EGFP作为报道基因在研究WT1基因调控元件中的应用

本研究调查EGFP基因作为报道基因在研究WT1基因调控功能中应用的可行性。利用基因重组技术分别将WT1基因启动子和增强子的功能片段构建到质粒pEGFP-1载体中,转化感受态E.coli菌细胞,筛选了阳性转化菌落,通过限制性酶切鉴定重组质粒插入片段正确的菌落,抽提重组质粒DNA;同时将重组质粒转染K562细胞,通过荧光显微镜检测重组质粒的功能。结果表明：通过基因重组技术分别构建了含有WT1基因启动子的载体（pEWP）和含有WT1基因启动子和增强子的载体（pEWPE、pEWPA和pEWPD）。转染48小时后,pEWP、pEWPE、pEWPA和pEWPD的K562细胞在荧光显微镜下能够显示荧光,而转染了空载体pEGFP-1的K562细胞未出现荧光。结论：EGFP基因作为报道基因可以用于WT1基因调控功能的研究,为进一步开展基因治疗创造了条件。     

GFP和HSV—TK基因在四种细胞株中的共表达

目的：研究新型报道基因——绿色荧光蛋白（green fluorescent protein，GFP）和目的基因HSV-TK基因在哺乳动物细胞内的表达。方法：将同时表达GFP和HSV-TK的多右反子重组逆转录病毒GCGPTKSN，分别转染K562、NS-1、EL4、Sp2/0细胞株，并在体外应用荧光显微镜检测GFP的表达，同时GCV杀伤试验检测HSV-TK基因表达。结果：GFP在四种细胞系中高效表达。高浓度的GCV能有效抑制转染细胞的生长。结论：逆转录病毒载体能介导GFP和HSV-TK基因进入多种细胞系，并在那里共表达。GFP基因是一种很有前途的报道基因，这为基因转移的检测以及转基因细胞的筛选提供了一种新的方法。     

利用重组慢病毒载体建立稳定表达绿色荧光蛋白细胞系

目的：利用重组慢病毒载体将绿色荧光蛋白基因（GFP）导入293T细胞,建立稳定表达GFP的细胞系,将GFP作为靶基因观察不同方法调节基因表达的效力的差异。方法：以载体质粒pHR＇-pCMV-GFP、包装质粒pCMV-ΔR8.2及包膜质粒pCMV-VSVG用磷酸钙共沉淀法共转染包装细胞系293T,收集病毒颗粒再次感染293T细胞,用克隆环筛选出荧光亮度较强的细胞克隆。多次传代后,流式细胞术检测细胞荧光表达的稳定性。同时进行靶向GFP的RNA干扰,观察GFP表达下调情况,验证该细胞系用于研究基因调控方法的有效性。结果：利用重组慢病毒载体可有效建立GFP稳定表达的新细胞系,此细胞系中GFP阳性细胞占99%以上,12次传代前后GFP表达无明显差异;靶向GFP的RNAi可以显著下调GFP表达。结论：成功建立了表达GFP的G4细胞系,并可有效地用于基因调节方法的研究。     

HCCR-2干扰真核表达载体的构建及其稳定转染细胞系的建立

目的构建HCCR-2干扰真核表达载体,获得干扰质粒稳定转染的HepG2细胞系。方法人工合成HCCR-2基因干扰序列并定向插入到RNAi真核表达载体pGenesil-1,通过测序鉴定。将干扰质粒用脂质体法转染HepG2细胞,经G418持续压力选择和有限稀释法获得稳定转染的细胞系。RT-PCR检测筛选克隆HCCR-2 mRNA的表达水平。结果测序表明HCCR-2干扰序列及读框完全正确,干扰质粒稳定转染的HepG2细胞系在倒置荧光显微镜下呈绿色荧光。RT-PCR结果显示RNAi-H1、RNAi-H3序列对HCCR-2 mRNA有较好的抑制效果。结论成功构建了HCCR-2干扰真核表达载体,HCCR-2干扰质粒稳定转染的HepG2细胞系的建立为进一步研究HCCR-2在肝癌细胞中的作用奠定了基础。     

