Novel Promoters Derived from Chinese Hamster Ovary Cells via In Silico and In Vitro Analysis

For the industrial production of recombinant proteins in mammalian cell lines, a high rate of gene expression is desired. Therefore, strong viral promoters are commonly used. However, these have several drawbacks as they override cellular responses, are not integrated into the cellular network, and thus can induce stress and potentially epigenetic silencing. Endogenous promoters potentially have the advantage of a better response to cellular state and thus a lower stress level by uncontrolled overexpression of the transgene. Such fine‐tuning is typically achieved by endogenous enhancers and other regulatory elements, which are difficult to identify purely based on the genomic sequence. Here, Chinese hamster ovary (CHO) endogenous promoters and enhancers are identified using histone marks and chromatin states, ranked based on expression level and tested for normalized promoter strength. Successive truncation of these promoters at the 5′‐ and 3′‐end as well as the combination with enhancers are identified in the vicinity of the promoter sequence further enhance promoter activity up to threefold. In an initial screen within stable cell lines, the strongest CHO promoter appears to be more stable than the human cytomegalovirus promoter with enhancer, making it a promising candidate for recombinant protein production and cell engineering applications. A deeper understanding of promoter functionality and response elements will be required to take full advantage of such promoters for cell engineering, in particular, for multigene network engineering applications.     

Promoter influence on baculovirus-mediated gene expression in permissive and nonpermissive insect cell lines.

The activities of viral and insect promoters were examined in a range of insect cell lines permissive and nonpermissive for the replication of the baculovirus Autographa californica nuclear polyhedrosis virus. Recombinant baculoviruses were constructed to place the bacterial chloramphenicol acetyltransferase gene under the control of promoters strongly active in the early, late, or very late stages of virus replication. In fully permissive cells, expression from a very late promoter was 2- to 3-fold higher than expression from a late promoter and 10- to 20-fold higher than expression from an early promoter or from a virus-borne insect promoter. In cell lines that do not support the efficient production of viral progeny, late-promoter-driven expression was similar to or surpassed very late promoter-driven expression. In nonpermissive insect cell lines, expression driven by an insect promoter derived from Drosophila melanogaster was higher than expression from the three viral promoters and was especially high in the Drosophila cell line tested. Surprisingly, late-promoter-driven expression, which is dependent on DNA replication, was higher than early-promoter-driven expression in three of four nonpermissive lines. In contrast, very late promoter-driven expression was quite limited in nonpermissive cell lines. The results indicate that the promoter used to drive foreign-gene expression strongly influences the range of insect cells which can efficiently support the production of the foreign protein during infection with recombinant baculoviruses.     

Polymerase II promoter strength determines efficacy of microRNA adapted shRNAs

Since the discovery of RNAi and microRNAs more than 10 years ago, much research has focused on the development of systems that usurp microRNA pathways to downregulate gene expression in mammalian cells. One of these systems makes use of endogenous microRNA pri-cursors that are expressed from polymerase II promoters where the mature microRNA sequence is replaced by gene specific duplexes that guide RNAi (shRNA-miRs). Although shRNA-miRs are effective in directing target mRNA knockdown and hence reducing protein expression in many cell types, variability of RNAi efficacy in cell lines has been an issue. Here we show that the choice of the polymerase II promoter used to drive shRNA expression is of critical importance to allow effective mRNA target knockdown. We tested the abundance of shRNA-miRs expressed from five different polymerase II promoters in 6 human cell lines and measured their ability to drive target knockdown. We observed a clear positive correlation between promoter strength, siRNA expression levels, and protein target knockdown. Differences in RNAi from the shRNA-miRs expressed from the various promoters were particularly pronounced in immune cells. Our findings have direct implications for the design of shRNA-directed RNAi experiments and the preferred RNAi system to use for each cell type.     

Gene transfer into established and primary fibroblast cell lines: comparison of transfection methods and promoters.

The stable transfection of immortalized Rat-1 and rat skin primary fibroblast cell lines by calcium phosphate precipitation, lipofection and electroporation methods have been examined. The lipofection method was found to be better than the other methods in terms of higher transfection efficiency and convenient use. Expression of beta-galactosidase from two different viral promoters showed that the level of transgene expression depends on the promoter strength in a particular cell type. The results presented here show that the transgene expression is extremely variable among different colonies generated from individually transfected cells. Therefore, it is necessary to examine individual colonies of cells for the production of reporter gene to obtain cell lines expressing high amounts of gene products.     

