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Transgenic mouse model expressing P53(R172H), luciferase, EGFP, and KRAS(G12D) in a single open reading frame for live imaging of tumor.

Ju HL, Calvisi DF, Moon H, Baek S, Ribback S, Dombrowski F, Cho KJ, Chung SI, Han KH, Ro SW - Sci Rep (2015)

Bottom Line: However, differential transcriptional and translational regulation between the tumor-initiating gene and the reporter gene can result in inconsistency between the actual tumor size and the size indicated by the imaging assay.A strong correlation was found between the bioluminescent signal and actual tumor size.Interestingly, all liver tumors induced by P53(R172H) and KRAS(G12D) in the model were hepatocellular adenomas.

View Article: PubMed Central - PubMed

Affiliation: 1] Liver Cirrhosis Clinical Research Center, Yonsei University College of Medicine, Seoul, Korea [2] Brain Korea 21 Project for Medical Science College of Medicine, Yonsei University, Seoul, Korea.

ABSTRACT
Genetically engineered mouse cancer models allow tumors to be imaged in vivo via co-expression of a reporter gene with a tumor-initiating gene. However, differential transcriptional and translational regulation between the tumor-initiating gene and the reporter gene can result in inconsistency between the actual tumor size and the size indicated by the imaging assay. To overcome this limitation, we developed a transgenic mouse in which two oncogenes, encoding P53(R172H) and KRAS(G12D), are expressed together with two reporter genes, encoding enhanced green fluorescent protein (EGFP) and firefly luciferase, in a single open reading frame following Cre-mediated DNA excision. Systemic administration of adenovirus encoding Cre to these mice induced specific transgene expression in the liver. Repeated bioluminescence imaging of the mice revealed a continuous increase in the bioluminescent signal over time. A strong correlation was found between the bioluminescent signal and actual tumor size. Interestingly, all liver tumors induced by P53(R172H) and KRAS(G12D) in the model were hepatocellular adenomas. The mouse model was also used to trace cell proliferation in the epidermis via live fluorescence imaging. We anticipate that the transgenic mouse model will be useful for imaging tumor development in vivo and for investigating the oncogenic collaboration between P53(R172H) and KRAS(G12D).

No MeSH data available.


Related in: MedlinePlus

Characteristics of liver tumors harvested from 2PLEASE mice.(A) A typical focus of altered hepatocytes detected in the liver of a 2PLEASE mouse (H&E staining, upper left panel) already presenting as a mass forming hepatocellular adenoma. Higher magnification (upper middle panel) reveals enlarged hepatocytes with small lipid vacuoles. Massive glycogen storage is evident in the PAS staining (upper right panel). Another example of a glycogen-storing focus in the liver (PAS staining, lower left panel). Fluorescence imaging shows GFP expression in cells in the nodule (lower middle panel), confirming transgene expression following Cre-mediated DNA excision. Beneath the region containing green fluorescent cells are normal hepatocytes that are GFP negative. Hepatocytes in this focus are also PAS-reactive (lower right panel). Scale bars, 250 μm for left panels and 50 μm for middle and right panels. (B) Comparison of mRNA levels of selected genes. Quantitative RT-PCR was performed to compare the expression levels of the indicated genes between normal liver tissue (white bar) and liver tumors (black bar). Relative expression levels are shown. Single asterisks indicate p < 0.05 and double asterisks indicate p < 0.01.
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f3: Characteristics of liver tumors harvested from 2PLEASE mice.(A) A typical focus of altered hepatocytes detected in the liver of a 2PLEASE mouse (H&E staining, upper left panel) already presenting as a mass forming hepatocellular adenoma. Higher magnification (upper middle panel) reveals enlarged hepatocytes with small lipid vacuoles. Massive glycogen storage is evident in the PAS staining (upper right panel). Another example of a glycogen-storing focus in the liver (PAS staining, lower left panel). Fluorescence imaging shows GFP expression in cells in the nodule (lower middle panel), confirming transgene expression following Cre-mediated DNA excision. Beneath the region containing green fluorescent cells are normal hepatocytes that are GFP negative. Hepatocytes in this focus are also PAS-reactive (lower right panel). Scale bars, 250 μm for left panels and 50 μm for middle and right panels. (B) Comparison of mRNA levels of selected genes. Quantitative RT-PCR was performed to compare the expression levels of the indicated genes between normal liver tissue (white bar) and liver tumors (black bar). Relative expression levels are shown. Single asterisks indicate p < 0.05 and double asterisks indicate p < 0.01.

Mentions: Histological examination of liver tumors harvested from the 2PLEASE mice revealed mass-forming foci of altered hepatocytes. The lesions showed the typical morphology of glycogen-storing foci. Hepatocytes in the foci were enlarged, owing to massive glycogen storage (as indicated by a positive periodic acid-Schiff (PAS) reaction) and the presence of fat vacuoles (Fig. 3A). All tumor lesions examined were diagnosed as hepatocellular adenomas, based on published criteria31. Progression to carcinoma was not observed, even in tumors of ~1 cm in diameter that were harvested at 8 months post-adenovirus administration.


