Lipid Transfection Reagent Research Articles

Predicting Transiently Expressed Protein Yields: Comparison of Transfection Methods in CHO and HEK293.

Therapeutic proteins are currently at the apex of innovation in pharmaceutical medicine. However, their industrial production is technically challenging and improved methods for transient transfection of mammalian cell cultures are necessary. We aimed to find a fast, microliter-scale transfection assay that allows the prediction of protein expression in the transient production settings. We used an array of lipid, polymeric and cell-penetrating peptide transfection reagents, and compared their performance in various high throughput transfection assays to their performance in protein (antibody) expression in professional protein-producer cell lines. First, we show that some of the most frequently used microliter-scale transfection efficacy assays fail to predict performance in the protein production in milliliter and liter scale settings. We found that CHO suspension culture post-transfection EGFP(+) population and SEAP quantitation correlate with large-scale protein production, whereas the adhesion culture assays and transfection of pLuc are non-predictive. Second, we demonstrated that cell-penetrating peptide-based transfection achieves significantly higher protein yields compared to PEI and lipoplex methods in both CHO and HEK293 producer cell lines. In this work we demonstrate a CPP-based transient protein expression approach that significantly outperformed the current industry standard workhorse method of PEI.

Retraction of "Synthesis of Phosphorodiamidate Morpholino Oligonucleotides and Their Chimeras Using Phosphoramidite Chemistry".

Phosphorodiamidate morpholinos (PMOs) and PMO–DNA chimeras have been prepared on DNA synthesizers using phosphoramidite chemistry. This was possible by first generating boranephosphoroamidate morpholino internucleotide linkages followed by oxidative substitution with four different amines: N,N-dimethylamine, N-methylamine, ammonia, and morpholine. When compared to a natural DNA duplex, the amino modified PMO was found to have a higher melting temperature with either complementary DNA or RNA, whereas the remaining PMO analogues having morpholino, dimethylamino, or N-methylamino phosphorodiamidate linkages had melting temperatures that were either comparable or reduced. Additionally the N,N-dimethylamino PMO–DNA chimeras were found to stimulate RNaseH1 activity. Treatment of HeLa cells with fluorescently labeled PMO chimeras demonstrated that these analogues were efficiently taken up by cells in the presence of a lipid transfection reagent. Because of the simplistic synthesis procedures, various PMO analogu...

Effective usage of cationic derivatives of polyprenols as carriers of DNA vaccines against influenza virus

BackgroundCationic derivatives of polyprenols (trimethylpolyprenylammonium iodides – PTAI) with variable chain length between 6 and 15 isoprene units prepared from naturally occurring poly-cis-prenols were tested as DNA vaccine carriers in chickens and mice. This study aimed to investigate if PTAI could be used as an efficient carrier of a DNA vaccine.MethodsSeveral vaccine mixtures were prepared by combining different proportions of the vaccine plasmid (carrying cDNA encoding a vaccine antigen, hemagglutinin from H5N1 influenza virus) and various compositions of PTAI. The vaccines were delivered by intramuscular injection to either chickens or mice. The presence of specific antibodies in sera collected from the immunized animals was analyzed by enzyme-linked immunosorbent assay (ELISA) and hemagglutination inhibition (HI) test.ResultsThe mixtures of PTAI with helper lipids, such as DOPE (1,2-dioleoyl-sn-glycero-3-phosphatidylethanolamine), DC-cholesterol [{3ß-[N-(N′,N′-dimethylaminoethane)-carbamoyl] cholesterol} hydrochloride] or DOPC (1,2-dioleoyl-sn-glycero-3-phosphatidylcholine) induced strong humoral response to the antigen encoded by the DNA vaccine plasmid.ConclusionThe animal immunization results confirmed that PTAI compositions, especially mixtures of PTAI with DOPE and DC-cholesterol, do work as effective carriers of DNA vaccines, comparable to the commercially available lipid transfection reagent.

Synthesis of Phosphorodiamidate Morpholino Oligonucleotides and Their Chimeras Using Phosphoramidite Chemistry.

