Name: inducible and reversible dominant-negative (dn) protein inhibition
Uploaded: 2019-01-07T14:00:00
Description: Watch this Scientific Journal Video about Inducible and Reversible Dominant-negative DN Protein Inhibition at JoVE.com

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 School of Medicine and grants GM103427 and GM110768 from the NIH/NIGMS. The facility was constructed with support from grants from the National Center for Research Resources (RR016469) and the NIGMS (GM103427). The mouse lines generated in this study were maintained at Creighton University's Animal Resource Facility, whose infrastructure was improved through a grant by NIH/NCRR G20RR024001. This work received past support through an NIH/NCRR 5P20RR018788-/NIH/NIGMS 8P20GM103471 COBRE grant (to Shelley D. Smith), NIH/ORIP R21OD019745-01A1 (S.M.R.-S.), and an emerging research grant from the Hearing Health Foundation (to S. Tarang). The content of this research is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health.

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&#160;1A and 1B).
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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...

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.
The method consists of fusing the lysosomal protease CB to a subunit of a multimeric POI (CB fusion complex). The resultant CB fusion complex can interact with and proteolytically digest the endogenous protein or divert the entire CB-POI complex to the lysosome to be degraded3. Moreover, a combination of the CB fusion complex with the inducible nature of the tetracycline-controlled transcriptional activation (TetO) system allows for an inducible and controlled expression of the transgene in a reversible fashion2. Although useful in many circumstances, the complete deletion of genes or proteins in vivo can result in lethality4,5,6. Likewise, tissue-specific, conditional deletion of some genes or proteins using the Cre/Lox system may not be straightforward, as it will, ultimately, lead to the permanent loss of critical genomic elements7. Therefore, depending on the gene or POI, neither of these approaches will be effective in providing a useful model for subsequent studies, particularly genes&#39; or proteins&#39; functional studies in late postnatal and adult mice.
To circumvent the problems associated with such approaches and provide proof-of-principle on the effectiveness of the proposed method, we have chosen to test the method presented here by generating a conditional DN version of the Retinoblastoma 1 (RB1) protein. Several options have been proposed8,9,10 to abolish the function of endogenous RB1; however, all of them faced some of the same limitations discussed above: permanent germline deletion of RB1 is embryonically lethal, and, consistent with its tumor suppressor role, permanent RB1 conditional deletion leads to a variety of tumors11. Even though a DN version of RB1 does not seem to occur naturally, a better alternative to the currently available strategies should allow for a temporally controlled inactivation of the endogenous RB1 and provide an alternative mechanism to eventually restore its function. The basis for such a construct was described over two decades ago1. However, due to technological limitations, it lacked a mechanism to control transgene activation, response, and tissue specificity. This study is the first to combine the elegance of the doxycycline (Dox)-dependent transcriptional system with an engineered transgenic construct of lysosomal protease CB and Rb1 proteins. The resulting CBRb mouse model allows for a temporarily regulated Dox-mediated RB1 regulation2. The advantage of using such a proteome-based approach to study gene function is that it can be adopted for any gene of interest, with minimal information on its activity.
The proposed DN transgene strategy offers many advantages over traditional approaches. First, DN protein inhibition leads to only a partial ablation in protein activity, thus preserving a residual endogenous expression. Such an outcome is highly desirable in situations where a complete elimination of protein activity leads to embryonic lethality, greatly limiting any investigation to study gene function in a live mouse. Second, the presence of the TetO system enables transgene activation only in the presence of an antibiotic, which allows for an efficient and reversible control of the transgene activity. Thus, by ceasing the antibiotic administration, the transgenic system can be deactivated, and a normal RB1 expression is back in place. Third, the specificity of the transgene expression can vary depending on the promoter of choice. While we have chosen the ubiquitous ROSA-CAG promoter for proof-of-principle, placing the transgene under a tissue-specific promoter is likely to restrict unwanted transgene expression and facilitate studies on the therapeutic application of this transgene methodology.