Transgenic mouse model expressing P53(R172H), luciferase, EGFP, and KRAS(G12D) in a single open reading frame for live imaging of tumor.

Ju HL, Calvisi DF, Moon H, Baek S, Ribback S, Dombrowski F, Cho KJ, Chung SI, Han KH, Ro SW - Sci Rep (2015)

Characteristics of liver tumors harvested from 2PLEASE mice.(A) A typical focus of altered hepatocytes detected in the liver of a 2PLEASE mouse (H&E staining, upper left panel) already presenting as a mass forming hepatocellular adenoma. Higher magnification (upper middle panel) reveals enlarged hepatocytes with small lipid vacuoles. Massive glycogen storage is evident in the PAS staining (upper right panel). Another example of a glycogen-storing focus in the liver (PAS staining, lower left panel). Fluorescence imaging shows GFP expression in cells in the nodule (lower middle panel), confirming transgene expression following Cre-mediated DNA excision. Beneath the region containing green fluorescent cells are normal hepatocytes that are GFP negative. Hepatocytes in this focus are also PAS-reactive (lower right panel). Scale bars, 250 μm for left panels and 50 μm for middle and right panels. (B) Comparison of mRNA levels of selected genes. Quantitative RT-PCR was performed to compare the expression levels of the indicated genes between normal liver tissue (white bar) and liver tumors (black bar). Relative expression levels are shown. Single asterisks indicate p < 0.05 and double asterisks indicate p < 0.01.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4306974&req=5

f3: Characteristics of liver tumors harvested from 2PLEASE mice.(A) A typical focus of altered hepatocytes detected in the liver of a 2PLEASE mouse (H&E staining, upper left panel) already presenting as a mass forming hepatocellular adenoma. Higher magnification (upper middle panel) reveals enlarged hepatocytes with small lipid vacuoles. Massive glycogen storage is evident in the PAS staining (upper right panel). Another example of a glycogen-storing focus in the liver (PAS staining, lower left panel). Fluorescence imaging shows GFP expression in cells in the nodule (lower middle panel), confirming transgene expression following Cre-mediated DNA excision. Beneath the region containing green fluorescent cells are normal hepatocytes that are GFP negative. Hepatocytes in this focus are also PAS-reactive (lower right panel). Scale bars, 250 μm for left panels and 50 μm for middle and right panels. (B) Comparison of mRNA levels of selected genes. Quantitative RT-PCR was performed to compare the expression levels of the indicated genes between normal liver tissue (white bar) and liver tumors (black bar). Relative expression levels are shown. Single asterisks indicate p < 0.05 and double asterisks indicate p < 0.01.
Mentions: Histological examination of liver tumors harvested from the 2PLEASE mice revealed mass-forming foci of altered hepatocytes. The lesions showed the typical morphology of glycogen-storing foci. Hepatocytes in the foci were enlarged, owing to massive glycogen storage (as indicated by a positive periodic acid-Schiff (PAS) reaction) and the presence of fat vacuoles (Fig. 3A). All tumor lesions examined were diagnosed as hepatocellular adenomas, based on published criteria31. Progression to carcinoma was not observed, even in tumors of ~1 cm in diameter that were harvested at 8 months post-adenovirus administration.

Bottom Line: However, differential transcriptional and translational regulation between the tumor-initiating gene and the reporter gene can result in inconsistency between the actual tumor size and the size indicated by the imaging assay.A strong correlation was found between the bioluminescent signal and actual tumor size.Interestingly, all liver tumors induced by P53(R172H) and KRAS(G12D) in the model were hepatocellular adenomas.

View Article: PubMed Central - PubMed

Affiliation: 1] Liver Cirrhosis Clinical Research Center, Yonsei University College of Medicine, Seoul, Korea [2] Brain Korea 21 Project for Medical Science College of Medicine, Yonsei University, Seoul, Korea.

ABSTRACT
Genetically engineered mouse cancer models allow tumors to be imaged in vivo via co-expression of a reporter gene with a tumor-initiating gene. However, differential transcriptional and translational regulation between the tumor-initiating gene and the reporter gene can result in inconsistency between the actual tumor size and the size indicated by the imaging assay. To overcome this limitation, we developed a transgenic mouse in which two oncogenes, encoding P53(R172H) and KRAS(G12D), are expressed together with two reporter genes, encoding enhanced green fluorescent protein (EGFP) and firefly luciferase, in a single open reading frame following Cre-mediated DNA excision. Systemic administration of adenovirus encoding Cre to these mice induced specific transgene expression in the liver. Repeated bioluminescence imaging of the mice revealed a continuous increase in the bioluminescent signal over time. A strong correlation was found between the bioluminescent signal and actual tumor size. Interestingly, all liver tumors induced by P53(R172H) and KRAS(G12D) in the model were hepatocellular adenomas. The mouse model was also used to trace cell proliferation in the epidermis via live fluorescence imaging. We anticipate that the transgenic mouse model will be useful for imaging tumor development in vivo and for investigating the oncogenic collaboration between P53(R172H) and KRAS(G12D).

No MeSH data available.


Related in: MedlinePlus