Weisheng Zhang

Rapid in vivo functional analysis of transgenes in mice using whole body imaging of luciferase expression.

The use of transgenic animals in biomedical research is increasing rapidly and may be the best means of determining gene function. Generating transgenic animals typically requires time-consuming screening processes, and gene function is assessed by an array of difficult phenotypic and biochemical assays performed ex vivo. To address the unmet need in transgenic research for functional assays performed with ease in living animals, we demonstrate here that in vivo detection of luciferase enzyme as a transcriptional reporter facilitates rapid screening for both the presence and function of transgenes in intact living mice. Using this approach we identified three bioluminescent transgenic founders where the transgene consisted of the heme oxygenase promoter fused to the modified coding sequence of the luciferase gene. These founders were identified from 183 pups and confirmed by PCR analysis. Identification of HO-1-luc homozygotes from back-crossed F2 littermates was then accelerated by in vivo imaging. In another transgenic mouse line, where the transgene was comprised of the bone morphogenic-4 (BMP4) promoter fused to the modified luciferase gene, we were able to identify transgenic animals and in each line we were able to visualize patterns of expression in living animals over time. The light production from these transgenic mice indicated that the desired DNA fragment was functional and different expression profiles apparent at different ages and after gene induction.

Selection of potential therapeutics based on in vivo spatiotemporal transcription patterns of heme oxygenase-1

Abstract. Heme oxygenase (HO), a key catabolic enzyme in the conversion of heme to bilirubin, is an ideal target for reducing bilirubin production and preventing pathological jaundice in newborn infants. Metalloporphyrins (Mps) have been well characterized as competitive inhibitors of HO and have been evaluated as potential chemopreventive agents for neonatal jaundice. However, in addition to reducing HO activity, many Mps have been shown to increase HO-1 transcription, which would likely reduce their potential therapeutic utility. The differential effects of Mps on the transcription of HO-1 were therefore evaluated in living transgenic (Tg) reporter mice. Of the compounds evaluated, we observed that zinc bis-glycol porphyrin (ZnBG), a potent inhibitor of HO enzyme activity, did not alter HO-1 transcription patterns in Tg mice. Whole body images of HO-1 transcription patterns did, however; reveal increases in HO-1 transcription in Tg mice after treatment with other Mps, heme and cadmium chloride (CdCl2). Intravenous injections of CdCl2 resulted in expression patterns that differed in tempo and location from those observed in Tg mice treated with intraperitoneal injections. Spatiotemporal analyses of transcriptional regulation in living animals accelerated the assessment of an adverse effect of Mps by revealing different patterns of HO-1 transcription. Among the known inhibitors of HO enzyme activity that were evaluated in this study, ZnBG did not significantly affect HO-1 transcription and therefore may be well suited for the prevention of neonatal jaundice.

Bioluminescence for biological sensing in living mammals.

Noninvasive in vivo assays are required for the study of biological processes that are dynamic involving intact organ systems and complex physiologic changes, such as tissue oxygenation. Optical methods that use external light sources in the near infrared (nir) have been developed for oxygenation determinations and have widespread application in medicine and biomedical research (Jobsis, 1977; Benaron et al., 1997). Further refinements in these methods are providing powerful tools for research; however, access to in vivo information relating to some physiologic changes can not be assessed using nir monitoring and imaging. To address this unmet need for additional real-time bioassays, we have developed an in vivo method utilizing bioluminescent reporters, or photoproteins, as indicators of biological functions (Contag et al., 1995). In contrast to other optical methods which use external sources of light, the photoproteins provide an internal source of light that can be monitored externally as an indicator of biological processes such as infection and gene expression (Contag et al., 1995, 1996, 1997).

