含抗凋亡基因bcl-2重组逆转录病毒重组体的构建及鉴定

目的:构建并鉴定含鼠bcl-2基因重组逆转录病毒。方法:将抗凋亡基因bcl-2片段从载体pcDNA3.1中切下,定向插入逆转录病毒载体PLNCX2中,酶切鉴定;脂质体法将重组逆转录病毒转入包装细胞系PT67,G418筛选建立稳定表达bcl-2的细胞株。结果:双酶切鉴定,成功构建重组逆转录病毒载体;重组逆转录病毒转入包装细胞PT67,经G418筛选,形成了抗性克隆,并测得病毒滴度为3×1011cfu/L,提示构建的含鼠bcl-2基因重组逆转录病毒载体及稳定的包装细胞系成功。结论:含抗凋亡基因bcl-2重组逆转录病毒载体构建成功。     

复制缺陷型逆转录病毒载体介导基因工程化视网膜色素上皮细胞的制备

目的制备稳定高表达生物活性神经生长因子（nerve growth factor,NGF）工程视网膜色素上皮(retinal pigment epithelium,RPE)细胞,拟将转基因基因治疗与视网膜移植技术结合,用于视网膜变性疾病的治疗。方法首先经亚克隆和克隆构建复制缺陷性逆转录病毒质粒重组体pLXSN-NGF,然后经包装细胞pT67包装,获得含有NGF基因的复制缺陷性逆转录病毒重组体;NIH3T3细胞测定其滴度,然后转导培养RPE细胞;反转录聚合酶链反应和Western blot分析NGF基因在RPE细胞中的表达;PC12细胞测定表达产物的生物活性。结果复制缺陷性逆转录病毒滴度为1.2×10     

重组人神经生长因子腺相关病毒载体的构建及病毒滴度的测定

目的克隆人神经生长因子-β(nerve growth factor-β,NGF-β)基因,重组于腺相关病毒(adeno-associated virus,AAV)载体,构建表达人NGF-β的重组腺相关病毒,检测滴度,为NGF-β的真核表达及临床应用提供实验基础。方法从正常人基因组中扩增出人NGF-β全长开放读框,将其克隆于pAAV-MCS载体,双酶切和测序鉴定重组病毒载体。将该质粒与腺相关病毒包装质粒(pAAV-RC)、腺相关病毒辅助质粒(pAAV-Helper)共转染AAV-293细胞,通过包装得到重组腺相关病毒并回收病毒原液。以pAAV-LacZ和pAAV-RC、pHelper共转染AAV-293细胞,重组病毒AAV-LacZ感染AAV-HT1080细胞,经对报告基因LacZ的X-Gal染色,通过蓝染的细胞计算病毒滴度。结果PCR产物经电泳证明大小正确,重组质粒pAAV-NGF-β经双酶切和测序证实NGF-β基因正确插入载体且序列正确。通过AAV-LacZ感染AAV-HT1080细胞后X-gal染色计数,测定重组腺相关病毒的滴度为10×109pfu.L-1。结论成功构建了表达人NGF-β基因的重组腺相关病毒载体,为进一步研究NGF-β基因治疗角膜病打下基础。     

携带sFlt-1基因片段的慢病毒载体的构建及表达

目的分别建立携带可溶性血管内皮生长因子受体-1（sFlt-1）不同基因片段的慢病毒表达质粒，为进一步研究sFlt-1不同基因片段表达的蛋白生物学功能奠定基础。方法以人新鲜胎盘组织提取的cDNA为模板，进行RT—PCR扩增，制备sFlt-1基因片段sFlt-1（2-3）和sFlt-1（2-4）。扩增片断经限制性内切酶BamH1和Sall双酶切后，插入慢病毒载体pRRL-GFP中，构建慢病毒表达质粒pRRL／sFlt．1（2-3）及pRRL／sFlt-1（2—4），与慢病毒包装载体pMDLg／pRRE、包被载体PRSV／REV、pMD2．G共转染人胚肾细胞（HEK293T），获得重组慢病毒Lenti．sFlt-1（2—3）及Lenti-sFlt-1（2-4），并感染人视网膜色素上皮（RPE）细胞，通过RT-PCR和Westernblotting验证Lenti-sFlt-1（2—3）及LentisFlt-1（2-4）在人RPE细胞的表达。结果经酶切鉴定及基因测序证实pRRL／sFlt-1（2-3）和pRRL／sFlt-1（2-4）的序列与GenBank中的序列完全一致，并且包装成病毒后能有效感染人RPE细胞。结论本实验成功构建了分别携带sFlt-1基因第2-3和第2-4免疫球蛋白样区域编码序列的慢病毒载体，包装成病毒后能有效感染人RPE细胞。     

