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Transgenic Mouse Expressing Optical MicroRNA Reporter for Monitoring MicroRNA-124 Action during Development.

Choi Y, Hwang do W, Kim MY, Kim JY, Sun W, Lee DS - Front Mol Neurosci (2016)

Bottom Line: A method to monitor the action of miRNAs in vivo shall help understand their dynamic behavior during development.Bioluminescence dramatically decreased in the brain between embryonic day 13 and 16 as endogenous miR-124 expression increased, which sustained into adulthood.The inverse relationship of miR-124 expression was observed with luciferase bioluminescence and activity ex vivo as well as in vivo.

View Article: PubMed Central - PubMed

Affiliation: Department of Nuclear Medicine, College of Medicine, Seoul National University Seoul, South Korea.

ABSTRACT
MicroRNAs (miRNAs) fine-tune target protein synthesis by suppressing gene expression, temporally changing along development and possibly in pathological conditions. A method to monitor the action of miRNAs in vivo shall help understand their dynamic behavior during development. In this study, we established a transgenic mouse harboring miR-124 responsive element in their luciferase-eGFP reporter transgenes which enabled monitoring the action of miR-124 in the brain and other organs in vivo by the bioluminescence imaging. The mouse model was produced and verified by imaging ex vivo so that luminescence by luciferase shone and then reduced during development with miR-124 expression. Bioluminescence dramatically decreased in the brain between embryonic day 13 and 16 as endogenous miR-124 expression increased, which sustained into adulthood. The inverse relationship of miR-124 expression was observed with luciferase bioluminescence and activity ex vivo as well as in vivo. Taken together, one can use this microRNA-transgenic mouse to investigate the temporal changes of microRNA action in vivo in the brain as well as in other organs.

No MeSH data available.


Related in: MedlinePlus

Bioluminescence images of candidate miR-124 reporter-expressing transgenic mice. (A) Whole body bioluminescence images of wt and transgenic mice harboring effluc-eGFP-miR-124_3 × PT acquired with D-luciferin injection. The acquisition time of line 67 (left) and line 18 (right) was 10 s and 600 s, respectively. (B)Ex vivo images of effluc reporter activity of fetuses of wt mouse and transgenic mice (line 67) at E16 period. (C)Ex vivo images of major organs excised from a wt and a transgenic mouse (line 67).
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Figure 2: Bioluminescence images of candidate miR-124 reporter-expressing transgenic mice. (A) Whole body bioluminescence images of wt and transgenic mice harboring effluc-eGFP-miR-124_3 × PT acquired with D-luciferin injection. The acquisition time of line 67 (left) and line 18 (right) was 10 s and 600 s, respectively. (B)Ex vivo images of effluc reporter activity of fetuses of wt mouse and transgenic mice (line 67) at E16 period. (C)Ex vivo images of major organs excised from a wt and a transgenic mouse (line 67).

Mentions: Transgenic mice were produced by pronuclear injection of effluc-eGFP-miR-124_3 × PT gene using the CMV promoter to drive the expression reporter transgenes in high amount. We tested whether the transgenic mouse was able to carry out stable germ-line transmission and inheritance. After D-luciferin injection, we found two positive transgenic mice (line 18 and line 67) among 11 founders emitting bioluminescence signals. The origin mouse of transgenic strain C57BL/6 mouse (wild type: wt) was used as negative control (Figure 2A). High bioluminescence signal was observed in the areas of hairless skin such as snout, ears, tail, and feet. In whole body imaging, the bioluminescence was greater in line 67 than in line 18 (Figure 2A). As shown in Figure 2B, the bioluminescence was prominent throughout the whole body of E16. Luciferase signals were also observed in the major isolated organs of transgenic mice, compared to those of wt mouse, which revealed that CMV promoter let the transgenes express in a variety of mouse tissues and cells (Figure 2C). Interestingly, in young adults, lung activity was the most prominent and removal of hairs from the skin made skins shine (Figure 2C). Line 67 was used for further experiment of miR-124 imaging. In ex vivo measurement of isolated organs of line 18, luciferase signals were shown only in the skin but not in the major organs (data not shown). There was no difference in miR-124 expression between wt and transgenic mice in their various areas of the brains on in situ hybridization analysis (Supplementary Figure S1). We concluded that a line of transgenic mouse containing effluc-eGFP-miR-124_3 × PT transgene was established.


