Development of the transgenic cyan fluorescent protein (CFP)‐expressing nude mouse for “Technicolor” cancer imaging

A major goal for in vivo biology is to develop models which can express multiple colors of fluorescent proteins in order to image many processes simultaneously in real time. Towards this goal, the cyan fluorescent protein (CFP) nude mouse was developed by crossing non‐transgenic nude mice with the transgenic CK/ECFP mouse in which the β‐actin promoter drives expression of CFP in almost all tissues. In crosses between nu/nu CFP male mice and nu/+ CFP female mice, approximately 50% of the embryos fluoresced blue. In the CFP nude mice, the pancreas and reproductive organs displayed the strongest fluorescent signals of all internal organs which vary in intensity. Orthotopic implantation of XPA‐1 human pancreatic cancer cells expressing red fluorescent protein (RFP); or green fluorescent protein (GFP) in the nucleus and RFP in the cytoplasm, was performed in female nude CFP mice. Color‐coded fluorescence imaging of these human pancreatic cancer cells implanted into the bright blue fluorescent pancreas of the CFP nude mouse afforded novel insight into the interaction of the pancreatic tumor and the normal pancreas, in particular the strong desmoplastic reaction of the tumor. The naturally enhanced blue fluorescence of the pancreas in the CFP mouse serves as an ideal background for color‐coded imaging of the interaction of implanted cancer cells and the host. The CFP nude mouse will provide unique understanding of the critical interplay between the cancer cells and their microenvironment. J. Cell. Biochem. 107: 328–334, 2009. © 2009 Wiley‐Liss, Inc.     

Simultaneous color‐coded imaging to distinguish cancer “stem‐like” and non‐stem cells in the same tumor

In this study, we demonstrate that the differential behavior, including malignancy and chemosensitivity, of cancer stem‐like and non‐stem cells can be simultaneously distinguished in the same tumor in real time by color‐coded imaging. CD133⁺ Huh‐7 human hepatocellular carcinoma (HCC) cells were considered as cancer stem‐like cells (CSCs), and CD133^? Huh‐7 cells were considered as non‐stem cancer cells (NSCCs). CD133⁺ cells were isolated by magnetic bead sorting after Huh‐7 cells were genetically labeled with green fluorescent protein (GFP) or red fluorescent protein (RFP). In this scheme, CD133⁺ cells were labeled with GFP and CD133^? cells were labeled with RFP. CSCs had higher proliferative potential compared to NSCCs in vitro. The same number of GFP CSCs and the RFP NSCCs were mixed and injected subcutaneously or in the spleen of nude mice. CSCs were highly tumorigenic and metastatic as well as highly resistant to chemotherapy in vivo compared to NSCCs. The ability to specifically distinguish stem‐like cancer cells in vivo in real time provides a visual target for prevention of metastasis and drug resistance. J. Cell. Biochem. 111: 1035–1041, 2010. © 2010 Wiley‐Liss, Inc.     

Multi-color palette of fluorescent proteins for imaging the tumor microenvironment of orthotopic tumorgraft mouse models of clinical pancreatic cancer specimens

Pancreatic-cancer-patient tumor specimens were initially established subcutaneously in NOD/SCID mice immediately after surgery. The patient tumors were then harvested from NOD/SCID mice and passaged orthotopically in transgenic nude mice ubiquitously expressing red fluorescent protein (RFP). The primary patient tumors acquired RFP-expressing stroma. The RFP-expressing stroma included cancer-associated fibroblasts (CAFs) and tumor-associated macrophages (TAMs). Further passage to transgenic nude mice ubiquitously expressing green fluorescent protein (GFP) resulted in tumors that acquired GFP stroma in addition to their RFP stroma, including CAFs and TAMs as well as blood vessels. The RFP stroma persisted in the tumors growing in the GFP mice. Further passage to transgenic nude mice ubiquitously expressing cyan fluorescent protein (CFP) resulted in tumors acquiring CFP stroma in addition to persisting RFP and GFP stroma, including RFP- and GFP-expressing CAFs, TAMs and blood vessels. This model can be used to image progression of patient pancreatic tumors and to visually target stroma as well as cancer cells and to individualize patient therapy.     