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		<tr height="35">
			<td height="35" style="height:35px;width:255px;">Dulbecco&#39;s Modified Eagle&#39;s Medium, DMEM</td>
			<td style="width:228px;">GIBCO-BRL</td>
			<td style="width:119px;">11965-084</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:255px;">Minimum Essential Medium Eagle, MEME&#160;</td>
			<td style="width:228px;">Sigma&#160;</td>
			<td style="width:119px;">M8042</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Fetal bovine serum</td>
			<td style="width:228px;">Sigma&#160;</td>
			<td style="width:119px;">F2442&#160;</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:255px;">Lipofectamine&#160;</td>
			<td style="width:228px;">DharmaFECT</td>
			<td style="width:119px;">T-2010-03</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:255px;">Sal I</td>
			<td style="width:228px;">Roche</td>
			<td style="width:119px;">11745622</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:255px;">EcoRV-HF</td>
			<td style="width:228px;">New England BioLabs</td>
			<td style="width:119px;">R3195S</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:255px;">NotI</td>
			<td style="width:228px;">Roche</td>
			<td style="width:119px;">13090730</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:255px;">CaspaTag&#160;</td>
			<td style="width:228px;">Millipore</td>
			<td style="width:119px;">APT523</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:255px;">DAPI</td>
			<td style="width:228px;">Sigma&#160;</td>
			<td style="width:119px;">D9542</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:255px;">Staurosporine</td>
			<td style="width:228px;">Sigma&#160;</td>
			<td style="width:119px;">S4400</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:255px;">CyQuant NF cell proliferation kit&#160;</td>
			<td style="width:228px;">Invitrogen</td>
			<td style="width:119px;">C35007</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:255px;">Retinoblastoma 1 antibody</td>
			<td style="width:228px;">Abcam</td>
			<td>Ab6075</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:255px;">c-Myc antibody</td>
			<td style="width:228px;">Sigma&#160;</td>
			<td>M5546</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:255px;">b-actin</td>
			<td style="width:228px;">Sigma&#160;</td>
			<td>A5316</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:255px;">Ki-67&#160;</td>
			<td style="width:228px;">Thermofisher scientific</td>
			<td style="width:119px;">MA5-14520</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:255px;">Phallodin</td>
			<td style="width:228px;">Thermofisher scientific</td>
			<td style="width:119px;">A12379</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Fluorescence microplate reader</td>
			<td style="width:228px;">FLUOstar OPTIMA, BMG Labtech</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:255px;">Epifluorescence microscope&#160;</td>
			<td style="width:228px;">NikonEclipse80i</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:601px;">The TetO-DN-CB-myc6-Rb1 (DN-CBRb) mouse line is available from the Jackson Laboratory as JAX#032011.&#160;</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
	</tbody>
</table>

inducible and reversible dominant-negative (dn) protein inhibition

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.

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...

Watch this Scientific Journal Video about Inducible and Reversible Dominant-negative DN Protein Inhibition at JoVE.com

Inducible and Reversible Dominant-negative DN Protein Inhibition

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.

Inducible and Reversible Dominant-negative (DN) Protein Inhibition

Dominant-negative (DN) protein inhibition is a powerful method to manipulate protein function and offers several advantages over other genome-based ...

