Green Fluorescent Protein Knockdown Research Articles

Modification of Branched Poly(ethylene imine) with d-Fructose for Selective Delivery of siRNA into Human Breast Cancer Cells.

Branched poly(ethylene imine) (bPEI) is frequently used in RNA interference (RNAi) experiments as a cationic polymer for the delivery of small interfering RNA (siRNA) because of its ability to form stable polyplexes that facilitate siRNA uptake. However, the use of bPEI in gene delivery is limited by its cytotoxicity and a need for target specificity. In this work, bPEI is modified with d-fructose to improve biocompatibility and target breast cancer cells through the overexpressed GLUT5 transporter. Fructose-substituted bPEI (Fru-bPEI) is accessible in three steps starting from commercially available protected fructopyranosides and bPEI. Several polymers with varying molecular weights, degrees of substitution, and linker positions on d-fructose (C1 and C3) are synthesized and characterized with NMR spectroscopy, size exclusion chromatography, and elemental analysis. In vitro biological screenings show significantly reduced cytotoxicity of 10kDa bPEI after fructose functionalization, specific uptake of siRNA polyplexes, and targeted knockdown of green fluorescent protein (GFP) in triple-negative breast cancer cells (MDA-MB-231) compared to noncancer cells (HEK293T).

Evaluating the Mechanisms of Light-Triggered siRNA Release from Nanoshells for Temporal Control Over Gene Regulation.

The ability to regulate intracellular gene expression with exogenous nucleic acids such as small interfering RNAs (siRNAs) has substantial potential to improve the study and treatment of disease. However, most transfection agents and nanoparticle-based carriers that are used for the intracellular delivery of nucleic acids cannot distinguish between diseased and healthy cells, which may cause them to yield unintended widespread gene regulation. An ideal delivery system would only silence targeted proteins in diseased tissue in response to an external stimulus. To enable spatiotemporal control over gene silencing, researchers have begun to develop nucleic acid-nanoparticle conjugates that keep their nucleic acid cargo inactive until it is released from the nanoparticle on-demand by externally applied near-infrared laser light. This strategy can overcome several limitations of other nucleic acid delivery systems, but the mechanisms by which these platforms operate remain ill understood. Here, we perform a detailed investigation of the mechanisms by which silica core/gold shell nanoshells (NSs) release conjugated siRNA upon excitation with either pulsed or continuous wave (CW) near-infrared (NIR) light, with the goal of providing insight into how these nanoconjugates can enable on-demand gene regulation. We demonstrate that siRNA release from NSs upon pulsed laser irradiation is a temperature-independent process that is substantially more efficient than siRNA release triggered by CW irradiation. Contrary to literature, which suggests that only pulsed irradiation releases siRNA duplexes, we found that both modes of irradiation release a mixture of siRNA duplexes and single-stranded oligonucleotides, but that pulsed irradiation results in a higher percentage of released duplexes. To demonstrate that the siRNA released from NSs upon pulsed irradiation remains functional, we evaluated the use of NSs coated with green fluorescent protein (GFP)-targeted siRNA (siGFP-NS) for on-demand knockdown of GFP in cells. We found that GFP-expressing cells treated with siGFP-NS and irradiated with a pulsed laser experienced a 33% decrease in GFP expression compared to cells treated with no laser. Further, we observed that light-triggered gene silencing mediated by siGFP-NS is more potent than using commercial transfection agents to deliver siRNA into cells. This work provides unprecedented insight into the mechanisms by which plasmonic NSs release siRNA upon light irradiation and demonstrates the importance of thoroughly characterizing photoresponsive nanosystems for applications in triggered gene regulation.

Formulation of Nonionic Surfactant Vesicles (NISV) Prepared by Microfluidics for Therapeutic Delivery of siRNA into Cancer Cells.

Small interfering RNAs (siRNA) have a broad potential as therapeutic agents to reversibly silence any target gene of interest. The clinical application of siRNA requires the use of safe and effective delivery systems. In this study, we investigated the use of nonionic surfactant vesicles (NISV) for the delivery of siRNA. Different types of NISV formulations were synthesized by microfluidic mixing and then evaluated for their physiochemical properties and cytotoxicity. The ability of the NISV to carry and transfect siRNA targeting green fluorescent protein (GFP) into A549 that stably express GFP (copGFP-A549) was evaluated. Flow cytometry and Western blotting were used to study the GFP expression knockdown, and significant knockdown was observed as a result of siRNA delivery to the cells by NISV. This occurred in particular when using Tween 85, which was able to achieve more than 70% GFP knockdown. NISV were thus demonstrated to provide a promising and effective platform for therapeutic delivery of siRNA.

