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Besides neuro-imaging, the Thy1-YFP mouse could serve for visualizing experimental tumours, inflammation and wound-healing.

Jósvay K, Winter Z, Katona RL, Pecze L, Marton A, Buhala A, Szakonyi G, Oláh Z, Vizler C - Sci Rep (2014)

Bottom Line: The Thy1 (CD90) cell surface glycoprotein is present on many cell types besides neurons, and is known to be involved in cell adhesion, migration and signal transduction.We hypothesized that Thy1-activating conditions could probably activate the truncated Thy1 regulatory sequences used in the Thy1-YFP construct, resulting in YFP transgene expression outside the nervous system.In the tumour mass, the yellow-green fluorescent stromal cells were clearly distinguishable from 4T1 carcinoma cells stably transfected with red fluorescent protein.

View Article: PubMed Central - PubMed

Affiliation: 1] Institute of Biochemistry, Biological Research Centre of the Hungarian Academy of Sciences, Szeged, Hungary [2] Institute of Pharmaceutical Analysis, Faculty of Pharmacy, University of Szeged, Szeged, Hungary.

ABSTRACT
The B6.Cg-Tg(Thy1-YFP)16Jrs/J transgenic mouse strain, widely used to study neuronal development and regeneration, expresses the yellow fluorescent protein (YFP) in the peripheral nerves and the central nervous system under the control of regulatory sequences of the Thy1 gene. The Thy1 (CD90) cell surface glycoprotein is present on many cell types besides neurons, and is known to be involved in cell adhesion, migration and signal transduction. We hypothesized that Thy1-activating conditions could probably activate the truncated Thy1 regulatory sequences used in the Thy1-YFP construct, resulting in YFP transgene expression outside the nervous system. We demonstrated that the stroma of subcutaneous tumours induced by the injection of 4T1 or MC26 carcinoma cells into BALB/c(Thy1-YFP) mice, carrying the same construct, indeed expressed the YFP transgene. In the tumour mass, the yellow-green fluorescent stromal cells were clearly distinguishable from 4T1 carcinoma cells stably transfected with red fluorescent protein. Local inflammation induced by subcutaneous injection of complete Freund's adjuvant, as well as the experimental wound-healing milieu, also triggered YFP fluorescence in both the BALB/c(Thy1-YFP) and B6.Cg-Tg(Thy1-YFP)16Jrs/J mice, pointing to eventual overlapping pathways of wound-healing, inflammation and tumour growth.

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Examination of the tumor cell and Thy1 promoter activity co-localization.(A–C): Transmitted light, pseudo-green and red fluorescent microscopic image of the tumor formed 35 days after 4T1-red tumor cell injection into the ear pinna of BALB/c(Thy1-YFP) transgenic mice. Photographs of the mice ears were made under light anaesthesia, using a Nikon Eclipse E600 fluorescent microscope. The YFP fluorescence in BALB/c(Thy1-YFP) mice shows co-localization with the red fluorescence of RFP-expressing tumor cells (4× magnification objective lens). (D–G): Transmitted light (F), green (D), red (E) fluorescent and combined (G) microscopic image of the sections of the Thy1-YFP mice ear 7 days after tdTomato expressing 4T1 tumor cell injection into the ear pinna. The red fluorescence of tumor cells is only partly co-localized with YFP fluorescence revealing the different origins of the cells comprising the tumor body. Photographs of the sections were made using a Zeiss Axiovision Z1 fluorescent microscope (20× magnification objective lens).
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f2: Examination of the tumor cell and Thy1 promoter activity co-localization.(A–C): Transmitted light, pseudo-green and red fluorescent microscopic image of the tumor formed 35 days after 4T1-red tumor cell injection into the ear pinna of BALB/c(Thy1-YFP) transgenic mice. Photographs of the mice ears were made under light anaesthesia, using a Nikon Eclipse E600 fluorescent microscope. The YFP fluorescence in BALB/c(Thy1-YFP) mice shows co-localization with the red fluorescence of RFP-expressing tumor cells (4× magnification objective lens). (D–G): Transmitted light (F), green (D), red (E) fluorescent and combined (G) microscopic image of the sections of the Thy1-YFP mice ear 7 days after tdTomato expressing 4T1 tumor cell injection into the ear pinna. The red fluorescence of tumor cells is only partly co-localized with YFP fluorescence revealing the different origins of the cells comprising the tumor body. Photographs of the sections were made using a Zeiss Axiovision Z1 fluorescent microscope (20× magnification objective lens).

