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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

Robust transgene expression in the skin following Cre-mediated DNA excision.(A) Bioluminescence imaging of the dorsal skin of 2PLEASE; R26-Cre-ERT2 double-transgenic mice at 1 week post-treatment with vehicle (left panel) and tamoxifen (right panel). (B) Basal cells in the dorsal skin of these mice were imaged via in vivo green fluorescence imaging. Numerous skin cells in the basal layer expressed EGFP following tamoxifen treatment, while no detectable signals were found in the layer of vehicle-treated mice (Tile-scanned images, scale bars, 200 μm). (C) Average bioluminescent signals from the dorsal skin of mice at the indicated time points after topical treatment with tamoxifen or vehicle. Note that no increases in bioluminescence signals were observed from the tamoxifen-treated mice over several months.
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f6: Robust transgene expression in the skin following Cre-mediated DNA excision.(A) Bioluminescence imaging of the dorsal skin of 2PLEASE; R26-Cre-ERT2 double-transgenic mice at 1 week post-treatment with vehicle (left panel) and tamoxifen (right panel). (B) Basal cells in the dorsal skin of these mice were imaged via in vivo green fluorescence imaging. Numerous skin cells in the basal layer expressed EGFP following tamoxifen treatment, while no detectable signals were found in the layer of vehicle-treated mice (Tile-scanned images, scale bars, 200 μm). (C) Average bioluminescent signals from the dorsal skin of mice at the indicated time points after topical treatment with tamoxifen or vehicle. Note that no increases in bioluminescence signals were observed from the tamoxifen-treated mice over several months.

Mentions: To investigate whether expression of P53R172H and KRASG12D is capable of inducing skin cancer and whether tumor growth in skin can be monitored via in vivo imaging of the co-expressed reporters, 2PLEASE mice were crossed with R26-Cre-ERT2 mice and the double transgenic offspring (2PLEASE; R26-Cre-ERT2) were treated topically with tamoxifen on the dorsal skin following depilation. Bioluminescence imaging performed at one week post-treatment revealed strong signals on the depilated skin of the tamoxifen-treated mice, while background signals were detected from vehicle-treated mice (Fig. 6A). Furthermore, in vivo fluorescence imaging showed that epidermal cells on the basal layer of tamoxifen-treated mice exhibited green fluorescence (Fig. 6B). Thus, bioluminescence and in vivo fluorescence imaging confirmed robust expression of the transgenes in the skin following treatment with tamoxifen. However, no increases in bioluminescent signals were observed over several months in the tamoxifen-treated skin, strongly suggesting that the tumor did not develop in the skin (Fig. 6C). Consistent with the bioluminescence imaging data, no tumors were detected on the skin of 11 double-transgenic mice until 8 months post-tamoxifen treatment (data not shown). Interestingly, one mouse, excluded from the in vivo imaging experiment due to a severe injury to the depilated skin, developed skin tumors at about 2 months post-tamoxifen treatment (see Supplementary Fig. S7 online). Live imaging showed strong bioluminescence and green fluorescence signals from the tumor (Supplementary Fig. S7 online). Histo-pathologic examination revealed that the tumor was a well-differentiated squamous cell carcinoma of the skin. These data suggest that co-expression of KRASG12D and P53R172H is not sufficient to induce skin tumors, which might require an additional tumor-promoting condition, such as inflammation, for efficient development of skin cancer3536.


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)

Robust transgene expression in the skin following Cre-mediated DNA excision.(A) Bioluminescence imaging of the dorsal skin of 2PLEASE; R26-Cre-ERT2 double-transgenic mice at 1 week post-treatment with vehicle (left panel) and tamoxifen (right panel). (B) Basal cells in the dorsal skin of these mice were imaged via in vivo green fluorescence imaging. Numerous skin cells in the basal layer expressed EGFP following tamoxifen treatment, while no detectable signals were found in the layer of vehicle-treated mice (Tile-scanned images, scale bars, 200 μm). (C) Average bioluminescent signals from the dorsal skin of mice at the indicated time points after topical treatment with tamoxifen or vehicle. Note that no increases in bioluminescence signals were observed from the tamoxifen-treated mice over several months.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
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getmorefigures.php?uid=PMC4306974&req=5

f6: Robust transgene expression in the skin following Cre-mediated DNA excision.(A) Bioluminescence imaging of the dorsal skin of 2PLEASE; R26-Cre-ERT2 double-transgenic mice at 1 week post-treatment with vehicle (left panel) and tamoxifen (right panel). (B) Basal cells in the dorsal skin of these mice were imaged via in vivo green fluorescence imaging. Numerous skin cells in the basal layer expressed EGFP following tamoxifen treatment, while no detectable signals were found in the layer of vehicle-treated mice (Tile-scanned images, scale bars, 200 μm). (C) Average bioluminescent signals from the dorsal skin of mice at the indicated time points after topical treatment with tamoxifen or vehicle. Note that no increases in bioluminescence signals were observed from the tamoxifen-treated mice over several months.
Mentions: To investigate whether expression of P53R172H and KRASG12D is capable of inducing skin cancer and whether tumor growth in skin can be monitored via in vivo imaging of the co-expressed reporters, 2PLEASE mice were crossed with R26-Cre-ERT2 mice and the double transgenic offspring (2PLEASE; R26-Cre-ERT2) were treated topically with tamoxifen on the dorsal skin following depilation. Bioluminescence imaging performed at one week post-treatment revealed strong signals on the depilated skin of the tamoxifen-treated mice, while background signals were detected from vehicle-treated mice (Fig. 6A). Furthermore, in vivo fluorescence imaging showed that epidermal cells on the basal layer of tamoxifen-treated mice exhibited green fluorescence (Fig. 6B). Thus, bioluminescence and in vivo fluorescence imaging confirmed robust expression of the transgenes in the skin following treatment with tamoxifen. However, no increases in bioluminescent signals were observed over several months in the tamoxifen-treated skin, strongly suggesting that the tumor did not develop in the skin (Fig. 6C). Consistent with the bioluminescence imaging data, no tumors were detected on the skin of 11 double-transgenic mice until 8 months post-tamoxifen treatment (data not shown). Interestingly, one mouse, excluded from the in vivo imaging experiment due to a severe injury to the depilated skin, developed skin tumors at about 2 months post-tamoxifen treatment (see Supplementary Fig. S7 online). Live imaging showed strong bioluminescence and green fluorescence signals from the tumor (Supplementary Fig. S7 online). Histo-pathologic examination revealed that the tumor was a well-differentiated squamous cell carcinoma of the skin. These data suggest that co-expression of KRASG12D and P53R172H is not sufficient to induce skin tumors, which might require an additional tumor-promoting condition, such as inflammation, for efficient development of skin cancer3536.

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