αA晶状体蛋白重组腺病毒载体的构建和鉴定

目的构建携带鼠αA晶状体蛋白的腺病毒表达载体,为进一步研究αA晶状体蛋白高表达对损伤条件下神经细胞的保护作用提供实验基础。方法采用RT-PCR扩增的方法获取鼠源性的αA晶状体蛋白基因,并克隆入穿梭质粒pAdTrack-CMV,构建携带外源性αA晶状体蛋白编码基因的重组腺病毒载体pAd-CRYAA。脂质体法转染人胚肾293细胞,包装产生重组腺病毒Ad-CRYAA。应用PCR鉴定重组腺病毒,空斑传代纯化病毒并反复冻融扩增病毒。以腺病毒感染滴度快速测定试剂盒测定病毒滴度。结果 PCR、序列测定以及限制性酶切证实αA晶状体蛋白基因正确插入腺病毒表达载体,并在人胚肾293细胞中包装出重组腺病毒。收获病毒的滴度为1.143×1012ifu·L-1。结论成功构建Ad-CRYAA重组腺病毒表达载体,可作为后续研究中可靠的基因转染工具。     

应用转基因技术体外培养表达人碱性成纤维细胞生长因子的视网膜色素上皮细胞

目的 探讨应用转基因技术体外培养稳定表达人碱性成纤维细胞生长因子(basic fibroblast growth factor,bFGF)的视网膜色素上皮(retinal pigment epithelium,RPE)细胞的可行性。方法 构建以绿色荧光蛋白(green fluorescent protein,GFP)作为标记基因的人bFGF真核表达载体pcFG。应用脂质体介导法转染人RPE细胞,以G418筛选出表达bFGF的RPE细胞,并进行细胞克隆,传代培养4周。于荧光显微镜下观察GFP的表达情况,用原位杂交和免疫组织化学染色法检测bFGF在RPE细胞的表达情况。结果 酶切结果证实含有GFP的真核表达载体pcFG构建正确。在荧光显微镜下可见RPE细胞表达绿色荧光蛋白。经原位杂交和免疫组织化学染色证实,转染pcFG后的RPE细胞内有大量bFGF—mRNA的转录蛋白表达。结论 应用转基因技术可体外培养稳定表达bFGF的RPE细胞。     

逆转录病毒载体携带报告基因在色素上皮细胞的转染与表达

目的 观察逆转录病毒载体携带报告基因在视网膜色素上皮(retinal pigment epithelium,RPE)细胞的表达情况,以探讨基因疗法用于抑制增生性玻璃体视网膜病变的可行性.方法 选第3～4代培养的已长满融合的RPE细胞,以1:3的分种率分种传代.随机分为对照组与实验组,每组12孔.实验组用绿色荧光蛋白基因作为报告基因,以逆转录病毒载体携带来转染RPE细胞,重组合绿色荧光蛋白基因的逆转录病毒上清液浓度为1.2×109 cfu·L-1.对照组RPE细胞以同样方法直接转染绿色荧光蛋白基因质粒DNA.观察绿色荧光蛋白基因在2组RPE细胞的表达情况.结果 基因转染后可见实验组RPE细胞的绿色荧光蛋白基因表达率可达56%～72%,对照组未见有任何表达.结论 逆转录病毒载体可以携带目的基因转染于RPE细胞,并达到较高的转染率.     

