Figure 1

A mouse model system with EL4-Gax cells. A. The structural organization of the R3Gaxbsr genome in EL4-Gax cells [29]. The EGFP coding sequence was fused with tax cDNA at the 5'-end, resulting in Gax. The Gax gene was linked with a drug resistance gene, bsr, by an internal ribosome entry site (IRES). The U3 region in the MLV LTR was replaced with that in the HTLV-1 LTR. 1 × 10⁶ of EL4-Gax cells cultured in vitro (in vitro, a) were injected into peritoneal cavity of a syngenic C57BL/6J mouse. 3 weeks after challenge, cells were collected from ascitic fluids (in vivo, b) and transferred to the in vitro culture condition for 48 hours (ex vivo, c). B. Left, the expression of Gax protein in living cells was monitored as the intensity of GFP fluorescence by fluorescent-activated cell sorter (FACS); Right, statistical analysis of the GFP mean fluorescent intensity (GFP mfi), after deducting EL4 cells background level. EL4, parent cell line. C. Left, cell lysates were subjected to Western blot analysis with anti-Tax serum, PARP, an indicator of apoptosis or necrosis exhibiting the signature 89 kDa or 50 kDa fragment respectively to see if the in vivo cells were healthy or not, or anti-β-actin antibody as loading control; Right, quantification of Gax and normalized to β-actin with a densitometry software program (NIH-image). D. Left, RT-PCR analysis of several viral and cellular mRNAs.; Right, Real-time PCR analysis of Gax mRNA expression and normalized to 18S ribosomal RNA in EL4-Gax cells with SYBR Green. Error bars indicate SEMs. Data were obtained from four independent experiments analyzing one mouse per experiment, and statistical analysis of the data was performed between the in vivo or ex vivo against the in vitro. *; p < 0.01. **; p < 0.05.