Systemic delivery of shRNA by AAV9 provides highly efficient knockdown of ubiquitously expressed GFP in mouse heart, but not liver.

AAV9 is a powerful gene delivery vehicle capable of providing long-term gene expression in a variety of cell types, particularly cardiomyocytes. The use of AAV-delivery for RNA interference is an intense area of research, but a comprehensive analysis of knockdown in cardiac and liver tissues after systemic delivery of AAV9 has yet to be reported. We sought to address this question by using AAV9 to deliver a short-hairpin RNA targeting the enhanced green fluorescent protein (GFP) in transgenic mice that constitutively overexpress GFP in all tissues. The expression cassette was initially tested in vitro and we demonstrated a 61% reduction in mRNA and a 90% reduction in GFP protein in dual-transfected 293 cells. Next, the expression cassette was packaged as single-stranded genomes in AAV9 capsids to test cardiac GFP knockdown with several doses ranging from 1.8×1010 to 1.8×1011 viral genomes per mouse and a dose-dependent response was obtained. We then analyzed GFP expression in both heart and liver after delivery of 4.4×1011 viral genomes per mouse. We found that while cardiac knockdown was highly efficient, with a 77% reduction in GFP mRNA and a 71% reduction in protein versus control-treated mice, there was no change in liver expression. This was despite a 4.5-fold greater number of viral genomes in the liver than in the heart. This study demonstrates that single-stranded AAV9 vectors expressing shRNA can be used to achieve highly efficient cardiac-selective knockdown of GFP expression that is sustained for at least 7 weeks after the systemic injection of 8 day old mice, with no change in liver expression and no evidence of liver damage despite high viral genome presence in the liver.

Bimodal Gold Nanoparticle Therapeutics for Manipulating Exogenous and Endogenous Protein Levels in Mammalian Cells

A new advance in cell transfection protocol using a bimodal nanoparticle agent to selectively manipulate protein expression levels within mammalian cells is demonstrated. The nanoparticle based transfection approach functions by controlled release of gene regulatory elements from a 6 nm AuNP (gold nanoparticle) surface. The endosomal release of the regulatory elements from the nanoparticle surface results in endogenous protein knockdown simultaneously with exogenous protein expression for the first 48 h. The use of fluorescent proteins as the endogenous and exogenous signals for protein expression enables the efficiency of codelivery of siRNA (small interfering RNA) for GFP (green fluorescent protein) knockdown and a dsRed-express linearized plasmid for induction to be optically analyzed in CRL-2794, a human kidney cell line expressing an unstable green fluorescent protein. Delivery of the bimodal nanoparticle in cationic liposomes results in 20% GFP knockdown within 24 h of delivery and continues exhibiting knockdown for up to 48 h for the bimodal agent. Simultaneous dsRed expression is observed to initiate within the same time frame with expression levels reaching 34% after 25 days although cells have divided approximately 20 times, implying daughter cell transfection has occurred. Fluorescence cell sorting results in a stable colony, as demonstrated by Western blot analysis. The simultaneous delivery of siRNA and linearized plasmid DNA on the surface of a single nanocrystal provides a unique method for definitive genetic control within a single cell and leads to a very efficient cell transfection protocol.

Critical Role of Monoubiquitination of Histone H2AX Protein in Histone H2AX Phosphorylation and DNA Damage Response

DNA damage response is an important surveillance mechanism used to maintain the integrity of the human genome in response to genotoxic stress. Histone variant H2AX is a critical sensor that undergoes phosphorylation at serine 139 upon genotoxic stress, which provides a docking site to recruit the mediator of DNA damage checkpoint protein 1 (MDC1) and DNA repair protein complex to sites of DNA breaks for DNA repair. Here, we show that monoubiquitination of H2AX is induced upon DNA double strand breaks and plays a critical role in H2AX Ser-139 phosphorylation (γ-H2AX), in turn facilitating the recruitment of MDC1 to DNA damage foci. Mechanistically, we show that monoubiquitination of H2AX induced by RING finger protein 2 (RNF2) is required for the recruitment of active ataxia telangiectasia mutated to DNA damage foci, thus affecting the formation of γ-H2AX. Importantly, a defect in monoubiquitination of H2AX profoundly enhances ionizing radiation sensitivity. Our study therefore suggests that monoubiquitination of H2AX is an important step for DNA damage response and may have important clinical implications for the treatment of cancers.