疱疹性口腔炎病毒G蛋白/胸苷激酶逆转录病毒载体转移人视网膜色素上皮细胞及其表达

目的　应用新型逆转录病毒载体介导单纯疱疹病毒胸苷激酶 (herpessimplexvirus thymidinekinase ,HSV TK)基因转移 ,探讨丙氧鸟苷 (ganciclovir,GCV)对HSV TK基因修饰人视网膜色素上皮细胞 (retinalpigmentepithelialcells,RPE)的杀伤抑制作用。方法　生产制备高滴度疱疹性口腔炎病毒G蛋白 (vesicularstomatitisvirus Gprotein ,VSV G) /胸苷激酶 (thymidinekinase ,TK)和VSV G/LacZ病毒 ,对NIH3T3细胞进行VSV G/TK滴度测定 ;VSV G/LacZ感染CRL2 30 2细胞后 ,以X gal染色估计感染率 ;对CRL2 30 2细胞、分离培养的RPE细胞及NIH3T3细胞感染不同病毒滴度的VSV G/TK ,结合GCV作用 ,观察 3种细胞生长抑制率。结果　VSV G/TK滴度为 1 2× 10 8克隆形成单位 /ml;VSV G/LacZ感染CRL2 30 2细胞的X gal染色蓝染细胞率为 5 8% ;当感染复数为 2 0 0时可最大的抑制细胞生长 ,抑制率为 4 5 %。结论　VSV G逆转录病毒可以介导LacZ、HSV TK基因在离体NIH3T3细胞和人RPE细胞中高效转移和表达。被HSV TK基因修饰的细胞对GCV作用敏感 ,其生长受到抑制。     

携带Tum5基因的慢病毒表达载体的构建及其在人脐静脉血管内皮细胞中的表达

目的:构建及鉴定Tum5基因慢病毒表达载体,并观察在体外人血管内皮细胞中的表达。方法:采用PCR技术从含有tumstatin基因的质粒克隆模板pSPORT1-Sfi钓取Tum5基因,并将基因克隆到慢病毒载体表达质粒pGC-FU(含EGFP基因)中,构建慢病毒载体表达质粒pGC-FU-Tum5,通过酶切、测序验证Tum5基因后,将pGC-FU-Tum5质粒和包装质粒pHelper1.0,pHelp-er2.0共同转染人胚胎肾上皮细胞系293T细胞,获得携带Tum5基因和EGFP基因的重组慢病毒GC-FU-Tum5,并转染靶细胞人脐静脉血管内皮细胞。通过检测标志蛋白-增强型绿色荧光蛋白(EGFP)和目的蛋白Tum5进一步验证pGC-FU-Tum5在靶细胞中Tum5的表达。结果:(1)pGC-FU-Tum5中携有正确的Tum5基因,并能在人类细胞中表达;pGC-FU-Tum5共转染包装细胞293T能产生重组病毒GC-FU-Tum5;(2)目的基因Tum5能被重组慢病毒高效地转导入靶细胞人脐静脉血管内皮细胞,并达到稳定的表达,荧光显微镜下能直接观察到GFP,Western blotting能检测到Tum5蛋白在靶细胞中的表达。结论:成功构建了携带Tum5基因的重组慢病毒载体,转染人脐静脉血管内皮细胞后能够稳定表达Tum5基因,为进一步研究Tum5的功能和眼部新生血管疾病的治疗奠定基础。     

HTLV-1 Tax 基因真核表达载体的构建及对 RPE 细胞的影响

目的构建人T淋巴细胞白血病病毒-1(human T-cell leukemia virus type1,HTLV-1)Tax基因真核表达载体,在人视网膜色素上皮(retinal pigment epithelium,RPE)细胞表达Tax蛋白,观察其对细胞的影响。方法应用基因重组技术构建重组载体PIRES2-EGFP-Tax(PT),鉴定后将重组载体转染入RPE细胞,应用RT-PCR、Western-blot和免疫荧光法检测细胞内Tax基因和蛋白的表达;通过MTT和流式细胞仪检测质粒(空白载体、PT和Green-Tax)转染RPE细胞24h和48h后对RPE细胞生长活力及细胞周期的影响。结果经双酶切鉴定和测序分析证实成功地构建了HTLV-1Tax真核表达载体;通过RT-PCR、Western-blot和免疫荧光法检测到转染Tax重组载体的RPE细胞中有Tax基因和蛋白的表达。质粒转染RPE细胞后,细胞生长活力受到抑制,G1期减少而S期增加。但转染Tax质粒和空白载体,对RPE细胞的生长活力和细胞周期产生相似影响。结论成功构建了HTLV-1Tax基因真核表达载体,并在RPE细胞中表达Tax蛋白;Tax质粒对RPE细胞的影响不排除是转染试剂所致。     

