Transient gene expression in mammalian cells grown in serum-free suspension culture

In order to establish a simple and scaleable transfection system we have used the cationic polymer polyethylenimine (PEI) to study transient transfection in HEK293 and 293(EBNA) cells grown in serum-free suspension culture. The transfection complexes were made directly within the cell culture by consecutively adding plasmid and PEI (direct method). Alternatively, the DNA-PEI transfection complexes were prepared in fresh medium (1/10 culture volume) and then added to the cells (indirect method). The results of this study clearly show that the ratio of PEI nitrogen to DNA phosphate is very important for high expression levels. The precise ratio is dependent on the DNA concentration. For example, using 1 μg/ml DNA by the indirect method, the ratio of optimal PEI:DNA was about 10–13:1. However, the ratio increases to 33:1 for 0.1–0.2 μg/ml DNA. By testing several different molecular weights of the polycationic polymer we could show that the highest transfection efficiency was obtained with the PEI 25 kDa. Using PEI 25 kDa the indirect method is superior to the direct addition because significantly lower DNA concentrations are needed. The expression levels of the soluble human TNF receptor p55 are even higher at low DNA compared to 1 μg/ml plasmid. The EBV-based pREP vectors gave better transient gene expression when used in 293(EBNA) cells compared to HEK293 cells in suspension culture. No differences in expression levels in the two cell lines were observed when the pC1 (CMV)-TNFR was used. In conclusion, PEI is a low-toxic transfection agent which provides high levels of transient gene expression in 293(EBNA) cells grown in serum-free suspension culture. This system allows highly reproducible, cost-effective production of milligram amounts of recombinant proteins in 2–5 l spinner culture scale within 3–5 days. Fermentor scale experiments, however, are less efficient because the PEI-mediated transient tranfection is inhibited by conditioned medium. This revised version was published online in July 2006 with corrections to the Cover Date.     

Carrier-free cellular uptake and the gene-silencing activity of the lipophilic siRNAs is strongly affected by the length of the linker between siRNA and lipophilic group

The conjugation of siRNA to molecules, which can be internalized into the cell via natural transport mechanisms, can result in the enhancement of siRNA cellular uptake. Herein, the carrier-free cellular uptake of nuclease-resistant anti-MDR1 siRNA equipped with lipophilic residues (cholesterol, lithocholic acid, oleyl alcohol and litocholic acid oleylamide) attached to the 5'-end of the sense strand via oligomethylene linker of various length was investigated. A convenient combination of H-phosphonate and phosphoramidite methods was developed for the synthesis of 5'-lipophilic conjugates of siRNAs. It was found that lipophilic siRNA are able to effectively penetrate into HEK293, HepG2 and KB-8-5 cancer cells when used in a micromolar concentration range. The efficiency of the uptake is dependent upon the type of lipophilic moiety, the length of the linker between the moiety and the siRNA and cell type. Among all the conjugates tested, the cholesterol-conjugated siRNAs with linkers containing from 6 to 10 carbon atoms demonstrate the optimal uptake and gene silencing properties: the shortening of the linker reduces the efficiency of the cellular uptake of siRNA conjugates, whereas the lengthening of the linker facilitates the uptake but retards the gene silencing effect and decreases the efficiency of the silencing.     

Inhibition of thimet oligopeptidase by siRNA alters specific intracellular peptides and potentiates isoproterenol signal transduction

Mammalian cells have a large number of intracellular peptides that are generated by extralysosomal proteases. In this study, the enzymatic activity of thimet oligopeptidase (EP24.15) was inhibited in human embryonic kidney (HEK) 293 cells using a specific siRNA sequence. The semi-quantitative intracellular peptidome analyses of siRNA-transfected HEK293 cells shows that the levels of specific intracellular peptides are either increased or decreased upon EP24.15 inhibition. Decreased expression of EP24.15 was sufficient to potentiate luciferase gene reporter activation by isoproterenol (1-10 μM). The protein kinase A inhibitor KT5720 (1 μM) reduced the positive effect of the EP24.15 siRNA on isoproterenol signaling. Thus, EP24.15 inhibition by siRNA modulates the levels of specific intracellular peptides and isoproterenol signal transduction.     