Imaging the recruitment of cancer-associated fibroblasts by liver-metastatic colon cancer

The tumor microenvironment (TME) is critical for tumor growth and progression. However, the formation of the TME is largely unknown. This report demonstrates a color-coded imaging model in which the development of the TME can be visualized. In order to image the TME, a green fluorescent protein (GFP)-expressing mouse was used as the host which expresses GFP in all organs but not the parenchymal cells of the liver. Non-colored HCT-116 human colon cancer cells were injected in the spleen of GFP nude mice which led to the formation of experimental liver metastasis. TME formation resulting from the liver metastasis was observed using the Olympus OV100 small animal fluorescence imaging system. HCT-116 cells formed tumor colonies in the liver 28 days after cell transplantation to the spleen. GFP-expressing host cells were recruited by the metastatic tumors as visualized by fluorescence imaging. A desmin positive area increased around and within the liver metastasis over time, suggesting cancer-associated fibroblasts (CAFs) were recruited by the liver metastasis which have a role in tumor progression. The color-coded model of the TME enables its formation to be visualized at the cellular level in vivo, in real-time. This imaging model of the TME should lead to new visual targets in the TME.     

Color-coded intravital imaging demonstrates a transforming growth factor-β (TGF-β) antagonist selectively targets stromal cells in a human pancreatic-cancer orthotopic mouse model

Pancreatic cancer is a recalcitrant malignancy, partly due to desmoplastic stroma which stimulates tumor growth, invasion, and metastasis, and inhibits chemotherapeutic drug delivery. Transforming growth factor-β (TGF-β) has an important role in the formation of stromal desmoplasia. The present study describes the ability of color-coded intravital imaging to demonstrate the efficacy of a TGF-β inhibitor to target stroma in an orthotopic mouse model of pancreatic cancer. The BxPC-3 human pancreatic adenocarcinoma cell line expressing green fluorescent protein (GFP), which also has a high TGF-β expression level, was used in an orthotopic model in transgenic nude mice ubiquitously expressing red fluorescent protein (RFP). Fourteen mice were randomized into a control group (n = 7, vehicle, i.p., weekly, for 3 weeks) and a treated group (n = 7, SB431542 [TGF-β receptor type I inhibitor] 0.3 mg, i.p., weekly, for 3 weeks). Stromal cells expressing RFP and cancer cells expressing GFP were observed weekly for 3 weeks by real-time color-coded intravital imaging. The RFP fluorescence area from the stromal cells, relative to the GFP fluorescence area of the cancer cells, was significantly decreased in the TGF-β-inhibitor-treatment group compared to the control group. The present study demonstrated color-coded imaging in an orthotopic pancreatic-cancer cell-line mouse model can readily detect the selective anti-stromal-cell targeting of a TGF-β inhibitor.     

Color-coded fluorescence imaging of tumor-host interactions

Fluorescent proteins have the properties of being very bright with high quantum yield and are available in many colors. Tumor-host models consist of transgenic mice expressing green fluorescent protein (GFP) in essentially all cells and tissues or expressing GFP selectively in specific tissues such as blood vessels. Particularly useful are the corresponding nude mice transgenic for GFP expression, as they can accept human tumors. When tumor cells expressing red fluorescent protein are implanted in mice expressing GFP, various types of tumor-host interactions can be observed, including those involving host blood vessels, lymphocytes, tumor-associated fibroblasts, macrophages, dendritic cells and others. The 'color-coded' tumor-host models enable imaging and therefore a deeper understanding of the host cells involved and their function in tumor progression. Approximately 4-8 weeks are needed for these procedures.     

