Over the last 20 years, transgenic and knockout mouse models have significantly contributed to the remarkable progress on the in vivo understanding of molecules and pathways underlying human health, and for pre-clinical testing of potential therapeutics. One unvavoidable issue with most commonly used approaches is that once a gene has been targeted for deletion or mutation, such genetic change is permanent in the specific cell lineage(s). Targeting of the protein rather than the gene, in the cell lineage of choice, would be hugely useful for a great number of investigations. Furthermore, if the protein loss were reversible, this would avoid another common problem faced in genetic deletions, adaptations to a particular gene deletion in the cells or cell lineages being examined. Here, we propose to generate an in vivo method to achieve inducible and resersible protein degradation (denoted IRIP). IRIP combines the power of two approaches: a recently described auxin-induced degron (AID) system for reversible protein degradation and lineage specific targeting via the vast number of existing Cre-lines. Auxins are a class of plant hormones that bind to the normally `inert' plant TIR1 protein; the auxin-induced activation of TIR1 induces rapid degradation of proteins containing a specific targeting region for TIR1. This auxin-Induced protein degradation can be adapted for mammalian cells expressing an exogenous TIR1 protein, and can cause degradation of an endogenous target protein engineered to carry the TIR1 targeting sequence. Upon withdrawal of auxin, TIR1 becomes inactive, and the target protein gets re-expressed. Here, based on the preliminary success in using this system in vitro and ex vivo, we propose to develop an auxin-induced degradation sytem/IRIP in a mouse model. Specifically, we will target SHP-1, a critical negative regulator of T cell signaling. We propose to engineer and combine two mouse strains to achieve inducible and reversible SHP-1 protein deletion in vivo. These mice will be used to study the role of SHP-1 in autoimmunity and cancer immune therapy. Moreover, since such a murine model of inducible and reversible protein degradation (IRIP) should be easily adaptable to other proteins and with the ever-growing number of Cre strains, we believe creating a new tool for in vivo studies should be of significant benefit to the larger research community.

Public Health Relevance

A balanced immune system is critical to human health. To generate a healthy functional immune system, many essential components are needed. A dysfunction of these components can throw the system out of balance leading to immunodeficiency or autoimmune diseases. Gaining a better mechanistic understanding of the players and processes has shown to be critical for the development of therapeutic options. Much knowledge has been acquired from the use of genetically modified mice that carry targeted mutations in proteins. However even with the advancement of using inducible gene targeting, one limitation has been that once a protein was modified or deleted, it would be a permanent change throughout the lifespan of the cell lineage, since any modifications were at the DNA level. Here, we propose to target the protein itself and trigger its degradation in a reversible manner, which will provide insights in temporal protein functions during immune processes as well as an alternative pre-clinical option to test therapeutics. Moreover, use of this reversible protein degradation system is expected to be widely usable by researchers and will provide a new tool for research beyond the immune system with potential implications for many areas of human health.

National Institute of Health (NIH)
National Institute of Allergy and Infectious Diseases (NIAID)
Exploratory/Developmental Grants (R21)
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Cellular and Molecular Immunology - A Study Section (CMIA)
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Mallia, Conrad M
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University of Virginia
Schools of Medicine
United States
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