Phosphorodiamidate morpholinos (PMOs) and PMO-DNA chimeras have been prepared on DNA synthesizers using phosphoramidite chemistry. This was possible by first generating boranephosphoroamidate morpholino internucleotide linkages followed by oxidative substitution with four different amines: N,N-dimethylamine, N-methylamine, ammonia, and morpholine. When compared to a natural DNA duplex, the amino modified PMO was found to have a higher melting temperature with either complementary DNA or RNA, whereas the remaining PMO analogues having morpholino, dimethylamino, or N-methylamino phosphorodiamidate linkages had melting temperatures that were either comparable or reduced. Additionally the N,N-dimethylamino PMO-DNA chimeras were found to stimulate RNaseH1 activity. Treatment of HeLa cells with fluorescently labeled PMO chimeras demonstrated that these analogues were efficiently taken up by cells in the presence of a lipid transfection reagent. Because of the simplistic synthesis procedures, various PMO analogues are now readily available and should therefore open new pathways for research into the antisense, diagnostic, and nanotechnology oligonucleotide fields.

Immune response of healthy horses to DNA constructs formulated with a cationic lipid transfection reagent.

BackgroundDeoxyribonucleic acid (DNA) vaccines are used for experimental immunotherapy of equine melanoma. The injection of complexed linear DNA encoding interleukin (IL)-12/IL-18 induced partial tumour remission in a clinical study including 27 grey horses. To date, the detailed mechanism of the anti-tumour effect of this treatment is unknown.ResultsIn the present study, the clinical and cellular responses of 24 healthy horses were monitored over 72 h after simultaneous intradermal and intramuscular application of equine IL-12/IL-18 DNA (complexed with a transfection reagent) or comparative substances (transfection reagent only, nonsense DNA, nonsense DNA depleted of CG).Although the strongest effect was observed in horses treated with expressing DNA, horses in all groups treated with DNA showed systemic responses. In these horses treated with DNA, rectal temperatures were elevated after treatment and serum amyloid A increased. Total leukocyte and neutrophil counts increased, while lymphocyte numbers decreased. The secretion of tumour necrosis factor alpha (TNFα) and interferon gamma (IFNγ) from peripheral mononuclear blood cells ex vivo increased after treatments with DNA, while IL-10 secretion decreased. Horses treated with DNA had significantly higher myeloid cell numbers and chemokine (C-X-C motif) ligand (CXCL)-10 expression in skin samples at the intradermal injection sites compared to horses treated with transfection reagent only, suggesting an inflammatory response to DNA treatment.In horses treated with expressing DNA, however, local CXCL-10 expression was highest and immunohistochemistry revealed more intradermal IL-12-positive cells when compared to the other treatment groups.In contrast to non-grey horses, grey horses showed fewer effects of DNA treatments on blood lymphocyte counts, TNFα secretion and myeloid cell infiltration in the dermis.ConclusionTreatment with complexed linear DNA constructs induced an inflammatory response independent of the coding sequence and of CG motif content. Expressing IL-12/IL-18 DNA locally induces expression of the downstream mediator CXCL-10.The grey horses included appeared to display an attenuated immune response to DNA treatment, although grey horses bearing melanoma responded to this treatment with moderate tumour remission in a preceding study. Whether the different immunological reactivity compared to other horses may contributes to the melanoma susceptibility of grey horses remains to be elucidated.Electronic supplementary materialThe online version of this article (doi:10.1186/s12917-015-0452-3) contains supplementary material, which is available to authorized users.

