Immunofluorescence analysis confirmed the multifocal expression of Afp in the liver, in what appeared to be hepatocytes

Immunofluorescence analysis confirmed the multifocal expression of Afp in the liver, in what appeared to be hepatocytes. expression profile oflucwas identical to that of endogenousAfpgene during development. As early as two month after exposure to DEN, BLI revealed multifocal signals in the liver, long before the appearance of histologically apparent neoplastic lesions. By six months, BL and PET dual imaging showed strong signals in malignant HCC. By serendipity, strong BL signal was also detected in adult testes, a previously unknown site of Afp expression. == Conclusion == TheHCRmodel enables longitudinal monitoring of liver tumor development and progression, providing a powerful tool in developing chemoprevention and therapeutic strategies for HCC. Keywords:positron emission tomography, bioluminescence imaging, hepatocarcinogenesis, hepatocellular carcinoma, testes == Introduction == Hepatocellular carcinoma (HCC) is the fifth most common type of human cancer and the third most common 6-Bnz-cAMP sodium salt cause of cancer death worldwide. In North America, it is one of the 6-Bnz-cAMP sodium salt few tumor types whose incidence is usually increasing, bucking the pattern of continuing reduction for many other tumors. Common HCC arises in a setting of chronic hepatitis with contamination by hepatitis B and C viruses responsible for most HCC cases in humans [1,2]. Although the pathogenic causes are well known, no effective treatment is usually available for most HCC patients except for surgical resection [3]. Historically, studying naturally occurring HCC in model organisms, particularly mice, have been instrumental in advancing our understanding of etiology CSMF of HCC. Genetically defined mouse models are increasingly used as powerful tools to evaluate genetic factors and therapeutic strategies for HCC [4-6]. However, because of the stochastic and non-visible nature of HCC occurrence, the detection and analysis of HCC from these models often require the termination of the animals, precluding the real time assessment of the behavior of individual HCC tumor under a natural microenvironment and/or over a defined period of time. In vivo imaging in small laboratory 6-Bnz-cAMP sodium salt animals is an emerging and promising technology that can be used to facilitate noninvasive, longitudinal monitoring of naturally occurring tumors. In mice, two types of reporter-based imaging modalities, the luciferase (Luc) reporter-based in vivo bioluminescence imaging (BLI) and the thymidine kinase (TK) -based Positron Emission Tomography (PET) imaging, respectively, are particularly useful. BLI has been widely used in cancer imaging often with multiple xenograft tumor-based systems where tumor cells express Luc. It provides a reliable, sensitive, and rapid modality for noninvasively tracking of tumor status in mice [7-9]. On the other hand, PET is usually a highly quantitative detection method that also provides three-dimensional information for the imaging target in the context of its surrounding [10-12]. In order to monitor HCC in mice by BLI and PET imaging, we decided to produce a mouse model in which Luc and TK are expressed specifically in HCC by placing the coding sequences of Luc and TK under the control of the promoter of Alpha-fetoprotein (Afp) encoding gene. Afp, a secreted polypeptide, is usually a serum tumor marker for HCC. It is highly expressed in fetal liver in both rodents and humans but is not expressed in normal adult liver. Interestingly, this protein is usually dramatically re-expressed in vast majority of HCCs [13]. Thus, it is a unique HCC tumor specific marker in both mice and humans. Here we took advantage of the dynamic regulation of Afp expression and generated a novel reporter mouse model that enables dual modality imaging of early and late stages of hepatocarcinogenesis in vivo. == Materials and Methods == == Gene Targeting in Mouse Embryonic Stem (ES) Cells and Generation of Knock-in Mice == A targeting vector,pAfp-tv1, was constructed using theBACmodification system [14,15] as described [16]. 6-Bnz-cAMP sodium salt This vector was designed to introduce a fusion gene cassette, designatedPuroTK-Luc, consisting of the coding sequences for both the fruit travel luciferase (Luc) and thePuroTK[19], linked by an internal ribosomal entry site (IRES) along with aloxP-PGKneo-loxPcassette [20] in between the A and T of the translation start codon of theAfpgene (Detail information for the plasmid construction will be made available upon request). This vector was electroporated into ES cells and targeted clones were identified by the standard mini-Southern approach. Two of these targeted clones were injected into blastocytes. Heterozygous mice carrying the targeted allele were obtained. Once heterozygous knock-in.