Rapid Generation of Stable Cell Lines Expressing Corticotropin-Releasing Hormone Receptor for Drug Discovery

Horlick, Robert A.; Sperle, Karen; Breth, Leah A.; Reid, Christian; Shen, Emily S.; Robbins, A. K.; Cooke, Gary M.; Largent, Brian L.

doi:10.1006/prep.1996.0701

Cited by 18 publications

(8 citation statements)

References 20 publications

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“…Full-length human cDNAs for human CRF 1 and CRF 2␣ receptors were subcloned into plasmids and transfected into HEK293EBNA (HEK293e) cells (Invitrogen) using lipofectamine (Invitrogen). The details of the plasmid construct were described in previous studies (Horlick et al, 1997;Kostich et al, 1998). HEK293e cells stably expressing human CRF 1 or CRF 2␣ receptors were grown in Dulbecco's modified Eagle's medium containing 10% (v/v) fetal bovine serum at 37°C in a humid environment (5% CO 2 ) for 10 days.…”

Section: Methodsmentioning

confidence: 99%

Pharmacological Characterization of a Novel Nonpeptide Antagonist Radioligand, (±)-N-[2-Methyl-4-methoxyphenyl]-1-(1-(methoxymethyl) propyl)-6-methyl-1H-1,2,3-triazolo[4,5-c]pyridin-4-amine ([³H]SN003) for Corticotropin-Releasing Factor₁Receptors

Zhang¹,

Huang²,

Li³

et al. 2003

J Pharmacol Exp Ther

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The in vitro pharmacological profile of a novel small molecule corticotropin-releasing factor 1 (CRF 1 ) receptor antagonist, (Ϯ)- N-[2-methyl-4-methoxyphenyl]-1-(1-(methoxymethyl)propyl)-6-methyl-1H-1,2,3-triazolo [4,5-c] Corticotropin-releasing factor (CRF) was first isolated from ovine hypothalamus (Vale et al., 1981) and identified as a key secretagogue for ACTH release from the anterior pituitary.In the past 10 years, considerable progress has been made in understanding the physiological and potential pathological roles of the CRF system. In addition to its endocrine role in the regulation of the hypothalamic-pituitary-adrenal axis in response to stress, CRF appears to be implicated in a variety of other central and peripheral functions including arousal, anxiety-like behaviors, learning and memory, feeding, imArticle, publication date, and citation information can be found at

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Section: Methodsmentioning

confidence: 99%

Pharmacological Characterization of a Novel Nonpeptide Antagonist Radioligand, (±)-N-[2-Methyl-4-methoxyphenyl]-1-(1-(methoxymethyl) propyl)-6-methyl-1H-1,2,3-triazolo[4,5-c]pyridin-4-amine ([³H]SN003) for Corticotropin-Releasing Factor₁Receptors

Zhang¹,

Huang²,

Li³

et al. 2003

J Pharmacol Exp Ther

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“…Final extension was 72 Њ C for 7 minutes. PCR fragments were digested with XhoI and XbaI, and cloned into an EBV ori-P vector (Horlick et al 1997). This plasmid has the cytomegalovirus (CMV) immediate early promoter to drive receptor expression, Epstein Barr virus origin of replication (EBV ori-P) for the maintenance as an episome in cells expressing EBV nuclear antigen 1 (EBNA1), and the E. coli hygromycin B resistance gene.…”

Section: Human Rna and Cdna Cloning Of Edited Isoformsmentioning

confidence: 99%

Messenger RNA Editing of the Human Serotonin 5-HT2C Receptor

Fitzgerald

Iyer

Conklin

et al. 1999

Neuropsychopharmacology

102

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“…For this purpose, the novel vector was tested in 293-EBNA cells, which are routinely used for fermentation processes and have been shown to support EBV-mediated DNA replication (16). Initial tests were done on adherent cells in 75 cm 2 T flasks.…”

