A retroviral vector for siRNA expression in mammalian cells

Utilization of RNA interference (RNAi) for knockdown of gene expression has become a standard tool for the study of gene function. Short hairpin RNAs (shRNAs) expressed from RNA polymerase III promoters are widely used to achieve stable knockdown of gene expression by RNAi. We have constructed a retroviral-based shRNA expression vector, pSiRPG, as a tool for shRNA-based functional genomic studies. This vector is based on a widely used shRNA expression system and was modified to harbor an enhanced green fluorescent protein (EGFP) and a puromycin selection marker. The functionality of the elements in the pSiRPG vector was validated. The H1(TetO₂) promoter in the vector facilitates doxycycline-inducible shRNA expression, which was demonstrated in cells expressing the Tet repressor (TetR). However, we also demonstrated limited efficiency of the inhibition of shRNA expression in an uninduced TetR-expressing cell line. This observation strongly indicates that the H1(TetO₂) promoter, which is used in a wide range of vectors, is not optimal for tightly regulated shRNA expression. Stable repression of the NDRG1 protein level was observed when introducing pSiRPG constructs expressing shRNAs targeting NDRG1 into two mammary epithelial cell lines by retroviral delivery. This vector should therefore facilitate functional studies in breast cell lines that are hard to transfect with conventional plasmid-based methods.     

High epitope expression levels increase competition between T cells

Both theoretical predictions and experimental findings suggest that T cell populations can compete with each other. There is some debate on whether T cells compete for aspecific stimuli, such as access to the surface on antigen-presenting cells (APCs) or for specific stimuli, such as their cognate epitope ligand. We have developed an individual-based computer simulation model to study T cell competition. Our model shows that the expression level of foreign epitopes per APC determines whether T cell competition is mainly for specific or aspecific stimuli. Under low epitope expression, competition is mainly for the specific epitope stimuli, and, hence, different epitope-specific T cell populations coexist readily. However, if epitope expression levels are high, aspecific competition becomes more important. Such between-specificity competition can lead to competitive exclusion between different epitope-specific T cell populations. Our model allows us to delineate the circumstances that facilitate coexistence of T cells of different epitope specificity. Understanding mechanisms of T cell coexistence has important practical implications for immune therapies that require a broad immune response.     

Interactions between the non-seed region of siRNA and RNA-binding RLC/RISC proteins,Ago and TRBP,in mammalian cells

Small interfering RNA (siRNA)-based RNA interference (RNAi) is widely used for target gene silencing in various organisms. We previously showed that 8-nt-long 5′ proximal nucleotides, which include seed sequence (positions 2–8 from the 5′ end of guide strand), and the complementary sequence of the passenger strand are capable of being simultaneously replaced with cognate deoxyribonucleotides without any substantial loss of gene silencing. In the present study, examination was made of RNA requirements in the non-seed region of siRNA. The non-seed region of siRNA was found to be subdivided into four domains, in which two nucleotide pairs (positions 13 and 14) were replaceable with cognate deoxyribonucleotides without reducing RNAi activity. However, RNA sequences at positions 9-12 and 15-18 were essential for effective gene silencing, and these two double-stranded RNA cores are required for binding of the trans-activation response RNA-binding protein (TRBP). The terminal RNA (positions 19–21) provided Argonaute protein binding sites. Argonaute binding was enhanced by the presence of RNAs at positions 15–18. Knockdown experiments showed that, unlike Argonaute and TRBP, Dicer was dispensable for RNAi. Based on these observations, we discuss possible RNA/protein and protein/protein interactions in RNA-induced silencing complex formation.     

Reduced expression of GSTM2 and increased oxidative stress in spontaneously hypertensive rat

Human essential hypertension is a complex polygenic trait with underlying genetic components that remain unknown. The spontaneously hypertensive rat (SHR) is a well-characterized experimental model for essential hypertension. By comparative proteomics, we previously identified glutathione S-transferase, mu 2 (GSTM2), a protein involved in detoxification of reactive oxygen species, which had a significant reduction in left ventricles of 16-week-old SHR compared with WKY rats. In parallel, Western blotting and RT-PCR showed a similar reduction of GSTM2 in left ventricles and aortas of 4-, 8-, and 16-week-old SHR, which is before the onset of hypertension. This suggests that differential expression is not attributable to long-term changes in blood pressure. Meanwhile, the activities of GSTM2 were significantly decreased in different ages old SHR. Conversely, there was an enhanced generation of superoxide anion and activation of NADPH oxidase in SHR, which was accompanied by an increase in the protein expression of p47phox, a subunit of NADPH oxidase. These data suggest that it maybe a reduction in antioxidant defenses, evident by a reduced expression and activity of GSTM2, in the left ventricles and aortas of SHR that leads to increased levels of superoxide anion and activation of NADPH oxidase.     