Differential expression of mammalian or viral promoter-driven gene in adherent versus suspension cells

Although expression vectors using viral and mammalian promoters constitutively express genes of interest in adherent cells, few studies have examined whether the function of these vectors in suspended cells, such as in over-agar or soft agar assay (an in vitro cell transformation assay), is as robust as when they are in adherent cells. The selection of appropriate expression vector to optimally express genes in suspended cells would be useful in determining whether these genes play a critical role in maintaining colony formation or cell transformation. To compare promoter-driven expression vector function in adherent versus suspension cells, we performed transient transfection assays using viral (simian virus 40 [SV40] and cytomegalovirus [CMV]) and mammalian (beta-actin) promoters fused to luciferase or beta-galactosidase reporter gene. Over-agar assay was used to suspend cells on top of agar, which allowed cell retrieval and analysis. We found that beta-actin and SV40 promoters exhibited suppressed gene expression of 70 and 56%, respectively, in cells suspended on agar compared with those attached on plates. The suppressed response by the exogenous beta-actin promoter in suspension was consistent with the response of the endogenous beta-actin promoter activity because the steady-state level of beta-actin messenger ribonucleic acid in suspended cells was significantly reduced by 50% relative to that expressed in attached cells. In contrast to SV40 promoter, CMV promoter activity was not decreased in cells suspended in over-agar when compared with adherent cells. These studies show that regardless of mammalian or viral vectors, one cannot assume that all expression vectors behave similarly in both suspension and adherent state.     

Functional analyses of promoter elements responsible for the differential expression of the human metallothionein (MT)-IG and MT-IF genes

The sequences responsible for heavy metal-inducible expression are situated within the proximal 437 and 160 base pairs (bp) of MT-IF and MT-IG 5'-flanking sequence, respectively. Only 105 bp of proximal MT-IG 5'-flanking sequence containing a TATA box, two metal responsive elements (MREs), and three GC motifs and 147 bp of proximal MT-IF 5'-flanking sequence containing a TATCA box, four MREs, and two GC motifs were required for heavy metal-inducible expression. However, the proximal 111 bp of MT-IF 5'-flanking sequences (a TATCA box, two MREs, and two GC motifs) was not responsive to heavy metals and competes less efficiently than the 105-bp MT-IG fragment in a competition transfection analysis. The MT-IF promoter fragment containing MREc and MREd is substantially stronger and a more efficient competitor than the MT-IG promoter fragment containing MREc and MREd. Furthermore, the proximal 160 bp of MT-IG 5'-flanking sequence functions as a strong metal-inducible promoter but not as a metal-inducible enhancer. Mobility shift analysis of MT-IF and MT-IG promoter subregions suggests a correlation between protein binding to MRE sequences and MT gene expression. These data illustrate that the overall structural and functional organization of the MT-IF and MT-IG promoters are very different and that the molecular mechanisms governing differential expression levels of human MT genes are quite complex.     

Efficient gene delivery into mammalian cells mediated by a recombinant baculovirus containing a whispovirus ie1 promoter, a novel shuttle promoter between insect cells and mammalian cells

Recombinant baculoviruses have emerged as a new gene delivery vehicle for mammalian cells. Thus, a shuttle promoter that mediates gene expression in both insect and mammalian cells will facilitate the development of a baculovirus vector-based mammalian cell gene delivery vehicle. This study described the generation of three recombinant baculoviruses with an EGFP reporter gene under the control of the white spot syndrome virus (WSSV) ie1 promoter, or either of two control promoters, the baculovirus early-to-late (ETL) promoter and polyhedrin promoter. The resulting recombinant baculoviruses were used to infect insect Sf9 cells and transduce several mammalian cell lines to test the expression of EGFP. We found that the WSSV ie1 promoter displayed a strong promoter activity in both insect and mammalian cells, and showed a stronger promoter activity than the ETL promoter in some mammalian cell lines. The activity of the WSSV ie1 promoter, but not the ETL promoter, can be enhanced by sodium butyrate, a histone deacetylase inhibitor. A transient plasmid transfection assay indicated that the WSSV ie1 promoter activity in mammalian cells is independent of baculovirus gene expression, differing from the ETL promoter, which was shown to be baculovirus-dependent. This study demonstrates, for the first time, that the WSSV ie1 promoter can function as a baculovirus-independent shuttle promoter between insect cells and mammalian cells. This novel shuttle promoter will facilitate the application of baculovirus-based vectors in gene expression, gene therapy, and non-replicative vector vaccines.