Cell Penetrating Peptides as an Alternative Transformation Method in Citrus

Huanlongbing (HLB) has caused the loss of thousands of trees in Florida’s multi-billion dollar citrus industry. An effective, long-term strategy to controlling this disease will be by the incorporation of genetic resistance into commercial genotypes. Because conventional breeding is limited by the lack of natural resistance in citrus to HLB, genetic engineering is now considered a significant alternative to incorporating such characteristics. In fact, despite general concerns from the public against genetically modified organisms (GMOs), one National Academy report1 stated that genetic engineering will be the way to fully exterminate HLB, while growers’ support of a transgenic approach for disease resistant traits also continues to rise. The primary transformation method of citrus typically uses Agrobacterium, in which explants are suspended with the bacterium and subsequently placed on selection media. After treatment, the explants produce shoots that can ultimately lead to stable transgenic plants. Due to the slow growth and lengthy maturation, this process takes several years to produce reproductive trees and must be optimized for each cultivar. Consequently, transformation efficiency is substantially less than other model systems. The commercialization of transgenic disease resistant cultivars is even slower due to regulations limiting GMOs worldwide. In order to decrease the dependence upon bacterial vectors and increase transformation efficiency, we have researched an alternative method for introducing nucleic acids into plants that does not involve Agrobacterium and instead uses cell penetrating peptides (CPPs). CPPs are short, positively charged amino acid sequences that bind to negatively charged molecules and subsequently translocate across cellular membranes. Most surprisingly plant cell walls can also be bypassed, as CPPs are currently used in plants in transient expression and gene silencing assays. Until now, CPPs have not been examined in citrus or other woody crops for stable transformation protocols. We have developed a method for the transient expression of reporter genes (GUS and GFP) using plasmid DNA and CPPs. Our data indicate that up to 50% of treated explants express GUS when CPPs are used alone. Several optimization steps have been tested and the expression efficiency can be increased up to 100% when CPPs are used in conjunction with a lipid transfection reagent. We have also produced hypocotyl segments which survived kanamycin selection. Some produced shoots that rooted and were planted in soil and have been maintained in a growth chamber. PCR and reporter gene analysis will confirm if stable integration has occurred. Our novel protocol could have far reaching effects for the successful integration of disease resistant GMOs in global markets by limiting the perceived negative effect of bacterial vectors.

Tracking of mesenchymal stem cells labeled with gadolinium diethylenetriamine pentaacetic acid by 7T magnetic resonance imaging in a model of cerebral ischemia.

Progress in the development of stem cell and gene therapy requires repeatable and non-invasive techniques to monitor the survival and integration of stem cells in vivo with a high temporal and spatial resolution. The purpose of the present study was to examine the feasibility of using the standard contrast agent gadolinium diethylenetriamine pentaacetic acid (Gd-DTPA) to label rat mesenchymal stem cells (MSCs) for stem cell tracking. MSCs, obtained from the bilateral femora of rats, were cultured and propagated. The non-liposomal lipid transfection reagent effectene was then used to induce the intracellular uptake of Gd-DTPA. Electron microscopy was used to detect the distribution of Gd-DTPA particles in the MSCs. The labeling efficiency of the Gd-DTPA particles in the MSCs was determined using spectrophotometry, and MTT and trypan blue exclusion assays were used to evaluate the viability and proliferation of the labeled MSCs. T1-weighted magnetic resonance imaging (MRI) was used to observe the labeled cells in vitro and in the rat brain. Gd-DTPA particles were detected inside the MSCs using transmission electron microscopy and a high labeling efficiency was observed. No difference was observed in cell viability or proliferation between the labeled and unlabeled MSCs (P>0.05). In the in vitro T1-weighted MRI and in the rat brain, a high signal intensity was observed in the labeled MSCs. The T1-weighted imaging of the labeled cells revealed a significantly higher signal intensity compared with that of the unlabeled cells (P<0.05) and the T1 values were significantly lower. The function of the labeled MSCs demonstrated no change following Gd-DTPA labeling, with no evident adverse effect on cell viability or proliferation. Therefore, a change in MR signal intensity was detected in vitro and in vivo, suggesting Gd-DTPA can be used to label MSCs for MRI tracking.

Innate adjuvant receptor Toll-like receptor 3 can promote breast cancer through cell surface.

