Cancer represents one of the greatest health risks worldwide. Consequently, there is a growing need for developing novel therapeutics and new advances in animal tumour modelling. However, despite much progress in this field, the development of clinically relevant animal models that permit rapid and sensitive monitoring of early tumour growth and subsequent metastasis remains an on-going challenge 
Many conventional animal tumour models used in the development of anticancer treatments involve injection of human tumour cells into immunocompromised mice 
followed by standard calliper measurements to assess tumour size, usually as an end-point measurement, after the animal has been sacrificed. These models are fairly limited and research has been on-going to develop a genetically marked tumour that would enable non-invasive monitoring of the tumour parameters by in vivo
imaging based on light emission from luciferase-expressing cells or fluorescence from GFP-expressing cells 
. The use of genetically marked tumour cells in an animal cancer model has a number of advantages. Primarily, it allows one to monitor the efficacy of therapeutic interventions such as drug, gene or cell therapies more easily than with conventional models. It facilitates tracking of tumour parameters, such as size and development, as well as enables highly sensitive visualisation of early metastasis and the evaluation of minimal residual disease after therapy 
. It also permits the use of sequential measurements to follow tumour size during treatment so that longitudinal studies can be performed to analyse the effects of therapies over time giving more reliable information and reducing the number of experimental animals 
In past studies, a variety of different methods have been employed to endow tumour cells with detectable markers 
. The most effective method for delivering genes to cells is the use of vectors derived from modified viruses 
. However, despite the advantages of this gene delivery system there are also significant limitations, mainly related to integration of the vector into the cell genome, the potential immunogenicity of viral encoding genes as well as loss of long-term expression of the reporter gene. It would be of great interest, therefore, to develop a non-viral gene delivery system that can mediate prolonged reporter gene expression in an animal tumour model. An effective way to achieve this goal is to use a plasmid DNA (pDNA) expression system which can be maintained as a functional, episomal entity once it has been delivered to cells of the tumour model and provide them with good detectable levels of marker gene expression throughout their lifetime 
Previous in vivo
studies involving pDNA vectors have shown that viral promoters, such as the cytomegalovirus (CMV) promoter is able to provide the highest levels of transgene expression initially 
but is followed with a subsequent decline in expression within two months 
. This decline in expression is promoter-dependent and likely to be the result of transcriptional silencing of the promoter 
. Indeed, CpG methylation of the CMV promoter in various plasmid vectors has been found to have a negative effect on transgene expression both in vitro
and in vivo
Recently, we and others have shown that a pDNA vector comprising a combination of a mammalian, tissue-specific promoter with a nuclear scaffold/matrix attachment region (S/MAR) element can promote long-term episomal expression in vitro
and in vivo
. The S/MAR element provides a specific association of the vector with the nuclear matrix via scaffold attachment factor-A (SAF-A), tethering the vector to the chromosome scaffold during mitosis and bringing the plasmid into close contact with the cell’s replication machinery, therefore creating mitotic stability and maintaining the plasmid as an epigenetic entity through hundreds of cell divisions 
. The S/MAR element has been shown to have a protective effect on methylation-sensitive sites in the α1-antitrypsin (AAT) liver-specific promoter 
, but has no such effect on the CMV promoter, highlighting that a mammalian rather than a viral promoter is more suitable for long-term transgene expression with this vector.
An S/MAR-containing plasmid has been developed for application to the liver by the utilisation of a liver-specific promoter, AAT, and has been shown to persist and express the luciferase transgene episomally over 6 months in hepatocytes 
. Given the long-term expression of these episomally maintained plasmids, an S/MAR based vector in combination with a mammalian promoter would appear to be ideal for use as a genetic marker of tumour cells.
Plasmids containing an S/MAR sequence and a CMV promoter have previously been successfully transfected into CHO 
, HaCat 
, HeLa 
, K562 leukaemia cells, U251 glioma 
and primary fibroblast 
and have been shown to replicate and to be maintained as extra-chromosomal episomes.
The work described here shows, for the first time, the use of an episomally maintained, pUbC-S/MAR plasmid, mediating persistent luciferase transgene expression to generate genetically labelled tumour cell lines which give rise to different cancers when applied in vivo. The cell lines used are a human hepatocellular carcinoma cell-line Huh7, which is derived from a patient with hepatocellular carcinoma and a human pancreatic carcinoma cell-line, MIA-PaCa2.