Production of lentiviral vectors in suspension cells using low proportion of supercoiled circular plasmid DNA

The supercoiled circular (SC) topology form of plasmid DNA has been regarded to be advantageous over open circular or linearized analogue in transfection and expression efficiency, and therefore are largely demanded in the biopharmaceutical manufacturing. However, production of high-purity SC plasmid DNA would result in high manufacturing cost. The effect of SC proportion in plasmid DNA on the quality of packaged lentiviral vectors has never been reported. In this study, we established an efficient system for production of high-titer lentiviral vectors using suspension HEK293SF cells in serum-free media, and the lentiviral titer was not associated with the proportion of SC plasmid DNA. Plasmids DNA with different proportion of SC, open-circular, and linearized forms were prepared using the thermal denaturation method, and were transfected to adherent HEK293T or suspension HEK293SF cells for packaging of lentiviral vectors. The titer of lentiviral vectors from HEK293T cells, but not from HEK293SF cells, was significantly impaired when the proportion of SC plasmid DNA decreased from 60–80% to 30–40%. Further decrease of SC plasmid proportion to 3% led to a dramatic reduction of lentiviral titer no matter the packaging cell line was. However, lentiviral vectors from HEK293SF cells still showed a high titer even when the proportion of SC plasmid DNA was 3%. This study demonstrated that extremely high proportion of SC plasmid DNA was not required for packaging of high-titer lentiviral vector in HEK293SF cells, at least under our manufacturing process.     

Selective gene silencing of rat ATP-binding cassette G2 transporter in an in vitro blood-brain barrier model by short interfering RNA

The aim of the present study was to specifically silence the rat ATP-binding cassette transporter G2 (rABCG2) gene in brain capillary endothelial cells by transfection of short interfering RNA (siRNA). Four different siRNAs designed to target rABCG2 were each transfected into HEK293 cells with myc-tagged rABCG2 cDNA. Quantitative real-time PCR and western blot analyses revealed that three of the siRNAs were able to reduce exogenous rABCG2 mRNA and protein levels in HEK293 cells. Moreover, rABCG2-mediated mitoxantrone efflux transport was suppressed by the introduction of these three siRNAs into HEK293 cells. In contrast, the other siRNA and non-specific control siRNA did not significantly affect the mRNA expression, the protein level or the transport activity. Endogenous rABCG2 mRNA and protein expression in a conditionally immortalized rat brain capillary endothelial cell line (TR-BBB13) was suppressed by the most potent siRNA among the four siRNAs tested. Furthermore, this siRNA did not affect the mRNA levels of other ABC transporters, such as ABCB1, ABCC1 and ABCG1, and the protein level of ABCB1 in TR-BBB13 cells, suggesting that it can selectively silence rABCG2 at the blood-brain barrier. This should be a useful and novel strategy for clarifying the contribution of rABCG2 to brain-to-blood transport of substrate drugs and endogenous compounds across the blood-brain barrier.     

A simple and visualized method to screen for effective siRNAs by using green fluorescence protein (GFP) as a reporter

To screen for effective small interference RNA (siRNA), a simple and visualized method was developed using the green fluorescence protein (GFP) as a reporter. Candidate siRNAs targeting macrophage migration inhibition factor genes (MIF) were identified. By using the pEGFP-N3 vector, the MIF-GFP expression plasmid, pEGFP-MIF, was constructed with the same Kozak consensus translation initiation site and start code ATG for the MIF-EGFP coding sequence. Based on the siRNA expression vector pSilencer-4.1, 3 candidate MIF siRNA expression plasmids were constructed and co-transfected with the pEGFP-MIF into the HEK293 cells, respectively. The GFP expression in HEK293 cells could be viewed by fluorescence microscopy and the MIF mRNA expressions were determined by real-time quantitative PCR. The 3 candidate MIF siRNA expression plasmids were also co-transfected with the MIF expression plasmid into the HEK293 cells, respectively, and the MIF mRNA expressions were determined by real-time quantitative PCR. The results show that the down-regulated expression of the MIF mRNA was consistent with the GFP expression and the same effective MIF siRNAs were screened by using the pEGFP-MIF or MIF expression plasmid with the candidate MIF siRNAs expression plasmids. Therefore, by using the GFP as a reporter, a useful method was provided to screen for effective siRNAs targeting specific genes co-expressed with the GFP. This may be a good strategy for screening for effective siRNAs targeting different genes. __________ Translated from Chinese Journal of Biochemistry and Molecular Biology, 2007, 23(3): 231–235 [译自: 中国生物化学与分子生物学报]     

Antigenicity of human melanoma cells transfected to express the B7-1 co-stimulatory molecule (CD80) varies with the level of B7-1 expression

