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.     

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.     

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.     

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.     

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.     

Role of the metastasis‐promoting protein osteopontin in the tumour microenvironment

Osteopontin (OPN) is a secreted protein present in bodily fluids and tissues. It is subject to multiple post‐translational modifications, including phosphorylation, glycosylation, proteolytic cleavage and crosslinking by transglutamination. Binding of OPN to integrin and CD44 receptors regulates signalling cascades that affect processes such as adhesion, migration, invasion, chemotaxis and cell survival. A variety of cells and tissues express OPN, including bone, vasculature, kidney, inflammatory cells and numerous secretory epithelia. Normal physiological roles include regulation of immune functions, vascular remodelling, wound repair and developmental processes. OPN also is expressed in many cancers, and elevated levels in patients’ tumour tissue and blood are associated with poor prognosis. Tumour growth is regulated by interactions between tumour cells and their tissue microenvironment. Within a tumour mass, OPN can be expressed by both tumour cells and cellular components of the tumour microenvironment, and both tumour and normal cells may have receptors able to bind to OPN. OPN can also be found as a component of the extracellular matrix. The functional roles of OPN in a tumour are thus complex, with OPN secreted by both tumour cells and cells in the tumour microenvironment, both of which can in turn respond to OPN. Much remains to be learned about the cross‐talk between normal and tumour cells within a tumour, and the role of multiple forms of OPN in these interactions. Understanding OPN‐mediated interactions within a tumour will be important for the development of therapeutic strategies to target OPN.     

A knock‐in mouse line conditionally expressing the tumor suppressor WTX/AMER1

WTX/AMER1 is an important developmental regulator, mutations in which have been identified in a proportion of patients suffering from the renal neoplasm Wilms' tumor and in the bone malformation syndrome Osteopathia Striata with Cranial Sclerosis (OSCS). Its cellular functions appear complex and the protein can be found at the membrane, within the cytoplasm and the nucleus. To understand its developmental and cellular function an allelic series for Wtx in the mouse is crucial. Whereas mice carrying a conditional knock out allele for Wtx have been previously reported, a gain‐of‐function mouse model that would allow studying the molecular, cellular and developmental role of Wtx is still missing. Here we describe the generation of a novel mouse strain that permits the conditional activation of WTX expression. Wtx fused to GFP was introduced downstream a stop cassette flanked by loxP sites into the Rosa26 locus by gene targeting. Ectopic WTX expression is reported after crosses with several Cre transgenic mice in different embryonic tissues. Further, functionality of the fusion protein was demonstrated in the context of a Wtx null allele.     

实验动物准备对裸鼠移植瘤模型microPET 显像的影响

目的 探讨实验动物准备条件对18 F-FDG microPET 裸鼠移植瘤模型显像的影响,以选择最佳的实验动物准备条件.方法 36 只人表皮样癌细胞A431 裸鼠皮下移植瘤模型.随机分为6 组（6 只/组）;A 组：无禁食、室温（20 ～22）℃、无麻醉（注射18 F-FDG 后60 min 清醒状态）、尾静脉注射18 F-FDG;B 组：禁食（6 ～8）h、加温（30 ～32）℃、麻醉（吸入2%异氟烷麻醉）、尾静脉注射18 F-FDG;C 组：无禁食、加温、麻醉、尾静脉注射18 F-FDG;D组：禁食、室温、麻醉、尾静脉注射18 F-FDG;E 组：禁食、加温、无麻醉、尾静脉注射18 F-FDG;F 组：禁食、加温、麻醉、腹腔注射18 F-FDG.注射18 F-FDG 约1 h 后,行microPET 显像,测量皮下移植瘤、颈部肌肉、棕色脂肪、脑、肝脏、肾脏、心脏、哈氏腺最大每克组织摄取率（%ID/gmax ）.扫描前裸鼠均测血糖.结果 （1）B 组、C 组、F 组裸鼠的血糖水平与肿瘤摄取之间均呈直线负相关.（2）棕色脂肪：A 组摄取最高（8.03 ±1.29）,B 组摄取降低71.98%（P ＝0.000）.颈部肌肉：A 组摄取最高（16.07 ±5.20）,B 组摄取降低最多达81.84%（P ＝0.000）.各组脑、心脏、肝脏、肾脏、哈氏腺摄取差异无统计学意义.（3）A 组皮下移植瘤/组织或器官的摄取率最低.B 组移植瘤/颈部肌肉,移植瘤/肝脏,移植瘤/棕色脂肪的摄取率较A 组分别升高6.50 倍、1.29 倍、4.76 倍（P 均＜0.05）,肿瘤与组织或器官的图像对比度明显改善.（4）第1 次microPET 显像,尾静脉注射与腹腔注射皮下移植瘤摄取值差别无统计学意义（P ＝0.364）.第2 次microPET 显像,腹腔注射腹腔可见不同程度显像剂浓聚,其他正常组织、器官及皮下移植瘤的摄取均减低.腹腔注射方式,两次皮下移植瘤的摄取值差异有统计学意义（P ＝0.025）.结论实验动物准备明显影响18 F-FDG 在裸鼠正常组织的分布及皮下移植瘤的摄取.禁食、加温、麻醉及尾静脉注射方式,可以改善肿瘤对18 F-FDG 的摄取,保证图像有较好的稳定性及可重复性.     