Mentions: The effect of tumour growth was tested in BALB/c(Thy1-YFP) mice created by back-crossing the original B6.Cg-Tg(Thy1-YFP)16Jrs/J mouse strain to BALB/c genetic background. Two tumour cell lines were tested, the 4T1 mammary carcinoma and the MC26 colon carcinoma, respectively. In the case of s.c. tumour injections in the ear pinnae, in the first few days the injection site was surrounded by a yellow fluorescent “halo” of cells. From about day 5, a more circumscribed fluorescence was strongly co-localized with the tumour mass. The 4T1 and the MC26 tumours behaved similarly in this respect. YFP expression was detected at an unchanged intensity up to the termination of the experiments, i.e. for at least 35 days (Figure 1). Surprisingly, no YFP fluorescence was detected in the case of metastases of the lung, kidney or peritoneum, probably as an indication of tissue-specific differences in the expression pattern even for the truncated Thy1 gene. For visual partitioning of the tumour cells from the stroma, we established a red fluorescent 4T1 cell line, stably transfected with pEF1α-tdTomato plasmid (Tsien Lab). We injected these cells s.c. into the ears of mice and, before the tumour could cause any considerable discomfort, the mice were sacrificed. Frozen sections from the tumour area were then examined by fluorescent microscopy. The red tumour cells were clearly distinguishable from the yellow-green tumour matrix, suggesting that the fluorescence-based cell sorting of tumour- derived stromal cells for functional studies, including adoptive transfer experiments, or in vivo analyses of tumour stroma formation, would be possible. Importantly this model also could be used for the in vivo imaging of a wide array of non-genetically labelled tumours in immunocompetent or immunodeficient transgenic mice (Figure 2).


Besides neuro-imaging, the Thy1-YFP mouse could serve for visualizing experimental tumours, inflammation and wound-healing.

Jósvay K, Winter Z, Katona RL, Pecze L, Marton A, Buhala A, Szakonyi G, Oláh Z, Vizler C - Sci Rep (2014)

Examination of the tumor cell and Thy1 promoter activity co-localization.(A–C): Transmitted light, pseudo-green and red fluorescent microscopic image of the tumor formed 35 days after 4T1-red tumor cell injection into the ear pinna of BALB/c(Thy1-YFP) transgenic mice. Photographs of the mice ears were made under light anaesthesia, using a Nikon Eclipse E600 fluorescent microscope. The YFP fluorescence in BALB/c(Thy1-YFP) mice shows co-localization with the red fluorescence of RFP-expressing tumor cells (4× magnification objective lens). (D–G): Transmitted light (F), green (D), red (E) fluorescent and combined (G) microscopic image of the sections of the Thy1-YFP mice ear 7 days after tdTomato expressing 4T1 tumor cell injection into the ear pinna. The red fluorescence of tumor cells is only partly co-localized with YFP fluorescence revealing the different origins of the cells comprising the tumor body. Photographs of the sections were made using a Zeiss Axiovision Z1 fluorescent microscope (20× magnification objective lens).
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Related In: Results  -  Collection

