Recombinant carcinoembryonic antigen as a reporter gene for molecular imaging.
Recombinant carcinoembryonic antigen as a reporter gene for molecular imaging.
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DOI：
10.1007/s00259-008-0921-z
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发表时间：
2009-01
影响因子：
9.1
通讯作者：
Wu, Anna M.
中科院分区：
文献类型：
--
作者：
Kenanova, Vania;Barat, Bhaswati;Olafsen, Tove;Chatziioannou, Arion;Herschman, Harvey R.;Braun, Jonathan;Wu, Anna M.
关键词：
报告基因可以提供一种非侵入性地评估体内基因活性的方法。然而，目前的报告基因策略可能受到外源报告蛋白的免疫原性、内源性表达或不需要的生物活性的限制。我们已经开发了一个报告基因的基础上癌胚抗原（CEA），人类蛋白质与有限的正常组织表达。为了构建用于PET的CEA报告基因，将CEA小基因（N-A3）融合到人FcγRIIb受体的胞外和跨膜结构域。将CMV启动子驱动的NA 3-FcγRIIb重组基因转染Jurkat（人T细胞白血病）细胞。通过流式细胞术、免疫组织化学（IHC）和microPET成像分析表达。流式细胞术鉴定了以低、中和高水平稳定表达NA 3-FcγRIIb的Jurkat克隆。Western blot检测到NA 3-FcγRIIb的高表达和中表达。报告基因阳性和阴性Jurkat细胞用于在无胸腺小鼠中建立异种移植物。IHC显示肿瘤染色，报告基因高表达;未检测到中等和低N-A3表达。使用抗CEA 124 I标记的scFv-Fc抗体片段的MicroPET成像证明仅可检测到高N-A3表达。早在4小时就在N-A3阳性肿瘤处观察到活性的特异性积累。MicroPET图像定量显示在4 h、20 h和48 h时肿瘤活性分别为1.8（±0.2）、15.2（±1.3）和4.6（±1.2）%ID/g。48小时的生物分布显示肿瘤摄取为4.8（±0.8）%ID/g。CEA N-A3小基因具有用作体内细胞成像的报告基因的潜力。
Reporter genes can provide a way of non-invasively assessing gene activity in vivo. However, current reporter gene strategies may be limited by the immunogenicity of foreign reporter proteins, endogenous expression or unwanted biological activity. We have developed a reporter gene based on carcinoembryonic antigen (CEA), a human protein with limited normal tissue expression. To construct a CEA reporter gene for PET, a CEA minigene (N-A3) was fused to the extracellular and transmembrane domains of the human FcγRIIb receptor. The NA3-FcγRIIb recombinant gene, driven by a CMV promoter, was transfected in Jurkat (human T cell leukemia) cells. Expression was analyzed by flow cytometry, immunohistochemistry (IHC), and microPET imaging. Flow cytometry identified Jurkat clones stably expressing NA3-FcγRIIb at low, medium, and high levels. High and medium NA3-FcγRIIb expression could also be detected by Western blot. Reporter gene positive and negative Jurkat cells were used to establish xenografts in athymic mice. IHC showed staining of the tumor with high reporter gene expression; medium and low N-A3 expression was not detected. MicroPET imaging, using an anti-CEA 124I-labeled scFv-Fc antibody fragment, demonstrated that only high N-A3 expression could be detected. Specific accumulation of activity was visualized at the N-A3 positive tumor as early as 4h. MicroPET image quantitation showed tumor activity of 1.8(±0.2), 15.2(±1.3) and 4.6(±1.2) %ID/g at 4h, 20h and 48h, respectively. Biodistribution at 48h, demonstrated tumor uptake of 4.8(±0.8) %ID/g. The CEA N-A3 minigene has the potential to be used as a reporter gene for imaging cells in vivo.
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影响因子：
3.2
作者：
Ajjan, RA;Kamaruddin, NA;Weetman, AP
通讯作者：
Weetman, AP
影响因子：
4.8
作者：
Smanik, PA;Ryu, KY;Jhiang, SM
通讯作者：
Jhiang, SM
影响因子：
3.9
作者：
Gangopadhyay, A;Thomas, P
通讯作者：
Thomas, P
影响因子：
5.1
作者：
MacLaren, DC;Gambhir, SS;Herschman, HR
通讯作者：
Herschman, HR
DOI：
10.1084/jem.170.4.1369
发表时间：
1989-10-01
期刊：
The Journal of experimental medicine
影响因子：
--
作者：
Brooks DG;Qiu WQ;Luster AD;Ravetch JV
通讯作者：
Ravetch JV