The NOD/Scid mouse demonstrates immunodeficiency that is severer than that in the nude mouse

The NOD/Scid mouse demonstrates immunodeficiency that is severer than that in the nude mouse. humanized mouse model [5, 10, 16, 17]. Growth and progression of transplanted cells are dependent on the formation of a suitable microenvironment [1,2,3,4, 14, 21]. For example, host stromal tissue components, such as vessels and fibroblasts, interact with engraftment derivatives and provide oxygen and other essential nutrients. To elucidate the mechanisms that form such microenvironments, various fluorescent protein-expressing murine models have been established [20, 22, 23] by mating systemically florescent protein-expressing mice with nude mice, which lack T cell production because of thymic deficiency, but retain production of B cells and other immunocompetent cells. The survivability of transplanted cells is dependent on the immune status of the recipient. It was previously exhibited that nude mice showed a little resistance to xenotransplantation [10]. Another group reported the nonobese diabetic/severe combined immunodeficient (NOD/Scid) transgenic mouse expressing enhanced green fluorescent protein (EGFP) [11]. NOD/Scid mice lack B and T lymphocytes and have low natural killer (NK) cells and hemolytic complement activity, defects in myeloid development, and poor antigen-presenting-cell function. The NOD/Scid mouse demonstrates immunodeficiency that is severer than that in the nude mouse. However, it was reported that lymphoma developed in the NOD/Scid mouse with high incidence [7]. We previously established a nonobese diabetic/severe combined immunodeficiency interleukin-2 receptor null(NOD/Shi-mutation from C57BL/6-mice by backcross mating A-484954 with NOD/Shi-mice [6]. NOG mice have no lymphocytes or NK cells and have impaired dendritic cells and macrophage function [9, 13]. Additionally, we previously reported an EGFP-expressing NOG mouse line (NOG-EGFP mouse) [18]. Since the NOG-EGFP mouse demonstrates severe immunodeficiency and systemic EGFP expression, it is considered the most appropriate recipient mouse for analyzing the microenvironment of xenograft tissues [15]. The NOG-EGFP mouse exhibited strong EGFP expression especially in the skin, muscle, and pancreas. However, EGFP expression in the hepatic parenchymal cells was not observed (Fig. S1). This suggests that the NOG-EGFP mouse is not suitable as a recipient for liver metastatic model. In the present study, we established a novel EGFP-expressing NOG mouse that A-484954 expressed EGFP under the control of the phosphoglycerate kinase promoter (PgkEGFP-NOG mouse). The PgkEGFP-NOG mouse retains equivalent immunodeficiency to the NOG mouse, and whole-body immunohistochemical analysis revealed that PgkEGFP-NOG mice exhibited strong EGFP expression especially in the liver. Xenograft colonies in the PgkEGFP-NOG mice were clearly identified as EGFP-negative colonies, and the tumor microenvironment in xenograft colonies was composed of EGFP-expressing fibroblasts and vessels. Moreover, a similar microenvironment was observed in human induced pluripotent stem (iPS) cell-derived teratomas. These results exhibited that PgkEGFP-NOG mice present a useful host model for analysis of the microenvironment of xenograft tissues. Materials and Methods DNA construction The gene expression unit was constructed as follows. The promoter region (621 bp) of the murine phosphoglycerate kinase 1 gene was amplified by polymerase chain reaction (PCR) at an annealing temperature of 60C with the primers mPGK-F-I-HI restriction site (pmPgkEGFP). A vector-free 1.6-kb expression fragment was prepared by cleavage of the pmPgkEGFP plasmid DNA at unique I and I restriction sites. Generation of the PgkEGFP-NOG mouse The PgkEGFP-NOG construct was microinjected into fertilized NOD/Shi strain mouse eggs using standard methods. Transgenic offspring were identified by PCR (annealing temperature 60C) using GFPF1 forward and reverse primers (5-CTGGTCGAGCTGGACGGCGACG-3 and 5-CACGAACTCCAGCAGGACCATG-3, respectively). Genomic DNA extracted from tail tissue was amplified in a 20 and mutations to the Rabbit polyclonal to ZNF394 offspring. The mutations were genotyped using a previously described PCR method [6]. The transgene was microinjected into fertilized NOD/Shi strain mouse eggs and confirmed by genomic PCR, and the sequence of the tandem transgene junction A-484954 was defined (Figs. 1 A and B). Transgenic offspring were mated with NOG mice, and insertion of the and mutations was confirmed as previously described [6]. Next, we confirmed EGFP expression in the PgkEGFP-NOG mice (Figs. S1CS3). Although EGFP expression in.