Phenotypic changes of transformed cells.

<p>A. Morphological properties of transgenic GFF cells. Phase contrast micrographs were shown at 200× magnification. B. Proliferation curves of wild-type GFF cells, LSL-hK-ras^G12D-IRES-HSV1-tk and Lox-hK-ras^G12D-IRES-HSV1-tk GFF cells to indicate enhanced proliferation. 2000 cells per well were seeded in the 96-well plate and incubated for indicated time. The cells were determined by MTT assay. C. Quantification of colony formation between LSL-hK-ras^G12D-IRES-HSV1-tk and Lox-hK-ras^G12D-IRES-HSV1-tk GFF cells. 1, LSL-hK-ras^G12D-IRES-HSV1-tk GFF cells. 2, Lox-hK-ras^G12D-IRES-HSV1-tk GFF cells. Columns, average of five random fields per dish from two dishes; bar, standard deviation (*, p<0.01; chi-square test). D. The anchorage-independent growth of transgenic cells. Cells were suspended in methylcellulose to evaluate anchorage-independent growth potential over 21 days. Bright field micrographs were shown at 100× magnification (2 weeks) and 200× magnification (3 weeks).</p

Activating hK-ras expression in LSL-hK-ras^G12D-IRES-HSV1-tk GFF cells by Adenovirus -Cre-GFP.

<p>A. Morphology of GFP positive cells 48-Cre-GFP. Phase contrast (Left panel) and florescent micrographs (Right panel) were shown at 100× magnification. B. Efficient excision of the LSL cassette in the cell culture. PCR analysis of genomic DNA prepared from the different GFF cells. The wild type allele was devoted the 279 bp fragment. The LSL-hKras^G12D-IRES-HSV1-TK allele was devoted 2.5 kb fragment. The Lox- hKras^G12D-IRES-HSV1-TK allele was devoted the 320 bp fragment. 1: Lox- hK-ras^G12D-IRES-HSV1-tk GFF cells, 2: LSL-hK-ras^G12D-IRES-HSV1-tk GFF cells, 3: wild-type GFF cells. C. mRNA of HSV1-tk in GFF cells. Marker: 100 bp ladder, 1: LSL-hK-ras^G12D-IRES-HSV1-tk GFF was infected with Adenovirus-Cre-GFP 48 h. 2: LSL-hK-ras^G12D-IRES-HSV1-tk GFF cells. 3: Lox- hK-ras^G12D-IRES-HSV1-tk GFF cells, 4: wild-type GFF cells, N: negative control (H₂O as template). D. Dependence of ganciclovir concentrations against GFF cell viability using MTT assays. The results were expressed as the percentage of living cells in treated conditions at various concentrations of ganciclovir with respect to ganciclovir-free cultures. E. Specific reduction of mutant hK-ras transcripts in GFF cells. BstNI digestion cuts the WT K-ras allele to produce a 156-bp DNA fragment, whereas the mutant hK-ras allele remains uncut to produce a 186-bp DNA fragment. 1: wild-type GFF cells, 2: LSL-hK-ras^G12D-IRES-HSV1-tk GFF cells, 3: Lox- hK-ras^G12D-IRES-HSV1-tk GFF cells, 4: LSL-hK-ras^G12D-IRES-HSV1-tk 48 h post infection.</p

Schematic representation of the conditional hK-Ras^G12D construct.

<p>A, Targeting strategy of knock-in of the hK-ras^G12D to goat K-ras exon 1 locus and its activation by Cre; B, Schematic outline of the cloning strategy for the construction of targeting vector: pKO2.1-LSL-hK-ras^G12D-IRES-HSV1-tk.</p

Tumor formation in mice inoculated with Lox-hK-ras^G12D-IRES-HSV1-tk GFF cells.

<p>A. Tumor growth curves of mice bearing Lox-hK-ras^G12D-IRES-HSV1-tk GFF cells. Inset. Dissected tumor masses from the 5 mice. B. Coronal microPET images of nude mouse inoculated with Lox-hK-ras^G12D-IRES-HSV1-tk GFF cells after injection of ¹⁸F-FDG. Red arrow indicates palpable tumor mass. Mouse inoculated with LSL-hK-ras^G12D-IRES-HSV1-tk GFF cells was used as a control.</p