Multimodality imaging of T-cell hybridoma trafficking in collagen-induced arthritic mice: image-based estimation of the number of cells accumulating in mouse paws

Appropriate targeting of therapeutic cells is essential in adoptive cellular gene therapy (ACGT). Imaging cell trafficking in animal models and patients will guide development of ACGT protocols. Collagen type II (C-II)-specific T cell hybridomas are transduced with a lentivirus carrying a triple fusion reporter gene (TFR) construct consisting of a fluorescent reporter gene (RG), a bioluminescent RG (hRluc), and a positron emission tomography (PET) RG. Collagen-induced arthritic (CIA) mice are scanned with a bioluminescence imaging camera before and after implantation of various known cell quantities in their paws. Linear regression analysis yields equations relating two parameters of image signal intensity in mice paws to the quantity of hRluc expressing cells in the paws. Afterward, trafficking of intravenously injected cells is studied by quantitative analysis of bioluminescence images. Comparison of the average cell numbers does not demonstrate consistently higher accumulation of T-cell hybridomas in the paws with higher inflammation scores, and injecting more cells does not cause increased accumulation. MicroPET images illustrate above background signal in the inflamed paws and chest areas of CIA mice. The procedures described in this study can be used to derive equations for cells expressing other bioluminescent RGs and in other animal models.     

A paper-based invasion assay: Assessing chemotaxis of cancer cells in gradients of oxygen

This work describes a 3D, paper-based assay that can isolate sub-populations of cells based on their invasiveness (i.e., distance migrated in a hydrogel) in a gradient of concentration of oxygen (O₂). Layers of paper impregnated with a cell-compatible hydrogel are stacked and placed in a plastic holder to form the invasion assay. In most assays, the stack comprises a single layer of paper containing mammalian cells suspended in a hydrogel, sandwiched between multiple layers of paper containing only hydrogel. Cells in the stack consume and produce small molecules; these molecules diffuse throughout the stack to generate gradients in the stack, and between the stack and the bulk culture medium. Placing the cell-containing layer in different positions of the stack, or modifying the permeability of the holder to oxygen or proteins, alters the profile of the gradients within the stack. Physically separating the layers after culture isolates sub-populations of cells that migrated different distances, and enables their subsequent analysis or culture. Using this system, three independent cell lines derived from A549 cancer cells are shown to produce distinguishable migration behavior in a gradient of oxygen. This result is the first experimental demonstration that oxygen acts as a chemoattractant for cancer cells.     

Generation of Mechanical and Biofunctional Gradients in PEG Diacrylate Hydrogels by Perfusion-Based Frontal Photopolymerization

The spatial presentation of soluble growth factors, immobilized extracellular matrix molecules, as well as matrix rigidity, plays an important role in directed and guided cell migration. Synthetic hydrogel scaffolds offer the ability to systematically introduce gradients of these factors contributing to our understanding of how the 3D arrangement of biochemical and mechanical cues influence cell behavior. Using a novel photopolymerization technique, perfusion-based frontal photopolymerization (PBFP), we have engineered poly(ethylene glycol) diacrylate (PEGDA) hydrogel scaffolds with gradients of mechanical properties and immobilized biofunctionality. The controlled delivery of a buoyant photoinitiator, eosin Y, through a glass frit filter results in the formation and subsequent propagation of a polymer reaction front that is self-sustained and able to propagate through the monomeric mixture. Propagation of this front results in monomer depletion, leading to variations in cross-linking, as well as spatial gradients of elastic modulus and immobilized concentrations of the YRGDS cell adhesion ligand within PEGDA hydrogels. Furthermore, the magnitudes of the resulting gradients are controlled through alterations in polymerization conditions. Preliminary in vitro cell-culture studies demonstrate that the gradients generated stimulate directed 2D cell growth on the surface of PEGDA hydrogels. By day 14, fibroblast aggregates spread roughly twice as far in the direction parallel to the slope of the gradient as compared to the perpendicular direction. The presented technique has great potential in controlling gradients of mechanical properties and immobilized biofunctionality for directing and guiding 3D cell behavior within tissue-engineered scaffolds.     

