Transgenic Mice Expressing Luciferase as an Inducible Reporter of Gene Expression

Arguably, whole-cell assays with either constitutive expression of luciferase for cell tracking or inducible expression of luciferase for monitoring gene expression are higher

Fig. 1. p53-RE luciferase induction with doxorubicin in vivo. (Courtesy of Dr. Lidia Sambuccetti, Xenogen Corporation; see Color Plate 7 following p. 302.)

throughput, more simply performed, and easily interpreted because of fewer experimental variables than any in vivo experiment. This deconstructive mode of experimentation has given us a hook into gene function that made sequencing and genomic the technological advance that it represents today. After all, what would be the point of knowing the sequence if pairing that sequence with a gene function or disease state remained a mystery.

However, now is the time to press on and reach though our basic understanding of genomics and animal models and begin to piece together complex biology that surround the process of oncogenesis. One elegant way to approach complex systems biology is to pair in vivo bioluminescent imaging with genetically modified animals and label with a lucifer-ase reporter gene. By building these models, pathways that cross therapeutic areas and the order of genes in these pathways can be determined. By cross-referencing gene function among pathways and therapeutic areas, we can begin to understand gene and protein interactions and their role in disease.

Luciferase has been added to both transgenic mice and rats and knockout or gene-targeted animals. Both endogenous mouse promoter and human promoters and receptors have been labeled with luciferase in these transgenic models (18-20). By creating transgenic animals with genes in the same pathway labeled, it is possible to build datasets around time and location of expression of multiple genes in one pathway.

One example of this would be analysis of VegF and VegFR2. The binding of vascular endothelial growth factor (VEGF) to its receptor (VegFR2) on the surface of endothelial cell membrane triggers a mitogenic signal in the endothelial cells and promotes the angiogenesis process to the proliferation/invasion stage (see Fig. 2). At this stage, endothelial cells start to proliferate to form vascular sprouts. Both endothelial cells and stromal cells secrete a broad range of proteolytic enzymes, which degrade the extracellular matrix and allow growth of blood vessels into the tumor mass. The establishment of circulation to the tumor triggers rapid tumor growth. The endothelial cells in the newly

Fig. 2. Angiogenesis plays a critical role during tumor development. The tumor angiogenesis process is divided into three stages: initiation, proliferation/invasion, and maturation/differentiation. During the initiation stage, tumor cells secret many growth factors, such as VEGF. (Illustration courtesy of Dr. Ning Zhang, Xenogen Corporation.)

Fig. 2. Angiogenesis plays a critical role during tumor development. The tumor angiogenesis process is divided into three stages: initiation, proliferation/invasion, and maturation/differentiation. During the initiation stage, tumor cells secret many growth factors, such as VEGF. (Illustration courtesy of Dr. Ning Zhang, Xenogen Corporation.)

formed blood vessels go through a final stage of maturation/differentiation. Endothelial cells start to differentiate and become quiescence. The new established blood vessels ensure efficient blood flow and nutrient supply to the tumor. This process is an important target of antiagents. (See Fig. 3 and Color Plate 7 following p. 302.)

Another of the most promising transgenic models to date is the spontaneous-tumor-forming mouse model. These genetically modified animals allow researchers to address questions of the environmental effects on gene expression (epigenetic changes), changes as a result of mutation, and tissue- or organ-specific tumors.

Fig. 3. Inducible Vegfr2-luc during tumor development. The Vegfr2-luc transgenic mice were generated in a FVB background. We generated hybrid transgenic mice using FVB Vegfr2-luc and C57 BL/ 6 albino mice. We implanted Lewis lung carcinoma cells (LL/2) into the hybridVegfr2-luc transgenic mice and monitored Vegfr2-luc expression during tumor development using an IVIS™ imaging system (Xenogen, Alameda, CA). We were able to detect tumor-induced Vegfr2-luc expression through imaging. At d 7, the tumor was only a few millimeters in diameter. The Vegfr2-luc expression was ready detectable, indicating that the endothelial cells started to proliferate to form new blood vessels. The signals last through the entire tumor development period because of constant endothelial cell proliferation and growth/reorganization of new formed blood vessels. As the tumor grew larger, the Vegfr2-luc signals started to localize in the tumor periphery, perhaps as a result of necrosis in the center of the tumor mass, especially at the late stage of tumor development. (See Color Plate 7 following p. 302.) (Courtesy of Drs. Tony Purchio, Darlene Jenkins, and Ning Zhang, Xenogen Corporation.)

