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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

MiR-124 reduces luciferase activity in the cortical neurons extracted and cultured from transgenic mouse. (A) Cortical neurons from E16 of transgenic mouse were cultured for 10 days in vitro (DIV). Images were displayed with phase-contrast microscope using 4× or 100× magnification (inset). The scale bars represent 200 μm (low-scaled panel) and 50 μm (magnified panel). (B) Immunocytochemistry with anti-MAP2 (microtubule-associated protein-2, red, and labeling neurons) and anti-GFAP (Glial fibrillary acid protein, green, and labeling astrocytes) antibodies counterstained with DAPI were shown with confocal microscope using a 100× magnification. The scale bars represent 100 μm. 95% were MAP2 positive and the other 5% were GFAP positive. (C) Primary cultured cortical neurons which were transfected with scramble, miR-124, or miR-124 inhibitor at DIV 10, showed reduction of luciferase activity only with miR-124. The results were normalized as percent ratios to the scramble treatment. Data were represented by means ± SD (n = 6, ∗∗p < 0.01).
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Figure 5: MiR-124 reduces luciferase activity in the cortical neurons extracted and cultured from transgenic mouse. (A) Cortical neurons from E16 of transgenic mouse were cultured for 10 days in vitro (DIV). Images were displayed with phase-contrast microscope using 4× or 100× magnification (inset). The scale bars represent 200 μm (low-scaled panel) and 50 μm (magnified panel). (B) Immunocytochemistry with anti-MAP2 (microtubule-associated protein-2, red, and labeling neurons) and anti-GFAP (Glial fibrillary acid protein, green, and labeling astrocytes) antibodies counterstained with DAPI were shown with confocal microscope using a 100× magnification. The scale bars represent 100 μm. 95% were MAP2 positive and the other 5% were GFAP positive. (C) Primary cultured cortical neurons which were transfected with scramble, miR-124, or miR-124 inhibitor at DIV 10, showed reduction of luciferase activity only with miR-124. The results were normalized as percent ratios to the scramble treatment. Data were represented by means ± SD (n = 6, ∗∗p < 0.01).

Mentions: Cortical neurons extracted from E16 transgenic mice were cultured in vitro, and confirmed to have transgene genotypes (data not shown). Under phase contrast microscope, these neurons showed neuronal features along with lapsing in vitro days until 10 days in vitro (DIV) (Figure 5A). On immunocytochemistry using antibodies against MAP2 and GFAP, which labeled mature neurons and astrocytes, respectively, the proportion of primary cortical neurons was 94.9 ± 2.5% (Figure 5B). When these cortical neurons were transfected with scramble, miR-124 or miR-124 inhibitor for 24 h, miR-124 transfection alone significantly decreased the luciferase activity by 55.3%, compared to scramble transfection (Figure 5C). MiR-124 inhibitor did not change (increase) luciferase activity.


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)

MiR-124 reduces luciferase activity in the cortical neurons extracted and cultured from transgenic mouse. (A) Cortical neurons from E16 of transgenic mouse were cultured for 10 days in vitro (DIV). Images were displayed with phase-contrast microscope using 4× or 100× magnification (inset). The scale bars represent 200 μm (low-scaled panel) and 50 μm (magnified panel). (B) Immunocytochemistry with anti-MAP2 (microtubule-associated protein-2, red, and labeling neurons) and anti-GFAP (Glial fibrillary acid protein, green, and labeling astrocytes) antibodies counterstained with DAPI were shown with confocal microscope using a 100× magnification. The scale bars represent 100 μm. 95% were MAP2 positive and the other 5% were GFAP positive. (C) Primary cultured cortical neurons which were transfected with scramble, miR-124, or miR-124 inhibitor at DIV 10, showed reduction of luciferase activity only with miR-124. The results were normalized as percent ratios to the scramble treatment. Data were represented by means ± SD (n = 6, ∗∗p < 0.01).
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Figure 5: MiR-124 reduces luciferase activity in the cortical neurons extracted and cultured from transgenic mouse. (A) Cortical neurons from E16 of transgenic mouse were cultured for 10 days in vitro (DIV). Images were displayed with phase-contrast microscope using 4× or 100× magnification (inset). The scale bars represent 200 μm (low-scaled panel) and 50 μm (magnified panel). (B) Immunocytochemistry with anti-MAP2 (microtubule-associated protein-2, red, and labeling neurons) and anti-GFAP (Glial fibrillary acid protein, green, and labeling astrocytes) antibodies counterstained with DAPI were shown with confocal microscope using a 100× magnification. The scale bars represent 100 μm. 95% were MAP2 positive and the other 5% were GFAP positive. (C) Primary cultured cortical neurons which were transfected with scramble, miR-124, or miR-124 inhibitor at DIV 10, showed reduction of luciferase activity only with miR-124. The results were normalized as percent ratios to the scramble treatment. Data were represented by means ± SD (n = 6, ∗∗p < 0.01).
Mentions: Cortical neurons extracted from E16 transgenic mice were cultured in vitro, and confirmed to have transgene genotypes (data not shown). Under phase contrast microscope, these neurons showed neuronal features along with lapsing in vitro days until 10 days in vitro (DIV) (Figure 5A). On immunocytochemistry using antibodies against MAP2 and GFAP, which labeled mature neurons and astrocytes, respectively, the proportion of primary cortical neurons was 94.9 ± 2.5% (Figure 5B). When these cortical neurons were transfected with scramble, miR-124 or miR-124 inhibitor for 24 h, miR-124 transfection alone significantly decreased the luciferase activity by 55.3%, compared to scramble transfection (Figure 5C). MiR-124 inhibitor did not change (increase) luciferase activity.

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