视网膜下间隙移植经基因修饰的视网膜色素上皮细胞对大鼠光感受器变性及视功能的保护作用

目的 以RCS大鼠作模型,研究经基因修饰的永生化视网膜色素上皮细胞(RPE)视网膜下移植对光感受器变性的保护作用。方法 在绿色荧光蛋白基因逆转录病毒感染的基础上,利用脂质体介导节状神经生长因子(CNTF)表达质粒转移,修饰成人RPE细胞系CRL-2302。将1×105个表达绿色荧光蛋白(GFP)或GFP及CNTF的RPE细胞移植到4～5周龄RCS大鼠右眼视网膜下间隙,左眼不移植或注射。PBS作为对照。术后2、4、6、8、10和12周作荧光显微镜、光镜、电镜及电生理检查。结果 荧光显微镜观察,术后1周移植的人RPE细胞在RCS大鼠视网膜下间隙已扩散到几乎整个眼底,但随时间延长移植的细胞逐渐减少,术后6周仅残留少量移植细胞。光镜及电镜观察显示移植眼保留的光感受器数量明显较对照眼多,凋亡细胞则较对照跟少。此外,移植眼宿主RPE细胞形态较正常,并可见吞噬小体。视网膜电图(ERG)检查结果表明部分移植眼视网膜功能明显较对照眼好。结论 经过基因修饰的RPE细胞移植可延缓RCS大鼠视网膜光感受器变性,为治疗视网膜变性提供了新的途径。(中华眼科杂志,2004,40:552-556)     

逆转录病毒及睫状神经营养因子联合诱导成人视网膜色素上皮细胞转分化成为神经细胞

目的 观察逆转录病毒及睫状神经营养因子(CNTF)诱导的成人视网膜色素上皮（RPE）细胞转分化过程中的形态特征与标志物变化。 方法 成人RPE细胞经携带绿色荧光蛋白(GFP)基因的逆转录病毒感染后，进一步用脂质体介导CNTF 表达质粒进行转染.倒置荧光显微镜观察细胞形态变化,酶联免疫吸附实验、免疫组织化学染色及Western blotting方法检测细胞产生和分泌CNTF的能力、CNTF受体(CNTFR)、多种神经细胞标志物及信号传导分子一类蛋白酪氨酸激酶（JAK）的表达水平。 结果 体外培养的成人RPE细胞经GFP逆转录病毒感染后，RPE细胞的形态无明显变化，但开始表达神经元和胶质细胞的某些标志物，如神经丝蛋白(NF)和胶质纤维酸性蛋白(GFAP)；经CNTF表达质粒转染后，持续高水平表达CNTF的RPE细胞出现明显的神经细胞分化,CNTFR的表达量虽无明显变化，但其分布发生改变，主要分布在细胞膜上; 此外，信号传导分子JAK表达明显上调。 结论 体外培养的成人RPE细胞在逆转录病毒及CNTF的影响下可出现明显的神经细胞形态分化，转分化与CNTF-CNTFR-JAK信号传导通路有关。 (中华眼底病杂志，2006，22：400-403）     

Bax过表达诱导培养的人视网膜色素 上皮细胞凋亡观察

目的研究外源性促凋亡基因bax过表达对培养人的视网膜色素上皮（retinal pigment epithelium ,RPE）细胞凋亡的影响。 方法通过脂质体Lipofectin介导法用含人全长bax 基因片段的真核表达载体P^MDNA3-hbax质粒转化体外培养的RPE细胞,用流式细胞仪检测细胞周期的变化,提取实验组细胞DNA进行DNA梯度电泳。结果Zn^2+可诱导P^MDNA3-hbax转化组RPE细胞出现明显的凋亡峰 ,凋亡峰位于单倍体和二倍体之间,凋亡率为36%,DNA电泳可见DNA梯状条带,P^MDNA3空载体组及未转染组RPE细胞未见亚二倍峰和DNA梯状条带出现。结论外源性促凋亡基因bax过表达可诱导RPE细胞凋亡。(中华眼底病杂志,2001,17:132-134)     

Development and evaluation of the specificity of a cathepsin D proximal promoter in the eye