 The aim of this study was to compare the antigenicity of human melanoma cells molecularly modified by particle-mediated gene transfer to have transient or stable expression of the B7-1 co-stimulatory molecule (CD80). The unmodified melanoma cells (mel5, m21) had no constitutive expression of B7-1, but 22%–28% of cells had transient B7-1 expression 24 h following transfection with cDNA for B7-1 (mel5-B7, m21-B7). In addition, 85%–90% of cells had stable B7-1 expression following transfection with cDNA for B7-1 and in vitro culture under selection conditions (mel5-B7neo, m21-B7neo). Allogeneic HLA-unmatched normal donor peripheral blood mononuclear cells (PBMC) secreted greater amounts of granulocyte/macrophage-colony-stimulating factor (GM-CSF) when incubated for 3 days with m21-B7neo than did PBMC incubated with m21-B7, which, in turn, secreted greater amount of GM-CSF than PBMC incubated with m21. Similarly, cell-mediated cytotoxicity against unmodified melanoma cells by PBMC co-cultured for 5 days with the modified or unmodified melanoma cells was proportional to the level of B7-1 expression on the stimulating cells. This cytolytic activity had both an HLA-class-I-restricted and an HLA-class-I-unrestricted component. Following 5 days of co-culture, PBMC expression of CD28, the ligand for B7-1, was down-regulated in proportion to the level of B7-1 expression on the stimulating melanoma cells. Thus, particle-mediated gene delivery of cDNA for B7-1 into human melanoma cells increased expression of functional B7-1 and enhanced the antigenicity of the gene-modified cells in proportion to their level of B7-1 expression. Received: 14 October 1999 / Accepted: 3 December 1999     

Improving NK1R-targeted gene delivery of stearyl-antimicrobial peptide CAMEL by conjugating it with substance P

Specificity is a crucial condition that hampers the application of non-viral vectors for cancer gene therapy. In a previous study, we developed an efficient gene vector, stearyl-CAMEL, using N-terminal stearylation of the antimicrobial peptide CAMEL. Substance P (SP), an 11-residue neuropeptide, rapidly enters cells after binding to the neurokinin-1 receptor (NK1R), which is expressed in many cancer cell lines. In this study, the NK1R-targeted gene vector stearyl-CMSP was constructed by conjugating SP to the C-terminus of stearyl-CAMEL. Our results indicated that stearyl-CMSP displayed significant transfection specificity for NK1R-expressing cells compared with that shown by stearyl-CAMEL. Accordingly, the stearyl-CMSP/p53 plasmid complexes had significantly higher antiproliferative activity against HEK293-NK1R cells than they did against HEK293 cells, while the stearyl-CAMEL/p53 plasmid complexes did not show this specificity in antiproliferative activity. Consequently, conjugation of the NK1R-targeted ligand SP is a simple and successful strategy to construct efficient cancer-targeted non-viral gene vectors.     

A guanosine 3',5'-cyclic monophosphate (cGMP) reporter system based on the G-kinase/CREB/CRE signal transduction pathway

Guanylate cyclases constitute a gene family of enzymes that synthesize the second messenger guanosine 3′,5′-cyclic monophosphate (cGMP) and play important roles in diverse physiological functions. Here we report a novel, simple and highly sensitive method for measurement intracellular cGMP concentrations using a cAMP-responsive element (CRE) and cGMP-dependent protein kinase (cGK). Transient transfection of the CRE reporter plasmid, encoding a luciferase reporter gene under the control of a modified promoter containing a CRE, and a cGK expression vector into HEK293 cells followed by treatment with 8-bromo-cGMP showed a dose dependent increase in luciferase activity. Moreover, HEK293 cells expressing GC-A or GC-B natriuretic peptide receptors and harboring this reporter system responded to specific ligands in a dose dependent manner. Our results indicate that this reporter gene method enables high throughput screening of receptor-type GC selective agonists in the treatment of cardiovascular diseases and homeostatic dysfunctions.     

Carboxymethyl dextran-trimethyl chitosan coated superparamagnetic iron oxide nanoparticles: An effective siRNA delivery system for HIV-1 Nef

Gene therapy, including small interfering RNA (siRNA) technology, is one of the leading strategies that help to improve the outcomes of the current therapeutic systems against HIV-1 infection. The successful therapeutic application of siRNAs requires their safe and efficient delivery to specific cells. Here, we introduce a superparamagnetic iron oxide nanoparticle (SPION) for delivering siRNA against HIV-1 nef (anti-nef siRNA) into two cell lines, HEK293 and macrophage RAW 264.7. SPIONs were coated with trimethyl chitosan (TMC), and thereafter, different concentrations of SPION–TMC were coated with different ratios of a carboxymethyl dextran (CMD) to modify the physicochemical properties and improve the biological properties of the nanocarriers. The nanoparticles exhibited a spherical shape with an average size of 112 nm. The obtained results showed that the designed delivery route enhanced the uptake of siRNA into both HEK293 and RAW 264.7 cells compared with control groups. Moreover, CMD–TMC–SPIONs containing anti-nef siRNA significantly reduced the expression of HIV-1 nef in HEK293 stable cells. The modified siRNA-loaded SPIONs also displayed no toxicity or apoptosis-inducing effects on the cells. The CMD–TMC–SPIONs are suggested as potential nanocarriers for siRNA delivery in gene therapy of HIV-1 infection.     

