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Generation and characterization of DSPP-Cerulean/DMP1-Cherry reporter mice.

ABSTRACT: To gain a better understanding of the progression of progenitor cells in the odontoblast lineage, we have examined and characterized the expression of a series of GFP reporters during odontoblast differentiation. However, previously reported GFP reporters (pOBCol2.3-GFP, pOBCol3.6-GFP, and DMP1-GFP), similar to the endogenous proteins, are also expressed by bone-forming cells, which made it difficult to delineate the two cell types in various in vivo and in vitro studies. To overcome these difficulties we generated DSPP-Cerulean/DMP1-Cherry transgenic mice using a bacterial recombination strategy with the mouse BAC clone RP24-258g7. We have analyzed the temporal and spatial expression of both transgenes in tooth and bone in vivo and in vitro. This transgenic animal enabled us to visualize the interactions between odontoblasts and surrounding tissues including dental pulp, ameloblasts and cementoblasts. Our studies showed that DMP1-Cherry, similar to Dmp1, was expressed in functional and fully differentiated odontoblasts as well as osteoblasts, osteocytes and cementoblasts. Expression of DSPP-Cerulean transgene was limited to functional and fully differentiated odontoblasts and correlated with the expression of Dspp. This transgenic animal can help in the identification and isolation of odontoblasts at later stages of differentiation and help in better understanding of developmental disorders in dentin and odontoblasts.

SUBMITTER: Vijaykumar A

PROVIDER: S-EPMC6939995 | biostudies-literature | 2019 Oct

REPOSITORIES: biostudies-literature

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Generation and characterization of DSPP-Cerulean/DMP1-Cherry reporter mice.

Vijaykumar Anushree A Ghassem-Zadeh Sean S Vidovic-Zdrilic Ivana I Komitas Karren K Adameyko Igor I Krivanek Jan J Fu Yu Y Maye Peter P Mina Mina M

Genesis (New York, N.Y. : 2000) 20190704 10

To gain a better understanding of the progression of progenitor cells in the odontoblast lineage, we have examined and characterized the expression of a series of GFP reporters during odontoblast differentiation. However, previously reported GFP reporters (pOBCol2.3-GFP, pOBCol3.6-GFP, and DMP1-GFP), similar to the endogenous proteins, are also expressed by bone-forming cells, which made it difficult to delineate the two cell types in various in vivo and in vitro studies. To overcome these diffi ...[more]

PMID: 31271259

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Project description:The emergence of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the pathogen responsible of coronavirus disease 2019 (COVID-19), has devastated public health services and economies worldwide. Despite global efforts to contain the COVID-19 pandemic, SARS-CoV-2 is now found in over 200 countries and has caused an upward death toll of over 1 million human lives as of November 2020. To date, only one Food and Drug Administration (FDA)-approved therapeutic drug (Remdesivir) and a monoclonal antibody, MAb (Bamlanivimab) are available for the treatment of SARS-CoV-2. As with other viruses, studying SARS-CoV-2 requires the use of secondary approaches to detect the presence of the virus in infected cells. To overcome this limitation, we have generated replication-competent recombinant (r)SARS-CoV-2 expressing fluorescent (Venus or mCherry) or bioluminescent (Nluc) reporter genes. Vero E6 cells infected with reporter-expressing rSARS-CoV-2 can be easily detected via fluorescence or luciferase expression and display a good correlation between reporter gene expression and viral replication. Moreover, rSARS-CoV-2 expressing reporter genes have comparable plaque sizes and growth kinetics to those of wild-type virus, rSARS-CoV-2/WT. We used these reporter-expressing rSARS-CoV-2 to demonstrate their feasibility to identify neutralizing antibodies (NAbs) or antiviral drugs. Our results demonstrate that reporter-expressing rSARS-CoV-2 represent an excellent option to identify therapeutics for the treatment of SARS-CoV-2, where reporter gene expression can be used as valid surrogates to track viral infection. Moreover, the ability to manipulate the viral genome opens the feasibility of generating viruses expressing foreign genes for their use as vaccines for the treatment of SARS-CoV-2 infection.IMPORTANCE Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the pathogen that causes coronavirus disease 2019 (COVID-19), has significantly impacted the human health and economic status worldwide. There is an urgent need to identify effective prophylactics and therapeutics for the treatment of SARS-CoV-2 infection and associated COVID-19 disease. The use of fluorescent- or luciferase-expressing reporter expressing viruses has significantly advanced viral research. Here, we generated recombinant (r)SARS-CoV-2 expressing fluorescent (Venus and mCherry) or luciferase (Nluc) reporter genes and demonstrate that they represent an excellent option to track viral infections in vitro. Importantly, reporter-expressing rSARS-CoV-2 display similar growth kinetics and plaque phenotype that their wild-type counterpart (rSARS-CoV-2/WT), demonstrating their feasibility to identify drugs and/or neutralizing antibodies (NAbs) for the therapeutic treatment of SARS-CoV-2. Henceforth, these reporter-expressing rSARS-CoV-2 can be used to interrogate large libraries of compounds and/or monoclonal antibodies (MAb), in high-throughput screening settings, to identify those with therapeutic potential against SARS-CoV-2.

Dataset Information

Generation and characterization of DSPP-Cerulean/DMP1-Cherry reporter mice.

Publications

Generation and characterization of DSPP-Cerulean/DMP1-Cherry reporter mice.

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