Mutated polyadenylation signals for controlling expression levels of multiple genes in mammalian cells

A set of mutated SV40 early polyadenylation signals (SV40pA) with varying strengths is generated by mutating the AATAAA sequence in the wild-type SV40pA. They are shown to control the expression level of a gene over a 10-fold range using luciferase reporter genes in transient transfection assays. The relative strength of these SV40pA variants remains similar under three commonly used mammalian promoters and in five mammalian cell lines. Application of SV40pA variants for controlling expression level of multiple genes is demonstrated in a study of monoclonal antibody (mAb) synthesis in mammalian cells. By using SV40pA variants of different strengths, the expression of light chain (LC) and heavy chain (HC) genes encoded in a single vector is independently altered which results in different ratios of LC to HC expression spanning a range from 0.24 to 16.42. The changes in gene expression are determined by measuring mRNA levels and intracellular LC and HC polypeptides. It is found that a substantial decrease of HC expression, which increases the LC/HC mRNA ratio, only slightly reduces mAb production. However, reducing the LC expression by a similar magnitude, which decreases the LC/HC mRNA ratio results in a sharp decline of mAb production to trace amounts. This set of SV40pA variants offers a new tool for accurate control of the relative expression levels of multiple genes. It will have wide-ranging applications in fields related to the study of biosynthesis of multi-subunit proteins, proteomic research on protein interactions, and multi-gene metabolic engineering.     

Reduced placental docosahexaenoic acid levels associated with increased levels of sFlt-1 in preeclampsia

Our earlier studies, in preeclamptic women have shown altered levels of long chain polyunsaturated fatty acids (LCPUFA), essential constituents of the cell membrane lipids responsible for membrane stability as one of the key factors contributing to the pathophysiology of preeclampsia. We have also reported elevated levels of sFlt-1 in preeclampsia. The present study examines the levels of LCPUFA and their association with sFlt-1 levels in 69 pre-eclamptic women and 40 normotensive women. DHA and omega 3 fatty acid levels were lower (p<0.001) while arachidonic acid and omega 6 fatty acid levels were higher (p<0.05) in preeclamptic women as compared to normotensive women. Maternal plasma sFlt-1 levels were higher (p<0.05) in preeclamptic women and were negatively associated with DHA (p=0.008) and omega 3 fatty acids concentrations (p=0.031). Our results suggest that altered placental LCPUFA may result in altered membrane lipid fatty acid composition leading to increased release of sFlt-1 in circulation.     

A tightly regulated and reversibly inducible siRNA expression system for conditional RNAi-mediated gene silencing in mammalian cells

BACKGROUND: RNA interference (RNAi) is a powerful and widely used gene silencing strategy for studying gene function in mammalian cells. Transient or constitutive expression of either small interfering RNA (siRNA) or short hairpin RNA (shRNA) results in temporal or persistent inhibition of gene expression, respectively. A tightly regulated and reversibly inducible RNAi-mediated gene silencing approach could conditionally control gene expression in a temporal or spatial manner that provides an extremely useful tool for studying gene function involved in cell growth, survival and development. MATERIAL AND METHODS: In this study, we have developed a lactose analog isopropyl thiogalactose (IPTG)-responsive lac repressor-operator-controlled RNA polymerase III (Pol III)-dependent human RNase P RNA (H1) promoter-driven inducible siRNA expression system. To demonstrate its tight regulation, efficient induction and reversible inhibition, we have used this system to conditionally control the expression of firefly luciferase and human tumor suppressor protein p53 in both transient transfection cells and established stable clones. RESULTS: The results showed that this inducible siRNA expression system could efficiently induce conditional inhibition of these two genes in a dose- and time-dependent manner by administration of the inducing agent IPTG as well as being fully reverted after withdrawal of IPTG. In particular, this system could conditionally inhibit the expression of both the genes in not only established stable clones but also transient transfection cells, which should greatly increase its usefulness and convenience. CONCLUSIONS: The results presented in this study clearly indicate that this inducible siRNA expression system could efficiently, conditionally and reversibly inhibit gene expression with only very low or undetectable background silencing effects under non-inducing condition. Thus, this inducible siRNA expression system provides an ideal genetic switcher allowing the inducible and reversible control of specific gene activity in mammalian cells.     

Reduced immune effector cell NKG2D expression and increased levels of soluble NKG2D ligands in multiple myeloma may not be causally linked

Background

There is limited understanding of the dysregulation of the innate immune system in multiple myeloma (MM). We analysed the expression of the activating receptor NKG2D on NK cells and T cells of MM patients and investigated the impact of soluble versus membrane-bound NKG2D ligands on the expression of NKG2D.

Design

NKG2D expression on NK cells and CD8+ αβ T cells from patients with MM or monoclonal gammopathy of uncertain significance and healthy controls was examined flow-cytometrically. Sera from patients and controls were analysed for soluble NKG2D ligands (sNKG2D ligands).

