Limits...
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

Validation of luciferase expression of a miR-124 reporter transgenic mouse. (A) In a young adult mouse, quantitative RT-PCR of tissue RNAs of brain and major organs revealed prominent expression of miR-124 normalized to that of U6. The expression of other organs was represented using a fold increase relative to the value 1 of the brain [means ± SD (n = 3)]. (B) At E16 period of a transgenic mouse, luciferase transgene expressed as shown and counterstained by DAPI on whole body immunohistochemistry (C). Tissue sections of major organs of a transgenic mouse showed distribution of cells counterstained with DAPI (blue) and propensity of luciferase staining (red). Compared with highest lung and moderate heart/kidney activities, luciferase of brain and liver was scarce on confocal microscopy at 400× magnification. The scale bars represent 50 μm.
© Copyright Policy
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC4940420&req=5

Figure 3: Validation of luciferase expression of a miR-124 reporter transgenic mouse. (A) In a young adult mouse, quantitative RT-PCR of tissue RNAs of brain and major organs revealed prominent expression of miR-124 normalized to that of U6. The expression of other organs was represented using a fold increase relative to the value 1 of the brain [means ± SD (n = 3)]. (B) At E16 period of a transgenic mouse, luciferase transgene expressed as shown and counterstained by DAPI on whole body immunohistochemistry (C). Tissue sections of major organs of a transgenic mouse showed distribution of cells counterstained with DAPI (blue) and propensity of luciferase staining (red). Compared with highest lung and moderate heart/kidney activities, luciferase of brain and liver was scarce on confocal microscopy at 400× magnification. The scale bars represent 50 μm.

Mentions: Real-time PCR was done with the total RNA of the brain and other organs to determine the expression of miR-124. In young adult mouse, miR-124 expression was approximately 100-fold higher in the brain, compared to the other organs (lung, heart, liver, kidney, and spleen) (Figure 3A). Luciferase was detected using immunohistochemistry with transgenic embryo sections and with major organs of the young adult mouse (Figures 3B,C). In the whole body of E16, luciferase transgene was expressed throughout the entire body (Figure 3B). The distribution was uneven, but due to the difference of cell density between organs, the relative density or rarity could not be documented. In the young adults, luciferase counter-stained with DAPI showed evenly distributed immunoreactivity in major organs, among which the lungs showed the highest expression (Figure 3C). As we expected, the luciferase was rarely seen in the brain and was also barely seen in the liver in young adult mice. This ex vivo study showed that luciferase was expressed in other organs with the least miR-124 expression and luciferase was suppressed in the brain (and liver) in young adult periods.


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)

Validation of luciferase expression of a miR-124 reporter transgenic mouse. (A) In a young adult mouse, quantitative RT-PCR of tissue RNAs of brain and major organs revealed prominent expression of miR-124 normalized to that of U6. The expression of other organs was represented using a fold increase relative to the value 1 of the brain [means ± SD (n = 3)]. (B) At E16 period of a transgenic mouse, luciferase transgene expressed as shown and counterstained by DAPI on whole body immunohistochemistry (C). Tissue sections of major organs of a transgenic mouse showed distribution of cells counterstained with DAPI (blue) and propensity of luciferase staining (red). Compared with highest lung and moderate heart/kidney activities, luciferase of brain and liver was scarce on confocal microscopy at 400× magnification. The scale bars represent 50 μm.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4940420&req=5

Figure 3: Validation of luciferase expression of a miR-124 reporter transgenic mouse. (A) In a young adult mouse, quantitative RT-PCR of tissue RNAs of brain and major organs revealed prominent expression of miR-124 normalized to that of U6. The expression of other organs was represented using a fold increase relative to the value 1 of the brain [means ± SD (n = 3)]. (B) At E16 period of a transgenic mouse, luciferase transgene expressed as shown and counterstained by DAPI on whole body immunohistochemistry (C). Tissue sections of major organs of a transgenic mouse showed distribution of cells counterstained with DAPI (blue) and propensity of luciferase staining (red). Compared with highest lung and moderate heart/kidney activities, luciferase of brain and liver was scarce on confocal microscopy at 400× magnification. The scale bars represent 50 μm.
Mentions: Real-time PCR was done with the total RNA of the brain and other organs to determine the expression of miR-124. In young adult mouse, miR-124 expression was approximately 100-fold higher in the brain, compared to the other organs (lung, heart, liver, kidney, and spleen) (Figure 3A). Luciferase was detected using immunohistochemistry with transgenic embryo sections and with major organs of the young adult mouse (Figures 3B,C). In the whole body of E16, luciferase transgene was expressed throughout the entire body (Figure 3B). The distribution was uneven, but due to the difference of cell density between organs, the relative density or rarity could not be documented. In the young adults, luciferase counter-stained with DAPI showed evenly distributed immunoreactivity in major organs, among which the lungs showed the highest expression (Figure 3C). As we expected, the luciferase was rarely seen in the brain and was also barely seen in the liver in young adult mice. This ex vivo study showed that luciferase was expressed in other organs with the least miR-124 expression and luciferase was suppressed in the brain (and liver) in young adult periods.

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