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Abstract:
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We have used Drosophila melanogaster Schneider 2 (S2) cell line for heterologous expression of human store-operated calcium channel components: Orai1, Orai3 and STIM1. Orai1 and 3 proteins are believed to constitute the pore subunits of store operated channels whereas STIM1 is the calcium sensor in endoplasmic reticulum membrane that signals calcium store emptying to the channel subunits located in the plasma membrane. We have generated stable S2 cells lines expressing these genes by transfecting with Orai1+STIM1 and Orai3 using constructs in Drosophila expression vectors. The vector used pUChygMT vector contains an inducible promoter, the Mtn (metallothionein) promoter, and only at specific concentration of copper does the protein expression occur. Additionally, the plasmid carries resistance to Hygromycin B antibiotic. The S2 cells lines were created by selection in presence of Hygromycin B over the period of several months. After each cell line is induced with Cu2+, with controls/uninduced lines held on the side, RT-PCR reactions were conducted from the collected total RNA. The STIM1, Orai1, and Orai3 genes are downstream of the Mtn promoter and it is expected that Orai-s and STIM1 gene expression will only occur upon induction with copper but not in its absence. Hygromycin B resistance gene, on the other hand, will be expected to express both in induced and uninduced cell lines. We present the results of our RT-PCR experiments demonstrating that stable S2 cell lines have been created. |
