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. 1999 Oct;1(4):315-20.
doi: 10.1038/sj.neo.7900053.

A general approach to the non-invasive imaging of transgenes using cis-linked herpes simplex virus thymidine kinase

J G Tjuvajev  1 , A Joshi, J Callegari, L Lindsley, R Joshi, J Balatoni, R Finn, S M Larson, M Sadelain, R G Blasberg
Affiliations

A general approach to the non-invasive imaging of transgenes using cis-linked herpes simplex virus thymidine kinase

J G Tjuvajev et al. Neoplasia. 1999 Oct.

Abstract

Non-invasive imaging of gene expression opens new prospects for the study of transgenic animals and the implementation of genetically based therapies in patients. We have sought to establish a general paradigm to enable whole body non-invasive imaging of any transgene. We show that the expression and imaging of HSV1-tk (a marker gene) can be used to monitor the expression of the LacZ gene (a second gene) under the transcriptional control of a single promoter within a bicistronic unit that includes a type II internal ribosomal entry site. In cells bearing a single copy of the vector, the expression of the two genes is proportional and constant, both in vitro and in vivo. We demonstrate that non-invasive imaging of HSV1-tk gene accurately reflects the topology and activity of the other cis-linked transgene.

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Figures

Figure 1
Figure 1
Genetic analysis ofsubcloned W256 tumor cells transduced with the retroviral vector STLEO. (A) The retroviral vector STLEO. (B) Southern blot analysis of subclones of STLEO-transduced W256 tumor cells show single-copy integration of the intact proviral structure (EB, endogenous bands; LacZ/NeoR probe). (C) Integration analysis (Xhol digest) shows unique integration sites in each clone (WS 3 and 5, as well as 6 and 7, are distinct after digestion with BgIII, data not shown).
Figure 2
Figure 2
IRES-mediated coexpression of the HSV1-tk and lacZ genes in vitro. LacZ gene expression (β-galactosidase enzyme activity, U/g cells) was measured using the ONPG spectrophotometric assay for (A) RG2STLEO and (B) W256STLEO transduced single cell clones, and plotted against measurements of the HSV1-tk gene expression (FIAU/TdR accumulation ratio). The relationship between the two measurements was defined by regression analysis. (C) The same relationship was observed when the RG2STLEO and W256STLEO data sets were combined.
Figure 3
Figure 3
IRES-mediated coexpression of the HSV1-tk and lacZ genes in vivo. (A) Gamma camera images of [131I]FIAU accumulation in W256SLEO and RG2STLEO subcutaneous tumors derived from clonal cell lines, 24 hours after tracer administration reflect HSV1-tk expression. The white dashed line outlines the contours of the animal and tumors with relatively low activity and wild-type control tumors (lowest tumor on the right thigh). Some residual bladder activity is still present in RG2STLEO tumor-bearing rats. Note the difference in [131I]FIAU accumulation between different tumors. The [131I]FIAU images of HSV1-tk expression were also converted into parametric images of lacZ gene expression based on the relationship defined in panel D, and this is indicated by the color-coded intensity bar showing units of β-galactosidase activity (U/g) in panel A. The levels of HSV1-tk gene expression measured in different tumors (% dose/g [131I]FIAU) were plotted against measures of lacZ gene expression (U/g) obtained in corresponding tumor samples for different (B) W256SLEO and (C) RG2STLEO clonal tumors. The relationship between the two measures was defined by regression analysis. (D) The same relationship was observed when the RG2STLEO and W256STLEO data were combined.
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References

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