Synthetic poly(ester amine) and poly(amido amine) nanoparticles for efficient DNA and siRNA delivery to human endothelial cells

Biodegradable poly(ester amine) (PEA)-based and poly(amido amine) (PAA)-based nanoparticles were developed for efficient in vitro siRNA delivery to human umbilical vein endothelial cells (HUVECs). They were screened, characterized, and compared with traditionally studied DNA-containing particles. Several of the polymeric nanoparticles tested were found to be effective for delivering functional siRNA to green fluorescent protein (GFP) + HUVECs, achieving 60%–75% GFP knockdown while maintaining high viability. While PEAs have been used previously to form polyplexes or nanoparticles for DNA delivery, highly effective siRNA delivery in hard-to-transfect human cell types has not been previously reported. PEAs and linear nondendrimeric PAAs were also found to be effective for DNA delivery to HUVECs using GFP-encoding plasmid DNA (up to 50%–60% transfection efficiency). PEAs and PAAs can be separated into groups that form polymeric nanoparticles effective for siRNA delivery, for DNA delivery, or for both.

Neuron‐specific RNA interference using lentiviral vectors

Viral vectors have been used in several different settings for the delivery of small hairpin (sh) RNAs. However, most vectors have utilized ubiquitously-expressing polymerase (pol) III promoters to drive expression of the hairpin as a result of the strict requirement for precise transcriptional initiation and termination. Recently, pol II promoters have been used to construct vectors for RNA interference (RNAi). By embedding the shRNA into a micro RNA-context (miRNA) the endogenous miRNA processing machinery is exploited to achieve the mature synthetic miRNA (smiRNA), thereby expanding the possible promoter choices and eventually allowing cell type specific down-regulation of target genes. In the present study, we constructed lentiviral vectors expressing smiRNAs under the control of pol II promoters to knockdown gene expression in cell culture and in the brain. We demonstrate robust knockdown of green fluorescent protein using lentiviral vectors driving RNAi from the ubiquitously-expressing promoter of the cytomegalovirus (CMV) and, in addition, we show for the first time neuron-specific knockdown in the brain using a neuron-specific promoter. Furthermore, we show that the expression pattern of the presumed ubiquitously-expressing CMV promoter changes over time from being expressed initially in neurons and glial cells to being expressed almost exclusively in neurons in later stages. In the present study, we developed vectors for cell-specific RNAi for use in the brain. This offers the possibility of specifically targeting RNAi to a subset of cells in a complex tissue and may prove to be of great importance in the design of future gene therapeutic paradigms.

Triggered release of siRNA from poly(ethylene glycol)-protected, pH-dependent liposomes

The ability of small interfering RNA (siRNA) to regulate gene expression has potential therapeutic applications, but its use is limited by inefficient delivery. Triggered release of adsorbed poly(ethylene glycol) (PEG)-b-polycation polymers from pH-dependent (PD) liposomes enables protection from immune recognition during circulation (pH 7.4) and subsequent intracellular delivery of siRNA within the endosome (pH ~5.5). Polycationic blocks, based on either poly[2-(dimethylamino)ethyl methacrylate] (31 or 62 DMA repeat units) or polylysine (21 K repeat units), act as anchors for a PEG (113 ethylene glycol repeat units) protective block. Incorporation of 1,2-dioleoyl-3-dimethylammonium-propane (DAP), a titratable lipid, increases the liposome's net cationic character within acidic environments, resulting in polymer desorption and membrane fusion. Liposomes encapsulating siRNA demonstrate green fluorescent protein (GFP) silencing in genetically-modified, GFP-expressing HeLa cells and glyceraldehyde-3-phosphate dehydrogenase (GAPD) knockdown in human umbilical vein endothelial cells (HUVEC). Bare and PD liposomes coated with PEG113-DMA31 exhibit a 0.16 ± 0.2 and 0.32 ± 0.3 fraction of GFP knockdown, respectively. In contrast, direct siRNA administration and Oligofectamine complexed siRNA reduce GFP expression by 0.06 ± 0.02 and 0.14 ± 0.02 fractions, respectively. Our in vitro data indicates that polymer desorption from PD liposomes enhances siRNA-mediated gene knockdown.