Color-coded imaging of splenocyte-pancreatic cancer cell interactions in the tumor microenvironment

In spite of advances in surgical and medical care pancreatic cancer remains a leading cause of cancer-related death in the United States. An understanding of cancer cell interactions with host cells is critical to our ability to develop effective antitumor therapeutics for pancreatic cancer. We report here a color-coded model system for imaging cancer cell interactions with host immune cells within the native pancreas. A human pancreatic cancer cell line engineered to express green fluorescent protein (GFP) in the nucleus and red fluorescent protein (DsRed2) in the cytoplasm was orthotopically implanted into the pancreas of a nude mouse. After 10-14 days red or green fluorescent splenocytes from immune-competent donors were delivered systemically to the pancreatic cancer-bearing nude mice. Animals were imaged after splenocyte delivery using high-resolution intravital imaging systems. At 1 day after iv injection red or green fluorescent spleen cells were found distributed in lung, liver, spleen and pancreas. By 4 days after cell delivery, however, the immune cells could be clearly imaged surrounding the tumor cells within the pancreas as well as collecting within lymphatic tissues such as lymph nodes and spleen. With the high-resolution intravital imaging afforded by the Olympus IV100 and OV100 systems the interactions of the dual-colored cancer cells and the red fluorescent spleen cells could be clearly imaged in this orthotopic pancreatic cancer model. This color-coded in vivo imaging technology offers a novel approach to imaging the interactions of cancer and immune cells in the tumor microenvironment (TME).     

Nestin‐expressing interfollicular blood vessel network contributes to skin transplant survival and wound healing

Using nestin‐driven green fluorescent protein (ND‐GFP) transgenic mice, we previously demonstrated an inter‐hair‐follicle blood vessel network that expresses ND‐GFP and appears to originate from ND‐GFP expressing hair‐follicle stem cells. We report here that angiogenesis of transplanted skin or healing wounds originates from this ND‐GFP‐expressing microvasculature network. ND‐GFP‐expressing blood vessels were visualized growing from the ND‐GFP‐expressing hair‐follicle stem cell area and re‐establishing the dermal microvasculature network after skin transplantation or wound healing. When the ND‐GFP stem cell area from the vibrissa (whisker) from ND‐GFP mice was transplanted to transgenic mice ubiquitously expressing RFP, we observed chimeric ND‐GFP‐RFP blood vessels, suggesting the joining of inter‐follicular blood vessel networks from the transplant and host. These observations suggest that the inter‐hair‐follicle blood‐vessel network contributes to skin transplant survival and wound healing. J. Cell. Biochem. 110: 80–86, 2010. © 2010 Wiley‐Liss, Inc.     

In vivo imaging of nuclear-cytoplasmic deformation and partition during cancer cell death due to immune rejection

In this report, we investigated the in vivo cell biology of cancer cells during immune rejection. The use of nestin-driven green fluorescent protein (ND-GFP) transgenic mice as hosts, in which nascent blood vessels express GFP, and implanted dual-color mouse mammary tumor 060562 (MMT) cells, in which the cytoplasm expresses red fluorescent protein (RFP) and the nuclei express GFP, allowed very important novel observations of angiogenesis and subcellular death pathways during immune rejection of a tumor. Nascent blood vessels did not form in the initially-growing mouse mammary tumor in ND-GFP immunocompetent mice. In contrast, in ND-GFP immunodeficient nude mice, numerous GFP-expressing nascent blood vessels grew into the tumor. The results suggest that insufficient nascent tumor angiogenesis was important in tumor rejection. During immune rejection, the cancer cells deformed their cytoplasm and nuclei, which were readily imaged by RFP and GFP, respectively. The nuclear membrane of the cancer cells ruptured, and chromatin extruded during partition of cytoplasm and nuclei. T lymphocytes infiltrated into the initially-growing tumor in the nestin-GFP transgenic immunocompetent mice. The cytotoxic role of the sensitized T lymphocytes was confirmed in vitro when they were co-cultured with MMT cells. The CD8a-positive lymphocytes attached to the cancer cells and caused nuclear condensation, deformation, and partition from their cytoplasm, similar to what occurred in vivo. The color-coded subcellular fluorescence-imaging model of immune rejection of cancer cells can provide a comprehensive system for further testing of immune-based treatment for cancer.     

