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Knock-in Luciferase Reporter Mice for In Vivo Monitoring of CREB Activity.

Akhmedov D, Rajendran K, Mendoza-Rodriguez MG, Berdeaux R - PLoS ONE (2016)

Bottom Line: To overcome disadvantages of the currently used strategies, we created a ROSA26 knock-in CREB reporter mouse line (ROSA26-CRE-luc). cAMP-inducing ligands stimulate the reporter in primary hepatocytes and myocytes from ROSA26-CRE-luc animals.In vivo, these animals exhibit little hepatic CREB activity in the ad libitum fed state but robust induction after fasting.The ROSA26-CRE-luc mouse line is a useful resource to study dynamics of CREB activity longitudinally in vivo and can be used as a source of primary cells for analysis of CREB regulatory pathways ex vivo.

View Article: PubMed Central - PubMed

Affiliation: Department of Integrative Biology and Pharmacology, McGovern Medical School at the University of Texas Health Science Center at Houston (UTHealth), Houston, Texas, United States of America.

ABSTRACT
The cAMP response element binding protein (CREB) is induced during fasting in the liver, where it stimulates transcription of rate-limiting gluconeogenic genes to maintain metabolic homeostasis. Adenoviral and transgenic CREB reporters have been used to monitor hepatic CREB activity non-invasively using bioluminescence reporter imaging. However, adenoviral vectors and randomly inserted transgenes have several limitations. To overcome disadvantages of the currently used strategies, we created a ROSA26 knock-in CREB reporter mouse line (ROSA26-CRE-luc). cAMP-inducing ligands stimulate the reporter in primary hepatocytes and myocytes from ROSA26-CRE-luc animals. In vivo, these animals exhibit little hepatic CREB activity in the ad libitum fed state but robust induction after fasting. Strikingly, CREB was markedly stimulated in liver, but not in skeletal muscle, after overnight voluntary wheel-running exercise, uncovering differential regulation of CREB in these tissues under catabolic states. The ROSA26-CRE-luc mouse line is a useful resource to study dynamics of CREB activity longitudinally in vivo and can be used as a source of primary cells for analysis of CREB regulatory pathways ex vivo.

No MeSH data available.


Related in: MedlinePlus

Validation of a ROSA26-CRE-luc knock-in mouse.(A) Schematic of ROSA26-CRE-luc knock-in construct. (B) Bioluminescence imaging of male ROSA26-CRE-luc knock-in mice, ad libitum fed (ZT0) and after a 6-h fast (ZT6) (5 sec exposure). (C) Quantification of hepatic bioluminescence in indicated region of interest in ROSA26-CRE-luc mice shown in B and four additional littermates (median, 25th and 75th percentile and range indicated, n = 10; ***, p = 0.002 by paired, 2-tailed t-test). (D) Luciferase activity in primary hepatocytes from ROSA26-CRE-luc knock-in mice treated with FSK/ IBMX or glucagon (Glgn) for indicated times. (n = 3 per time point; ***, p<0.001 to un-stimulated control). Panel D is representative of three independent experiments performed in triplicate.
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pone.0158274.g001: Validation of a ROSA26-CRE-luc knock-in mouse.(A) Schematic of ROSA26-CRE-luc knock-in construct. (B) Bioluminescence imaging of male ROSA26-CRE-luc knock-in mice, ad libitum fed (ZT0) and after a 6-h fast (ZT6) (5 sec exposure). (C) Quantification of hepatic bioluminescence in indicated region of interest in ROSA26-CRE-luc mice shown in B and four additional littermates (median, 25th and 75th percentile and range indicated, n = 10; ***, p = 0.002 by paired, 2-tailed t-test). (D) Luciferase activity in primary hepatocytes from ROSA26-CRE-luc knock-in mice treated with FSK/ IBMX or glucagon (Glgn) for indicated times. (n = 3 per time point; ***, p<0.001 to un-stimulated control). Panel D is representative of three independent experiments performed in triplicate.

Mentions: To create ROSA26 knock-in CREB reporter mice, we obtained a validated CREB-activated luciferase reporter plasmid from Promega that contains two full and two half cAMP response elements (CRE) and codon-optimized luc2 fused to a destabilization sequence (PEST) (Fig 1A). We sub-cloned CRE-luc2PEST into the Ai9 ROSA26 targeting vector, which also encodes a CAG-lox-stop-lox-tdTomato transgene that is commonly used as a Cre recombinase reporter [15]. The final allele retains the CAG-LSL-tdTomato cassette and can be simultaneously used as a Cre recombinase reporter when crossing to other tissue-specific knockout lines. We confirmed that tdTomato is expressed in primary hepatocytes from ROSA26-CRE-luc mice upon expression of Cre recombinase in vitro using an adenoviral vector (Ad-Cre) but not Ad-GFP control (S1A Fig). The CREB-activated luciferase transgene is not contingent upon Cre recombinase expression.