PURPOSE: Recombinant adeno-associated virus (rAAV)-mediated gene delivery has emerged as a valuable tool for alternative treatment of ocular diseases. Cellular specificity of transgene expression could be influenced by either the viral capsid or the choice of promoter. The use of cellular promoter, cathepsin D (CatD) proximal promoter, and its potential for application in rAAV-based gene therapy are evaluated in this study. MATERIALS AND METHODS: Different sizes of CatD proximal promoter fragments -769 to -1 (CD768), -366 to -1 (CD365), -253 to -1 (CD252), and -124 to -1 (CD123) were subcloned upstream of the green fluorescent protein (GFP) gene. The specificity and activity of the promoter were tested in vitro using human retinal pigment epithelium (RPE) cell lines (RPE51, D407), with the human fibroblast cell line (F2000) used as control. The promoter fragment that showed higher activity in RPE cells was chosen to generate rAAV vector based on AAV serotype 2. The ability of CatD promoter to target transgene expression to RPE in vivo was determined following subretinal delivery of rAAV particles into nonpigmented RCS/rdy+ rats. RESULTS: In vitro studies showed that the proximal promoter fragment CD365 targeted high GFP expression in RPE cells. This fragment was then used to generate the AAV.CD365.gfp construct. It was shown in vivo that following subretinal injection, the CD365 fragment in AAV.CD365.gfp directed GFP expression preferentially into RPE cells. Relatively lower level of GFP expression was detected in the neuroretina. In contrast, injection of control virus (AAV.CMV.gfp) resulted in equal levels of transduction and fluorescence signal intensity in both the RPE and photoreceptor cells. CONCLUSIONS: The results of our study demonstrate that the promoter fragment CD365 has the potential to target preferential gene expression in the RPE following subretinal injection in rAAV-mediated gene therapy.     

Tracking RPE transplants labeled by retroviral gene transfer with green fluorescent protein.

PURPOSE: To determine whether human retinal pigment epithelium (RPE) can be modified by retroviral-mediated gene transfer and to monitor the human RPE cells in the subretinal space of living rabbits with scanning laser ophthalmoscopy (SLO). METHODS: Cultured human fetal retinal pigment epithelium (HFRPE) was exposed to green fluorescent protein (GFP)-transducing retroviral vectors, Moloney murine leukemia virus, and lentivirus. The cultured cells were followed by fluorescence microscopy. Suspensions of GFP-expressing HFRPE were transplanted into the subretinal space of pigmented rabbits, and the transplant sites were examined by SLO for fluorescence, including fluorescein and indocyanine green angiography. The rabbits were euthanatized at different times after transplantation, and the retinas were studied histologically. RESULTS: Retroviral gene transfer can introduce a foreign gene such as GFP into cultured HFRPE. Gene expression is maintained in cultured RPE for at least 3 months. The lentiviral vector transduced both nondividing and dividing cells; the Moloney vector only transduced the latter. GFP-expressing cells can be followed in the living retina. Their changes reflect the rejection response followed histologically. CONCLUSIONS: Cultured HFRPE could be transduced to express GFP for long periods of time by retroviral gene transfer. GFP allowed retinal transplants and gene expression to be monitored in vivo. These results provide a model for potential ex vivo gene therapy in the subretinal space.     

Characterisation of the advanced glycation endproduct receptor complex in the retinal pigment epithelium

AIMS: Advanced glycation endproducts (AGEs) accumulate with ageing and may have a significant impact on age related dysfunction of the retinal pigment epithelium (RPE). Many of the cellular effects of AGEs in other cell types are mediated through AGE binding proteins. The aim of this study was to characterise the AGE receptor complex in RPE cells in vitro and to focus on the role of the R3 component (galectin-3) as the primary effector of the complex. METHODS: Primary cultures of bovine RPE cells and the human D407 RPE cell line were exposed to AGE modified albumin. Receptor expression was determined using mRNA analysis by quantitative real time RT-PCR and protein characterisation by western blotting. Immunocytochemical analysis examined the cellular localisation of the various components of the AGE receptor complex. The role of the galectin-3 receptor component was examined by transfection and overexpression using the D407 cell line and analysis of soluble AGE-R3 by ELISA. RESULTS: All three components of the AGE receptor complex were expressed by bovine and human RPE cells. AGE exposure upregulated two components of the receptor complex and also induced significant RPE expression of VEGF mRNA (p<0.05). RPE D407 cells stably transfected to overexpress galectin-3 showed less VEGF induction. In non-transfected RPE which were exposed to AGEs, there was less soluble galectin-3 protein released into the medium (p<0.05), a response that was not evident in transfected cells. CONCLUSION: A conserved AGE receptor complex is evident in primary cultures of bovine RPE cells and also in a human cell line. These cells show a pathological response to AGE exposure, an effect which appears to be modulated by the galectin-3 component of the receptor complex.     