RNA干扰靶向抑制EGFR对鼻咽癌细胞凋亡及坏死的影响

目的:观察RNA干扰(RNA interference,RNAi)靶向抑制鼻咽癌CNE2细胞表皮生长因子受体(epidermal growth factor receptor,EGFR)表达对CNE2细胞辐射诱导的凋亡及坏死的影响.方法:用shRNA-EGFR转染人鼻咽癌细胞系CNE2,应用X射线照射转染EGFRSiRNA前后的细胞,MTT实验比较转染干扰质粒前后照射后细胞增殖变化,Annexin V/PI双标记法检测凋亡及坏死,并进行转染干扰质粒前后照射后凋亡及坏死率的比较.结果:shRNA-EGFR成功转染CNE2细胞.CNE2细胞干扰后照射较干扰前照射增殖减低.CNE2细胞干扰后照射较干扰前照射凋亡及坏死均增加;凋亡所占比例高过坏死.结论:抑制鼻咽癌细胞EGFR表达后,主要是通过提高辐射诱导的凋亡来抑制鼻咽癌细胞增殖.     

葡萄糖调节蛋白78真核表达载体及稳定转染人视网膜色素上皮细胞株的构建

背景:葡萄糖调节蛋白78 是存在于内质网上最多的分子伴侣蛋白,具有保护细胞对抗细胞调亡的作用.目的:构建携带并正确表达外源性人葡萄糖调节蛋白78 基因的重组真核表达载体,建立葡萄糖调节蛋白78 基因稳定转染的人视网膜色素上皮细胞系.方法:扩增人葡萄糖调节蛋白78 全长编码序列,将该目的基因与真核表达载体PEGFP-N1 进行定向连接,其产物转化细菌感受态细胞,以PCR 方法鉴定阳性克隆,并进行测序和分析比对,获得融合蛋白表达质粒载体pEGFP-grp78.通过阳离子脂质体介导将重组质粒pEGFP-grp78 转染入体外培养的人视网膜色素上皮细胞,经G418 筛选,建立稳定表达细胞系.结果与结论:体外培养的人视网膜色素上皮细胞株作为真核细胞表达系统,经荧光显微镜和RT-PCR 方法检测,G418 筛选的稳定转染细胞系中GFP大量表达,葡萄糖调节蛋白78 mRNA 的表达量是正常视网膜色素上皮细胞的4 倍左右,表示脂质体介导的pEGFP-grp78 质粒成功转染视网膜色素上皮细胞,并高效稳定表达葡萄糖调节蛋白78.     

小干扰RNA沉默HIF-1α基因表达对对人鼻咽癌细胞系CNE1 凋亡的影响

目的 探讨CNE1对人鼻咽癌细胞系CNE1凋亡的影响。方法 利用阳离子脂质体LipofectamineTM2000将HIF-1αsiRNA转染入CNE13 细胞中。WB法测定CNE1 细胞内HIF-1α的表达。流式细胞仪及Hochest 荧光染色测定细胞凋亡率的变化。WB法测定CNE1细胞内BCL-2的表达。结果 干扰HIF-1α能有效地促进CNE1细胞的凋亡,同时可下调BCL-2的表达。结论 体外实验初步证明HIF-1α基因在人鼻咽癌细胞系CNE1凋亡方面扮演重要角色,可通过下调BCL-2的表达来诱导凋亡。     

骨髓间质干细胞绿色荧光蛋白基因的转染和克隆化研究

目的 建立能稳定表达绿色荧光蛋白(GFP)并连续传代培养的骨髓间质干细胞(MSCs)株。方法 采用电穿孔法将带有GFP的质粒(pGFP-N1)在不同条件下转染MSCs,经过G418筛选,有限稀释法筛选阳性克隆,荧光显微镜及流式细胞仪检测GFP的表达情况。结果 电穿孔以电容量1050μF、电压260V时效果最好,转染GFP的MSCs能在含有G418的培养基中生长,未转染的骨髓间质干细胞不断死亡。转染24h后即能观察到少量荧光细胞,一个月后大部分荧光细胞呈集落样生长,荧光表达较强,转染率为50％-60％。体外克隆后,能稳定表达至10代,10代后荧光强度明显减弱。结论 GFP基因能在MSCs中进行有效的复制、转录和翻译,并可持续、稳定、高效表达;转染GFP的MSCs能作为研究MSCs的诱导分化及生物细胞工程治疗的载体细胞。     