Knock-in Luciferase Reporter Mice for In Vivo Monitoring of CREB Activity.

Akhmedov D, Rajendran K, Mendoza-Rodriguez MG, Berdeaux R - PLoS ONE (2016)

Validation of a ROSA26-CRE-luc knock-in mouse.(A) Schematic of ROSA26-CRE-luc knock-in construct. (B) Bioluminescence imaging of male ROSA26-CRE-luc knock-in mice, ad libitum fed (ZT0) and after a 6-h fast (ZT6) (5 sec exposure). (C) Quantification of hepatic bioluminescence in indicated region of interest in ROSA26-CRE-luc mice shown in B and four additional littermates (median, 25th and 75th percentile and range indicated, n = 10; ***, p = 0.002 by paired, 2-tailed t-test). (D) Luciferase activity in primary hepatocytes from ROSA26-CRE-luc knock-in mice treated with FSK/ IBMX or glucagon (Glgn) for indicated times. (n = 3 per time point; ***, p<0.001 to un-stimulated control). Panel D is representative of three independent experiments performed in triplicate.
© Copyright Policy
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC4940169&req=5

pone.0158274.g001: Validation of a ROSA26-CRE-luc knock-in mouse.(A) Schematic of ROSA26-CRE-luc knock-in construct. (B) Bioluminescence imaging of male ROSA26-CRE-luc knock-in mice, ad libitum fed (ZT0) and after a 6-h fast (ZT6) (5 sec exposure). (C) Quantification of hepatic bioluminescence in indicated region of interest in ROSA26-CRE-luc mice shown in B and four additional littermates (median, 25th and 75th percentile and range indicated, n = 10; ***, p = 0.002 by paired, 2-tailed t-test). (D) Luciferase activity in primary hepatocytes from ROSA26-CRE-luc knock-in mice treated with FSK/ IBMX or glucagon (Glgn) for indicated times. (n = 3 per time point; ***, p<0.001 to un-stimulated control). Panel D is representative of three independent experiments performed in triplicate.
Mentions: To create ROSA26 knock-in CREB reporter mice, we obtained a validated CREB-activated luciferase reporter plasmid from Promega that contains two full and two half cAMP response elements (CRE) and codon-optimized luc2 fused to a destabilization sequence (PEST) (Fig 1A). We sub-cloned CRE-luc2PEST into the Ai9 ROSA26 targeting vector, which also encodes a CAG-lox-stop-lox-tdTomato transgene that is commonly used as a Cre recombinase reporter [15]. The final allele retains the CAG-LSL-tdTomato cassette and can be simultaneously used as a Cre recombinase reporter when crossing to other tissue-specific knockout lines. We confirmed that tdTomato is expressed in primary hepatocytes from ROSA26-CRE-luc mice upon expression of Cre recombinase in vitro using an adenoviral vector (Ad-Cre) but not Ad-GFP control (S1A Fig). The CREB-activated luciferase transgene is not contingent upon Cre recombinase expression.

Bottom Line: To overcome disadvantages of the currently used strategies, we created a ROSA26 knock-in CREB reporter mouse line (ROSA26-CRE-luc). cAMP-inducing ligands stimulate the reporter in primary hepatocytes and myocytes from ROSA26-CRE-luc animals.In vivo, these animals exhibit little hepatic CREB activity in the ad libitum fed state but robust induction after fasting.The ROSA26-CRE-luc mouse line is a useful resource to study dynamics of CREB activity longitudinally in vivo and can be used as a source of primary cells for analysis of CREB regulatory pathways ex vivo.

View Article: PubMed Central - PubMed

Affiliation: Department of Integrative Biology and Pharmacology, McGovern Medical School at the University of Texas Health Science Center at Houston (UTHealth), Houston, Texas, United States of America.

ABSTRACT
The cAMP response element binding protein (CREB) is induced during fasting in the liver, where it stimulates transcription of rate-limiting gluconeogenic genes to maintain metabolic homeostasis. Adenoviral and transgenic CREB reporters have been used to monitor hepatic CREB activity non-invasively using bioluminescence reporter imaging. However, adenoviral vectors and randomly inserted transgenes have several limitations. To overcome disadvantages of the currently used strategies, we created a ROSA26 knock-in CREB reporter mouse line (ROSA26-CRE-luc). cAMP-inducing ligands stimulate the reporter in primary hepatocytes and myocytes from ROSA26-CRE-luc animals. In vivo, these animals exhibit little hepatic CREB activity in the ad libitum fed state but robust induction after fasting. Strikingly, CREB was markedly stimulated in liver, but not in skeletal muscle, after overnight voluntary wheel-running exercise, uncovering differential regulation of CREB in these tissues under catabolic states. The ROSA26-CRE-luc mouse line is a useful resource to study dynamics of CREB activity longitudinally in vivo and can be used as a source of primary cells for analysis of CREB regulatory pathways ex vivo.

No MeSH data available.


Related in: MedlinePlus