In vivo activation of the human CYP3A4 promoter in mouse liver and regulation by pregnane X receptors

Human cytochrome P450 3A4 (CYP3A4) is responsible for the metabolism of numerous xenobiotics in the human liver. We have examined the activation of the human CYP3A4 promoter in mouse liver by using in vivo bioluminescent imaging (BLI). Transcription of the CYP3A4 promoter occurs as a result of a ligand binding to a nuclear orphan receptor, pregnane X receptor (PXR), followed by dimerization with another nuclear receptor, retinoid X receptor (RXR). Since this heterodimer then binds to xenobiotic response elements to activate transcription of CYP3A4, we examined a 13kb promoter region of CYP3A4 for responsiveness to dexamethasone and rifampicin. A reporter vector CYP3A4-luc was constructed consisting of the CYP3A4 promoter driving the firefly luciferase gene. This DNA was injected into the tail veins of mice, and reporter gene expression was monitored in the liver region using BLI. Treatment of transfected mice with dexamethasone resulted in a 188-fold induction of luciferase, whereas treatment with rifampicin resulted in a 68-fold induction. Co-injection with a human PXR expression vector resulted in a dramatic increase in rifampicin-induced activity and a smaller increase of dexamethasone-induced activity. Co-injection of an antisense murine PXR construct with the CYP3A4-luc reduced both the dexamethasone- and rifampicin-induced responses, thus demonstrating that the murine PXR receptor can participate in the regulation of the human CYP3A4 promoter in mice. The approach described here will be of general use in studying the regulation of nuclear receptors in vivo.

Effects of Metalloporphyrins on Heme Oxygenase-1 Transcription: Correlative Cell Culture Assays Guide In Vivo Imaging

Heme oxygenase (HO) is the rate-limiting step in the heme degradation pathway and is a potential target for the control, or prevention, of pathologic jaundice in neonates. Metalloporphyrins (Mps), a diverse set of synthetic derivatives of heme, can competitively inhibit the HO enzymes. However, certain Mps are phototoxic and some increase transcription of HO-1, the inducible HO isozyme. Therefore, effective development of this class of compounds as therapeutics for treating pathologic jaundice will require rapid and integrated biological screens to identify the most efficacious and safe Mps. To study the safety of these compounds, we assessed their cytotoxic effects and measured luciferase activity by bioluminescent imaging (BLI) as an index of HO-1 transcription, first in live cell cultures and then in living transgenic reporter mice. A total of 12 Mps were first evaluated in the correlative cell culture assay. Based on results from this study, 2 Mps, zinc protoporphyrin (ZnPP) and zinc bis glycol porphyrin (ZnBG), were selected for further studies in the live animal model. In vitro BLI showed ZnPP to be a strong inducer of HO-1 transcription in comparison to ZnBG, which showed minimal induction. Cytotoxicity studies revealed that ZnPP was phototoxic, whereas ZnBG had no effect on cell viability. In vivo BLI showed that both ZnPP and ZnBG had minimal effects on the levels of HO-1 transcription in the animals. Furthermore, serum enzyme assays indicated that neither caused detectable liver toxicity. These findings, and especially those with ZnBG, support the use of selected Mps as therapies for pathologic jaundice. Coupling the high throughput advantage of cell culture with the capability of imaging for whole-body temporal analyses could accelerate and refine the preclinical phases of drug development. Thus, this study serves as a model for understanding the effects of specific compounds in relation to defined targets using an integrated approach.

PERSISTENT INDUCTION OF CYTOCHROME P450 (CYP)1A ENZYMES BY 3-METHYLCHOLANTHRENE IN VIVO IN MICE IS MEDIATED BY SUSTAINED TRANSCRIPTIONAL ACTIVATION OF THE CORRESPONDING PROMOTERS

There is significant human exposure to polycyclic aromatic hydrocarbons (PAHs), many of which are potent carcinogens. Cytochrome P450 (CYP)1A enzymes play key roles in the metabolic activation of PAHs to carcinogenic metabolites. We previously showed persistent induction of CYP1A enzymes by 3-methylcholanthrene (MC) in vivo in rodents. In this study, we tested the hypothesis that MC elicits persistent induction of CYP1A1 and 1A2 in vivo by mechanisms entailing sustained transcriptional activation of the corresponding promoters. Adult male wild type (WT) (Cd-1) mice, transgenic mice expressing the human CYP1A1 promoter or the mouse CYP1A2 promoter were treated with the vehicle corn oil (CO) or the carcinogenic PAH, 3-methylcholanthrene (MC), once daily for 4days, and luciferase reporter gene expression was determined at 1, 8, 15, and 22days after MC withdrawal by bioluminescent imaging. Pulmonary and hepatic endogenous expression of CYP1A1 and 1A2 was also determined at the enzymatic, protein, and mRNA levels. The major findings were that MC elicited marked enhancement in the luciferase expression in the CYP1A1-luc as well CYP1A2-luc transgenic mice that was sustained for up to 22days, the magnitude of induction being more pronounced in the CYP1A1-luc mice. MC also caused persistent induction of endogenous CYP1A1 and 1A2 expression in the WT, CYP1A1-luc, and 1A2-luc mice for up to 22days. In conclusion, our results support the hypothesis that MC elicits sustained CYP1A1 and 1A2 expression by sustained transcriptional activation of the corresponding promoters. Thus, these novel transgenic models should be very useful for further understanding of the molecular mechanisms of persistent CYP1A induction, in relation to PAH-mediated carcinogenesis.