Results

Significantly fewer NK cells and CD8+ αβ T cells from patients expressed NKG2D compared to healthy controls (NK cells: median 54% interquartile range (IQR) 32–68 versus 71% IQR 44–82%, P = 0.017, CD8+ αβ T cells: median 63% IQR 52–81 versus 77% IQR 71–90%, P = 0.018). The sNKG2D ligand sMICA was increased in patients [median 175 (IQR 87–295) pg/ml] versus controls [median 80 (IQR 32–129) pg/ml, P < 0.001], but levels of sMICA did not correlate with NKG2D expression on effector cells. To elucidate the mechanism of NKG2D down-regulation, we incubated lymphocytes from healthy donors in the presence of sNKG2D ligands or in co-culture with MM cell lines. sNKG2D ligands in clinically relevant concentrations did not down-regulate NKG2D expression, but co-culture of effector cells with myeloma cells with high surface expression of NKG2D ligands reduced NKG2D expression significantly.

Conclusions

These results indicate that MM is associated with a significant reduction in NKG2D expression which may be contact-mediated rather than caused by soluble NKG2D ligands.     

Elevated expression of the V-ATPase D2 subunit triggers increased energy metabolite levels in KrasG12D-driven cancer cells

Mutations of the Ras oncogene are frequently detected in human cancers. Among Ras-mediated tumorigenesis, Kras-driven cancers are the most dominant mutation types. Here, we investigated molecular markers related to the Kras mutation, which is involved in energy metabolism in Kras mutant-driven cancer. We first generated a knock-in Kras^G12D cell line as a model. The genotype and phenotype of the Kras ^G12D-driven cells were first confirmed. Dramatically elevated metabolite characterization was observed in Kras ^G12D-driven cells compared with wild-type cells. Analysis of mitochondrial metabolite-related genes showed that two of the 84 genes in Kras ^G12D-driven cells differed from control cells by at least twofold. The messenger RNA and protein levels of ATP6V0D2 were significantly upregulated in Kras ^G12D-driven cells. Knockdown of ATP6V0D2 expression inhibited motility and invasion but did not affect the proliferation of Kras ^G12D-driven cells. We further investigated ATP6V0D2 expression in tumor tissue microarrays. ATP6V0D2 overexpression was observed in most carcinoma tissues, such as melanoma, pancreas, and kidney. Thus, we suggest that ATP6V0D2, as one of the V-ATPase (vacuolar-type H ⁺-ATPase) subunit isoforms, may be a potential therapeutic target for Kras mutation cancer.     

Targeting the kinesin Eg5 to monitor siRNA transfection in mammalian cells

RNA interference, the inhibition of gene expression by double-stranded RNA, provides a powerful tool for functional studies once the sequence of a gene is known. In most mammalian cells, only short molecules can be used because long ones induce the interferon pathway. With the identification of a proper target sequence, the penetration of the oligonucleotides constitutes the most serious limitation in the application of this technique. Here we show that a small interfering RNA (siRNA) targeting the mRNA of the kinesin Eg5 induces a rapid mitotic arrest and provides a convenient assay for the optimization of siRNA transfection. Thus, dose responses can be established for different transfection techniques, highlighting the great differences in response to transfection techniques of various cell types. We report that the calcium phosphate precipitation technique can be an efficient and cost-effective alternative to Oligofectamine in some adherent cells, while electroporation can be efficient for some cells growing in suspension such as hematopoietic cells and some adherent cells. Significantly, the optimal parameters for the electroporation of siRNA differ from those for plasmids, allowing the use of milder conditions that induce less cell toxicity. In summary, a single siRNA leading to an easily assayed phenotype can be used to monitor the transfection of siRNA into any type of proliferating cells of both human and murine origin.     

The effect of L-2-oxothiazolidine on glutathione levels in cultured mammalian cells

Chinese hamster ovary cells were cultured with increasing concentrations (7 X 10(-8) M-7 X 10(-2) M) of L-2-oxothiazolidine in an attempt to increase intracellular glutathione levels in these multiply-passaged mammalian cells. In a series of 7 Expts., intracellular glutathione levels increased significantly at the highest concentrations tested and were protective against oxygen radical-induced genetic toxicity.     

The IE2 promoter/enhancer region from mouse CMV provides high levels of therapeutic protein expression in mammalian cells

Protein expression in mammalian cells is key for the production and manufacturing of bio-therapeutics with human-like properties and activities. As a molecular basis for reaching high protein expression levels, efficient promoter/enhancer systems are a prerequisite. Here we identify a novel enhancer from the mouse cytomegalovirus (CMV) immediate early 2 (IE2) region as a strong expression-promoting element. We further demonstrate its activity in bi-directional promoter architecture and apply it to generate production clones for IL-18BP, a protein with therapeutic indications in autoimmune diseases. These data show that the IE region from mouse CMV, and the IE2 enhancer/promoter in particular, have a broad potential for application in novel gene expression systems for research, development, and manufacturing of protein drugs.