Fig 1.
Sub-cloning of HrasV12 and LTg into pMSCV plasmids.
(A) Genes of interest (i.e. HrasV12 or LTg) were inserted in between MSCV LTRs, and either GFP or RFP gene was used as a tracer gene. (B) Inserts cloned into pMSCV plasmids were confirmed by enzymatic digestions with either BamHI or EcoRI. M: DNA ladder, 1: pMSCV-GFP; 2: pMSCV-HrasV12-GFP; 3: pMSCV-GFPcut; 4: pMSCV-HrasV12-GFPcut; 5:pBABE-HrasV12cut (+ control); 6: pMSCV-RFP; 7: pMSCV-SV40 LTg-RFP; 8: pMSCV-RFPcut 9: pMSCV-SV40 LTg-RFPcut; 10: pBABE-SV40 LTgcut (+ control). White arrows indicate inserts. Sequences of insert were also verified by DNA sequencing.
Fig 2.
Establishment and characterization of GP2-293 derivatives for stable generation of retrovirus.
(A) Confirmation of successful GFP expression in GP2-293 cells, and (B) Confirmation of successful RFP expression in GP2-293 cells after introduction of pMSCV plasmids. BF: bright field image; FL: fluorescence image. Scale bare is 400 μm. (C) Immunoblot analysis illustrating stable expression of HrasV12 and SV40 LTg in GP2-293 cell derivatives; 1. BL: GP2-293 blank cell; 2. GFP: GFP containing GP2-293 cell 3. HrasV12-GFP: Hras containing GP2-293 cell; 4. BL: GP2-293 blank cell; 5. RFP: RFP containing GP2-293 cell 6. SV40LTg-RFP: SV40LTg and RFP containing GP2-293 cell; M: Protein ladder. (D) Flow cytometry analysis (FACS) of 1x 105 NIH-3T3 mouse fibroblast after infection with retrovirus produced from GP2-293 derivatives.
Fig 3.
Characterization of genetically modified, retrovirally transduced mESCs.
Representative images from immunoblot analysis: (A) HrasV12, (B) SV40 LTg. (C) CCK cell proliferation assay for 7 days of proliferation period of mESCs and transformed mESCs. (D) Comparison of proliferations at day 7. Mean ± S.D. (n = 3), *, **, and #, ## p<0.05. ANOVA test was performed with Tukey’s post-test using the GraphPad Prism software.
Fig 4.
Characterization of stem cell properties of genetically modified, retrovirally transduced mESCs.
(A) Alkaline phosphatase (AP) staining. Red-colored areas indicate alkaline phosphatase activity. (a). Bright field image of mESCs; AP staining: (b). mESCs; (c). mESC-HrasV12; (d). mESC-SV40 LTg; (e). mESC-HrasV12/SV40 LTg. Scale bare is 10 μm. (B) Immunocytochemistry staining of live-cells expressing SSEA1. Pairs of bright filed and SSEA1 images: (a). mESCs; (b). mESC-HrasV12; (c).mESC-SV40 LTg; (d). mESC-HrasV12/SV40 LTg. The representative images were obtained from fluorescent microscope with TRITC filter. Scale bar is 100 μm.
Fig 5.
Ovarian mass at 31 days after orthotopic inoculation of mESCs transformed by both retroviral HrasV12 and SV40 LTg (mESC-HrasV12/SV40 LTg) into ovarian bursa of C57BL/6 mice.
(A). Abdominal site with ovarian mass (blue circle); (B). Cervix with Uterus, right normal ovary (blue arrow) and left ovarian mass (white arrow); (C). Left ovary with partially intact capsule and mass breaking through the ovarian surface (blue circle).
Fig 6.
Histo-Pathological analysis of representative images (hematoxylin-eosin (H&E) tissue stain).
(A) Ovarian Panel: (a). Normal right (non-injected) ovary (100X). Scale bar is 100 μm; (b). Left Ovarian mass (following orthotopic inoculation with mESC) showing signs of a mature teratoma (small window, 100X) characterized with a focus of mature, keratinizing squamous epithelium within the area depicted inside the box (200X). Scale bar is 100 μm; (c). Ovarian mass (following orthotopic inoculation with mESC-HrasV12/SV40-LTg) showing signs of immature teratoma (small window, 100X) characterized with scattered foci of a high-grade malignant neoplasm within the area depicted inside the box (400X). Scale bar is 100 μm. (B) Breast Panel: (a). Normal murine mammary tissue (100X). Scale bar is 100 μm; (b). Breast mass (following orthotopic inoculation of mESC into cleared inguinal mammary fat pad) exhibiting signs of mature teratoma (small window, 100X) characterized with the respiratory-like epithelium having well formed cilia within the area depicted inside the box (200X). Scale bar is 100 μm.