This method can help answer key questions in a wide variety of fields requiring conditional and transient inactivation of a gene of interest by reversibly over-expressing a dominant negative version of it. The main advantage of this technique is that it allows only a partial ablation in protein activity, thus, preserving a residual endogenous expression. Although, here we describe the condition inactivation of RB protein, which functions in a multimeric assembly, this methodology can also be applied to monomeric proteins.Umesh Pyakurel, our laboratory techinician and a coauthor in the manuscript will demonstrate the procedures. To begin, grow NIH3T3 cells, following the vendor's specifications, at 37 degrees Celsius with 5%CO2, using the recommended cell culture medium containing 10%fetal bovine serum. To cotransfect the cells using pTet-Splice and pCMV-Tet3G vectors, first mix two to four microliters of the lipid-based transfection reagent per well, and one milliliter of incomplete DMEM per well in a 15 milliliter tube, and incubate for five minutes at room temperature.Add two to three micrograms of plasma DNA per well to this transfection reagent's DMEM mix. Mix and incubate for 20 minutes at room temperature. Add one milliliter of the mix containing DNA and the transfection reagent in DMEM to each well of six-well plate with NIH3T3 cells.After three to four hours of incubation at 37 degrees Celsius with 5%CO2, add one milliliter of complete medium. Then, add 2 microliters of doxycycline stock to each well and mark the plate as plus-doxycycline. Incubate the cells at 37 degrees Celsius with 5%CO2.To assess the functionality of the TetO-DN-CB-myc6-Rb1 construct in promoting unscheduled cell proliferation, culture HEI-OC1 cells in DMEM containing 10%FBS, under permissive conditions. For cell proliferation studies, count the HEI-OC1 cells using a cell counter. Plate 10, 000 HEI-OC1 cells per well on a 96-well plate in 200 microliters of volume each.Incubate the cells over night at 33 degrees Celsius with 5%CO2. To induce transgene expression, add one micrograms per milliliter of doxycycline to a subset of transfected HEI-OC1 cells. Use the other subset labeled as minus-doxycycline and the non-transfected HEI-OC1 cells as controls, and incubate the cells as 33 degrees Celsius with 10%CO2.To assess cell proliferation, 48 hours after transfection, remove the cell culture medium and add 100 microliters of 1x dye binding solution from a cell proliferation kit to each well of the micro-plate. Follow the manufacturer's protocol and incubate for one hour at 37 degrees Celsius. Use a fluorescence microplate reader to measure the fluorescence intensity of each sample.To further assess cell proliferation using immunocytochemistry, plate the HEI-OC1 cells on a cover glass placed in each well of a 12-well plate in DMEM. Incubate the cells overnight at 33 degrees Celsius. On the following day, cotransfect to the cells in two wells with the pTet-Splice and the pCMV-Tet3G vectors, and then perform the doxycycline treatment on one well as done with the NIH3T3 cells.Use one untransfected well as a control. To process the cells for Ki-67 labeling, fix them with 4%paraformaldehyde in PBS for 10 minutes at room temperature. After that, wash them three times with ice-cold PBS for five minutes each.Add 0.25%nonionic detergent in PBS, and incubate for 10 minutes. Repeat the wash three times in PBS for five minutes each. Use 10%serum to block the cells.In a humidified chamber for one hour at room temperature. Add 500 microliters of Ki-67 primary antibody and incubate overnight at four degrees Celsius. Wash the cells again three times in PBS for five minutes each.Add one milliliter of the secondary antibody and incubate the cells for one hour at room temperature in the dark. After removing the secondary antibody solution, wash the cells again three times for five minutes in PBS in the dark. To label the HEI-OC1 cells with phalloidin, incubate them in 1:200 phalloidin for 30 minutes at room temperature.To label the cell nuclei, incubate the cells with five micrograms per milliliter of DAPI For 10 minutes at room temperature. After delivery of the transgene mice, genotype the pups using a primer set specific to the CBRb1 fusion region as described in the manuscript. Breed adult TetO-DN-CB-myc6-Rb1 mice to the ROSA-CAG-rtTA and tetrecycline inducer line to generate experimental DN-CBRb mice, and genotype their offspring as described in the manuscript.NIH3T3 does not express endogenous RB1 protein, so the robust RB1 expression seen here is due to transgene induction in the presence of Dox, but not in its absence, confirming the efficient activation of the TetO-DN-CB-myc6-Rb1 mix RB1 construct. In contrast, HEK293 cells have endogenous RB1 expression. The addition of doxycycline led to TetO-DN-CB-myc6-Rb1 activation and RB1 down-regulation.RB1 expression was resumed 21 hours after doxycycline was removed from the media. HEI-OC1 cells cotransfected with the same vectors, displayed a modest but significant increase in cell number follow doxycycline treatment compared to transfected cells not treated with doxycycline, and the untransfected cells. Fluorescent in situ hybridization confirmed the genomic insertion of TetO-DN-CB-myc6-RB1 transgene in mice.There was an initial increase in total RB1 protein expression in cochlea of the transgenic mice treated with doxycycline for three days compared to the control group. However, in groups treated with doxocycline for seven and 10 days, a significant reduction in RB1 expression was observed. Regardless of the tissue analyzed, a significant reduction in RB1 protein was observed in transgenic mice treated with doxocycline, but not in the control group, confirming efficiency of the TetO-DN-CB-myc6-RB1 construct.While attempting this procedure, it's important to verify that each of the coding elements of the transgene are in frame with each other. If the coding regions of Cathepsin B and retinoblastoma described in this report were, by chance, connected out of frame, it wouldn't yield the intended fusion protein. Thus, DNA sequence verification must be done before injecting the transgene into single-celled mouse embryos.Following this procedure, the ubiquitous CAG promoter can be replaced with specific promoters in order to study gene function in a cell's specific manner.