Imaging the interaction of αv integrin-GFP in osteosarcoma cells with RFP-expressing host stromal cells and tumor-scaffold collagen in the primary and metastatic tumor microenvironment

Human osteosarcoma 143B cells were previously stably transfected with an α_v integrin green flourescent protein (GFP) vector. 143B cells expressing α_v integrin-GFP were transplanted orthotopically in the tibia of transgenic nude mice ubiquitously expressing red fluorescent protein (RFP). The primary tumors acquired RFP-expressing stroma and were passaged orthotopically in the tibia in noncolored nude mice, which maintained the RFP stroma. The interaction of α_v integrin-GFP expression in 143B cells with RFP-expressing host stromal cells was observed by confocal microscopy using the Olympus FV1000. Collagen fibers were imaged simultaneously in reflectance mode. The RFP-expressing stroma included cancer-associated fibroblasts (CAFs) and tumor-associated macrophages (TAMs) which persisted even 3 weeks after passage to nontransgenic nude mice. CAFs expressing RFP were aligned between collagen fibers and cancer cells expressing α_v integrin-GFP. Six weeks after transplantation, pulmonary metastases expressing α_v integrin-GFP could be identified. TAMs expressing RFP accompanied metastasized osteosarcoma cells expressing α_v integrin-GFP in the lung. The current study demonstrates the importance of α_v integrin interaction with stromal elements in osteosarcoma.     

In vivo longitudinal imaging of RNA interference‐induced endocrine therapy resistance in breast cancer

Endocrine therapy resistance in breast cancer is a major obstacle in the treatment of patients with estrogen receptor‐positive (ER+) tumors. Herein, we demonstrate the feasibility of longitudinal, noninvasive and semiquantitative in vivo molecular imaging of resistance to three endocrine therapies by using an inducible fluorescence‐labeled short hairpin RNA (shRNA) system in orthotopic mice xenograft tumors. We employed a dual fluorescent doxycycline (Dox)‐regulated lentiviral inducer system to transfect ER+ MCF7L breast cancer cells, with green fluorescent protein (GFP) expression as a marker of transfection and red fluorescent protein (RFP) expression as a surrogate marker of Dox‐induced tumor suppressor phosphatase and tensin homolog deleted on chromosome 10 (PTEN) knockdown. Xenografted MCF7L tumor‐bearing nude mice were randomized to therapies comprising estrogen deprivation, tamoxifen or an ER degrader (fulvestrant) and an estrogen‐treated control group. Longitudinal imaging was performed by a home‐built multispectral imaging system based on a cooled image intensified charge coupled device camera. The GFP signal, which corresponds to number of viable tumor cells, exhibited excellent correlation to caliper‐measured tumor size (P << .05). RFP expression was substantially higher in mice exhibiting therapy resistance and strongly and significantly (P < 1e‐7) correlated with the tumor size progression for the mice with shRNA‐induced PTEN knockdown. PTEN loss was strongly correlated with resistance to estrogen deprivation, tamoxifen and fulvestrant therapies.      

Real‐time imaging of single cancer‐cell dynamics of lung metastasis

We have developed a new in vivo mouse model to image single cancer‐cell dynamics of metastasis to the lung in real‐time. Regulating airflow volume with a novel endotracheal intubation method enabled controlling lung expansion adequate for imaging of the exposed lung surface. Cancer cells expressing green fluorescent protein (GFP) in the nucleus and red fluorescent protein (RFP) in the cytoplasm were injected in the tail vein of the mouse. The right chest wall was then opened in order to image metastases on the lung surface directly. After each observation, the chest wall was sutured and the air was suctioned in order to re‐inflate the lung, in order to keep the mice alive. Observations have been carried out for up to 8 h per session and repeated up to six times per mouse thus far. The seeding and arresting of single cancer cells on the lung, accumulation of cancer‐cell emboli, cancer‐cell viability, and metastatic colony formation were imaged in real‐time. This new technology makes it possible to observe real‐time monitoring of cancer‐cell dynamics of metastasis in the lung and to identify potential metastatic stem cells. J. Cell. Biochem. 109: 58–64, 2010. © 2009 Wiley‐Liss, Inc.     