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f2: Examination of the tumor cell and Thy1 promoter activity co-localization.(A–C): Transmitted light, pseudo-green and red fluorescent microscopic image of the tumor formed 35 days after 4T1-red tumor cell injection into the ear pinna of BALB/c(Thy1-YFP) transgenic mice. Photographs of the mice ears were made under light anaesthesia, using a Nikon Eclipse E600 fluorescent microscope. The YFP fluorescence in BALB/c(Thy1-YFP) mice shows co-localization with the red fluorescence of RFP-expressing tumor cells (4× magnification objective lens). (D–G): Transmitted light (F), green (D), red (E) fluorescent and combined (G) microscopic image of the sections of the Thy1-YFP mice ear 7 days after tdTomato expressing 4T1 tumor cell injection into the ear pinna. The red fluorescence of tumor cells is only partly co-localized with YFP fluorescence revealing the different origins of the cells comprising the tumor body. Photographs of the sections were made using a Zeiss Axiovision Z1 fluorescent microscope (20× magnification objective lens).
Mentions: The effect of tumour growth was tested in BALB/c(Thy1-YFP) mice created by back-crossing the original B6.Cg-Tg(Thy1-YFP)16Jrs/J mouse strain to BALB/c genetic background. Two tumour cell lines were tested, the 4T1 mammary carcinoma and the MC26 colon carcinoma, respectively. In the case of s.c. tumour injections in the ear pinnae, in the first few days the injection site was surrounded by a yellow fluorescent “halo” of cells. From about day 5, a more circumscribed fluorescence was strongly co-localized with the tumour mass. The 4T1 and the MC26 tumours behaved similarly in this respect. YFP expression was detected at an unchanged intensity up to the termination of the experiments, i.e. for at least 35 days (Figure 1). Surprisingly, no YFP fluorescence was detected in the case of metastases of the lung, kidney or peritoneum, probably as an indication of tissue-specific differences in the expression pattern even for the truncated Thy1 gene. For visual partitioning of the tumour cells from the stroma, we established a red fluorescent 4T1 cell line, stably transfected with pEF1α-tdTomato plasmid (Tsien Lab). We injected these cells s.c. into the ears of mice and, before the tumour could cause any considerable discomfort, the mice were sacrificed. Frozen sections from the tumour area were then examined by fluorescent microscopy. The red tumour cells were clearly distinguishable from the yellow-green tumour matrix, suggesting that the fluorescence-based cell sorting of tumour- derived stromal cells for functional studies, including adoptive transfer experiments, or in vivo analyses of tumour stroma formation, would be possible. Importantly this model also could be used for the in vivo imaging of a wide array of non-genetically labelled tumours in immunocompetent or immunodeficient transgenic mice (Figure 2).

Bottom Line: The Thy1 (CD90) cell surface glycoprotein is present on many cell types besides neurons, and is known to be involved in cell adhesion, migration and signal transduction.We hypothesized that Thy1-activating conditions could probably activate the truncated Thy1 regulatory sequences used in the Thy1-YFP construct, resulting in YFP transgene expression outside the nervous system.In the tumour mass, the yellow-green fluorescent stromal cells were clearly distinguishable from 4T1 carcinoma cells stably transfected with red fluorescent protein.

View Article: PubMed Central - PubMed

Affiliation: 1] Institute of Biochemistry, Biological Research Centre of the Hungarian Academy of Sciences, Szeged, Hungary [2] Institute of Pharmaceutical Analysis, Faculty of Pharmacy, University of Szeged, Szeged, Hungary.

ABSTRACT
The B6.Cg-Tg(Thy1-YFP)16Jrs/J transgenic mouse strain, widely used to study neuronal development and regeneration, expresses the yellow fluorescent protein (YFP) in the peripheral nerves and the central nervous system under the control of regulatory sequences of the Thy1 gene. The Thy1 (CD90) cell surface glycoprotein is present on many cell types besides neurons, and is known to be involved in cell adhesion, migration and signal transduction. We hypothesized that Thy1-activating conditions could probably activate the truncated Thy1 regulatory sequences used in the Thy1-YFP construct, resulting in YFP transgene expression outside the nervous system. We demonstrated that the stroma of subcutaneous tumours induced by the injection of 4T1 or MC26 carcinoma cells into BALB/c(Thy1-YFP) mice, carrying the same construct, indeed expressed the YFP transgene. In the tumour mass, the yellow-green fluorescent stromal cells were clearly distinguishable from 4T1 carcinoma cells stably transfected with red fluorescent protein. Local inflammation induced by subcutaneous injection of complete Freund's adjuvant, as well as the experimental wound-healing milieu, also triggered YFP fluorescence in both the BALB/c(Thy1-YFP) and B6.Cg-Tg(Thy1-YFP)16Jrs/J mice, pointing to eventual overlapping pathways of wound-healing, inflammation and tumour growth.

Show MeSH
Related in: MedlinePlus