Review Articles
Assays for noninvasive imaging of reporter gene expression

https://doi.org/10.1016/S0969-8051(99)00021-9Get rights and content

Abstract

Repeated, noninvasive imaging of reporter gene expression is emerging as a valuable tool for monitoring the expression of genes in animals and humans. Monitoring of organ/cell transplantation in living animals and humans, and the assessment of environmental, behavioral, and pharmacologic modulation of gene expression in transgenic animals should soon be possible. The earliest clinical application is likely to be monitoring human gene therapy in tumors transduced with the herpes simplex virus type 1 thymidine kinase (HSV1-tk) suicide gene. Several candidate assays for imaging reporter gene expression have been studied, utilizing cytosine deaminase (CD), HSV1-tk, and dopamine 2 receptor (D2R) as reporter genes. For the HSV1-tk reporter gene, both uracil nucleoside derivatives (e.g., 5-iodo-2′-fluoro-2′-deoxy-1-β-d-arabinofuranosyl-5-iodouracil [FIAU] labeled with 124I, 131I ) and acycloguanosine derivatives {e.g., 8-[18F]fluoro-9-[[2-hydroxy-1-(hydroxymethyl)ethoxy]methyl]guanine (8-[18F]-fluoroganciclovir) ([18F]FGCV), 9-[(3-[18F]fluoro-1-hydroxy-2-propoxy)methyl]guanine ([18F]FHPG)} have been investigated as reporter probes. For the D2R reporter gene, a derivative of spiperone {3-(2′-[18F]-Fluoroethyl)spiperone ([18F]FESP)} has been used with positron emission tomography (PET) imaging. In this review, the principles and specific assays for imaging reporter gene expression are presented and discussed. Specific examples utilizing adenoviral-mediated delivery of a reporter gene as well as tumors expressing reporter genes are discussed.

Introduction

The process of gene expression includes the activation of transcription through a specific promoter, transcription of a particular gene leading to pre-messenger RNA (pre-mRNA), processing to a mature mRNA, transport of mRNA from nucleus to cytoplasm, and eventual translation of mRNA to a protein product. Not all genes are expressed in all cells, leading to a wide variety of cellular functions or phenotypes. Many phenomena, including cellular development, maturation, proliferation, and oncogenesis, can be attributed to differential gene expression. For many applications in animals and humans, the introduction of a gene into target tissue(s) is needed. Radiotracer imaging assays have recently been developed to monitor the expression of reporter genes introduced into target tissue(s).

Molecular biologists have long used reporter genes to study promoter/regulatory elements involved in gene expression, inducible promoters to examine the induction of gene expression, and transgenes containing endogenous promoters fused to a reporter gene to study endogenous gene expression. Once a reporter gene driven by a promoter of choice is introduced into the desired tissue, expression of the reporter gene can be monitored by one of several methods. Conventional methods to monitor reporter gene expression include: (i) tissue biopsy followed by immunohistochemistry or histochemical staining for reporter gene protein, (ii) in situ hybridization with probes targeted for reporter gene mRNA, and (iii) sampling the blood of the living animal in cases in which the reporter gene product is a protein secreted into the animal blood stream (e.g., alkaline phosphatase).

Conventional methods to detect reporter gene expression are limited by their inability to noninvasively determine the location(s) or the magnitude of gene expression in living animals. Approaches using green fluorescent protein (GFP) 17, 43 and luciferase (38) as reporter genes whose products fluoresce when exposed to light, allow localization of reporter gene expression in some living animals. Animals that are transparent to light can be imaged with simple video cameras when these types of reporter genes are used. However, these imaging techniques are very limited because of their lack of generalizability (e.g., GFP would not work with humans) and detailed spatial resolution. In contrast, radiotracer imaging techniques offer the possibility of monitoring the detailed location, magnitude, and persistence of reporter gene expression (with potentially a very high sensitivity) for in vivo use in animals and humans.

