Dataset Information

Combined experience of six independent laboratories attempting to create an Ewing sarcoma mouse model.

ABSTRACT: Ewing sarcoma (ES) involves a tumor-specific chromosomal translocation that produces the EWS-FLI1 protein, which is required for the growth of ES cells both in vitro and in vivo. However, an EWS-FLI1-driven transgenic mouse model is not currently available. Here, we present data from six independent laboratories seeking an alternative approach to express EWS-FLI1 in different murine tissues. We used the Runx2, Col1a2.3, Col1a3.6, Prx1, CAG, Nse, NEFL, Dermo1, P0, Sox9 and Osterix promoters to target EWS-FLI1 or Cre expression. Additional approaches included the induction of an endogenous chromosomal translocation, in utero knock-in, and the injection of Cre-expressing adenovirus to induce EWS-FLI1 expression locally in multiple lineages. Most models resulted in embryonic lethality or developmental defects. EWS-FLI1-induced apoptosis, promoter leakiness, the lack of potential cofactors, and the difficulty of expressing EWS-FLI1 in specific sites were considered the primary reasons for the failed attempts to create a transgenic mouse model of ES.

SUBMITTER: Minas TZ

PROVIDER: S-EPMC5470957 | biostudies-literature | 2017 May

REPOSITORIES: biostudies-literature

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Combined experience of six independent laboratories attempting to create an Ewing sarcoma mouse model.

Minas Tsion Zewdu TZ Surdez Didier D Javaheri Tahereh T Tanaka Miwa M Howarth Michelle M Kang Hong-Jun HJ Han Jenny J Han Zhi-Yan ZY Sax Barbara B Kream Barbara E BE Hong Sung-Hyeok SH Çelik Haydar H Tirode Franck F Tuckermann Jan J Toretsky Jeffrey A JA Kenner Lukas L Kovar Heinrich H Lee Sean S Sweet-Cordero E Alejandro EA Nakamura Takuro T Moriggl Richard R Delattre Olivier O Üren Aykut A

Oncotarget 20170501 21

Ewing sarcoma (ES) involves a tumor-specific chromosomal translocation that produces the EWS-FLI1 protein, which is required for the growth of ES cells both in vitro and in vivo. However, an EWS-FLI1-driven transgenic mouse model is not currently available. Here, we present data from six independent laboratories seeking an alternative approach to express EWS-FLI1 in different murine tissues. We used the Runx2, Col1a2.3, Col1a3.6, Prx1, CAG, Nse, NEFL, Dermo1, P0, Sox9 and Osterix promoters to ta ...[more]

PMID: 27191748

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Project description:Interrupted adenylation (A) domains are fascinating examples of multifunctional enzymes. They are found in nonribosomal peptide synthetases (NRPSs), which biosynthesize nonribosomal peptides (NRPs), a major class of medically relevant natural products (NPs). Interrupted A domains contain the catalytic portion of another domain within them, typically a methylation (M) domain, thus combining both adenylation and methylation capabilities. In recent years, interrupted A domains have demonstrated tremendous enzyme engineering potential as they are able to be constructed artificially in a laboratory setting by combining the A and M domains of two separate NRPS proteins. A recent discovery and characterization of a naturally occurring interrupted A domain that harbored two M domains back-to-back, a trifunctional protein, showed the ingenuity of Nature to both N- and O-methylate amino acids, the building blocks of NRPs. Since we have shown that a single M domain could be added to an uninterrupted A domain to create an artificial interrupted A domain, we set out to investigate if: (i) an A domain could be engineered to contain two back-to-back M domains and (ii) the added M domains would have to reflect the pattern in Nature, a side chain (O-) methylating M domain (Ms) followed by a backbone (N-) methylating M domain (Mb), or if the order of the M domains could be reversed. To address these questions, we set out to create our own AMsMbA and AMbMsA engineered interrupted A domains. We evaluated these engineered proteins connected (in cis) and/or disconnected (in trans) from the native thiolation (T) domain, through a series of radiometric assays, high performance liquid chromatography (HPLC), and mass spectrometry (MS) for adenylation, loading, and methylation ability. We found that although adenylation activity was preserved in both versions (AMsMbA and AMbMsA), addition of the M domains, in natural and unnatural order, did not result in the desired added methylation capability. This study offers valuable insights into the limits of constructing engineered interrupted A domains as potential tools for modifications of NRPs.

Dataset Information

Combined experience of six independent laboratories attempting to create an Ewing sarcoma mouse model.

Publications

Combined experience of six independent laboratories attempting to create an Ewing sarcoma mouse model.

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