Toll-like receptor 3 has been targeted in different cancers for adjuvant therapy. The ligand-mediated effects of TLR-3 on cancer cells are discordant. In the present work, we have addressed the hypothesis possibility of cell membrane-bound action of TLR-3 in breast cancer to justify its pro-tumor effect. TLR-3 was stimulated by Poly (I:C) on the surface of human breast cancer cells MCF-7 and MDA-MB-231 for up to 72 h. To check the cell survival and growth, thiazol blue tetrazolium bromide (MTT) assay, apoptosis assay, and cell cycle analysis were carried out. For changes in the metastatic properties, in vitro colony formation assay, scratch-wound healing assay and adhesion assay were also done. Using real-time PCR and immunocytochemistry, expression of E-cadherin, was studied. To determine the affect of cytoplasmic stimulation, Poly (I:C) was delivered with lipid transfection reagent. The results of the aforesaid experiments showed that there was a gradual increase of cellular survivability, growth, and metastasis after the cell surface stimulation of TLR-3 with Poly (I:C). Interestingly, E-cadherin expression was increased both at transcriptional and translational level. On the other hand, when Poly (I:C) was delivered in the cytoplasm by lipid transfecting medium, the cells survivability was decreased. For the first time, in the present work, we are convincingly reporting the functional evidence that TLR-3 induces cell survivability and metastasis through cell surface. The present work may help for the proper understanding of the adjuvant therapy of breast cancer.

Efficient delivery of RNA interference oligonucleotides to polarized airway epithelia in vitro

Polarized and pseudostratified primary airway epithelia present barriers that significantly reduce their transfection efficiency and the efficacy of RNA interference oligonucleotides. This creates an impediment in studies of the airway epithelium, diminishing the utility of loss-of-function as a research tool. Here we outline methods to introduce RNAi oligonucleotides into primary human and porcine airway epithelia grown at an air-liquid interface and difficult-to-transfect transformed epithelial cell lines grown on plastic. At the time of plating, we reverse transfect small-interfering RNA (siRNA), Dicer-substrate siRNA, or microRNA oligonucleotides into cells by use of lipid or peptide transfection reagents. Using this approach we achieve significant knockdown in vitro of hypoxanthine-guanine phosphoribosyltransferase, IL-8, and CFTR expression at the mRNA and protein levels in 1-3 days. We also attain significant reduction of secreted IL-8 in polarized primary pig airway epithelia 3 days posttransfection and inhibition of CFTR-mediated Cl⁻ conductance in polarized air-liquid interface cultures of human airway epithelia 2 wk posttransfection. These results highlight an efficient means to deliver RNA interference reagents to airway epithelial cells and achieve significant knockdown of target gene expression and function. The ability to reliably conduct loss-of-function assays in polarized primary airway epithelia offers benefits to research in studies of epithelial cell homeostasis, candidate gene function, gene-based therapeutics, microRNA biology, and targeting the replication of respiratory viruses.

DNA-lipid-apatite composite layers enhance gene expression of mesenchymal stem cells

DNA-apatite composite layer (D-Ap layer) and DNA-lipid-apatite composite layer (DLp-Ap layer) were prepared on ceramic hydroxyapatite disk and scaffold using supersaturated calcium phosphate solutions supplemented with 0.5–5μg/mL plasmid and 0–10μL/mL lipid transfection reagent FuGENE®. Both in vitro and in vivo studies were carried out using mesenchymal stem cells (MSCs) and two kinds of gene (luciferase and bone morphogenetic protein (BMP)-2) for demonstrating potential application of the gene transfer system using the D-Ap and DLp-Ap layers in bone tissue engineering. In the in vitro study using luciferase gene, the DLp-Ap layers showed 1–2 orders of magnitudes higher gene transfer efficiency to MSCs than the D-Ap layer. In the in vivo study using BMP-2 gene, DLp-Ap layer slightly increased BMP-2 protein concentration than D-Ap layer, thereby enhancing their osteogenic differentiation than D-Ap layer. The present gene transfer system using the DLp-Ap layers, with the advantages of good biocompatibility, bone-bonding ability, and efficacy in in vitro and in vivo gene transfer to MSCs, would be useful in bone tissue engineering.

