Retrovector encoding a green fluorescent protein-herpes simplex virus thymidine kinase fusion protein serves as a versatile suicide/reporter for cell and gene therapy applications
Retrovector encoding a green fluorescent protein-herpes simplex virus thymidine kinase fusion protein serves as a versatile suicide/reporter for cell and gene therapy applications
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DOI：
10.1089/104303401450924
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发表时间：
2001-01-01
影响因子：
4.2
通讯作者：
Galipeau, J
中科院分区：
文献类型：
--
作者：
Paquin, A;Jaalouk, DE;Galipeau, J
编码单纯疱疹病毒胸苷激酶（HSVTK）的表达载体已广泛用于细胞和基因治疗应用中，作为抗癌“自杀”或作为过继免疫治疗应用中的“自毁”转基因。在这两种基因治疗应用中，需要在基因工程细胞中可靠地检测HSVTK转基因表达，HSVTK蛋白的直接荧光标记可能是补救措施。我们设计了一个逆转录载体编码嵌合GFP-HSVTK融合蛋白，可以作为一个双功能的自杀和报告基因转基因。将融合基因整合到VSV G-假型逆转录病毒载体（vGFPTKfus）中，产生高滴度稳定的逆转录病毒生产者（类似于3 × 10（6）逆转录病毒颗粒/ml）。以MOI 8转导3天的肿瘤细胞系导致>90%的基因转移效率。Southern印迹分析证实，未重排的前病毒基因组整合在染色体DNA中。用HSVTK抗血清进行的蛋白质提取物免疫印迹揭示了与HSVTK-GFP融合蛋白的预测大小一致的70-kDa蛋白的存在。荧光显微镜和流式细胞仪分析表明，GFPTKfus介导的荧光是核定位的，是30倍以上的双顺反子HSVTK-GFP载体中观察到的。表达vGFPTKfus的细胞系的生长在更昔洛韦的存在下被显著抑制。用vGFPTKfus转导DA 3小鼠乳腺癌细胞系并植入同基因BALB/c小鼠中。用vGFPTKfus转导正常人外周血T淋巴细胞，观察到核限制性绿色荧光。分选绿色荧光淋巴细胞允许选择工程化细胞。总之，我们证明了vGFPTKfus在体外和体内肿瘤细胞中作为自杀/报告基因转基因的效用。此外，其作为靶向人T淋巴细胞的分析和治疗工具的潜在用途被示出。
Expression vectors encoding herpes simplex virus thymidine kinase (HSVTK) have been extensively used in cell and gene therapy applications either as anticancer "suicide" or as "self-destruct" transgenes in adoptive immunotherapy applications. In both gene therapy applications, reliable detection of HSVTK transgene expression is required in genetically engineered cells, Direct fluorescent labeling of the HSVTK protein may be the remedy. We designed a retrovector encoding a chimeric GFP-HSVTK fusion protein that can serve as a bifunctional suicide and reporter transgene. The fusion gene was incorporated in a VSV G-pseudotyped retrovector (vGFPTKfus) and high-titer stable retroviral producer was generated (similar to3 x 10(6) retroparticles/ml). Tumor cell lines transduced at an MOI of 8 for 3 days led to >90% gene transfer efficiency. Southern blot analysis confirmed that unrearranged proviral genomes integrated in chromosomal DNA. Protein extract immunoblot with HSVTK antisera revealed the presence of a 70-kDa protein consistent with the predicted size of an HSVTK-GFP fusion protein. Fluorescence microscopy and FACS analysis revealed that GFPTKfus-mediated fluorescence was nuclear localized and was 30-fold greater than that observed in a bicistronic HSVTK-GFP vector. Growth of cell lines expressing vGFPTKfus was significantly suppressed in the presence of ganciclovir. The DA3 mouse mammary carcinoma cell line was transduced with vGFPTKfus and implanted in syngeneic BALB/c mice. Preestablished tumors completely regressed in seven of nine mice treated with ganciclovir.Normal human peripheral blood T lymphocytes were transduced with vGFPTKfus and nucleus-restricted green fluorescence was observed. Sorting of green fluorescent lymphocytes allowed for selection of engineered cells. In conclusion, we demonstrate the utility of vGFPTKfus as a suicide/reporter transgene in tumor cells in vitro and in vivo. Furthermore, its potential use as an analytical and therapeutic tool targeting human T lymphocytes is shown.