Transgenic Mouse Expressing Optical MicroRNA Reporter for Monitoring MicroRNA-124 Action during Development.

Choi Y, Hwang do W, Kim MY, Kim JY, Sun W, Lee DS - Front Mol Neurosci (2016)

Bioluminescence images of candidate miR-124 reporter-expressing transgenic mice. (A) Whole body bioluminescence images of wt and transgenic mice harboring effluc-eGFP-miR-124_3 × PT acquired with D-luciferin injection. The acquisition time of line 67 (left) and line 18 (right) was 10 s and 600 s, respectively. (B)Ex vivo images of effluc reporter activity of fetuses of wt mouse and transgenic mice (line 67) at E16 period. (C)Ex vivo images of major organs excised from a wt and a transgenic mouse (line 67).
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Figure 2: Bioluminescence images of candidate miR-124 reporter-expressing transgenic mice. (A) Whole body bioluminescence images of wt and transgenic mice harboring effluc-eGFP-miR-124_3 × PT acquired with D-luciferin injection. The acquisition time of line 67 (left) and line 18 (right) was 10 s and 600 s, respectively. (B)Ex vivo images of effluc reporter activity of fetuses of wt mouse and transgenic mice (line 67) at E16 period. (C)Ex vivo images of major organs excised from a wt and a transgenic mouse (line 67).
Mentions: Transgenic mice were produced by pronuclear injection of effluc-eGFP-miR-124_3 × PT gene using the CMV promoter to drive the expression reporter transgenes in high amount. We tested whether the transgenic mouse was able to carry out stable germ-line transmission and inheritance. After D-luciferin injection, we found two positive transgenic mice (line 18 and line 67) among 11 founders emitting bioluminescence signals. The origin mouse of transgenic strain C57BL/6 mouse (wild type: wt) was used as negative control (Figure 2A). High bioluminescence signal was observed in the areas of hairless skin such as snout, ears, tail, and feet. In whole body imaging, the bioluminescence was greater in line 67 than in line 18 (Figure 2A). As shown in Figure 2B, the bioluminescence was prominent throughout the whole body of E16. Luciferase signals were also observed in the major isolated organs of transgenic mice, compared to those of wt mouse, which revealed that CMV promoter let the transgenes express in a variety of mouse tissues and cells (Figure 2C). Interestingly, in young adults, lung activity was the most prominent and removal of hairs from the skin made skins shine (Figure 2C). Line 67 was used for further experiment of miR-124 imaging. In ex vivo measurement of isolated organs of line 18, luciferase signals were shown only in the skin but not in the major organs (data not shown). There was no difference in miR-124 expression between wt and transgenic mice in their various areas of the brains on in situ hybridization analysis (Supplementary Figure S1). We concluded that a line of transgenic mouse containing effluc-eGFP-miR-124_3 × PT transgene was established.

Bottom Line: A method to monitor the action of miRNAs in vivo shall help understand their dynamic behavior during development.Bioluminescence dramatically decreased in the brain between embryonic day 13 and 16 as endogenous miR-124 expression increased, which sustained into adulthood.The inverse relationship of miR-124 expression was observed with luciferase bioluminescence and activity ex vivo as well as in vivo.

View Article: PubMed Central - PubMed

Affiliation: Department of Nuclear Medicine, College of Medicine, Seoul National University Seoul, South Korea.

ABSTRACT
MicroRNAs (miRNAs) fine-tune target protein synthesis by suppressing gene expression, temporally changing along development and possibly in pathological conditions. A method to monitor the action of miRNAs in vivo shall help understand their dynamic behavior during development. In this study, we established a transgenic mouse harboring miR-124 responsive element in their luciferase-eGFP reporter transgenes which enabled monitoring the action of miR-124 in the brain and other organs in vivo by the bioluminescence imaging. The mouse model was produced and verified by imaging ex vivo so that luminescence by luciferase shone and then reduced during development with miR-124 expression. Bioluminescence dramatically decreased in the brain between embryonic day 13 and 16 as endogenous miR-124 expression increased, which sustained into adulthood. The inverse relationship of miR-124 expression was observed with luciferase bioluminescence and activity ex vivo as well as in vivo. Taken together, one can use this microRNA-transgenic mouse to investigate the temporal changes of microRNA action in vivo in the brain as well as in other organs.

No MeSH data available.


Related in: MedlinePlus