14-3-3σ逆转录病毒载体的构建及稳定转染HaCat细胞系的建立

目的:构建14-3-3σ逆转录病毒载体,并建立高表达14-3-3σ的HaCat细胞系.方法:以人基因组为模板,通过PCR扩增出14-3-3σ基因的编码序列,定向插入到pLEGFP-N1质粒中,并在STBL3内进行扩增,刷选阳性克隆,酶切和测序验证后,转染293vr细胞进行病毒包装、扩增、纯化、获取逆转录病毒栽体.将逆转录病毒载体感染HaCat细胞后Western、实时荧光定量PCR法检测14-3-3σ的表达情况.结果:连接重组后经酶切和测序筛选出pLEGFP-N1-14-3-3σ;稳定转染的HaCat细胞系在倒置荧光显微镜下呈绿色荧光.Western和实时荧光定量PCR法表明14-3-3σ表达明显增强.结论:成功构建了14-3-3σ逆转录病毒载体,并构建了英稳定转染的HaCat细胞系.     

胰腺十二指肠同源框蛋白1基因转染人骨髓间充质干细胞及向胰岛样细胞的分化

背景:随着人胰岛素基因及某些血糖调节酶基因的克隆,以及重组载体为基础的基因克隆技术的建立,使骨髓间充质干细胞更容易向胰岛素分泌细胞分化,且具有体外扩增明显而无致瘤性的优点.目的:利用胰腺十二指肠同源框蛋白1(pancreatic duodenal homeobox-1,PDX-1)基因转染人骨髓间充质干细胞,观察基因修饰向胰岛样细胞的分化情况.设计、时间及地点:细胞-基因学体外实验,于2008-01/07在青岛大学医学院附属医院中心实验室完成.材料:骨髓来源于糖尿病患者,由青岛大学医学院附属医院干细胞中心提供.方法:利用反转录聚合酶链技术构建PDX-1基因的cDNA,酶切后插入经相同酶切处理的含绿色荧光蛋白的空白载体PEGFP-N1,构成重组质粒,再进行Bgl Ⅱ、HindⅢ双酶切筛选、鉴定并测序.Percoll法分离培养入骨髓间充质干细胞,当细胞汇合至90%时胰蛋白酶消化传代.转染前1 d加入无抗生素培养基,将第3代细胞按5×105接种于6孔培养板,适量重组载体及脂质体溶解后混合形成DNA-脂质体混合物,培养5 h后更换普通培养液.同时设立对照组,在6孔板中进行空白质粒转染.主要观察指标:脂质体介导转染后,荧光显微镜观察细胞转染情况;免疫荧光染色检测PDX-1蛋白的表达;双硫腙染色鉴定细胞分化情况.结果:通过测序、凝胶电泳、酶切等确定PDX-1基因已正确插入重组质粒.荧光显微镜下,成功转染的骨髓间充质干细胞发出绿色荧光,质粒转染效率约70%.转染8 d的细胞PDX-1呈阳性表达,对照组细胞PDX-1呈阴性.转染后14 d可见半悬浮的胰岛样细胞团,双硫腙染色呈棕红色,而未转染的对照组细胞双硫腙染色不着色.结论:PDX-1基因重组载体能够导入人骨髓间充质干细胞且稳定表达,并可以转化为胰岛样细胞.     

分子影像学报告基因显像的研究进展

报告基因显像技术是分子影像学中重要的显像技术,其可对多种不同的生物学和分子遗传学过程进行显像,有望更快地应用于临床.本文综述分子影像学中报告基因显像的一般原则、分类、相关影像学技术及潜在的临床应用.     

