamine2000TM转染试剂将pIDO-EGFP质粒转入原代培养的小鼠关节软骨细胞,应用荧光显微镜和激光共聚焦显微镜观察其转染过程及瞬时表达情况,流式细胞术检测其转染效率。结果:质粒携带的增强型绿色荧光蛋白在转染后24h得到了明显表达,48h后流式细胞术检测其转染效率为36.43%,未影响软骨细胞贴壁过程。结论:经绿色荧光蛋白检测表明,脂质体成功地将IDO基因转染进入原代培养的软骨细胞。转染后的软骨细胞在体外仍能存活,在最优化的条件下能达到良好的瞬时转染效率,为组织工程化软骨细胞基因导入和基因修饰提供了思路。

抗日本血吸虫生殖功能分子SIEA26～28kDa单链抗体与EGFP的融合表达及靶向性研究

目的体外观察抗日本血吸虫生殖功能分子SIEA26-28kDa单链抗体与日本血吸虫各阶段的靶向部位，探讨单链抗体在抗血吸虫病疫苗研究中的应用价值。方法扩增增强型绿色荧光蛋白（EGFP）基因．克隆至PET32a／SIEA26～28kDa-scFv质粒，构建PET32a／EGFP-scFv质粒。转化至感受态E．coli BL21（DE3）中。并诱导表达单链抗体融合蛋白。分析单链抗体融合蛋白与SIEA26～28kDa的结合特异性。单链抗体融合蛋白与日本血吸虫成虫、虫卵切片孵育，荧光显微镜下观测GFP信号。确定特异性单链抗体的靶向性。结果重组质粒PET32a／EGFP-scFv构建成功．Trx-EGFP-scFv融合蛋白高效表达．与SIEA26-28kDa特异性结合。GFP信号主要集中在未成熟卵卵胚．雌虫卵巢、卵黄腺及与生殖系统邻近的肠腔组织。结论SIEA26-28kDa单链抗体与血吸虫未成熟卵胚胎及雌虫生殖系统具有较强的靶向性，具有潜在抗卵胚发育、抗雌虫生殖作用，为SIEA26-28kDa单链抗体在抗日本血吸虫病疫苗免疫靶向方面的研究奠定了基础。