The PiggyBac-Based Transgenic Drosophila Express Tetra-Cycline-Eependent Transactivator

Abstract

The Tetracycline regulatory system is based on the Tet repressor protein (TetR) of the Escherichia coli Tn10 tetracycline (Tc) resistance operon and its operator (tetO) (Hillen and Berens, 1994). TetR blocks transcription of these genes by binding to the tetO operator sequences in the absence of Tc. Two gene expression systems, TetOff and TetOn, have been derived from the bacterial Tet system. In the Tet-Off system, the bacterial TetR is fused with the C-terminal of the herpes simplex virus (VP16) activation domain (Triezenberg et al., 1988). The resulted hybrid protein that is known as the tetracycline-controlled transactivator (tTA), efficiently activates gene expression in the absence of Tc or doycycline (Dox). In contrast, the Tet-On system is based on a "reverse" Tet repressor (rTetR) which was created by changing four amino acid (aa) in TetR (Hillen and Berens, 1994; Gossen et al., 1995). Three of the four aa are located in the protein core (Hinrichs et al., 1994; Orth et al., 1998) at the Tc or Dox binding site. Therefore, the rTetR binds to tetO only in the presence of Tc or Dox. Fusion of rTetR to the transcriptional activation domain of herpes simplex virus protein VP16 produces aeukaryotic reverse tTA (rtTA) which is widely used in transformation of different eukaryotic organisms. However, rtTA regulatory systems showed some limitations, therefore, the rtTAs-M2 has been generated with increased induction capabilities and tighter regulation (Urlinger et al., 2000). Stebbins et al. (2001) modified both rtTA and rtTAs-M2 by deleting a putative cryptic splice site of TetR gene, adjusting codon usage of the TetR moiety and flanking the cassette with the Drosophila boundary sequences SCS and SCS', producing the altered rtTA (rtTA-alt) and rtTA-M2-alt. The transgenic Drosophila expressing the rtTA-M2-alt under the constitutive promoter actin5C demonstrated its utility in adults, embryos, and larvae. With this activator, a 70-fold of transgene induction level has been reported compared with the original rtTA transactivator (Stebbins et al., 2001).
The current work is a part of transformation experimental series to generate transgenic line of Drosophila melanogaster capable to process the N-glycan as mammalian pathways. Previously, we developed transgenic Drosophila ubiquitously expressing sialic acid synthase, CMP-sialic acid synthetase, α2,6-sialyltransferase, and α2,3-sialyl-transferase (Mohammed et al., 2009a&b). The constitutive expression of N-acetylglucosaminylteransferase II (GntII) and, β 1,4-galactosyltransferase (GalT), have an adverse effect on the insect. Therefore, strong tight induction system to express both GntII and GalT in Drosophila tissues is the recommended choice (see review for gene expression systems in Drosophila, McGuire et al., 2004). This study aimed to develop a transgenic Drosophila line expressing the rtTAM2- alt under the constitutive hr5-ie1 enhancer-promoter element. This transgenic line will be used to induce and tightly regulate the expression of such mammalian enzymes within the progeny flies by crossing with other transgenic lines bearing mammalian genes.