MicroRNA-Mediated Gene Regulation Effectively Restricts In Vivo Transgene Expression in Hematopoietic Stem Cell Progeny.

Stem cell engineering and targeted in vivo gene delivery increasingly require tight control of transgene expression. Lineage- and differentiation stage-specific gene regulation is classically afforded by pol-II-dependent transcript regulation. A super-imposed layer of post-transcriptional control would be valuable to correct undesirable expression patterns or fine-tune developmentally regulated or inducible gene expression. microRNAs (miRNAs) have recently emerged as potent repressors of gene expression at the post-transcriptional level. In this study, we investigate the potential of using miRNA regulation to provide lineage-restricted expression, exploiting miRNAs with distinctive expression patterns in hematopoietic tissue. miR-223 is preferentially expressed in granulocytes and monocytes (80-and 110-fold, respectively). miR-181a is highly expressed in B-lymphocytes and, particularly, in thymocytes, but down-regulated (∼1000-fold) in post-thymic T cells. We, therefore, constructed lentiviral vectors encoding either green fluorescent protein (GFP) or an antigen-specific receptor, placed under the transcriptional control of the ubiquitous EF1a promoter and tagged with miRNA-recognition elements (MREs) complementary to the mature miR-223 or miR-181a. In a panel of murine and human cell lines expressing varying levels of the two miRNAs we find that GFP knockdown is dependent on the presence of the miRNA and directly proportional to the number of MRE repeats. Four copies of the repeat permit better down-regulation than 2 copies (albeit depending on the level of endogenous miRNA). Two different MREs can be combined in tandem, resulting in additive down-regulation. In vivo, in mouse bone marrow chimeras harboring the miR-223-responsive vector, GFP expression is specifically repressed in myeloid cells (>85% compared to the control vector lacking miRNA target sequences). Reciprocally, chimeras harboring the miR-181a-regulated vector express GFP in myeloid and erythroid lineages, but transgene expression is profoundly repressed in thymocytes and B-cell progenitors. A vector harboring a composite MRE confers GFP expression almost exclusively confined to the erythroid lineage. These results demonstrate for the first time that transgene expression can be selectively regulated at the post-transcriptional level within the hematopoietic tissue. In mouse chimeras expressing a CD19-specific antigen receptor (which we and others are currently using in clinical trials in B cell malignancies), we analyze receptor expression in the 4 double negative subsets (DN1, DN2, DN3 and DN4), the double-positive subset (DP), and the CD4+ and CD8+ single positive (SP) subsets. We detail and quantify receptor knock-down at each developmental stage, and show that miR-181a-mediated regulation prevents receptor expression at critical stages of positive and negative thymic selection. Antigen receptor expression is dramatically repressed in DN and DP cells, while rising in CD4+ and CD8+ SP thymocytes. Importantly, expression is fully restored in post-thymic T cells, and maintained in activated T cells. miRNA-mediated post-transcriptional regulation is thus proving to be a powerful means to direct lineage- and differentiation stage-specific transgene expression in genetically modified stem cells.

147 GENE KNOCKDOWN OF MATERNAL- AND EMBRYONIC-EXPRESSED GREEN FLUORESCENT PROTEIN IN MURINE EMBRYOS BY THE INJECTION OF A SHORT INTERFERING RNA (siRNA)