绿色荧光裸鼠的建立和验证

目的建立系统性表达绿色荧光蛋白的裸鼠,接种人源肺癌细胞验证该模型是否具有免疫缺陷性,并观察双色荧光的成像效果。方法利用系统性表达绿色荧光蛋白的C57BL/6J小鼠与BALB/C裸小鼠多代杂交和互交,建立稳定表达绿色荧光蛋白的裸鼠。大体解剖观察胸腺生长情况,整体和器官荧光成像验证绿色荧光蛋白的表达情况。以2×106/只的剂量对其皮下腋下接种表达红色荧光蛋白的人类A549肺癌细胞（RFP-A549）,通过观测肿瘤生长来验证模型的免疫缺陷性。同时,利用红色荧光标记的肿瘤和绿色宿主鼠,对双色的整体成像效果进行观测。结果构建出系统性表达绿色荧光蛋白的裸鼠,大体解剖可见胸腺缺失。在激发光的激发下,绿色荧光裸鼠全身发出清晰的绿色荧光,脑、心脏、肺脏、肝脏、肾脏,肠胃及胰腺等主要器官可见明显绿色荧光。接种RFP-A549细胞后,成瘤率达到100%,整体动物荧光成像表现出清晰的双色。结论本研究构建出的绿色荧光裸鼠,动物整体可以清晰地表达绿色荧光并具有免疫缺陷性     

In vivo gene transfer between interacting human osteosarcoma cell lines is associated with acquisition of enhanced metastatic potential

We report here in vivo gene transfer between cancer cells is associated with acquisition of high metastatic behavior. The 143B‐GFP cell line with high metastatic potential and the MNNG/HOS‐RFP cell line with low metastatic potential, both derived from the TE85 human osteosarcoma cell line, were either co‐transplanted or transplanted alone in the tibia in nude mice. Upon mixed transplantation of the two differently labeled sublines, resulting metastatic colonies are single colored either red or green, thereby demonstrating their clonality and enabling facile color‐coded quantification. When MNNG/HOS‐RFP and 143B‐GFP were co‐transplanted in the tibia, the number of lung metastases of MNNG/HOS‐RFP increased eight‐fold compared to MNNG/HOS‐RFP transplanted alone (P < 0.01). In contrast, no enhancement of MNNG/HOS‐RFP metastases occurred when MNNG/HOS‐RFP and 143B‐GFP were transplanted separately in the right and left tibiae, respectively. This result suggests that the presence of 143B‐GFP increased the metastatic potential of MNNG/HOS‐RFP within the mixed tumor. We observed transfer of the Ki‐ras gene from 143B‐GFP to MNNG/HOS‐RFP after they were co‐implanted suggesting the Ki‐ras played a role in increasing the metastatic potential of MNNG/HOS‐RFP in the presence of 143B‐GFP. These data suggest the possible role of in vivo gene transfer in enhancing the metastatic potential of cancer cells. The data also further demonstrated the power of color‐coded imaging to visualize cancer‐cell/cancer‐cell interactions in vivo. J. Cell. Biochem. 108: 362–367, 2009. © 2009 Wiley‐Liss, Inc.     

