【摘要】 目的：探讨重组腺相关病毒基因（recombinant adenoassociated virus gene, rAAV）载体转导绿荧光蛋白基因（green fluorescent protein, gfp）基因至视网膜的可行性。方法：18只家兔随机选取一眼玻璃体内注射rAAVgfp，对侧眼作为对照。分别于注射后3,7,14d摘除眼球进行视网膜铺片观察视网膜的荧光。结果：家兔玻璃体注射rAAVgfp后视网膜细胞浆内可见荧光点，提示gfp基因被有效转导至视网膜并进行荧光表达。结论：rAAV是一个可靠、简便易行的目标基因转移视网膜的载体。
【关键词】 重组腺相关病毒；绿荧光蛋白基因；基因转染；视网膜
A feasibility study of recombinant adenoassociated virus (rAAV) as a vector for transferring a target gene to retina
JianMing Wang, YaZhi Fan, Na Hui, Lei Xiong, HaiXiao Feng, NaiXue Sun
Foundation item: Natural Science Foundation of Shaanxi Province, China (No. 2001SM66)
Department of Ophthalmology, the Second Affiliated Hospital of Medical College of Xian Jiaotong University, Xian 710004, Shaanxi Province, China
Correspondence to: JianMing Wang. the Second Affiliated Hospital of Medical College of Xian Jiaotong University, Xian 710004, Shaanxi Province, China. [email protected]
AbstractAIM: To study the feasibility of recombinant adenoassociated virus (rAAV) as a vector to transfer the green fluorescent protein (GFP) gene as a target gene into rabbit retina. METHODS: Intravitreal injection of rAAVgfp was performed in either eye for each rabbit with the other eye taken as control. At the 3rd, 7th, and 14th day after injection, the eyeballs were removed, and the retinas were flatmounted on glass slides to inspect the retinal fluorescence, respectively. RESULTS: After intravitreal injection of rAAVgfp, the presence of fluorescent spots in the cytoplasm of retinal cells indicated that GFP gene was efficiently transferred and expressed in the rabbit retina.
CONCLUSION: Recombinant adenoassociated virus is a reliable and simple vector for transferring target gene, e.g., GFP gene, to the retina.
KEYWORDS: recombinant adenoassociated virus; green fluorescent protein gene; gene transfer; retina
Wang JM, Fan YZ, Hui N, Xiong L, Feng HX, Sun NX.
A feasibility study of recombinant adenoassociated virus (rAAV) as a vector for transferring a target gene to retina. Int J Ophthalmol(Guoji Yanke Zazhi)2008;8(9):17401742
INTRODUCTION
Optic nerve lesion is an important factor which influences the therapeutic efficacy of glaucoma. As an alternative to drugs, gene therapy has been used in treating some kinds of the human diseases and may be equally effective in the treatment of glaucoma. Gene therapy fulfills two distinct purposes. First, it serves to replace the abnormal (diseased) gene in vivo with the normal gene constructed in vitro. Second, it increases the expression of a special gene that can not otherwise express enough corresponding protein such that therapeutic effect is achieved. Moreover, it has been demonstrated that genes extrinsic to the human body can be introduced by special vectors. Recombinant adenoassociated virus (rAAV) is such a gene vector[1]. Its molecular weight is small, and it has no pathogenicity. It can transfect cells that are in both the mitotic and quiescent stages. The gene carried by the adenoassociated virus（AAV） vector can be transferred into the chromosome of the target cell. In this way, the target gene can be expressed for a long time, particularly since rAAV has been proved to be a safe and effective vector[13]. To determine whether rAAV can carry and transfer a target gene, e.g., green fluorescent protein (GFP) gene, we injected rAAVgfp into the vitreous body of rabbits and observed the expression of the GFP gene in the retina.
MATERIALS AND METHODS
Titer of rAAVgfp rAAVgfp was supplied by Xian Huaguang Bioengineering Company Limited (Xian, China). The titer of the rAAVgfp was 2.25×1010cfu/mL. Experimental Animals and the Procedure: Eighteen New Zealand white rabbits, either male or female and each weighing 1.82。5kg, were supplied by the Experimental Animal Center of Xian Jiaotong University. One of the bilateral eyes of each rabbit was used as the test eye, and the other eye as control. After shearing the uppertemporal bulbar conjunctiva from 2mm behind the corneal limbus, a 6G needle was injected into the vitreous body at an angle of 4060 degrees. 10μL rAAVgfp was injected into the vitreous body in front of the posterior pole of the central retina by microsyringe, while 10μL physiological saline was injected into the vitreous body of control eyes. Antibiotic eye drops were used after the procedures for 3 days. With each time course consisting of a group of 6 rabbits, bilateral eyes were excised and flatmounted on glass slides to detect the retinal fluorescence intensity at the 3rd, 7th, and 14th day postinjection, respectively.
Retina Whole Flatmount Preparations and Fluorescence Observation Intramuscular anaesthesia was performed by using 50mg/kg ketamine hydrochloride. Saline was perfused via ascending aorta into left ventricle after thoracotomy. 40g/L paraform perfusion was done replacing saline perfusion after clear liquid flowed out from the heart. Bilateral eyes were enucleated after the rabbit expired. Cornea was cut at 2 mm posterior to the corneal limbus. The lens and vitreous body were removed. The residual eye cups were fixed in 40g/L paraform for 24 hours. After separation from the eye wall, the retina was partly cut radially from the edges at four positions: 1∶30, 4∶30, 7∶30, and 10∶30. Then the whole retina flatmount preparations were made on the slide and sealed with glycerine. The retina flatmount slides were observed and imaged with both fluorescence microscope (Leica Q550cw) and laser confocal scanning microscope (Leica Tcs SP2). The mean grey of the whole retina fluorescence was detected by the Leica Q550cw image analysis system.
RESULTS
Fluorescence Observation of GFP Expression on the Whole Retina Flatmount Preparations with the Fluorescence Microscope No fluorescence on the normal retina whole flatmount preparations was observed by fluorescence microscopy. Scattered hypofluorescence was found near the injection site on the retina at the 3rd day after intravitreal injection of rAAVgfp. GFP protein expression increased gradually 7 days later. Fluorescence spots were also more abundant during this period, and fluorescence intensity was stronger. At the 14th day after injection, fluorescence intensity of the GFP protein expression was strongest. The value of mean grey of GFP protein fluorescence on the whole retina is shown in Table 1.Table 1The mean grey value of the whole retina fluorescence (略)
Fluorescence Observation of GFP Expression on the Whole Retina Flatmount Preparations with the Laser Confocal Microscopy 14 days after intravitreal injection of rAAVgfp, the laser confocal scanning microscope identified obvious evidence of fluorescence spots in the cytoplasm of the retinal cells (the arrow in Figure 1).
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