Development of a Tet-On inducible expression system for the anhydrobiotic cell line, Pv11

Tokumoto, S.; Miyata, Y.; Usui, K.; Deviatiiarov, R.; Ohkawa, T.; Kondratieva, S.; Shagimardanova, E.; Gusev, O.; Cornette, R.; Itoh, M.; Hayashizaki, Y.; Kikawada, T.

doi:10.1101/2020.05.29.123570

bioRxiv · 10.1101/2020.05.29.123570

Development of a Tet-On inducible expression system for the anhydrobiotic cell line, Pv11

Tokumoto, S.·Miyata, Y.·Usui, K.·Deviatiiarov, R.·Ohkawa, T.·Kondratieva, S.·Shagimardanova, E.·Gusev, O.·Cornette, R.·Itoh, M.·Hayashizaki, Y.·Kikawada, T.

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Abstract

The Pv11 cell line established from an African chironomid, Polypedilum vanderplanki, is the only cell line tolerant to complete desiccation. In Pv11 cells, a constitutive expression system for Pv11 cells was previously exploited and several reporter genes were successfully expressed. Here we report the identification of an effective minimal promoter for Pv11 cells and its application to the Tet-On inducible expression system. First, using a luciferase reporter assay, we showed that a 202 bp deletion fragment derived from the constitutively active 121-promoter functions in Pv11 cells as an appropriate minimal promoter with the Tet-On inducible expression system. The AcGFP1 protein was also successfully expressed in Pv11 cells using the inducible system. In addition to these reporter genes, avian myeloblastosis virus reverse transcriptase subunit (AMV RT), which is one of the most widely commercially available RNA-dependent DNA polymerases, was successfully expressed through the inducible expression system and its catalytic activity was verified. These results demonstrate the establishment of an inducible expression system in cells that can be preserved in the dry state and highlight a possible application to the production of large and complex proteins. HighlightsO_LIA 202 bp-deletion fragment derived from a constitutively active promoter was identified as a minimal promoter in Pv11 cells. C_LIO_LIA Tet-On inducible expression system was developed for Pv11 cells using the minimal promoter. C_LIO_LITypical reporter genes (GFP and luciferase) and an enzyme with complex structure, i.e. a viral reverse transcriptase, were successfully and inducibly expressed in Pv11 cells using the Tet-On system. C_LI

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Tokumoto, S., Miyata, Y., Usui, K., Deviatiiarov, R., Ohkawa, T., Kondratieva, S., Shagimardanova, E., Gusev, O., Cornette, R., Itoh, M., Hayashizaki, Y., Kikawada, T.. 2020-05-31. Development of a Tet-On inducible expression system for the anhydrobiotic cell line, Pv11. https://doi.org/10.1101/2020.05.29.123570

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