Cancer metastasis directly eradicated by targeted therapy with a modified Salmonella typhimurium

Cancer metastasis is the life‐threatening aspect of cancer and is usually resistant to standard treatment. We report here a targeted therapy strategy for cancer metastasis using a genetically‐modified strain of Salmonella typhimurium. The genetically‐modified strain of S. typhimurium is auxotrophic for the amino acids arginine and leucine. These mutations preclude growth in normal tissue but do not reduce bacterial virulence in cancer cells. The tumor‐targeting strain of S. typhimurium, termed A1‐R, and expressing green fluorescent protein (GFP), was administered to both axillary lymph and popliteal lymph node metastasis of human pancreatic cancer and fibrosarcoma, respectively, as well as lung metastasis of the fibrosarcoma in nude mice. The bacteria were delivered via a lymphatic channel to target the lymph node metastases and systemically via the tail vein to target the lung metastasis. The cancer cells expressed red fluorescent protein (RFP) in the cytoplasm and GFP in the nucleus linked to histone H2B, enabling color‐coded real‐time imaging of the bacteria targeting the metastatic tumors. After 7–21 days of treatment, the metastases were eradicated without the need of chemotherapy or any other treatment. No adverse effects were observed. This new strategy demonstrates the clinical potential of targeting and curing cancer metastasis with engineered bacteria without the need of toxic chemotherapy. J. Cell. Biochem. 106: 992–998, 2009. © 2009 Wiley‐Liss, Inc.     

Rhodobacter sphaeroides,a novel tumor‐targeting bacteria that emits natural near‐infrared fluorescence

Several optical imaging techniques have been used to monitor bacterial tropisms for cancer. Most such techniques require genetic engineering of the bacteria to express optical reporter genes. This study investigated a novel tumor‐targeting strain of bacteria, Rhodobacter sphaeroides 2.4.1 (R. sphaeroides), which naturally emits near‐infrared fluorescence, thereby facilitating the visualization of bacterial tropisms for cancer. To determine the penetration depth of bacterial fluorescence, various numbers of cells (from 10⁸ to 10¹⁰ CFU) of R. sphaeroides and two types of Escherichia coli, which stably express green fluorescent protein (GFP) or red fluorescent protein (RFP), were injected s.c. or i.m. into mice. Bacterial tropism for cancer was determined after i.v. injection of R. sphaeroides (10⁸ CFU) into mice implanted s.c. with eight types of tumors. The intensity of the fluorescence signal in deep tissue (muscle) from R. sphaeroides was much stronger than from E. coli‐expressing GFP or RFP. The near‐infrared fluorescence signal from R. sphaeroides was visualized clearly in all types of human or murine tumors via accumulation of bacteria. Analyses of C‐reactive protein and procalcitonin concentrations and body weights indicated that i.v. injection of R. sphaeroides does not induce serious systemic immune reactions. This study suggests that R. sphaeroides could be used as a tumor‐targeting microorganism for the selective delivery of drugs to tumor tissues without eliciting a systemic immune reaction and for visualizing tumors.     

Zebrafish lines expressing UAS‐driven red probes for monitoring cytoskeletal dynamics

Actin filaments and microtubules are principal components of the cytoskeleton that regulate the basic cellular phenomena underlying many fundamental cellular processes. Therefore, analyzing their dynamics in living cells is important for understanding cellular events more precisely. In this article, we report two novel transgenic zebrafish lines expressing red fluorescent proteins tagged with Lifeact or EB1 that interact with actin filaments and microtubule plus ends, respectively, under the control of the GAL4‐UAS system. Using these transgenic lines, we could detect F‐actin and microtubule plus end dynamics in specific tissues of living zebrafish embryos by crossing with GAL4 driver lines. In addition, we could achieve multi‐color imaging using these transgenic lines with GFP‐expressing transgenic lines. Therefore, our transgenic lines that carry UAS‐driven red fluorescent cytoskeletal probes are useful tools for analyzing spatiotemporal changes of the cytoskeletal elements using multicolor live imaging.     