肿瘤内皮标志物8同型物Ⅰ基因真核表达载体的构建及其在HEK293F细胞中的表达

目的:构建肿瘤内皮标志物8(TEM8)基因真核表达载体,实现TEM8在HEK293F细胞中的外源表达。方法:用PCR技术扩增TEM8基因,经限制性酶切、连接、转化,插入pcDNA3.1(+)-EGFP真核表达载体,并通过脂质体将TEM8表达质粒转染至HEK293F细胞中,Western印迹检测TEM8的表达。结果:PCR扩增得到TEM8基因,构建了真核表达载体pcDNA3.1(+)-TEM8-EGFP并转染HEK293F细胞,经G418加压筛选及有限稀释法得到生长性状良好、表达效率高的单克隆细胞系TEM8-EGFP/HEK293F;Western印迹证明过表达细胞系TEM8-EGFP/HEK293F显著表达TEM8蛋白。结论:构建了表达TEM8的重组HEK293F工程细胞系TEM8-EGFP/HEK293F,为进一步研究TEM8的生理功能奠定了基础。     

Investigation of relationship between EBNA-1 expression level and specific foreign protein productivity in transient gene expression of HEK293 cells

In an attempt to determine the relationship between the Epstein–Barr virus nuclear antigen-1 (EBNA-1) expression level and specific foreign protein productivity (q_p), EBNA-1-amplifed HEK293 cells, which achieved a higher EBNA-1 expression level than that achieved by HEK293E cells, were established using dihydrofolate reductase (dhfr)-mediated gene amplification. Compared with a control culture in a null pool, Fc-fusion protein production by transient transfection in the EBNA-1-amplified pool showed a significant improvement. q_p was linearly correlated with the EBNA-1 expression level in the transient transfection of EBNA-1-amplified clones, as indicated by the correlation coefficient (R² = 0.7407). The Fc-fusion protein production and q_p in a transient gene expression-based culture with EBNA-1-amplified HEK293 cells, E-amp-68, were approximately 2.0 and 3.2 times, respectively, higher than those in a culture with HEK293E cells. The increase in q_p by EBNA-1 amplification mainly resulted from an enhancement in the amount of replicated DNA and level of mRNA expression but not an improved transfection efficiency. Taken together, it was found that EBNA-1 amplification could improve the therapeutic protein production in an HEK293 cell-based transient gene expression system.     

Activation of cloned BK<Subscript>Ca</Subscript> channels in nitric oxide-induced apoptosis of HEK293 cells

The large conductance Ca²⁺-activated K⁺ (BK_Ca) channels are highly expressed in vascular smooth muscle cells (VSMCs) and play an essential role in the regulation of various physiological functions. Besides its electrophysiological function in vascular relaxation, BK_Ca has also been reported to be implicated in nitric oxide (NO)-induced apoptosis of VSMCs. However, the molecular mechanism is not clear and has not been determined on cloned channels. The present study was designed to clarify whether activation of cloned BK_Ca channel was involved in NO-induced apoptosis in human embryonic kidney 293 (HEK293) cell. The cDNA encoding the α-subunit of BK_Ca channel, hSloα, was transiently transfected into HEK293 cells. The apoptotic death in HEK-hSloα cells was detected using immunocytochemistry, analysis of fragmented DNA by agarose gel electrophoresis, MTT test, and flow cytometry assays. Whole-cell and single-channel characteristics of HEK-hSloα cells exhibited functional features similar to native BK_Ca channel in VSMCs. Exposuring of HEK- hSloα cells to S-nitroso-N-acetyl-penicillamine increased the hSloα channel activities of whole-cell and single-channel, and then increased percentage of cells undergoing apoptosis. However, blocking hSloα channels with 1 mM tetraethylammonia or 100 nM iberiotoxin significantly decreased the NO-induced apoptosis, whereas 30 μM NS1619, the specific agonist of BK_Ca, independently increased hSloα currents and induced apoptosis. These results indicated that activation of cloned BK_Ca channel was involved in NO-induced apoptosis of HEK293 cells.