Fabrication of a DNA-lipid-apatite composite layer for efficient and area-specific gene transfer

A surface-mediated gene transfer system using biocompatible apatite-based composite layers has great potential for tissue engineering. Among the apatite-based composite layers developed to date, we focused on a DNA-lipid-apatite composite layer (DLp-Ap layer), which has the advantage of relatively high efficiency as a non-viral system. In this study, various lipid transfection reagents, including a newly developed reagent, polyamidoamine dendron-bearing lipid (PD), were employed to prepare the DLp-Ap layer, and the preparation condition was optimized in terms of efficiency of gene transfer to epithelial-like CHO-K1 cells in the presence of serum. The optimized DLp-Ap layer derived from PD had the highest gene transfer efficiency among all the apatite-based composite layers prepared in this study. In addition, the optimized DLp-Ap layer demonstrated higher gene transfer efficiency in the presence of serum than the conventional particle-mediated systems using commercially available lipid transfection reagents. It was also shown that the optimized DLp-Ap layer mediated the area-specific gene transfer on its surface, i.e., DNA was preferentially transferred to the cells adhering to the surface of the layer. The present gene transfer system using the PD-derived DLp-Ap layer, with the advantages of high efficiency in the presence of serum and area-specificity, would be useful in tissue engineering.

Manipulation of Cell Physiology Enables Gene Silencing in Well-differentiated Airway Epithelia

The application of RNA interference-based gene silencing to the airway surface epithelium holds great promise to manipulate host and pathogen gene expression for therapeutic purposes. However, well-differentiated airway epithelia display significant barriers to double-stranded small-interfering RNA (siRNA) delivery despite testing varied classes of nonviral reagents. In well-differentiated primary pig airway epithelia (PAE) or human airway epithelia (HAE) grown at the air–liquid interface (ALI), the delivery of a Dicer-substrate small-interfering RNA (DsiRNA) duplex against hypoxanthine–guanine phosphoribosyltransferase (HPRT) with several nonviral reagents showed minimal uptake and no knockdown of the target. In contrast, poorly differentiated cells (2–5-day post-seeding) exhibited significant oligonucleotide internalization and target knockdown. This finding suggested that during differentiation, the barrier properties of the epithelium are modified to an extent that impedes oligonucleotide uptake. We used two methods to overcome this inefficiency. First, we tested the impact of epidermal growth factor (EGF), a known enhancer of macropinocytosis. Treatment of the cells with EGF improved oligonucleotide uptake resulting in significant but modest levels of target knockdown. Secondly, we used the connectivity map (Cmap) database to correlate gene expression changes during small molecule treatments on various cells types with genes that change upon mucociliary differentiation. Several different drug classes were identified from this correlative assessment. Well-differentiated epithelia treated with DsiRNAs and LY294002, a PI3K inhibitor, significantly improved gene silencing and concomitantly reduced target protein levels. These novel findings reveal that well-differentiated airway epithelia, normally resistant to siRNA delivery, can be pretreated with small molecules to improve uptake of synthetic oligonucleotide and RNA interference (RNAi) responses.

A Neuronal Transcriptome Response Involving Stress Pathways is Buffered by Neuronal microRNAs

A single microRNA (miRNA) can inhibit a large number of mRNA transcripts. This widespread regulatory function has been experimentally demonstrated for a number of miRNAs. However, even when a multitude of targets is confirmed, function of a miRNA is frequently interpreted through a prism of a handful arbitrarily selected “interesting” targets. In this work we first show that hundreds of transcripts with target sites for two miRNAs expressed endogenously in neurons, miR-124 and miR-434-3p, are coordinately upregulated in a variety of neuronal stresses. This creates a landscape where these two miRNAs can exert their widespread inhibitory potential on stress-induced transcripts. Next, we experimentally demonstrate that overexpression of these two miRNAs indeed significantly inhibits expression of hundreds of stress-induced transcripts, thus confirming that these transcripts are enriched in true targets of examined miRNAs. A number of miRNAs were previously shown to have important roles in the regulation of stress responses, and our results suggest that these roles should be understood in light of a wide spread activation of miRNA targets during stresses. Importantly, a popular cationic lipid transfection reagent triggers such induction of miRNA targets. Therefore, when a transfection paradigm is employed to study miRNA function, the results of such studies should be interpreted with consideration for the inadvertent induction of miRNA targets.