Fig. 3. Inducible Vegfr2-luc during tumor development. The Vegfr2-luc transgenic mice were generated in a FVB background. We generated hybrid transgenic mice using FVB Vegfr2-luc and C57 BL/ 6 albino mice. We implanted Lewis lung carcinoma cells (LL/2) into the hybridVegfr2-luc transgenic mice and monitored Vegfr2-luc expression during tumor development using an IVIS™ imaging system (Xenogen, Alameda, CA). We were able to detect tumor-induced Vegfr2-luc expression through imaging. At d 7, the tumor was only a few millimeters in diameter. The Vegfr2-luc expression was ready detectable, indicating that the endothelial cells started to proliferate to form new blood vessels. The signals last through the entire tumor development period because of constant endothelial cell proliferation and growth/reorganization of new formed blood vessels. As the tumor grew larger, the Vegfr2-luc signals started to localize in the tumor periphery, perhaps as a result of necrosis in the center of the tumor mass, especially at the late stage of tumor development. (See Color Plate 7 following p. 302.) (Courtesy of Drs. Tony Purchio, Darlene Jenkins, and Ning Zhang, Xenogen Corporation.)

Conditional alleles containing LoxP recombination sites, in conjunction with Cre recom-binase (which can be delivered by a variety of means), allow for spatial and temporal control of gene expression in mouse models. Safran et al. (21) created a mouse strain in which the LoxP-stop-LoxP (L-S-L) cassette preceded the luciferase gene in the ubiquitously expressed ROSA26 locus. In these animals, when crossed with a mouse in which Cre was under the control of a zygotically expressed promoter (EIIA-Cre) or a liver-specific promoter (Albumin-Cre), luciferase was expressed either diffusely (EIIA-Cre) or in a liver-specific manner (Albumin-Cre). In this model, as the Cre-Recombinase is delivered to the cell containing the promoter/ L-S-L/luciferase construct in the genome, the L-S-L sequence is excised and luciferase is activated.

This idea is the basis for organ-specific spontaneous tumor formation that could be monitored noninvasively by bioluminescent imaging. Vooijs et al. (20) used a Cre recom-binase system to create a conditional mouse model for retinoblastoma-dependent sporadic cancer. The Rb tumor suppressor gene, if mutated in mice, causes animals to be predisposed to the development of pituitary tumors. Using the Cre/loxP (20) or the Flp-mediated system (21), the Rb gene can be conditionally inactivated in the pituitary, causing rapid development of melanotroph tumors. These mice were crossed with a transgenic line that carried a lobe-specific promoter (POMC) controlling both luciferase and the Cre recom-binase. Upon the inactivation of the Rb gene, these animals formed pituitary tumors that correlate with the extent that the Rb gene (heterozygous or homozygous inactivation) is inactivated. This method was applied to the generation of a mouse with the potential for the tissue-specific luciferase expression form and luciferase reporter expressed ubiquitously in mice. These authors used the B-actin promoter preceding the luciferase gene in transgenic animals made by pronuclear injection. A Cre-activatable polyadenylated transcription termination site was placed in between the promoter and the luciferase gene. These LucRep animals were then crossed with a conditional oncogenic Kras2 transgenic mouse to demonstrate that the LucRep allele could be used to image spontaneous lung tumor development in vivo (20). In both models, sensitive noninvasive detection facilitated the identification of these genes in the pathway of oncogenesis and the efficacy of therapeutic intervention.

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