After analysis of the 87 optimised expressions in vitro, 6 samples remained that were not expressed in vivo or in vitro (Table 2).
The codon usage of the dynein genes was optimised for expression in Sf9 cells, followed by their insertion into expression cassettes (Vijayachandran et al, 2013) containing a polyhedrin (PolH) promoter and an SV40 terminator sequence.
Accordingly, we designed and constructed a synthetic cyt2Aa1 gene (syncyt2Aa1)2 that was optimised for expression in this eukaryotic host.
The gene was codon optimised for expression in E. coli and cloned into pET28b via NcoI and XhoI restriction sites (Entelechon GmBH, Regensburg, Germany).
In the interplay with optimised gene expression [ 44] and product stabilisation [ 45], the utilisation of moss-endogenous secretion signals could greatly enhance the productivity of the moss-bioreactor system.
The following genes were codon optimised for expression in Sf9 cells and synthesised commercially (Epoch Life Science): DHC (DYNC1H1, accession number NM_001376.4), DIC (DYNC1I2, IC2C, AF134477), DLIC (DYNC1LI2, LIC2, NM_006141.2), Tctex (DYNLT1, Tctex1, NM_006519.2), LC8 (DYNLC1, LC8‐1, NM_003746.2) and Robl (DYNLRB1, Robl1, NM_014183.3).
To develop an optimised gene expression-based test, we have examined additional candidate gene expression markers.
The mouse Bicd2 (NM_029791.4) gene was codon optimised for Sf9 expression and synthesised commercially (Epoch Life Science).
These functions include insulin gene expression, optimised beta cell secretory function and blood glucose regulation, mediated by growth factors, collagen IV or other basement membrane components [ 14, 20].
Two-dimensional electrophoresis was optimised to compare expression patterns of proteins secreted from the cancer cell lines A431 and A549 treated with Gefitinib (Iressa) vs untreated or vehicle-only-treated samples.