Controlled microscale diffusion gradients in quiescent extracellular fluid

Microchannels offer a means of establishing concentration gradients of soluble factors over micron length scales representative of those in tissues. Here, we report the development of a microfluidic channel system wherein a hydrogel has been patterned to generate temporally and spatially stable concentration gradients of multiple solutes in quiescent extracellular fluid. The fluorophore Alexa Fluor 488 and a fluorescent glucose analog are used as probes to illustrate the generation of stable, reproducible, and linear probe concentration gradients. A method is described for estimating the diffusivity and hydrogel permeability of a solute from in situ imaging data. Concentration gradients are also generated in the presence of a mouse insulinoma cell line to demonstrate the compatibility of the system with living cells. The net transport and metabolism rate of the glucose analog is found to be heterogeneous and independent of the applied extracellular gradient. This system may be suitable for the study of cell response to various extracellular gradients of soluble factors.     

Rapid detection of bacterial growth in blood cultures by bioluminescent assay of bacterial ATP

A method for rapid detection of bacterial growth in blood cultures by bioluminescent assay of bacterial ATP was developed. Samples from blood cultures were treated with a blood-lysing detergent combined with an ATP-hydrolyzing enzyme to destroy blood cell ATP. Blood cell ATP which was bound to cell debris and escaped the ATPase activity was then separated from the bacteria on Percoll density gradients. Bacterial ATP from the bacterial layer was determined by the firefly bioluminescence system. In simulated blood cultures inoculated with 10 CFU of bacteria per ml of blood, bacterial ATP levels exceeded the detection limit (10(-10) M) after 6 to 10 h of incubation. This ATP level corresponds to approximately 10(4) CFU of bacteria per ml.     

Systemic trafficking of macrophages induced by bone cement particles in nude mice

Macrophages play an important role in the biological response to wear particles, which can result in periprosthetic osteolysis and implant loosening. In this study, we demonstrate that polymer particles induce systemic trafficking of macrophages by non-invasive in vivo imaging and immunohistochemistry. The distal femora of nude mice were injected with 10% (w/v) Simplex bone cement (BC) suspensions or saline (PBS). Reporter RAW264.7 macrophages which stably expressed the bioluminescent reporter gene fluc, and the fluorescence reporter gene gfp, were injected intravenously. Bioluminescence imaging was performed immediately and periodically at 2-day intervals until day 14. Compared to the non-operated contralateral femora, the bioluminescent signal of femora injected with BC suspension increased 4.7+/-1.6 and 7.8+/-2.9-fold at day 6 and 8, respectively. The same values for PBS group were 1.2+/-0.2 and 1.4+/-0.5, respectively. The increase of bioluminescence of the BC group was significantly greater than the PBS group at day 8 (p<0.05) and day 6 (p<0.1). Histological study confirmed the presence of reporter macrophages within the medullary canal of mice that received cement particles. Modulation of the signaling mechanisms that regulate systemic macrophage trafficking may provide a new strategy for mitigating the chronic inflammatory response and osteolysis associated with wear debris.     

Patterning and transferring hydrogel-encapsulated bacterial cells for quantitative analysis of synthetically engineered genetic circuits

We describe a hydrogel patterning and transferring (HPT) method that facilitates the quantitative analysis of synthetically engineered genetic circuits within bacterial cells. The HPT method encapsulates cells in the alginate hydrogel patterns by using polydimethylsiloxane (PDMS) template. Then, the hydrogel-encapsulated cell patterns are transferred onto an agarose hydrogel substrate that encapsulates inducer chemicals or bacterial cells. Using the HPT method, we demonstrate that inducers in the agarose hydrogel substrate regulate gene expression of the patterned cells for qualitative analysis by activating the promoters of fluorescence protein genes. In addition, we demonstrate that the HPT method can be used for the analysis of the cross-talk between genetic circuits and the concentration-dependent gene expression and regulation because the agarose hydrogel substrate can produce concentration gradients of inducers. Lastly, we demonstrate that the HPT method can be applied to investigating intercellular communication between neighboring cells with a wide range of cell densities. Since the HPT method is simple to deal with but versatile and powerful to quantitatively analyze genetic circuits in living cells in many controllable manners, we believe that the method can be widely used for the rapid advancement of synthetic, molecular, and systems biology.     

Construction of a bioluminescent mycobacterium and its use for assay of antimycobacterial agents.

