Involvement of RDR1/RDR6 genes in RNA silencing in Nicotiana tabacum BY-2 cells

Details

Supervisors

Chaumont, François ; Bouhon, Laurent

Faculty

Faculté des bioingénieurs

Degree label

Master [120] : bioingénieur en chimie et bioindustries, à finalité spécialisée

Abstract

Production of pharmaceutical recombinant proteins is expanding in recent years. Plant cells and especially Nicotiana tabacum BY-2 suspension cells are a promising expression platform. They have the advantages of higher eucaryotes for producing recombinant glycoproteins. They are easy to scale-up for manufacturing at low contamination risks compared to CHO or bacteria cultures. They have a rapid doubling time and are easily transformable via Agrobacterium tumefaciens. However, plant suspension cells usually have lower production rates compared to mammalian cell cultures. Many ways were investigated to improve the production yield of recombinant proteins, suitable with industrial demands. Increasing transcription rates with the use of strong promoters or many transgene copies is one of the most used methods. However, various studies have shown that RNA silencing occurs when the transgene expression rate reaches a certain gene-specific threshold. Above this threshold, the induction of RNA silencing results in a significant reduction in mRNA levels. RNA silencing prevents the production of excessive amounts of aberrant mRNA. Fortunately, some strategies can overcome this issue. The generation of cell lines mutated in RDR1 and RDR6 genes (rdr1/6) should inhibit the triggering of RNA silencing. RDR1 and RDR6 enzymes are involved in the very first steps of this mechanism. Thus, a multiplex CRISPR/Cas9 approach was used to silence the corresponding alleles in a cell line previously silenced for the mCherry fluorescent reporter transgene. In this master’s thesis, transformants were screened to search for rdr1/6 mutated cell lines. We showed that transcriptional gene silencing (TGS) was an issue for Cas9 expression and impairs the full edition of the genes. Removal of TGS gave positive results in the expression of Cas9. This enabled the full edition of several transformed lines. Then, a link between mCherry expression and rdr1/6 mutants was discussed. TGS mechanisms could also impair the mCherry expression even if RDR1 and RDR6 genes are knocked-out. Consequently, removal of methylations seems to be required to prevent the silencing. Finally, a plasmid was constructed to express the viral recombinant protein gB in these rdr1/6 mutated lines cells. After transfection, the expression level of gB will be compared with the expression in BY-2 cells. If positive results are obtained, these cell lines could be used for producing high amount of pharmaceutical recombinant proteins.