Section: Resultsmentioning

confidence: 99%

Hybrid Sindbis/Epstein-Barr Virus Episomal Expression Vector for Inducible Production of Proteins

Ivanova¹,

Brändli²,

Saudan³

et al. 2005

BioTechniques

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Alphavirus vectors are attractive as recombinant protein expression systems due to the high level of gene expression achieved. The combination of two mutations in the viral replicase, which render the replicase noncytopathic and temperature-sensitive, allowed the generation of a DNA-based vector (CytTs) that shows temperature inducible expression. This vector is of significant value for the production of toxic protein. However, like for other stable expression systems, tedious screening of individual cell clones are required in order to get a high producer cell clone. To circumvent this, we generated an episomally replicating vector by introducing an Epstein-Barr virus mini-replicon unit into CytTs. This novel vector allowed rapid generation of cell populations that showed tight regulation of expression and comparable expression levels to the ones achieved with high producer cell clones with CytTs. Moreover, protein production with selected cell populations could easily be scaled-up to a fermentation process.

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Rapid Generation of Stable Cell Lines Expressing Corticotropin-Releasing Hormone Receptor for Drug Discovery

Cited by 18 publications

References 20 publications

Pharmacological Characterization of a Novel Nonpeptide Antagonist Radioligand, (±)-N-[2-Methyl-4-methoxyphenyl]-1-(1-(methoxymethyl) propyl)-6-methyl-1H-1,2,3-triazolo[4,5-c]pyridin-4-amine ([³H]SN003) for Corticotropin-Releasing Factor₁Receptors

Pharmacological Characterization of a Novel Nonpeptide Antagonist Radioligand, (±)-N-[2-Methyl-4-methoxyphenyl]-1-(1-(methoxymethyl) propyl)-6-methyl-1H-1,2,3-triazolo[4,5-c]pyridin-4-amine ([³H]SN003) for Corticotropin-Releasing Factor₁Receptors

Messenger RNA Editing of the Human Serotonin 5-HT2C Receptor

Hybrid Sindbis/Epstein-Barr Virus Episomal Expression Vector for Inducible Production of Proteins

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Rapid Generation of Stable Cell Lines Expressing Corticotropin-Releasing Hormone Receptor for Drug Discovery

Cited by 18 publications

References 20 publications

Pharmacological Characterization of a Novel Nonpeptide Antagonist Radioligand, (±)-N-[2-Methyl-4-methoxyphenyl]-1-(1-(methoxymethyl) propyl)-6-methyl-1H-1,2,3-triazolo[4,5-c]pyridin-4-amine ([3H]SN003) for Corticotropin-Releasing Factor1Receptors

Pharmacological Characterization of a Novel Nonpeptide Antagonist Radioligand, (±)-N-[2-Methyl-4-methoxyphenyl]-1-(1-(methoxymethyl) propyl)-6-methyl-1H-1,2,3-triazolo[4,5-c]pyridin-4-amine ([3H]SN003) for Corticotropin-Releasing Factor1Receptors

Messenger RNA Editing of the Human Serotonin 5-HT2C Receptor

Hybrid Sindbis/Epstein-Barr Virus Episomal Expression Vector for Inducible Production of Proteins

Contact Info

Product

Resources

About

Pharmacological Characterization of a Novel Nonpeptide Antagonist Radioligand, (±)-N-[2-Methyl-4-methoxyphenyl]-1-(1-(methoxymethyl) propyl)-6-methyl-1H-1,2,3-triazolo[4,5-c]pyridin-4-amine ([³H]SN003) for Corticotropin-Releasing Factor₁Receptors

Pharmacological Characterization of a Novel Nonpeptide Antagonist Radioligand, (±)-N-[2-Methyl-4-methoxyphenyl]-1-(1-(methoxymethyl) propyl)-6-methyl-1H-1,2,3-triazolo[4,5-c]pyridin-4-amine ([³H]SN003) for Corticotropin-Releasing Factor₁Receptors