A Simple, Highly Efficient Method for Heterologous Expression in Mammalian Primary Neurons Using Cationic Lipid-mediated mRNA Transfection

Expression of heterologous proteins in adult mammalian neurons is a valuable technique for the study of neuronal function. The post-mitotic nature of mature neurons prevents effective DNA transfection using simple, cationic lipid-based methods. Adequate heterologous protein expression is often only achievable using complex techniques that, in many cases, are associated with substantial toxicity. Here, a simple method for high efficiency transfection of mammalian primary neurons using in vitro transcribed mRNA and the cationic lipid transfection reagent Lipofectamine™ 2000 is described. Optimal transfection conditions were established in adult mouse dissociated dorsal root ganglion (DRG) neurons using a 96-well based luciferase activity assay. Using these conditions, a transfection efficiency of 25% was achieved in DRG neurons transfected with EGFP mRNA. High transfection efficiencies were also obtained in dissociated rat superior cervical ganglion (SCG) neurons and mouse cortical and hippocampal cultures. Endogenous Ca2+ currents in EGFP mRNA-transfected SCG neurons were not significantly different from untransfected neurons, which suggested that this technique is well suited for heterologous expression in patch clamp recording experiments. Functional expression of a cannabinoid receptor (CB1R), a G protein inwardly rectifying K+ channel (GIRK4) and a dominant-negative G protein α-subunit mutant (GoA G203T) indicate that the levels of heterologous protein expression attainable using mRNA transfection are suitable for most functional protein studies. This study demonstrates that mRNA transfection is a straightforward and effective method for heterologous expression in neurons and is likely to have many applications in neuroscience research.

Lipid and cationic polymer based transduction of botulinum holotoxin, or toxin protease alone, extends the target cell range and improves the efficiency of intoxication

Botulinum neurotoxin (BoNT) heavy chain (Hc) facilitates receptor-mediated endocytosis into neuronal cells and transport of the light chain (Lc) protease to the cytosol where neurotransmission is inhibited as a result of SNARE protein cleavage. Here we show that the role of BoNT Hc in cell intoxication can be replaced by commercial lipid-based and polycationic polymer DNA transfection reagents. BoNT “transduction” by these reagents permits efficient intoxication of neuronal cells as well as some non-neuronal cell lines normally refractory to BoNT. Surprisingly, the reagents facilitate delivery of recombinant BoNT Lc protease to the cytosol of both neuronal and non-neuronal cells in the absence of BoNT Hc, and with sensitivities approaching that of BoNT holotoxin. Transduction of BoNT, as with natural intoxication, is inhibited by bafilomycin A1, methylamine and ammonium chloride indicating that both pathways require endosome acidification. DNA transfection reagents facilitate intoxication by holotoxins, or isolated Lc proteases, of all three BoNT serotypes tested (A, B, E). These results suggest that lipid and cationic polymer transfection reagents facilitate cytosolic delivery of BoNT holotoxins and isolated Lc proteases by an endosomal uptake pathway.

Combined genome-wide expression profiling and targeted RNA interference in primary mouse macrophages reveals perturbation of transcriptional networks associated with interferon signalling.