神经科学研究中绿色荧光蛋白报告基因的应用

随着研究手段的进步,人们对神经学的研究更加深入。绿色荧光蛋白作为一种新型的报告基因,具有荧光性质稳定、对生物体无毒性作用、检测时不需底物的特点,在神经细胞的基因表达、蛋白定位的研究上优于其他报告基因,尤其适合于活体细胞功能与形态相结合的研究。     

Design considerations for incorporating sodium iodide symporter reporter gene imaging into prostate cancer gene therapy trials

This study was done to aid in the design of a phase I gene therapy trial in patients with prostate cancer. We determined the dosimetric characteristics of our reporter gene system when coupled with intravenous administration of radioactive sodium pertechnetate (Na(99m) TcO(4)) and determined the feasibility of using human sodium iodide symporter (hNIS) as a reporter gene to study the dynamics of adenoviral transgene expression in a large animal tumor. A replication-competent Ad5-yCD/mutTK(SR39) rep-hNIS adenovirus was injected into the prostate gland of dogs for dosimetry purposes, and into a canine soft tissue sarcoma (STS) for imaging purposes. After resection of the prostate, the amount of (99m)TcO(4)() sequestered in the prostate was determined, the radiation dose absorbed by the prostate and nontarget critical organs was calculated, and hNIS reporter gene expression was imaged in the STS by single-photon emission computed tomography (SPECT). On the basis of the findings from 25 dogs, the amount of (99m)TcO (4)() sequestered in the prostate ranged from 13 to 276 muCi. Using the highest value observed, absorbed radiation dose to critical organs was calculated and found to be below U.S. Food and Drug Administration limits for diagnostic imaging. Also, (99m)TcO (4)() uptake was readily detected by SPECT and found to persist in vivo for at least 4 days. On the basis of our dosimetry calculations, up to five imaging procedures can be safely performed in humans after intraprostatic injection of the Ad5-yCD/mutTK(SR39)rep-hNIS adenovirus and the hNIS reporter gene system can be used to study the dynamics of adenoviral gene therapy vectors in large animal tumors.     

LCN2基因启动子区荧光素酶报告基因载体的构建及鉴定

目的 为进一步研究LCN2(Lipocalin 2)基因在急性肾损伤中的作用,克隆LCN2的编码序列,构建含LCN2基因的荧光素酶报告基因载体,并在肾小管上皮细胞系HK-2 中表达.方法 以HK-2细胞系提取的RNA为模板进行反转录-聚合酶链反应(RT-PCR),扩增产物用HindⅢ和SmaI双酶切后克隆到双荧光素酶报告基因载体PGL3-Basic中,用非脂质体法将构建质粒PGL3-Basic /LCN2导入HK-2 中,氨苄青霉素选择培养,经双荧光素酶鉴定其表达.结果 PCR和测序结果表明扩增的LCN2启动子序列正确,酶切检测证实重组荧光素酶报告基因载体构建成功.转染PGL3-Basic/LCN2 质粒的HK-2细胞荧光素酶活性比转染空载体的明显增加(P<0.000 1).结论 成功克隆了LCN2的编码序列,构建了其荧光素酶报告基因载体PGL3-Basic/LCN2,有助于进一步研究LCN2基因在急性肾损伤中的作用机制.     

Branched oligonucleotides induce in vivo gene conversion of a mutated EGFP reporter

Branched oligonucleotides (b-oligonucleotides) based on a novel branching monomer were used for site-specific sequence alteration in vivo. With a stable integrated mutated enhanced green fluorescent protein (EGFP) template in Chinese hamster ovary cells, up to 0.1% EGFP-positive cells were counted after transfection with b-oligonucleotides. The presence of EGFP protein in converted cells was demonstrated by anti-EGFP immunocytochemistry. Genomic sequencing of converted cells showed in 40% of the analysed clones the corrected wild-type codon, while 9.3% of the sequences showed a corrected wild-type sequence and an additional collateral mutation. Despite the stable corrected genomic locus, converted cells entered selective apoptosis after 3-6 days. The cell line Irs-1 that is deficient in the homologous recombination pathway showed a reduced frequency of b-oligonucleotide-induced site-specific sequence conversion. The reduced conversion rates in the mutant cell line could be partly rescued by complementation with XRCC2 cDNA.     