To show, as a model system, that mycobacteria can express heterologous luciferase genes and that bioluminescence can be a rapid method of measuring antimycobacterial activity, a bioluminescent form of Mycobacterium smegmatis was made by transformation with a Mycobacterium-Escherichia coli shuttle vector containing the luxAB genes from Vibrio harveyi. The antimycobacterial effects of antibiotics and biocides could be assayed in real time by using bioluminescent M. smegmatis.     

Developing MR reporter genes: promises and pitfalls

MR reporter genes have the potential to monitor transgene expression non-invasively in real time at high resolution. These genes can be applied to interrogate the efficacy of gene therapy, to assess cellular differentiation, cell trafficking, and specific metabolic activity, and also assess changes in the microenvironment. Efforts toward the development of MR reporter genes have been made for at least a decade, but, despite these efforts, the field is still in its early developmental stage. This reflects the fact that there are potential pitfalls, caused by the low sensitivity of detection, the need for substrates with their associated undesirable pharmacokinetics, and/or the difficult and, in some cases, delayed interpretation of signal changes. Nevertheless, significant progress has been made during the last few years. Whereas enzyme-based reporters were initially applied to NMR spectroscopic monitoring of changes in phosphor and fluorine metabolism, MRI-based approaches are now emerging that rely on: (1) enzyme-based cleavage of functional groups that block water (proton) exchange or protein binding of MR contrast agents; (2) expression of surface receptors that enable binding of specific MR contrast agents; (3) expression of para- and anti-ferromagnetic (metallo)proteins involved with iron metabolism, such as tyrosinase, transferrin receptor, and ferritin. After an introduction to the basic principles of designing promoters, expression vectors, and cloning of transgenes, a fresh look is provided on the use of reporter genes for optical (including bioluminescent) and nuclear imaging, with which MR reporter genes compete. Although progress in the use of MR reporter genes has been slow, newer strategies that use metalloproteins or alternative contrast mechanisms, with no need for substrates, promise rapid growth potential for this field.     

Acinetobacter bioreporter assessing heavy metals toxicity

This work was conducted to employ a whole cell-based biosensor to monitor toxicity of heavy metals in water and wastewater. An isolate of industrial wastewater bacterium, Acinetobacter sp. DF4, was genetically modified with lux reporter gene to create a novel bioluminescent bacterial strain, designated as DF4/PUTK2. This bioreporter can investigate the toxicity through light inhibition due to cell death or metabolic burden and the specific stress effects of the tested soluble materials simultaneously. The use of Acinetobacter DF4/PUTK2 as a bioluminescent reporter for heavy metal toxicity testing and for the application of wastewater treatment influent toxicity screening is presented in this study. Among eight heavy metals tested, the bioluminescence of DF4/PUTK2 was most sensitive to Zn, Cd, Fe, Co, Cr followed by Cu in order of decreasing sensitivity. The same pattern of sensitivity was observed when several contaminated water and wastewater effluents were assayed. This work suggested that luxCDABE -marked Acinetobacter bacterium DF4/PUTK2 can be used to bioassay the ecotoxicity of wastewater and effluent samples contaminated with heavy metals. Using this assay, it is possible to pre-select the more toxic samples for further chemical analysis and to discard wastewater samples with low or no inhibition because they are not toxic to the environment.     

Microfluidic assay of endothelial cell migration in 3D interpenetrating polymer semi-network HA-Collagen hydrogel

Cell migration through the extracellular matrix (ECM) is one of the key features for physiological and pathological processes such as angiogenesis, cancer metastasis, and wound healing. In particular, the quantitative assay of endothelial cell migration under the well-defined three dimensional (3D) microenvironment is important to analyze the angiogenesis mechanism. In this study, we report a microfluidic assay of endothelial cell sprouting and migration into an interpenetrating polymer semi-network HA-Collagen (SIPNs CH) hydrogel as ECM providing an enhanced in vivo mimicking 3D microenvironment to cells. The microfluidic chip could provide a well-controlled gradient of growth factor to cells, whereas the hydrogel could mimic a well-defined 3D microenvironment in vivo. (In addition/Furthermore, the microfluidic chip gives a well-controlled gradient of growth factor to cells) For this reason, three types of hydrogel, composed of semi-interpenetrating networks of collagen and hyaluronic acid were prepared, and firstly we proved the role of the hydrogel in endothelial cell migration. The diffusion property and swelling ratio of the hydrogel were characterized. It modulated the migration of endothelial cells in quantified manner, also being influenced by additional synthesis of Matrix metalloproteinase(MMP)-sensitive remodeling peptides and Arginine–glycine–lycinee (RGD) cell adhesion peptides. We successfully established a novel cell migration platform by changing major determinants such as ECM material under biochemical synthesis and under growth factor gradients in a microfluidic manner.     

