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

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 α subunit (AMV RTα), 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.HighlightsA 202 bp-deletion fragment derived from a constitutively active promoter was identified as a minimal promoter in Pv11 cells.A Tet-On inducible expression system was developed for Pv11 cells using the minimal promoter.Typical 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.

Sequence Analysis and Expression Study of LTP7 Promoter Isolated from Cotton (Gossypium hirsutum L.)

Lipid transfer proteins (LTPs) have role in transfer of phospholipids along biological membranes. A cotton LTP7 promoter was isolated using high throughput genomic sequences (HTGS) data base. Analysis of promoter nucleotide sequence revealed a number of crucial regulatory elements including core promoter elements. A 1.8 kb fragment of LTP7 promoter was isolated from genomic DNA of cotton and finally cloned in plant expression vector to characterize its functionality. Transient GUS assay revealed that promoter showed expression in cotton fibres during the time of elongation and different stages of secondary cell wall synthesis. Deletion analysis at 5' end showed that 1 kb promoter showed strong expression during stage of secondary cell wall synthesis. Whereas, 1.5 kb deletion fragment exhibited less strong expression in cotton fibres. Results of this present study, showed that 1 kb deletion fragment and 1.8 kb LTP7 promoter exhibits fibre specific expression and may be used to express fiber genes in cotton.