BackgroundInterferons (IFNs) are potent antiviral cytokines capable of reprogramming the macrophage phenotype through the induction of interferon-stimulated genes (ISGs). Here we have used targeted RNA interference to suppress the expression of a number of key genes associated with IFN signalling in murine macrophages prior to stimulation with interferon-gamma. Genome-wide changes in transcript abundance caused by siRNA activity were measured using exon-level microarrays in the presence or absence of IFNγ.ResultsTransfection of murine bone-marrow derived macrophages (BMDMs) with a non-targeting (control) siRNA and 11 sequence-specific siRNAs was performed using a cationic lipid transfection reagent (Lipofectamine2000) prior to stimulation with IFNγ. Total RNA was harvested from cells and gene expression measured on Affymetrix GeneChip Mouse Exon 1.0 ST Arrays. Network-based analysis of these data revealed six siRNAs to cause a marked shift in the macrophage transcriptome in the presence or absence IFNγ. These six siRNAs targeted the Ifnb1, Irf3, Irf5, Stat1, Stat2 and Nfkb2 transcripts. The perturbation of the transcriptome by the six siRNAs was highly similar in each case and affected the expression of over 600 downstream transcripts. Regulated transcripts were clustered based on co-expression into five major groups corresponding to transcriptional networks associated with the type I and II IFN response, cell cycle regulation, and NF-KB signalling. In addition we have observed a significant non-specific immune stimulation of cells transfected with siRNA using Lipofectamine2000, suggesting use of this reagent in BMDMs, even at low concentrations, is enough to induce a type I IFN response.ConclusionOur results provide evidence that the type I IFN response in murine BMDMs is dependent on Ifnb1, Irf3, Irf5, Stat1, Stat2 and Nfkb2, and that siRNAs targeted to these genes results in perturbation of key transcriptional networks associated with type I and type II IFN signalling and a suppression of macrophage M1 polarization.

Efficient in vitro labeling rabbit neural stem cell with paramagnetic Gd-DTPA and fluorescent substance

Objectives The aim of this study is to label rabbit neural stem cells (NSCs) by using standard contrast agents (Gd-DTPA) in combination with PKH26 and in vitro track them with MR imaging. Materials and methods NSCs from prenatal brains of rabbits were cultured and propagated. Intracellular uptake of Gd-DTPA was achieved by using a non-liposomal lipid transfection reagent (Effectene) as the transfection agent. After labeling with Gd-DTPA, cells were incubated with cellular membrane fluorescent dye PKH26. The labeling effectiveness and the longevity of Gd-DTPA maintenance were measured on a 1.5 T MR scanner. The influence of labeling on the cellular biological behaviors was assessed by cellular viability, proliferation and differentiation assessment. Results The labeling efficiency of Gd-DTPA was up to 90%. The signal intensity on T1-weighted imaging and T1 values of labeled cells were significantly higher than those of unlabeled cells ( P < 0.05). The minimal number of detectable cells for T1-weighted imaging was 5 × 10 3. Cellular uptake of Gd-DTPA was maintained until 15 days after initially labeling. There was no significant difference in the cellular viability and proliferation between the labeled and unlabeled NSCs ( P > 0.05). Normal glial and neuronal differentiation remained in labeled NSCs like unlabeled NSCs. Conclusion Highly efficient labeling NSCs with Gd-DTPA could be achieved by using Effectene. This method of labeling NSCs allows for tracking cells with MR imaging, and without alterations of cellular biological behaviors.

Durable Protection from Herpes Simplex Virus-2 Transmission Following Intravaginal Application of siRNAs Targeting Both a Viral and Host Gene

A vaginal microbicide should prevent pathogen transmission without disrupting tissue barriers to infection. Ideally, it would not need to be applied immediately before sexual intercourse, when compliance is a problem. Intravaginal administration of small interfering RNA (siRNA) lipoplexes targeting Herpes Simplex Virus Type 2 (HSV-2) genes protects mice from HSV-2. However, protection is short-lived, and the transfection lipid on its own unacceptably enhances transmission. Here, we show that cholesterol-conjugated (chol)-siRNAs without lipid silence gene expression in the vagina without causing inflammation or inducing interferons. A viral siRNA prevents transmission within a day of challenge, whereas an siRNA targeting the HSV-2 receptor nectin-1 protects for a week, but protection is delayed for a few days until the receptor is downmodulated. Combining siRNAs targeting a viral and host gene protects mice from HSV-2 for a week, irrespective of the time of challenge. Therefore, intravaginal siRNAs could provide sustained protection against viral transmission.