A mouse carrying the green fluorescent protein gene targeted to the Pdx1 locus facilitates the study of pancreas development and function

The pancreatic and duodenal homeobox gene 1 (Pdx1) has multiple roles in the specification and development of foregut endoderm-derived tissues. We report the characterization of a mouse line in which the gene encoding green fluorescent protein (GFP) has been targeted to the Pdx1 locus, allowing the visualization of Pdx1 expressing cells. Analysis of GFP expression during development showed that the reporter faithfully reproduced the known expression pattern of Pdx1. We demonstrate the utility of this mouse line for the isolation of Pdx1(+) cells by fluorescence-activated cell sorting and for the real-time observation of Pdx1(+) cells in an ex vivo embryonic pancreas culture system. This mouse model should prove useful for the study of pancreas development and regeneration.     

Dual‐color‐emitting green fluorescent protein from the sea cactus Cavernularia obesa and its use as a pH indicator for fluorescence microscopy

We isolated and characterized a green fluorescent protein (GFP) from the sea cactus Cavernularia obesa. This GFP exists as a dimer and has absorption maxima at 388 and 498 nm. Excitation at 388 nm leads to blue fluorescence (456 nm maximum) at pH 5 and below, and green fluorescence (507 nm maximum) at pH 7 and above, and the GFP is remarkably stable at pH 4. Excitation at 498 nm leads to green fluorescence (507 nm maximum) from pH 5 to pH 9. We introduced five amino acid substitutions so that this GFP formed monomers rather than dimers and then used this monomeric form to visualize intracellular pH change during the phagocytosis of living cells by use of fluorescence microscopy. The intracellular pH change is visualized by use of a simple long‐pass emission filter with single‐wavelength excitation, which is technically easier to use than dual‐emission fluorescent proteins that require dual‐wavelength excitation. Copyright © 2013 John Wiley & Sons, Ltd.     

Afp::mCherry, a red fluorescent transgenic reporter of the mouse visceral endoderm

Live imaging of genetically encoded fluorescent protein reporters is increasingly being used to investigate details of the cellular behaviors that underlie the large-scale tissue rearrangements that shape the embryo. However, the majority of mouse fluorescent reporter strains are based on the green fluorescent protein (GFP). Mouse reporter strains expressing fluorescent colors other than GFP are therefore valuable for co-visualization studies with GFP, where relative positioning and relationship between two different tissues or compartments within cells are being investigated. Here, we report the generation and characterization of a transgenic Afp::mCherry mouse strain in which cis-regulatory elements from the Alpha-fetoprotein (Afp) locus were used to drive expression of the monomeric Cherry red fluorescent protein. The Afp::mCherry transgene is based on and recapitulates reporter expression of a previously described Afp::GFP strain. However, we note that perdurance of mCherry protein is not as prolonged as GFP, making the Afp::mCherry line a more faithful reporter of endogenous Afp expression. Afp::mCherry transgenic mice expressed mCherry specifically in the visceral endoderm and its derivatives, including the visceral yolk sac, gut endoderm, fetal liver, and pancreas of the embryo. The Afp::mCherry reporter was also noted to be expressed in other documented sites of Afp expression including hepatocytes as well as in pancreas, digestive tract, and brain of postnatal mice.     

A Probasin‐MerCreMer BAC allows inducible recombination in the mouse prostate

Tissue‐specific transgene expression in the prostate epithelium has previously been achieved using short prostate‐specific promoters, rendering transgenic mouse lines susceptible to integration site‐dependent effects. Here we demonstrate the applicability of bacterial artificial chromosome (BAC) technology to transgene expression in the prostate epithelium. We present mouse lines expressing an inducible Cre protein (MerCreMer) under the control of regulatory elements of the probasin gene on a BAC. These mouse lines show high organ specificity, high transgene expression in anterior, dorsal and lateral prostate lobes, no background Cre recombination using a reporter strain and adjustable amounts of Cre‐induced recombination upon tamoxifen induction. Together with two recently reported transgenic lines expressing the Cre‐ERT2 protein from small prostate‐specific promoters, these mouse lines will be useful in research focused on prostate‐specific disorders such as benign hyperplasia or cancer. genesis 47:757–764, 2009. © 2009 Wiley‐Liss, Inc.     

