Inducible and Reversible Dominant-negative (DN) Protein Inhibition

Shikha Tarang, Umesh Pyakurel, Songila M.S.R. Doi, Michael Weston, Sonia Rocha-Sanchez

Research output: Contribution to journalArticle

Abstract

Dominant-negative (DN) protein inhibition is a powerful method to manipulate protein function and offers several advantages over other genome-based approaches. For example, although chimeric and Cre-LoxP targeting strategies have been widely used, the intrinsic limitations of these strategies (i.e., leaky promoter activity, mosaic Cre expression, etc.) have significantly restricted their application. Moreover, a complete deletion of many endogenous genes is embryonically lethal, making it impossible to study gene function in postnatal life. To address these challenges, we have made significant changes to an early genetic engineering protocol and combined a short (transgenic) version of the Rb1 gene with a lysosomal protease procathepsin B (CB), to generate a DN mouse model of Rb1 (CBRb). Due to the presence of a lysosomal protease, the entire CB-RB1 fusion protein and its interacting complex are routed for proteasome-mediated degradation. Moreover, the presence of a tetracycline inducer (rtTA) element in the transgenic construct enables an inducible and reversible regulation of the RB1 protein. The presence of a ubiquitous ROSA-CAG promoter in the CBRb mouse model makes it a useful tool to carry out transient and reversible Rb1 gene ablation and provide researchers a resource for understanding its activity in virtually any cell type where RB1 is expressed.

Original languageEnglish (US)
JournalJournal of visualized experiments : JoVE
Issue number143
DOIs
StatePublished - Jan 7 2019

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Genes
Proteins
Peptide Hydrolases
Genetic Engineering
Proteasome Endopeptidase Complex
Genetic engineering
Tetracycline
Ablation
Research Personnel
Genome
Fusion reactions
Degradation

All Science Journal Classification (ASJC) codes

  • Neuroscience(all)
  • Chemical Engineering(all)
  • Biochemistry, Genetics and Molecular Biology(all)
  • Immunology and Microbiology(all)

Cite this

Inducible and Reversible Dominant-negative (DN) Protein Inhibition. / Tarang, Shikha; Pyakurel, Umesh; Doi, Songila M.S.R.; Weston, Michael; Rocha-Sanchez, Sonia.

In: Journal of visualized experiments : JoVE, No. 143, 07.01.2019.

Research output: Contribution to journalArticle

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