Comparison of several promoters and polyadenylation signals for use in heterologous gene expression in cultured Drosophila cells

Parts used

Key findings

• In stably transfected polyclonal Drosophila melanogaster S2 cell lines (hygromycin B selection, SV40 early poly(A) signal, tPA or HIV-1 gp120 secreted reporters, Mtn promoter induced with 0.5 mM CuSO4 added on day 4 of a 10-day culture), the relative levels of reporter protein follow the order Mtn promoter = actin 5C distal promoter > alpha 1-tubulin promoter > Bombyx mori fibroin promoter, with the pattern holding for both reporters (the actin 5C distal promoter was tested only with gp120).
• In stable S2 cell lines, the Drosophila Mtn promoter (induced with 0.5 mM CuSO4) drives approximately three-fold more tPA and five-fold more HIV-1 gp120 than the alpha 1-tubulin promoter (-1300 to +120), and the actin 5C distal promoter (-2500 to +88) drives approximately the same level of gp120 as the induced Mtn promoter (-380 to +55).
• The Bombyx mori fibroin promoter (-238 to +16) produces no detectable tPA or gp120 protein or RNA in Drosophila S2 cells even though the integrated gene copy numbers of fibroin-promoter constructs are similar to those of Mtn-promoter constructs. Fibroin promoter activity is otherwise restricted to the silk gland of B. mori, and the failure in S2 cells likely reflects the absence of required transcription factors such as the homeodomain proteins EVE and ZEN, which are absent from S2 cells.
• In stable S2 cell lines driving tPA or gp120, the steady-state reporter RNA levels from the Mtn, actin 5C distal, alpha 1-tubulin and fibroin promoters and from the SV40 early, SV40 late and Mtn poly(A) signals correlate very well with the corresponding secreted protein levels, and the relative effects of each promoter and poly(A) signal are the same for both reporters.
• Stable polyclonal S2 cell lines generated by cotransfection of 19 ug of expression plasmid with 1 ug of the hygromycin B resistance plasmid pCOhygro carry an average of approximately 700 copies of the Mtn-promoter tPA construct, 1000 copies of the Mtn-promoter BaL gp120 construct and 700 copies of the Mtn-promoter BH10 gp120 construct, as measured by Southern blot against the endogenous actin 5C gene.
• After selection of stable S2 cell lines, constructs driven by the constitutive alpha 1-tubulin promoter are present at about four-fold lower gene copy number, and constructs driven by the constitutive actin 5C distal promoter at about three-fold lower copy number, than the analogous constructs driven by the Mtn promoter (inactive in the absence of copper). Constructs driven by the nonfunctional fibroin promoter have copy numbers very similar to those of Mtn constructs, and the pattern holds for both tPA and gp120 reporters.
• Per gene copy, the actin 5C distal promoter may be inherently stronger than the Mtn promoter in S2 cells because it produces comparable gp120 from fewer integrated copies, and per gene copy the alpha 1-tubulin promoter is only two- to three-fold less productive than the actin 5C distal promoter and comparable to the induced Mtn promoter.
• In stable S2 cell lines with the Mtn promoter, replacing the SV40 early poly(A) signal with the SV40 late poly(A) signal increases secreted protein approximately two-fold for tPA and three-fold for gp120, and raises reporter RNA three- to five-fold, while gene copy number is slightly lower with the SV40 late signal.
• In stable S2 cell lines with the Mtn promoter driving tPA, the Drosophila Mtn poly(A) signal gives approximately the same tPA protein and RNA levels as the SV40 early poly(A) signal, so mammalian (SV40) and Drosophila (Mtn) poly(A) signals are used with comparable efficiency, and the SV40 late signal is stronger than the SV40 early signal in S2 cells as it is in human and monkey cells.

Abstract

We have directly compared the ability of four promoters and three polyadenylation (poly(A)) signals to direct heterologous gene expression in stably transfected Drosophila melanogaster S2 cells. We compared two constitutive Drosophila promoters, the actin 5C distal promoter and the alpha 1-tubulin promoter, with the tightly regulated Drosophila metallothionein (Mtn) promoter and the Bombyx mori fibroin promoter. We find that the actin 5C and induced Mtn promoters generate comparable high levels of RNA and protein in this system. The alpha 1-tubulin promoter generates about four-fold lower levels, and the fibroin promoter shows no detectable activity in S2 cells. Interestingly, genes expressed from the constitutive actin 5C and alpha 1-tubulin promoters are consistently present at three- to four-fold lower copy numbers than genes expressed from the inducible Mtn promoter or the inactive fibroin promoter. Poly(A) signals of both mammalian (SV40) and Drosophila (Mtn) origin efficiently directed stable RNA synthesis in S2 cells, and, as in mammalian cells, the SV40 late poly(A) signal was more efficient than the SV40 early poly(A) signal. Thus the process of polyadenylation appears to be conserved between mammalian and Drosophila cells.