Yang Ning, Huang Bin, Tsinkalovsky Oleg, Brekkå Narve, Zhu Huaiyang, Leiss Lina, Enger Per Øyvind, Li Xingang, Wang Jian
Department of Neurosurgery, Qilu Hospital of Shandong University, Jinan, China ; Department of Biomedicine, University of Bergen, N-5009 Bergen, Norway ; Brain Science Research Institute, Shandong University, Jinan, China.
Department of Neurosurgery, Qilu Hospital of Shandong University, Jinan, China ; Brain Science Research Institute, Shandong University, Jinan, China.
Cancer Cell Int. 2014 Dec 21;14(1):541. doi: 10.1186/s12935-014-0146-0. eCollection 2014.
A key strategy for the study of the tumor microenvironment is to implant human tumor cells in an immunodeficient rodent strain ubiquitously expressing a fluorescent marker. Here, a novel nude rat expressing a green fluorescent protein (GFP) transgene was established and engrafted with primary human tumor tissue in order to separate tumor from stromal cell populations for subsequent molecular analysis.
SD-TG (GFP) 2BalRrrc transgenic rats were crossed with HsdHan™: rnu/rnu Rowett nude rats to develop a GFP expressing immunocompromised rat. PCR and flow cytometry were used to follow the GFP genotype and phenotype in newborns. After three to four generations, animals were implanted with 4 T1 dsRed murine breast cancer cells or primary human glioblastoma (GBM) biopsies to generate xenografts for subsequent separation by fluorescence-activated cell sorting (FACS).
Fluorecence microscopy and reverse transcription-PCR demonstrated that GFP, under the control of the human ubiquitin C promoter, was stably maintained and expressed in diverse organs over several generations. Immunophenotyping of blood samples by flow cytometry confirmed that the immunodeficient features of the parental rat strain, HsdHan™: rnu/rnu, were retained in the GFP nude rat. Both the murine cell line and human GBM biopsies engrafted, and stromal cell populations were isolated from dissociated xenografts by FACS to > 95% purity.
A GFP transgene was stably introduced into a nude rat background in which human and murine cancer cells successfully engrafted. This animal strain provides a novel in vivo system for detailed cellular and molecular characterization of tumor-stroma interactions.
肿瘤微环境研究的一个关键策略是将人类肿瘤细胞植入普遍表达荧光标记的免疫缺陷啮齿动物品系中。在此，建立了一种表达绿色荧光蛋白（GFP）转基因的新型裸大鼠，并将其植入原发性人类肿瘤组织，以便从基质细胞群体中分离肿瘤细胞，用于后续的分子分析。
将SD-TG（GFP）2BalRrrc转基因大鼠与HsdHan™:rnu/rnu Rowett裸大鼠杂交，培育出表达GFP的免疫缺陷大鼠。采用聚合酶链反应（PCR）和流式细胞术追踪新生大鼠的GFP基因型和表型。经过三到四代后，给动物植入4T1红色荧光蛋白（dsRed）小鼠乳腺癌细胞或原发性人类胶质母细胞瘤（GBM）活检组织，以生成异种移植物，随后通过荧光激活细胞分选（FACS）进行分离。
荧光显微镜和逆转录PCR表明，在人泛素C启动子的控制下，GFP在几代大鼠的不同器官中稳定维持并表达。通过流式细胞术对血样进行免疫表型分析证实，亲代大鼠品系HsdHan™:rnu/rnu的免疫缺陷特征在GFP裸大鼠中得以保留。小鼠细胞系和人类GBM活检组织均能成功植入，并且通过FACS从解离的异种移植物中分离出基质细胞群体，纯度>95%。
GFP转基因被稳定引入裸大鼠背景中，人类和小鼠癌细胞在其中成功植入。该动物品系为详细的肿瘤-基质相互作用的细胞和分子特征分析提供了一种新型的体内系统。
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