Research

JoVE Journal

Genetics

Inducible T7 RNA Polymerase-mediated Multigene Expression System, pMGX

Co-expression of multiple proteins is increasingly essential for synthetic biology, studying protein-protein complexes, and characterizing and harnessing ...

Generation of Fluorescent Protein Fusions in Candida Species

Generation of Fluorescent Protein Fusions in Candida Species

Candida species, prevalent colonizers of the intestinal and genitourinary tracts, are the cause of the majority of invasive fungal infections in humans. ...

Controllable Ion Channel Expression through Inducible Transient Transfection

Transfection, the delivery of foreign nucleic acids into a cell, is a powerful tool in protein research. Through this method, ion channels can be ...

Microinjection of CRISPR/Cas9 Protein into Channel Catfish, Ictalurus punctatus, Embryos for Gene Editing

Microinjection of CRISPR/Cas9 Protein into Channel Catfish, Ictalurus punctatus, Embryos for Gene Editing

The complete genome of the channel catfish, Ictalurus punctatus, has been sequenced, leading to greater opportunities for studying channel catfish gene ...

Constitutive and Inducible Systems for Genetic In Vivo Modification of Mouse Hepatocytes Using Hydrodynamic Tail Vein Injection

Constitutive and Inducible Systems for Genetic In Vivo Modification of Mouse Hepatocytes Using Hydrodynamic Tail Vein Injection

In research models of liver cancer, regeneration, inflammation, and fibrosis, flexible systems for in vivo gene expression and silencing are highly ...

Identification of Homologous Recombination Events in Mouse Embryonic Stem Cells Using Southern Blotting and Polymerase Chain Reaction

Relative to the issues of off-target effects and the difficulty of inserting a long DNA fragment in the application of designer nucleases for genome ...

Inducible, Cell Type-Specific Expression in Arabidopsis thaliana Through LhGR-Mediated Trans-Activation

Inducible, Cell Type-Specific Expression in Arabidopsis thaliana Through LhGR-Mediated Trans-Activation

Inducible, tissue-specific expression is an important and powerful tool to study the spatio-temporal dynamics of genetic perturbation. Combining the ...

Mass Spectrometry-Based Proteomics Analyses Using the OpenProt Database to Unveil Novel Proteins Translated from Non-Canonical Open Reading Frames

Genome annotation is central to today's proteomic research as it draws the outlines of the proteomic landscape. Traditional models of open reading frame ...

An Assay for Quantifying Protein-RNA Binding in Bacteria

In the initiation step of protein translation, the ribosome binds to the initiation region of the mRNA. Translation initiation can be blocked by binding ...

In Vitro Biochemical Assays using Biotin Labels to Study Protein-Nucleic Acid Interactions

Protein-nucleic acid interactions play important roles in biological processes such as transcription, recombination, and RNA metabolism. Experimental ...