Quantitation of cell number by a positron emission tomography reporter gene strategy.

PURPOSE: An important potential of positron emission tomography (PET) is the capacity for quantitation of cell signals in an anatomic regions of interest. However, little is known about the constraints and parameters for using PET signal detection to establish cell numbers in regions of interest. In this study, we determined the correlation of PET signal to cell number, and characterized the cellular limit of detection for PET imaging. PROCEDURES: Cells expressing the herpes simplex virus type I thymidine kinase PET reporter gene (HSV1-sr39TK) were detected following accumulation of [(18)F]FHBG (9-[4-[(18)F]-fluoro-3-(hydroxymethyl) butyl]guanine) by microPET scanning and quantitation. RESULTS: When cells were cultured with [(18)F]FHBG in vitro, and then transferred to a model vascularized site, 73% retention was observed one hour post-transfer. Using this information, and the measured attenuation of PET signal in whole mouse scans, we assessed the per-cell uptake of [(18)F]FHBG in the vascularized site following standard parenteral administration of the substrate. We observed a cell number-dependent signal, with a limit of detection calculated as 10(6) cells in a region of interest of 0.1 mL volume. Quantitatively similar parameters were observed with stably tranfected N2a glioma cells and retrovirally transduced primary T lymphocytes. CONCLUSION: These methods and findings provide a strategy for quantitation of cellularity using PET imaging that has implications for both experimental models and clinical diagnosis.     

DNA-loaded bacterial ghosts efficiently mediate reporter gene transfer and expression in macrophages.

There is a demand for efficient and safe DNA delivery vehicles mediating gene transfer and expression. We present bacterial ghosts as a novel platform technology for DNA delivery and targeting of macrophages. Bacterial ghosts are cell envelopes of gram-negative bacteria that are devoid of the cytoplasmic content. Escherichia coli ghosts were loaded with plasmid DNA and linear double-stranded DNA. Confocal laser scanning microscopy and flow cytometry confirmed that the DNA localized to the inner lumen of bacterial ghosts and was not associated with the outer surface of the bacteria. Up to approximately 6000 plasmids could be loaded per single ghost and the amount of loaded DNA correlated with the DNA concentration used for loading. E. coli ghosts loaded with plasmids encoding the enhanced green fluorescent protein (EGFP) targeted efficiently murine macrophages (RAW264.7) and mediated effective gene transfer. The EGFP was expressed by more than 60% of the macrophages as measured by flow cytometry detecting the green fluorescence and immunocytochemical staining with antibodies specific for EGFP.     

Tyrosinase as a dual reporter gene for both photoacoustic and magnetic resonance imaging

Reporter genes are useful scientific tools for analyzing promoter activity, transfection efficiency, and cell migration. The current study has validated the use of tyrosinase (involved in melanin production) as a dual reporter gene for magnetic resonance and photoacoustic imaging. MCF-7 cells expressing tyrosinase appear brown due to melanin. Magnetic resonance imaging of tyrosinase-expressing MCF-7 cells in 300 μL plastic tubes displayed a 34 to 40% reduction in T1 compared to normal MCF-7 cells when cells were incubated with 250 μM ferric citrate. Photoacoustic imaging of tyrosinase-expressing MCF-7 cells in 700 μm plastic tubes displayed a 20 to 57-fold increase in photoacoustic signal compared to normal MCF-7 cells. The photoacoustic signal from tyrosinase-expressing MCF-7 cells was significantly greater than blood at 650 nm, suggesting that tyrosinase-expressing cells can be differentiated from the vasculature with in vivo photoacoustic imaging. The imaging results suggest that tyrosinase is a useful reporter gene for both magnetic resonance and photoacoustic imaging.