The use of chitosan based hydrogel for enhancing the therapeutic benefits of adipose-derived MSCs for acute kidney injury

Transplantation of mesenchymal stem cells (MSCs) has been reported a great therapeutic potential for acute kidney injury (AKI). However, the therapeutic benefits are limited due to the low retention and survival of transplanted cells within target sites. In this study, thermosensitive chitosan chloride (CSCl) hydrogel was explored as injectable scaffold for adipose-derived MSCs (ADMSCs) delivery into ischemia/reperfusion (I/R) induced acute kidney injury (AKI). Thermosensitive CSCl hydrogels with/without ADMSCs were injected into the I/R site of rat AKI models. Dihydroethidium staining was used to detect the number of ROS in vivo. In order to track ADMSCs in vivo, ADMSCs were transfected with firefly luciferase and monomeric red fluorescent protein reporter genes (fluc-mrfp). The retention and survival of ADMSC were assessed using bioluminescence imaging, differentiation behaviors of ADMSCs were investigated using immunofluorescent and immunohistochemical staining. Proliferation and apoptosis of host renal cell in vivo were characterized by PCNA and TUNEL staining. Results suggested that CSCl hydrogels could improve the retention and survival of grafted ADMSCs, moreover, CSCl hydrogels could enhance the proliferation activity and reduce apoptosis of host renal cells. At 4 weeks, significant improvement of the renal function, microvessel density and tubular cell proliferation were observed in CSCl hydrogels with ADMSCs groups. Therefore, the application of thermosensitive CSCl hydrogel as scaffold for ADMSCs delivery into renal region could resolve the main obstacle of cell transplantation for acute kidney injury (AKI). Therefore, CSCl hydrogel is a potential cell carrier for treatment of AKI.     

压应力对破骨细胞活化的影响

目的建立3D水凝胶细胞模型,同时采用不同强度、频率、时间压应力作用于破骨细胞,观察压应力对破骨细胞分化的影响,探讨抑制破骨细胞分化的适宜压应力方案。方法 M-CSF和RANKL诱导骨髓单核细胞成破骨细胞,3D细胞水凝胶种板后,次日进行压应力干预,设计不同强度、频率压应力梯度方案对细胞水凝胶进行干预,对照组细胞不进行干预。分组如下:G0(对照组)、G1(1%,0.5 Hz,4 h)、G2(2%,0.5 Hz,4 h)、G3(3%,0.5 Hz,4 h)、G4(1%,1.0 Hz,4 h)、G5(2%,1.0 Hz,4 h)、G6(3%,1.0 Hz,4 h)。在此基础上,通过统计学计算出有效的强度和频率压应力方案后,按照不同干预时间施加压应力,分组如下:D1(4 h)、D2(8 h)、D3(12 h)、D4(16 h),每组两个孔。加压干预结束后即刻收集细胞,将RNA反转录为c DNA,再对样本中的Ctsk mRNA、NFATc1 mRNA、TRACP mRNA、M-CSF mRNA和RANK mRNA进行定量检测。结果RANK表达水平显著依赖于压应力的强度和频率(P0.01);TRACP的表达水平显著依赖于压应力的强度(P0.01),且强度和频率两者对TRACP的表达量出现交互作用(P0.01);Ctsk的表达量水平显著依赖于压应力的强度(P0.05)和频率(P0.01),且两者之间出现交互作用(P0.01)。加压8 h M-CSF的表达量比加压12 h(P0.01)和16 h(P0.05)的低。加压8 h RANK的表达量比加压12 h(P0.05)和16 h(P0.01)的低。加压16 h Ctsk和NFATc1的表达量比加压4 h和8 h的高(P0.05)。结论在3D水凝胶模型中,以1%强度、0.5 Hz频率、8 h的压应力干预方案能够抑制破骨细胞的分化。研究结果为通过适宜的运动预防骨质疏松、提高峰值骨量奠定理论基础。     

Evolution of Oxygen and Glucose Concentration Profiles in a Tissue-Mimetic Culture System of Embryonic Stem Cells