A new mouse model for temporal‐ and tissue‐specific control of extracellular superoxide dismutase

The extracellular isoform of superoxide dismutase (EC‐SOD, Sod3) plays a protective role against various diseases and injuries mediated by oxidative stress. To investigate the pathophysiological roles of EC‐SOD, we generated tetracycline‐inducible Sod3 transgenic mice and directed the tissue‐specific expression of transgenes by crossing Sod3 transgenic mice with tissue‐specific transactivator transgenics. Double transgenic mice with liver‐specific expression of Sod3 showed increased EC‐SOD levels predominantly in the plasma as the circulating form, whereas double transgenic mice with neuronal‐specific expression expressed higher levels of EC‐SOD in hippocampus and cortex with intact EC‐SOD as the dominant form. EC‐SOD protein levels also correlated well with increased SOD activities in double transgenic mice. In addition to enabling tissue‐specific expression, the transgene expression can be quickly turned on and off by doxycycline supplementation in the mouse chow. This mouse model, thus, provides the flexibility for on–off control of transgene expression in multiple target tissues. genesis 47:142–154, 2009. © 2009 Wiley‐Liss, Inc.     

A 900 bp genomic region from the mouse dystrophin promoter directs lacZ reporter expression only to the right heart of transgenic mice

In order to study the regulatory mechanism of developmental and tissue-specific expression of the muscle type dystrophin gene in mice, transgenic mice were generated carrying the 900 bp genomic fragment derived from the muscle type dystrophin promoter region fused to the bacterial lacZ gene. Six independent transgenic mouse lines showed specific reporter gene expression in the right heart, but not in skeletal or smooth muscle. The reporter gene expression was first detected in the presumptive right ventricle of the embryos at 8.5 days post coitum, and the expression continued only in the right ventricle throughout the development and at the adult stage. The results indicate that the 900 bp genomic fragment contains the regulatory element required for expression of dystrophin only in the right heart, suggesting that distinct elements are responsible for the expression in the left and right compartments of the heart, and/or in skeletal and smooth muscle cells. Based on these findings, the relationship between defects in muscle type promoter and the diseases caused by abnormal dystrophin expression is discussed.     

A set of fluorescent protein‐based markers expressed from constitutive and arbuscular mycorrhiza‐inducible promoters to label organelles,membranes and cytoskeletal elements in Medicago truncatula

Medicago truncatula is widely used for analyses of arbuscular mycorrhizal (AM) symbiosis and nodulation. To complement the genetic and genomic resources that exist for this species, we generated fluorescent protein fusions that label the nucleus, endoplasmic reticulum, Golgi apparatus, trans‐Golgi network, plasma membrane, apoplast, late endosome/multivesicular bodies (MVB), transitory late endosome/ tonoplast, tonoplast, plastids, mitochondria, peroxisomes, autophagosomes, plasmodesmata, actin, microtubules, periarbuscular membrane (PAM) and periarbuscular apoplastic space (PAS) and expressed them from the constitutive AtUBQ10 promoter and the AM symbiosis‐specific MtBCP1 promoter. All marker constructs showed the expected expression patterns and sub‐cellular locations in M. truncatula root cells. As a demonstration of their utility, we used several markers to investigate AM symbiosis where root cells undergo major cellular alterations to accommodate their fungal endosymbiont. We demonstrate that changes in the position and size of the nuclei occur prior to hyphal entry into the cortical cells and do not require DELLA signaling. Changes in the cytoskeleton, tonoplast and plastids also occur in the colonized cells and in contrast to previous studies, we show that stromulated plastids are abundant in cells with developing and mature arbuscules, while lens‐shaped plastids occur in cells with degenerating arbuscules. Arbuscule development and secretion of the PAM creates a periarbuscular apoplastic compartment which has been assumed to be continuous with apoplast of the cell. However, fluorescent markers secreted to the periarbuscular apoplast challenge this assumption. This marker resource will facilitate cell biology studies of AM symbiosis, as well as other aspects of legume biology.     

The H-2KbtsA58 transgenic mouse: A new tool for the rapid generation of novel cell lines

The ability to generate expanded populations of individual cell types able to undergo normal differentiationin vitro andin vivo is of critical importance in the investigation of the mechanisms that underly differentiation and in studies on the use of cell transplantation to repair damaged tissues. This review discusses the development of a strain of transgenic mice that allows the direct derivation of conditionally immortal cell lines from a variety of tissues, simply by dissociation of the tissue of interest and growth of cells in appropriate conditions. In these mice the tsA58 mutant of SV40 large T antigen is controlled by the interferon-inducible Class I antigen promoter. Cells can be grown for extended periodsin vitro simply by growing them at 33°C in the presence of interferon, while still retaining the capacity to undergo normal differentiationin vivo andin vitro. In addition, it appears that cell lines expressing mutant phenotypes can readily be generated by preparing cultures from appropriate offspring of matings between H-2K^btsA58 transgenic mice and mutant mice of interest.