UV light killing efficacy of fluorescent protein‐expressing cancer cells in vitro and in vivo

We investigated the cell‐killing efficacy of UV light on cancer cells expressing GFP in the nucleus and RFP in the cytoplasm (dual‐color cells). After exposure to various doses of UVA, UVB, or UVC, apoptotic and viable cells were quantitated under fluorescence microscopy using dual‐color 143B human osteosarcoma cells, HT‐1080 human fibrosarcoma cells, Lewis lung carcinoma (LLC), and XPA‐1 human pancreatic cancer cells in vitro. UV‐induced cancer cell death was wave‐length and dose dependent, as well as cell‐line dependent. After UVA exposure, most cells were viable even when the UV dose was increased up to 200 J/m². With UVB irradiation, cell death was observed with irradiation at 50 J/m². For UVC, as little as 25 J/m² UVC irradiation killed approximately 70% of the 143B dual‐color cells. This dose of UVB or UVA had almost no effect on the cancer cells. UV‐induced cancer cell death varied among the cell lines. Cell death began about 4 h after irradiation and continued until 10 h after irradiation. UVC exposure also suppressed cancer cell growth in nude mice in a model of minimal residual cancer (MRC). No apparent side effects of UVC exposure were observed. This study opens up the possibility of UVC treatment for MRC after surgical resection. J. Cell. Biochem. 110: 1439–1446, 2010. © 2010 Wiley‐Liss, Inc.     

Ubiquitous expression of the monomeric red fluorescent protein mcherry in transgenic mice

The use of the green fluorescent protein (GFP) to label specific cell types and track gene expression in animal models, such as mice, has evolved to become an essential tool in biological research. Transgenic animals expressing genes of interest linked to GFP, either as a fusion protein or transcribed from an internal ribosomal entry site (IRES) are widely used. Enhanced GFP (eGFP) is the most common form of GFP used for such applications. However, a red fluorescent protein (RFP) would be highly desirable for use in dual‐labeling applications with GFP derived fluorescent proteins, and for deep in vivo imaging of tissues. Recently, a new generation of monomeric (m)RFPs, such as monomeric (m)Cherry, has been developed that are potentially useful experimentally. mCherry exhibits brighter fluorescence, matures more rapidly, has a higher tolerance for N‐terminal fusion proteins, and is more photostable compared with its predecessor mRFP1. mRFP1 itself was the first true monomer derived from its ancestor DsRed, an obligate tetramer in vivo. Here, we report the successful generation of a transgenic mouse line expressing mCherry as a fluorescent marker, driven by the ubiquitin‐C promoter. mCherry is expressed in almost all tissues analyzed including pre‐ and post‐implantation stage embryos, and white blood cells. No expression was detected in erythrocytes and thrombocytes. Importantly, we did not encounter any changes in normal development, general physiology, or reproduction. mCherry is spectrally and genetically distinct from eGFP and, therefore, serves as an excellent red fluorescent marker alone or in combination with eGFP for labelling transgenic animals. genesis 48:723–729, 2010. © 2010 Wiley‐Liss, Inc.     

GFP转基因裸鼠脾脏组织学观察

目的探讨GFP基因导入对BALB/c荧光裸鼠脾脏组织学及免疫功能的影响。方法取不同日龄（14日龄、28日龄、49日龄、70日龄）BALB/c荧光裸鼠及BALB/c普通裸鼠各32只,雌雄各半,处死取脾脏,对脾脏的绝对重量、脾脏指数进行测量分析,对脾脏的组织学改变进行观察,并对脾脏淋巴细胞数进行统计分析。结果与14日龄荧光裸鼠相比,28日龄荧光裸鼠脾脏指数明显较高（P〈0.05）。与14日龄荧光裸鼠相比,49日龄、70日龄荧光裸鼠淋巴细胞数明显变少（P〈0.05）。与普通裸鼠（14日龄、28日龄、49日龄、70日龄）相比较,相同日龄荧光裸鼠（14日龄、28日龄、49日龄、70日龄）淋巴细胞数明显减少（P〈0.05）。结论 GFP基因对不同日龄荧光裸鼠的脾脏发育及其功能有一定影响。