A tissue-mimetic culture system (TMCS) in which cells are sandwiched between two glass slides provides an ideal microenvironment for studying the effects of oxygen and nutrient gradients on cells in culture. A mathematical model was utilized to predict the time course of the development of oxygen and glucose concentration gradients within the TMCS. Oxygen and glucose consumption rates of mouse embryonic stem cells were measured as parameters for the model. The model predicts oxygen and glucose concentration profiles directly using a single experimentally controlled variable, the seeding density of cells within the system. The model predicts that the time required for the gradients to reach steady state is inversely related to the cell density, and the penetration depth of the gradients into the TMCS is inversely related to the square root of the cell density. Experimental oxygen concentration measurements were performed at a cell density of 9.1×10⁶ cells cm⁻³, and the gradient was found to develop to a steady-state profile within 20 min and penetrate approximately 2 mm into the TMCS, consistent with the theoretical predictions. This model and the TMCS provide useful tools for investigating the effect of the metabolic microenvironment on cells in culture.     

The effect of synthetic oxygen carrier-enriched fibrin hydrogel on Schwann cells under hypoxia condition in vitro

Schwann cell (SC), which plays a key role in peripheral nerve regeneration, is one of the most classic supportive cells in neural tissue engineering. However, the biological activity of SCs seeded in nerve scaffolds decays subsequently due to local hypoxia induced by ischemia. Thus, we aimed to investigate whether a synthetic oxygen carrier-enriched fibrin gel would provide a sustained oxygen release to cultured SCs in vitro for overcoming a temporary (48 h) oxygen deprivation. In this study, perfluorotributylamine (PFTBA)-based oxygen carrying fibrin gel was prepared to provide oxygen for SCs under normoxic or hypoxic conditions. The dissolved oxygen within the culture media was measured by a blood-gas analyzer to quantify the time course of oxygen release from the PFTBA-enriched fibrin gel. SCs were cultured in the presence or absence of PFTBA-enriched fibrin gel under normoxic or hypoxic conditions. The tolerance of SCs to hypoxia was examined by a cell apoptosis assay. The growth of cells was characterized using S-100 staining and a CCK-8 assay. The migration of cells was examined using a Transwell chamber. The mRNA of brain-derived neurotrophic factor (BDNF), nerve growth factor (NGF), glial cell derived neurotrophic factor (GDNF), neural cell adhesion molecule (N-CAM) and vascular endothelial growth factor (VEGF) in SCs were assayed by RT-PCR. In addition, SCs cultured in 3D PFTBA-enriched hydrogel were characterized by Live/Dead staining and the mRNA levels of BDNF, NGF, GDNF, N-CAM and VEGF were assayed by RT-PCR. The results showed that the PFTBA-enriched fibrin hydrogel was able to promote cell adhesion, migration, and proliferation under hypoxic conditions. Interestingly, PFTBA applied through the fibrin hydrogel dramatically enhanced the mRNA of BDNF, NGF, GDNF, N-CAM and VEGF under hypoxic condition. These findings highlight the possibility of enhancing nerve regeneration in cellular nerve grafts through PFTBA increased neurotropic secretion in SCs.     

Quantitative Evaluation of Bioluminescence Imaging Based on Radiative Transfer Equation

Bioluminescence imaging is a kind of emerging detection technology at cellular,molecular and genetic level.The most popular bioluminescence imaging model is diffusion approximation(DA).However,because of the ill-posedness of the DA-based inverse problem and the instability of reconstruction algorithms,the location accuracy of the reconstructed sources is low.Radiative transfer equation(RTE),which considers the direction of the photon migration and the effect of absorption and scattering in tissues,can accurately express the transmission of bioluminescent photons through the tissues.In this paper,we studied the bioluminescence imaging based on the RTE.2D simulations were performed,and quantitative evaluation was given by the absolute source position error,the relative source area error and the minimum bounding box.The results of the experiment showed that the imaging quality based on RTE was better than that one based on DA.     