Visualizing Drug Efficacy In Vivo

Many enzymes are therapeutic targets for drug discovery, whereas other enzymes are important for understanding drug metabolism and pharmacokinetics during compound testing in animals. Testing of drug efficacy and metabolism in an animal model requires the measurement of disease endpoints as well as assays of enzyme activity in specific tissues at selected time points during treatment. This requires the removal of tissue and biochemical assays. Techniques to noninvasively assess drug effects on enzyme activity using imaging technology would facilitate understanding of drug efficacy, pharmacokinetics, and drug metabolism. Using a commercially available cytochrome P−450 3A substrate whose oxidized product is a luciferase substrate, we show for the first time that cytochrome P−450 enzyme activity can be measured in vivo in real time by bioluminescent imaging. This imaging approach could be applicable to study drug effects on therapeutic target enzymes, as well as drug metabolism enzymes.

Functional imaging: monitoring heme oxygenase-1 gene expression in vivo

The regulation of genetic elements can be monitored in living animals using photoproteins as reporters. Heme oxygenase (HO) is the key catabolic enzyme in the heme degradation pathway. Here, HO expression serves as a model for in vivo functional imaging of transcriptional regulation of a clinically relevant gene. HO enzymatic activity is inhibited by heme analogs, metalloporphyrins, but many members of this family of compounds also activate transcription of the HO-1 promoter. The degree of transcriptional activation by twelve metalloporphyrins, differing at the central metal and porphyrin ring substituents, was evaluated in both NIH 3T3 stable lines and transgenic animals containing HO-1 promoter-luciferase gene fusions. In the correlative cell culture assays, the metalloporphyrins increased transcription form the full length HO promoter fusion to varying degrees, but none increased transcription from a truncated HO-1 promoter. These results suggested that one or both of the two distal enhancer elements located at -4 and -10 Kb upstream from transcriptional start are required for HO-1 induction by heme and its analogs. The full-length HO-1-luc fusion was then evaluated as a transgene in mice. It was possible to monitor the effects of the metalloporphyrins, SnMP and ZnPP, in living animals over time. This spatiotemporal analyses of gene expression in vivo implied that alterations in porphyrin ring substituents and the central metal may affect the extent of gene activation. These data further indicate that using photoprotein reporters, subtle differences in gene expression can be monitored in living animals.

Functional analysis of tumor cell growth and clearance in living animals

Evaluation of antineoplastic therapies would be enhanced by sensitive methods that noninvasively asses both tumor location and neoplastic growth kinetics in living animals. Since light is transmitted through mammalian tissues, it was possible to externally monitor growth and regression of luciferase labeled murine tumor cells engrafted into immunodeficient mice. External quantification of tumor burden revealed the biological impact of the chemotherapeutic agent cyclophosphamide on the kinetics of tumor growth in living animals. Therapeutic activity was apparent but this drug did not eliminate the NIH 3T3 cell signal over the 28 d time course. This novel, noninvasive system allowed sensitive, real time spatiotemporal analyses of neoplastic cell growth and may facilitate rapid optimization of effective therapeutic treatment regimes.

An Systematic Approach for the Evaluation of Therapeutic Agents for Neonatal Hyperbilirubinemia † 1017

An Systematic Approach for the Evaluation of Therapeutic Agents for Neonatal Hyperbilirubinemia † 1017