SAMY , a novel mammalian reporter gene derived from Bacillus stearothermophilus α-amylase

Schlatter, Stefan; Rimann, Markus; Kelm, Jens M.; Fussenegger, Martin

doi:10.1016/s0378-1119(01)00824-1

Cited by 144 publications

(140 citation statements)

References 30 publications

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“…Production of the SEAP was quantified in cell culture supernatants (36) and mouse serum (37) as described previously. Luciferase was measured using the Tropix luciferase assay kit according to the manufacturer's protocol (Applied Biosystems).…”

Section: Methodsmentioning

confidence: 99%

Pharmaceutically controlled designer circuit for the treatment of the metabolic syndrome

Hamri

Zwicky

et al. 2012

Proc. Natl. Acad. Sci. U.S.A.

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Synthetic biology has significantly advanced the design of genetic devices that can reprogram cellular activities and provide novel treatment strategies for future gene-and cell-based therapies. However, many metabolic disorders are functionally linked while developing distinct diseases that are difficult to treat using a classic one-drug-one-disease intervention scheme. For example, hypertension, hyperglycemia, obesity, and dyslipidemia are interdependent pathologies that are collectively known as the metabolic syndrome, the prime epidemic of the 21st century. We have designed a unique therapeutic strategy in which the clinically licensed antihypertensive drug guanabenz (Wytensin) activates a synthetic signal cascade that stimulates the secretion of metabolically active peptides GLP-1 and leptin. Therefore, the signal transduction of a chimeric traceamine-associated receptor 1 (cTAAR1) was functionally rewired via cAMP and cAMP-dependent phosphokinase A (PKA)-mediated activation of the cAMP-response element binding protein (CREB1) to transcription of synthetic promoters containing CREB1-specific cAMP response elements. Based on this designer signaling cascade, it was possible to use guanabenz to dose-dependently control expression of GLP-1-Fc mIgG -Leptin, a bifunctional therapeutic peptide hormone that combines the glucagon-like peptide 1 (GLP-1) and leptin via an IgG-Fc linker. In mice developing symptoms of the metabolic syndrome, this three-in-one treatment strategy was able to simultaneously attenuate hypertension and hyperglycemia as well as obesity and dyslipidemia. Using a clinically licensed drug to coordinate expression of therapeutic transgenes combines drug-and genebased therapies for coordinated treatment of functionally related metabolic disorders. T he metabolic syndrome is a combination of disorders and risk factors including hypertension, hyperglycemia, obesity, and dyslipidemia that show an extremely complex and littleunderstood interdependent pathophysiology and collectively increase the risk for cardiovascular diseases that remain the prime cause of mortality worldwide (1-8). Currently available therapies independently target each of the individual disorders and risk factors, but a well-coordinated collective treatment strategy does not exist (2). We have therefore designed a synthetic signaling cascade in which a clinically licensed small-molecule drug finetunes expression of therapeutic transgenes. The combination of drug-and gene-based therapies enables simultaneous treatment of all key metabolic syndrome pathologies. Guanabenz (Wytensin) is a clinically licensed antihypertensive drug that is thought to activate alpha-2-selective adrenergic receptors in the central nervous system, thereby reducing the sympathetic outflow to heart, kidney, and peripheral vasculature and decreasing the systolic and diastolic blood pressure (9). Recently, guanabenz was also identified as an agonist of the trace amine-associated receptor 1 (TAAR1) (10). TAARs are G protein-coupled receptors located in the neu...

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Section: Methodsmentioning

confidence: 99%

Pharmaceutically controlled designer circuit for the treatment of the metabolic syndrome

Hamri

Zwicky

et al. 2012

Proc. Natl. Acad. Sci. U.S.A.

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“…Production of human placentalsecreted alkaline phosphatase (SEAP) was quantified using a p-nitrophenylphosphate-based light absorbance kinetic assay as previously described (Berger et al, 1988;Schlatter et al, 2002) with results expressed in units per liter (U/L). Levels of Bacillus stearothermophilus-derived secreted aamylase (SAMY) were quantified using a blue starch Phadebas 1 assay (Pharmacia Upjohn, Peapack, NJ, Cat.…”

Section: Quantification Of Reporter Gene Expressionmentioning

confidence: 99%

“…No. 10-5380-32) according to (Schlatter et al, 2002). Assessment of Bacillus subtilis-derived secreted xylanase A (SeXy), a recently developed mammalian reporter enzyme that converts xylan into glucose, was assessed using a fluorescence-based EnzCheck 1 xylanase assay (Molecular Probes, Eugene, OR, Cat.…”

Section: Quantification Of Reporter Gene Expressionmentioning

confidence: 99%

Multi‐gene engineering: Simultaneous expression and knockdown of six genes off a single platform

Greber

Fussenegger

2006

Biotech & Bioengineering

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Increases in our understanding of gene function have greatly expanded the repertoire of possible genetic interventions at our disposal with the consequence that many genetic engineering applications require multiple manipulations in which target genes can be both overexpressed and silenced in a simple and co-ordinated manner. Using synthetic introns as a source of encoding shortinterfering RNA (siRNA), we demonstrate that it is possible to simultaneously express both a transgene and siRNA from a single polymerase (Pol) II promoter. By encoding siRNA as an intron between two protein domains requiring successful splicing for functionality, it was possible to demonstrate that splicing was occurring, that the coding genes (exonic transgenes) resulted in functional protein, and that the spliced siRNA-containing lariat was capable of modulating expression of a separate target gene. We subsequently extended this concept to develop pTRIDENT-based multi-cistronic vectors that were capable of co-ordinated expression of up to three siRNAs and three transgenes off a single genetic platform. Such multi-gene engineering technology, enabling concomitant transgene overexpression and target gene knockdown, should be useful for therapeutic, biopharmaceutical production, and basic research applications.

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“…27. SEAP was quantified in cell culture supernatants by using a p-nitrophenylphosphate-based assay (30) and in mouse serum by employing a commercial chemiluminescence test (Roche Applied Science, catalog no. 11779842001).…”

Section: Methodsmentioning

confidence: 99%

A synthetic mammalian gene circuit reveals antituberculosis compounds

Weber

Schoenmakers

Keller

et al. 2008

Proc. Natl. Acad. Sci. U.S.A.

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SAMY , a novel mammalian reporter gene derived from Bacillus stearothermophilus α-amylase

Cited by 144 publications

References 30 publications

Pharmaceutically controlled designer circuit for the treatment of the metabolic syndrome

Pharmaceutically controlled designer circuit for the treatment of the metabolic syndrome

Multi‐gene engineering: Simultaneous expression and knockdown of six genes off a single platform

A synthetic mammalian gene circuit reveals antituberculosis compounds

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