A novel HIV-1 reporter virus with a membrane-bound Gaussia princeps luciferase

HIV-1 reporter viruses are a critical tool for investigating HIV-1 infection. By having a reporter gene incorporated into the HIV-1 genome, the expressed reporter protein acts as a specific tag, thus enabling specific detection of HIV-1 infected cells. Currently existing HIV-1 reporter viruses utilize reporters for the detection of HIV-1 infected cells by a single assay. A reporter virus enabling the detection of viral particles as well as HIV-1 infected cells by two assays can be more versatile for many applications. In this report, a novel reporter HIV-1 was generated by introducing a membrane-anchored form of the Gaussia princeps luciferase gene (mGluc) upstream of the nef gene in the HIV-1(NL4-3) genome using a picornaviral 2A-like sequence. The resulting HIV-1(NL4-3mGluc) virus expresses G. princeps luciferase efficiently on viral membrane and the cell surface of infected human T cell lines and primary peripheral blood mononuclear cells. This HIV-1 reporter is replication competent and the reporter gene mGluc is expressed during multiple rounds of infection. Importantly, viral particles can be detected by bioluminescence and infected cells can be detected simultaneously by bioluminescence and flow cytometric assays. With the versatility of two sensitive detection methods, this novel luciferase reporter has many applications such as cell-based screening for anti-HIV-1 agents or studies of HIV-1 pathogenicity.     

Rapid bioluminescent assay for determining netilmicm and tobramycin concentrations in serum

A rapid bioluminescent assay for determining netilmicin and tobramycin concentrations in serum based on the dose-dependent effect of these agents on the accumulation of extracellular ATP inEscherichia coli LU 14 cultures is presented. This strain ofEscherichia coli is unaffected by antibiotics used in combination with aminoglycosides and it lacks significant ATP-ase activity, which is a prerequisite for extracellular ATP accumulation. ATP was quantified by the firefly bioluminescence system. The accuracy of the bioluminescent assay expressed as mean coefficient of variation over the therapeutic range was 3.2 %; corresponding figures for EMIT and an agar disk diffusion assay were 4.2 % and 4.8 % respectively. All methods used correlated well (r=0.935?0.986) when they were evaluated on clinical serum specimens. The bioluminescent assay requires 25μl serum and results are available within 75 min.     

Bioluminescent Salmonella reverse mutation assay: a screen for detecting mutagenicity with high throughput attributes

Here, we describe the development and evaluation of a novel bioluminescent high-throughput Salmonella reverse mutation assay applicable to the screening of large numbers of small molecules. The bioluminescent Salmonella assay utilizes genetically engineered standard Salmonella tester strains TA98 and TA100 expressing the lux(CDABE) operon from Xenorhabdus luminescence. In principle, the assay employs bioluminescence as a sensor of changes in bacterial metabolism associated with starvation or energy depletion effectively identifying colonies of histidine-independent revertant cells in a high-throughput fashion. The assay provides highly concordant data with the outcome in the standard Salmonella plate incorporation reverse mutation assay. Since the results of the standard Salmonella plate assay are required by various regulatory agencies for approval of new drugs, the bioluminescent Salmonella assay can be effectively used for prioritization of compounds in pharmaceutical drug discovery as well as the evaluation of environmental and industrial chemicals. Because of its high throughput attributes, the assay permits effective, fast and economical screening of a large series of structural analogs enabling the investigation of structure-activity relationships.     

Transgenic reporter mouse for bioluminescence imaging of herpes simplex virus 1 infection in living mice

Bioluminescence imaging allows spatial and temporal progression of viral infection to be detected and quantified in living mice, thereby providing a new approach for studies of viral-host pathogenesis. It has been necessary to construct and validate recombinant reporter viruses that express firefly luciferase to investigate viral replication and spread with this imaging technology. This strategy greatly limits the ability to analyze multiple strains of virus and/or existing viral mutants, and reporter viruses also may be attenuated relative to the respective parental viruses. To facilitate bioluminescence imaging of herpes simplex virus type 1 (HSV-1), we developed a transgenic reporter mouse that uses the promoter from HSV-1 thymidine kinase to control expression of firefly luciferase. Infection with HSV-1 activated expression of firefly luciferase in corneal and flank models of infection, and amounts of bioluminescence increased in proportion to increasing input titers of virus. Imaging could detect infection with three different strains of HSV-1 with the following relative rank order of bioluminescence produced at the site of infection: McKrae > 17 > KOS. Corneal infection with as few as 1 x 10(3) pfu strain McKrae was detectable above background levels. By comparison, infection with vaccinia virus did not affect bioluminescence in the reporter mouse. Collectively, these data establish a new transgenic reporter mouse for infection with HSV-1, thereby enabling in vivo bioluminescence imaging studies of HSV-1 pathogenesis without constructing new reporter viruses.