TY - JOUR
T1 - Inducible and reversible dominant-negative (DN) protein inhibition
AU - Tarang, Shikha
AU - Pyakurel, Umesh
AU - Doi, Songila M.S.R.
AU - Weston, Michael D.
AU - Rocha-Sanchez, Sonia M.
N1 - Funding Information:
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.
Funding Information:
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.
PY - 2019/1
Y1 - 2019/1
N2 - Dominant-negative (DN) protein inhibition is a powerful method to manipulate protein function and offers several advantages over other genomebased 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.
AB - Dominant-negative (DN) protein inhibition is a powerful method to manipulate protein function and offers several advantages over other genomebased 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.
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U2 - 10.3791/58419
DO - 10.3791/58419
M3 - Article
C2 - 30663696
AN - SCOPUS:85060146832
VL - 2019
JO - Journal of Visualized Experiments
JF - Journal of Visualized Experiments
SN - 1940-087X
IS - 143
M1 - e58419
ER -