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Genetics

Inducible and Reversible Dominant-negative (DN) Protein Inhibition

Published: January 7th, 2019

DOI:

10.3791/58419

1Department of Oral Biology, Creighton University School of Dentistry

Here we present a protocol to develop a dominant-negative inducible system, in which any protein can be conditionally inactivated by reversibly overexpressing a dominant-negative mutant version of it.

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.

Most approaches aiming at the gene and protein ablations rely on permanent processes, which generally lead to the complete elimination or truncation of the gene, RNA sequences, or protein of interest (POI). The overall goal of this method is to engineer a recombinant protein to abolish the function of endogenous, wild-type protein. We have revisited and revamped an alternative strategy1,2, which allows for the temporary ablation of a POI through DN inhibition. This method works for both multimeric and monomeric peptides but is best suited for proteins that function in a multimeric assembly.

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Generation of the transgenic CBRb mouse and all animal care and experiments associated with the study were approved by the Creighton University Institutional Animal Care and Use Committee (IACUC) and performed by their guidelines.

1. Transgenic CB-Myc6-Rb1 Construct

NOTE: The cloning of CBRb into a pTet_Splice vector was done in a multi-step process (Figure 1A and 1B).

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Generally, designing a DN mutation requires a considerable amount of information on the structure and function of the POI. In contrast, the DN strategy presented here is particularly useful when the structural and functional information for the POI is limited. If the POI is a multimeric protein, a fusion of one subunit to a lysosomal protease can dominantly inhibit the assembled multimer and, potentially, other ligands through a combination of proteolysis of the endogenous subunits and su.......

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To circumvent the limitations associated with traditional transgenic strategies, we sought to generate a mouse model in which an endogenous POI can be conditionally inactivated by overexpressing a DN mutant form of it in a spatiotemporal manner. To abolish the function of endogenous POIs, several options have been proposed15,16,17. We have modified an earlier genetic strategy1 by combining the Dox-depende.......

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The pCS2+CB-Myc6 vector was a gift from Marshall Horwitz (University of Washington, Seattle, WA, USA). The HEI-OC1 cells were kindly provided by Fedrico Kalinec (David Geffen School of Medicine, UCLA, Los Angeles, CA, USA). Technical support was provided by the UNMC Mouse Genome Engineering Core (C.B. Gurumurthy, Don Harms, Rolen Quadros) and the Creighton University Integrated Biomedical Imaging Facility (Richard Hallworth, John Billheimer). The UNMC Mouse Genome Engineering was supported by an Institutional Development Award (IDea) from the NIH/NIGMS, grant number P20 GM103471. The Integrated Biomedical Imaging Facility was supported by the Creighton University Scho....

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Name Company Catalog Number Comments
Dulbecco's Modified Eagle's Medium, DMEM GIBCO-BRL 11965-084
Minimum Essential Medium Eagle, MEME  Sigma  M8042
Fetal bovine serum Sigma  F2442 
Lipofectamine  DharmaFECT T-2010-03
Sal I Roche 11745622
EcoRV-HF New England BioLabs R3195S
NotI Roche 13090730
CaspaTag  Millipore APT523
DAPI Sigma  D9542
Staurosporine Sigma  S4400
CyQuant NF cell proliferation kit  Invitrogen C35007
Retinoblastoma 1 antibody Abcam Ab6075
c-Myc antibody Sigma  M5546
b-actin Sigma  A5316
Ki-67  Thermofisher scientific MA5-14520
Phallodin Thermofisher scientific A12379
Fluorescence microplate reader FLUOstar OPTIMA, BMG Labtech
Epifluorescence microscope  NikonEclipse80i
The TetO-DN-CB-myc6-Rb1 (DN-CBRb) mouse line is available from the Jackson Laboratory as JAX#032011. 

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