Identification of Sensitizing Targets to the Hypomethylating Agent 5-Azacytidine in Acute Myeloid Leukemia Cells Using RNAi-Based Functional Genomics Screening

• Hypomethylating agents like 5-Azacytidine (5Aza) have become an effective therapy for myelodysplastic syndromes (MDS) and show promise in acute myeloid leukemia (AML). In AML, complimentary mechanisms including epigenetic silencing of growth controlling genes, i.e. tumor suppressors, and activation of kinases contribute to malignant transformation. In order to enhance the therapeutic potential of epigenetic therapies, we developed a high-throughput RNA interference (HT-RNAi) platform for large-scale transient gene silencing in acute myeloid leukemia cells. This assay allows for the first time to individually silence hundreds or thousands of genes in combination with 5Aza to identify molecular targets whose inhibition enhances the anti-leukemic effect of hypomethylating agents. As part of assay development for HT-RNAi, ten AML cell lines were used to determine the median inhibitory concentration (IC50) of 5Aza for each AML cell lines. Furthermore, the ten cell lines were tested with a panel of cationic lipid transfection reagents at varying weight to volume (wt:vol) ratios to determine the optimal siRNA transfection conditions. Results from these studies identified two AML cell lines TF1 and ML4, which were advanced into kinome-epigenetic RNAi screens.Using a lipid-based method, cells were reverse transfected for 48hrs with 2 different siRNA sequences per gene targeting a total of 572 kinases. After 48hrs, 5Aza at the calculated IC25 was added for an additional 72 hrs and cell proliferation was measured using a luminescence-based assay. Data was background corrected and analyzed using the B-score method to report the strength and statistical significance of growth inhibition compared to controls. A B-score of <−2 indicates statistical significance with p<0.05 (>95% confidence); a B-score <−1.5 provides >87% confidence and was used as lowest cutoff given that screens are focused and contain validated siRNA to kinases. Analysis of two independent RNAi kinome screens, one in TF1 and the other in ML4, in combination with 5Aza, identified six and eleven kinases respectively whose silencing by two different siRNA sequences (2× coverage) potentiated the effects of 5Aza at B-score <−1.5. In ML4 cells 2 kinases were highly significant with a B-score for both siRNA <−2. Six kinases were common targets in both cell lines with significant growth inhibition at a B-score for both siRNA of at least <−1.5 making these kinases potential important modifiers of response to 5Aza.In summary, initial kinome RNAi screens in myeloid cells identified specific kinases as potential sensitizing targets to hypomethylating agents. Moreover, functional genomic RNAi screens provide a fast and attractive approach to identify molecular targets in AML for the rational development of combination therapies with hypomethylating agents as well as other drug classes.

Non-Viral Delivery of the Gene for Glial Cell Line-Derived Neurotrophic Factor to Mesenchymal Stem Cells In Vitro Via a Collagen Scaffold

Recent advances in tissue engineering that combine an extracellular matrix-like scaffold with therapeutic molecules, cells, DNA encoding therapeutic proteins, or a combination of the three hold promise for treating defects in the brain resulting from a penetrating injury or tumor resection. The purpose of this study was to investigate a porous sponge-like collagen scaffold for non-viral delivery of a plasmid encoding for glial cell line-derived neurotrophic factor (pGDNF) to rat marrow stromal stem cells (also referred to as mesenchymal stem cells, MSCs). The effects of the following parameters on GDNF synthesis in the three-dimensional (3D) constructs were evaluated and compared with results in monolayer culture: initial plasmid load (2-50 microg pGDNF), ratio of a lipid transfection reagent to plasmid (5:10), culture environment during the transfection (static and dynamic), and cell density. The level of gene expression in the collagen scaffolds achieved therapeutic levels that had previously been found to support survival of dopaminergic and trigeminal neurons in vitro. For the highest loading of plasmid (50 microg), the level of GDNF protein remained six to seven times above the control level after 2 weeks, a significant difference. Cell density in the scaffold was of importance for an early increase in GDNF production, with accumulated GDNF being approximately 60% greater after 9 days of culture when scaffolds were initially seeded with 2 million rat MSCs compared to 500,000 cells. Application of orbital shaking during the 4 h of transfection had a positive effect on the production of GDNF on 3D constructs but not of the same magnitude as reported in monolayer studies. Overall, these results demonstrate that the combination of tissue engineering and non-viral transfection of MSCs for the over-expression of GDNF is a promising approach for the long-term production of GDNF and probably for neurotrophic factors in general.