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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 action reporter vector and its validation. (A) Schematic diagram of miR-124 reporter transgene of lentiviral vector. The lentiviral construct consists of CMV promoter, effluc, IRES, and 3 × PT (triple perfect targets) including three repeat perfect target sequences complementary to mature miR-124. (B) Luminescence produced after 24 h of transient transfection of scramble or miR-124 showed working of effluc in the effluc-eGFP-miR-124_3 × PT transfected HeLa cells. Luciferase activity (photons/second) was quantified using Living image software. Data are represented by the means ± SEM (n = 3).
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Figure 1: MiR-124 action reporter vector and its validation. (A) Schematic diagram of miR-124 reporter transgene of lentiviral vector. The lentiviral construct consists of CMV promoter, effluc, IRES, and 3 × PT (triple perfect targets) including three repeat perfect target sequences complementary to mature miR-124. (B) Luminescence produced after 24 h of transient transfection of scramble or miR-124 showed working of effluc in the effluc-eGFP-miR-124_3 × PT transfected HeLa cells. Luciferase activity (photons/second) was quantified using Living image software. Data are represented by the means ± SEM (n = 3).

Mentions: In order to produce reporter construct for monitoring miR-124 action (previous ID: miR-124a), we generated a construct containing three copies of a miR-124-perfectly matched sequence (miR-124_3 × PT) with the enhanced firefly luciferase (effluc) and enhanced GFP (eGFP) reporter genes in its 3′UTR. Using IRES (internal ribosome entry site) lentiviral vector, effluc-eGFP-miR-124_3 × PT construct co-expressed effluc and GFP genes from a single mRNA transcript. This allowed miR-124 to suppress the expression of two reporter proteins (Figure 1A). In order to validate the constructs, HeLa cells were co-transfected with reporter construct and synthetic scramble or miR-124 precursors. We found that miR-124 overexpression significantly affected luciferase activity of effluc-eGFP-miR-124_3 × PT-infected cells by luciferase assay (5.37 ± 1.85%, p < 0.01, Figure 1B). Effluc-eGFP-miR-124_3 × PT construct was considered suitable for monitoring miR-124 action as well as biogenesis, hence generation of a new transgenic mouse for in vivo monitoring of miR-124 using this construct was further investigated.


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 action reporter vector and its validation. (A) Schematic diagram of miR-124 reporter transgene of lentiviral vector. The lentiviral construct consists of CMV promoter, effluc, IRES, and 3 × PT (triple perfect targets) including three repeat perfect target sequences complementary to mature miR-124. (B) Luminescence produced after 24 h of transient transfection of scramble or miR-124 showed working of effluc in the effluc-eGFP-miR-124_3 × PT transfected HeLa cells. Luciferase activity (photons/second) was quantified using Living image software. Data are represented by the means ± SEM (n = 3).
© Copyright Policy
Related In: Results  -  Collection

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

Figure 1: MiR-124 action reporter vector and its validation. (A) Schematic diagram of miR-124 reporter transgene of lentiviral vector. The lentiviral construct consists of CMV promoter, effluc, IRES, and 3 × PT (triple perfect targets) including three repeat perfect target sequences complementary to mature miR-124. (B) Luminescence produced after 24 h of transient transfection of scramble or miR-124 showed working of effluc in the effluc-eGFP-miR-124_3 × PT transfected HeLa cells. Luciferase activity (photons/second) was quantified using Living image software. Data are represented by the means ± SEM (n = 3).
Mentions: In order to produce reporter construct for monitoring miR-124 action (previous ID: miR-124a), we generated a construct containing three copies of a miR-124-perfectly matched sequence (miR-124_3 × PT) with the enhanced firefly luciferase (effluc) and enhanced GFP (eGFP) reporter genes in its 3′UTR. Using IRES (internal ribosome entry site) lentiviral vector, effluc-eGFP-miR-124_3 × PT construct co-expressed effluc and GFP genes from a single mRNA transcript. This allowed miR-124 to suppress the expression of two reporter proteins (Figure 1A). In order to validate the constructs, HeLa cells were co-transfected with reporter construct and synthetic scramble or miR-124 precursors. We found that miR-124 overexpression significantly affected luciferase activity of effluc-eGFP-miR-124_3 × PT-infected cells by luciferase assay (5.37 ± 1.85%, p < 0.01, Figure 1B). Effluc-eGFP-miR-124_3 × PT construct was considered suitable for monitoring miR-124 action as well as biogenesis, hence generation of a new transgenic mouse for in vivo monitoring of miR-124 using this construct was further investigated.

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