Human gene therapy trials would be significantly aided by the ability to determine the location, magnitude, and change in magnitude over time of the expression of delivered therapeutic genes. The use of a reporter gene coupled to the therapeutic gene may allow for the indirect monitoring of expression of the therapeutic gene. Alternately, in some cases such as tumor gene therapy with the herpes simplex type 1 virus thymidine kinase (HSV1-tk) suicide gene, direct monitoring of the HSV1-tk suicide gene without the introduction of a separate reporter gene may also soon be possible.

Section snippets

Principles of imaging reporter gene expression

Figure 1 illustrates how a reporter gene can be used to develop an imaging assay. The accumulation of the reporter probe is dependent directly on expression of the reporter gene. The choice for a promoter for driving reporter gene expression include constitutive or inducible promoters, based on the intended application. Constitutive promoters can be used to produce continuous transcription of a reporter gene. Inducible promoters can be used to provide external control for varying the levels of

Delivery of reporter genes

Ex vivo gene delivery in which target cells are first removed from the host, transfected with a specific gene, and then redelivered to the host is one possible approach for gene delivery applications. Alternatively, gene delivery can be preformed in vivo by using one of several vectors to deliver the gene directly into specific tissue(s) of interest.

Introduction of genes into animals can be accomplished by one of two general methods. Transgenic animals can be made in which every cell in the

Characteristics of the ideal imaging reporter gene/reporter probe

The ideal reporter gene/reporter probe imaging system would have the following characteristics: (a) The reporter gene should be present in mammalian cells, but not expressed (this will prevent an immune response). (b) When expressed, the reporter gene protein should produce specific reporter probe accumulation only in those cells in which it is expressed. (c) When the reporter gene is not expressed, there should be no significant accumulation of reporter probe in cells. (d) There should be no

Cytosine deaminase (CD) reporter gene

CD was one of the first reporter genes to be studied for imaging reporter gene expression (Table 1). CD is found primarily in yeasts and bacteria and its expression is responsible for the deamination of cytosine to form uracil. Mammalian cells lack CD and therefore cannot convert cytosine to uracil. In those cells expressing CD, 5-fluorocytosine is converted to 5-fluorouracil, which is cytotoxic. CD has been used as a suicide gene in animal (48) and human (70) cancer therapy models with

Reporter genes for use with magnetic resonance imaging (MRI)

To be useful with MRI, a reporter gene would have to lead to a change in relaxivity in cells in which the reporter gene is expressed. Intrinsically, MRI has a sensitivity of ∼10−4 to 10−5 M, and does not compete well with PET where the sensitivity is ∼10−10 M. Therefore, a PET-based approach would need much less accumulation of a reporter probe than would an MRI-based approach for a given level of gene expression. However, an MRI-based approach might have an advantage of improved spatial and

Human gene therapy trials

There are many human gene therapy clinical trials being investigated throughout the world (1). Fifteen trials involve the retroviral transfer of the HSV1-tk gene into tumor cells followed by systemic treatment with the pro-drug ganciclovir. These suicide gene therapy protocols are highly dependent on the expression of HSV1-tk in tumor tissues. This approach leads to inhibition of cell replication by inhibiting DNA replication as described previously. No clinical methods currently exist to

Future directions

The development of assays for imaging reporter gene expression are still in their infancy. Much work remains to be done in the development of practical, highly sensitive, and specific quantitative assays for in vivo applications. An important arena of research is the maximization of specific signals from cells expressing low levels of reporter gene in vivo. A systematic search for substrates with enhanced affinity for reporter gene products will be needed. Alternatively, because reporter genes

Acknowledgements

We thank our growing body of undergraduate and graduate students, postdoctoral fellows, research scientists, and faculty who have joined us in bringing together different disciplines to form a new science of biological imaging targeted toward specific cellular events. We sincerely thank K. Akhoon, Ph.D., E. Bauer, M.S., A. Berk, Ph.D., A. Borghei, B.S., A. Chatziioannou, Ph.D., S. Cherry, Ph.D., R. Goldman, B.S., S. Golish, B.S., L.A. Green, M.S., M. Iyer, Ph.D., Q. Liang, Ph.D., D. Mac Laren

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