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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

Transgene expression from the 2PLEASE plasmid upon Cre-mediated DNA excision.(A) Schematic illustration of transgene expression following Cre-mediated DNA excision. The epitope-containing β-geo (β-geo-epi) is expressed from the 2PLEASE plasmid in the absence of Cre. Following Cre-mediated DNA recombination between the two loxP sites, P53R172H, luciferase, EGFP, and KRASG12D are expressed. (B) Fluorescence imaging of cells transfected with the 2PLEASE plasmid in the absence (left) and presence (right) of Cre. (C) Western blotting shows tight regulation of transgene expression of EGFP, P53 and Ras from the 2PLEASE plasmid. The blots were performed under the same experimental condition except for blotting with different primary antibodies. These blots were shown as cropped images (D) Luminescent signals from cells transfected with the 2PLEASE plasmid increased by ~1000-fold when Cre was co-expressed, compared to cells in which Cre was not co-expressed, which displayed a background level of luminescence.
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f1: Transgene expression from the 2PLEASE plasmid upon Cre-mediated DNA excision.(A) Schematic illustration of transgene expression following Cre-mediated DNA excision. The epitope-containing β-geo (β-geo-epi) is expressed from the 2PLEASE plasmid in the absence of Cre. Following Cre-mediated DNA recombination between the two loxP sites, P53R172H, luciferase, EGFP, and KRASG12D are expressed. (B) Fluorescence imaging of cells transfected with the 2PLEASE plasmid in the absence (left) and presence (right) of Cre. (C) Western blotting shows tight regulation of transgene expression of EGFP, P53 and Ras from the 2PLEASE plasmid. The blots were performed under the same experimental condition except for blotting with different primary antibodies. These blots were shown as cropped images (D) Luminescent signals from cells transfected with the 2PLEASE plasmid increased by ~1000-fold when Cre was co-expressed, compared to cells in which Cre was not co-expressed, which displayed a background level of luminescence.

Mentions: To express the two oncogenes and the two reporter genes (four transgenes in total) simultaneously in a single open reading frame, the DNA sequence encoding the Thosea asigna virus (TaV) 2A peptide was inserted between the transgenes, in-frame (Fig. 1A). Because expression of these oncogenes from conception would likely have a detrimental effect on animal development, we chose to control expression of the transgenes using the Cre-mediated recombination strategy. Insertion of the lacZ-neomycin fusion gene (β-geo), flanked by loxP sites, immediately prior to the four transgenes would allow β-geo to be expressed until Cre induces DNA recombination between the loxP sites (Fig. 1A). One concern with this Cre-mediated transgene expression strategy was that abrupt expression of the reporters EGFP and luciferase in an adult tissue following Cre-mediated DNA excision might induce an immune response, leading to a cytotoxic T-lymphocyte (CTL) response directed at cells expressing the proteins2223. To minimize the CTL response, a DNA sequence encoding the predicted immunodominant epitope regions of EGFP and luciferase in C57BL/6 mice2425 was inserted in-frame within the β-geo gene segment, generating the “β-geo-epi” gene, which was placed immediately prior to the four transgenes (Fig. 1A). The resulting plasmid is referred to as the “2PLEASE” plasmid.


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)

Transgene expression from the 2PLEASE plasmid upon Cre-mediated DNA excision.(A) Schematic illustration of transgene expression following Cre-mediated DNA excision. The epitope-containing β-geo (β-geo-epi) is expressed from the 2PLEASE plasmid in the absence of Cre. Following Cre-mediated DNA recombination between the two loxP sites, P53R172H, luciferase, EGFP, and KRASG12D are expressed. (B) Fluorescence imaging of cells transfected with the 2PLEASE plasmid in the absence (left) and presence (right) of Cre. (C) Western blotting shows tight regulation of transgene expression of EGFP, P53 and Ras from the 2PLEASE plasmid. The blots were performed under the same experimental condition except for blotting with different primary antibodies. These blots were shown as cropped images (D) Luminescent signals from cells transfected with the 2PLEASE plasmid increased by ~1000-fold when Cre was co-expressed, compared to cells in which Cre was not co-expressed, which displayed a background level of luminescence.
© Copyright Policy - open-access
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4306974&req=5

f1: Transgene expression from the 2PLEASE plasmid upon Cre-mediated DNA excision.(A) Schematic illustration of transgene expression following Cre-mediated DNA excision. The epitope-containing β-geo (β-geo-epi) is expressed from the 2PLEASE plasmid in the absence of Cre. Following Cre-mediated DNA recombination between the two loxP sites, P53R172H, luciferase, EGFP, and KRASG12D are expressed. (B) Fluorescence imaging of cells transfected with the 2PLEASE plasmid in the absence (left) and presence (right) of Cre. (C) Western blotting shows tight regulation of transgene expression of EGFP, P53 and Ras from the 2PLEASE plasmid. The blots were performed under the same experimental condition except for blotting with different primary antibodies. These blots were shown as cropped images (D) Luminescent signals from cells transfected with the 2PLEASE plasmid increased by ~1000-fold when Cre was co-expressed, compared to cells in which Cre was not co-expressed, which displayed a background level of luminescence.
Mentions: To express the two oncogenes and the two reporter genes (four transgenes in total) simultaneously in a single open reading frame, the DNA sequence encoding the Thosea asigna virus (TaV) 2A peptide was inserted between the transgenes, in-frame (Fig. 1A). Because expression of these oncogenes from conception would likely have a detrimental effect on animal development, we chose to control expression of the transgenes using the Cre-mediated recombination strategy. Insertion of the lacZ-neomycin fusion gene (β-geo), flanked by loxP sites, immediately prior to the four transgenes would allow β-geo to be expressed until Cre induces DNA recombination between the loxP sites (Fig. 1A). One concern with this Cre-mediated transgene expression strategy was that abrupt expression of the reporters EGFP and luciferase in an adult tissue following Cre-mediated DNA excision might induce an immune response, leading to a cytotoxic T-lymphocyte (CTL) response directed at cells expressing the proteins2223. To minimize the CTL response, a DNA sequence encoding the predicted immunodominant epitope regions of EGFP and luciferase in C57BL/6 mice2425 was inserted in-frame within the β-geo gene segment, generating the “β-geo-epi” gene, which was placed immediately prior to the four transgenes (Fig. 1A). The resulting plasmid is referred to as the “2PLEASE” plasmid.

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