逆转录病毒感染法对RP患者人体细胞向iPS细胞和iPS-RPE细胞诱导分化的研究

背景 视网膜色素上皮(RPE)细胞移植是目前人类尝试治疗视网膜色素变性(RP)的主要手段.诱导多能干细胞(iPSCs)将成为移植细胞的一个重要来源,人胚胎干细胞(hESCs)可以无限期地自我更新和分化,是RPE细胞移植的重要供体来源.此外,iPS-RPE细胞为患者自身细胞成为治疗源组织提供了个性化治疗平台. 目的 评估利用逆转录病毒将Oct4、Sox2、c-Myc和Klf44种基因导入RP患者皮肤成纤维细胞后诱导人iPSCs的可行性,并建立将正常人iPSCs诱导分化为RPE细胞的方法. 方法 分别采集基因突变点为SNRNP200 p.S1087L的RP患者及无SNRNP200 p.S1087L突变受试者的大腿皮肤组织块约2 cm×3 cm.采用胰蛋白酶消化和组织块培养法分离纯化得到成纤维细胞并进行传代,取第3代细胞用于研究,分别采用形态学和免疫荧光技术对培养细胞进行鉴定.采用含OCT4、SOX2、C-MYC和KLF4 4种cDNA的质粒病毒转染的成纤维细胞,并用hESCs条件培养液诱导iPSCs,采用细胞形态学、碱性磷酸酶(AP)染色、干细胞表面多潜能标志物表达对所得到的iPSCs和iPS-RPE细胞进行鉴定,并通过细胞在SCID小鼠的体内种植考察iPSCs的成瘤性.采用诱导分化拟胚体(EB)法将不含SNRNP200 p.S1087L突变的iPSCs诱导分化为RPE细胞,分别采用免疫荧光技术和实时荧光定量PCR法检测细胞中特异性蛋白的表达.结果 用皮肤组织块培养的成纤维细胞呈梭形和多角形,光学显微镜下可见细胞间排列紧密,细胞形态和大小趋于一致,细胞中特异性蛋白Vimentin表达阳性.经逆病毒转染的成纤维细胞培养后第5～7天可见小的细胞聚集,细胞形态由梭形变为圆形;培养后20d出现类似hESCs集落形态的iPSCs克隆;培养后25 ～ 30 d细胞中的标志物SSEA3、SSEA4、TRA-1-60、TRA-1-81及Nanog均呈阳性表达,培养后12周形成的iPSCs克隆中AP染色阳性,接种到SCID小鼠体内后形成畸胎瘤.免疫荧光染色显示,诱导分化后30 d时iPS-RPE细胞中RPE细胞特异性标志物RPE65、闭锁小带蛋白1(ZO-1)和卵磷脂视黄醇酰基转移酶(LRAT)蛋白均呈阳性表达.结论 利用逆转录病毒将Oct4、Sox2、c-Myc和Klf44种基因转入SNRNP200 p.S1087L突变的RP患者皮肤成纤维细胞后可以获得iPSCs,建立的iPSCs有ESCs的形态和多能分化的特征.采用同样的转染技术可在无SNRNP200 p.S1087L突变的人皮肤成纤维细胞中建立体外高分化、高效能的iPS-RPE细胞.     

Optimization of non-viral gene transfer to human primary retinal pigment epithelial cells

PURPOSE: To optimise the high efficiency, non-viral transfer of DNA to retinal pigment epithelial (RPE) cells in vitro. METHODS: A mammalian expression vector (pcDNA3.1) containing a firefly luciferase (luc) cDNA was used to transfect RPE cells using different chemical methods; calcium phosphate, DEAE-dextran and, liposomes-based transfection techniques. Transfection was optimised for both dose and time of exposure. The efficiency of gene transfer and cytotoxicity was measured 48 hours post-transfection using luciferase and MTT assays, respectively. The percentage of transfected cells (using optimal conditions) was determined with a construct expressing a jellyfish green fluorescent protein (GFP) using flow cytometery. RESULTS: Calcium phosphate and DEAE-dextran techniques failed to transfect the vector and led to high cytotoxicity. Liposomes-based methods successfully transferred the vector to RPE cells, but the efficiency varied for different liposomes; Tfx-50 > Lipofectin > Lipofectamine > Cellfectin > DMRIE-C. No significant cytotoxicity was observed with any of the liposome treatments. Optimal transfection was achieved with Tfx-50 at a 3:1 ratio of DNA:liposome; between 12-15% of cells being transfected. CONCLUSIONS: Efficient and non-toxic transfer of functional genes into primary RPE cells in vitro can be successfuly achieved by liposomes-based techniques. Tfx-50 appears to be a promising non-viral vector for RPE gene transfer.