Generation of stable Drosophila cell lines using multicistronic vectors

Abstract

Insect cell culture is becoming increasingly important for applications including recombinant protein production and cell-based screening with chemical or RNAi libraries. While stable mammalian cell lines expressing a protein of interest can be efficiently prepared using IRES-based vectors or viral-based approaches, options for stable insect cell lines are more limited. Here, we describe pAc5-STABLEs, new vectors for use in Drosophila cell culture to facilitate stable transformation. We show that viral-derived 2A-like (or "CHYSEL") peptides function in Drosophila cells and can mediate the multicistronic expression of two or three proteins of interest under control of the Actin5C constitutive promoter. The current vectors allow mCherry and/or GFP fusions to be generated for positive selection by G418 resistance in cells and should serve as a flexible platform for future applications.

Serotonergic Modulation Differentially Targets Distinct Network Elements within the Antennal Lobe of Drosophila melanogaster

“…In the case of the gene-trap lines, the MiMIC cassette is replaced with a cassette encoding a universal splice-acceptor and GAL4. In the case of the protein-trap lines, the MiMIC cassette is replaced with one that encodes a universal splice acceptor followed by a self-cleaving T2A peptide4546 fused to the GAL4 coding sequence (see42 for a detailed description of cassette insertion sites). Thus, the gene-trap and protein-trap 5-HTR lines represent endogenous 5-HTR gene transcription and translation, respectively.…”

Unique patterns of organization and migration of FGF-expressing cells during Drosophila morphogenesis

“…2A). T2A facilitates “ribosome skipping” (González et al, 2011) and independent translation initiation of LexA . Similarly, to disrupt co-translation of a truncated C terminal portion of Bnl from the hybrid transcript, the nls-lexA:p65 is followed by a stop codon.…”

Simultaneous Expression of Multiple Proteins Under a Single Promoter in Caenorhabditis elegans via a Versatile 2A-Based Toolkit

“…Because an uncleaved product can sometimes be detected, but not always, as already described in other species (Hasegawa et al 2007;Provost et al 2007;Gonzalez et al 2011;Kim et al 2011), we cannot exclude that, in rare cases, low amounts of an uncleaved product could be associated with a dominant negative activity. It should be noted that this has never been described, while the multi-subunit T-cell receptor, or cocktails of pluripotency factors, for example, have been delivered to cells with no adverse effects (Szymczak et al 2004;Carey et al 2009).…”

“…For these reasons we decided to develop the 2A viral peptide technology for C. elegans. 2As have been used to deliver multiple proteins from a single ORF for various vertebrate models and very recently in Drosophila (Gonzalez et al 2011;Diao and White 2012). Several studies have shown that the 2A system allows robust production of several proteins in a near-stochiometric manner (Szymczak et al 2004;Torres et al 2010).…”