RNA interference (RNAi) is now widely used for gene silencing in various biological systems. Injection of long double-stranded RNAs has been shown to specifically knock down gene expression in mammalian embryos. The utilization of short interfering RNAs (siRNA) to target specific embryonic genes would make this approach flexible and efficient enough for studying physiological functions in development. To demonstrate the feasibility of a single class of siRNA molecules for achieving long-lasting effects after injection into mammalian zygotes, we used siRNAs to knock down expression of the green fluorescent protein (GFP) in transgenic murine embryos of the OG2 transgenic line. Homozygous OG2-animals, carrying the Oct4-GFP transgene, were mated with NMRI animals to produce OG2 hemizygous zygotes, which show a parentally dependent expression pattern of the marker gene. Hemizygous zygotes with a maternally inherited Oct4-GFP gene continuously express the GFP marker; hemizygous zygotes with a paternally inherited Oct4-GFP start transcription of the GFP gene at the 4–8 cell stage, i.e., after onset of embryonic genomic activation. Thus efficacy and duration of gene silencing could be tested under 2 different conditions where (i) GFP mRNA was already present at the time point of injection, and (ii) GFP transcription started 2–3 cell cycles after siRNA injection. Zygotes were microinjected with either a 22-basepair GFP-siRNA or a control siRNA and then cultured in vitro. The siRNAs were conjugated with the fluorochome rhodamine to allow monitoring of injection and subsequent degradation of siRNAs. At the end of the in vitro culture, the developmental stage, the number of nuclei, GFP fluorescence (Table 1), and the GFP mRNA levels were determined by RT-PCR. In conclusion, results demonstrate that injection of a synthetic siRNA is sufficient to knock down a target gene transcript with either maternal or embryonic expression in mammalian embryos. Importantly, maternally derived GFP proteins showed a delayed functional knockdown of GFP for 2 days. The advantages of this approach are that (i) off-target effects of long double-stranded RNAs can be avoided, (ii) siRNAs against any known transcript can be rapidly designed and synthesized, and (iii) developmentally important genes can be silenced in embryos for at least 5 days following injection. Table 1.siRNA microinjection into zygotes with maternally and paternally inherited GFP

Stable RNA interference: comparison of U6 and H1 promoters in endothelial cells and in mouse brain

RNA interference (RNAi) is a post-transcriptional RNA degradation process, which has become a very useful tool in gene function studies and gene therapy applications. Long-term cellular expression of small interfering RNA (siRNA) molecules required for many gene therapy applications can be achieved by lentiviral vectors (LVs). The two most commonly used promoters to drive the short hairpin RNA (shRNA) expression are the human U6 small nuclear promoter (U6) and the human H1 promoter (H1). We investigated whether there is any significant difference between the efficiencies of U6 and H1 in LV-mediated RNAi using green fluorescent protein (GFP) as a target gene by flow cytometry and real-time reverse-transcription polymerase chain reaction (RT-PCR) in endothelial cells. Also, we compared the efficiencies of U6 and H1 in the GFP transgenic mouse brain after stereotactic LV injection. We show that the U6 promoter is more efficient than H1 in GFP silencing in vitro, leading to 80% GFP knockdown at an average of one integrated vector genome per target cell genome. The silencing is persistent for several months. In addition, the U6 promoter is superior to H1 in vivo and leads to stable GFP knockdown in mouse brain for at least 9 months. These results show that LV-mediated RNAi is a powerful gene-silencing method for the long-term inhibition of gene expression in vitro and in vivo.

98. Stable Lentivirus-Mediated RNA Interference: Comparison of U6 and H1 Promoters in Endothelial Cells and GFP Knockdown in Mouse Brain

RNA interference (RNAi) is a post-transcriptional RNA degradation process, which has become a useful tool in gene function studies and recently in gene therapy applications. Long-term expression of short hairpin RNA molecules (shRNA) required for many gene therapy applications can be achieved by lentiviral vectors (LVs). The two most commonly used promoters to drive the shRNA expression are the human U6 small nuclear promoter (U6) and human H1 promoter (H1). Our goal was to construct an efficient and optimal LV-based shRNA expression system and to explore LV-mediated RNAi for long-term gene silencing. For this purpose, we systematically compared the U6 and H1 promoters for optimal silencing of green fluorescent protein (GFP) in mouse endothelial cells. We show that the U6 promoter is more efficient than H1 in GFP silencing, leading to 80% GFP knockdown at the average of one integrated vector genome per target cell genome. In addition, the silencing was persistent for several months. Finally, as a proof of principle, we demonstrate that the GFP expression is silenced in vivo after stereotactic LV injection into GFP transgenic mice brain. In conclusion, LV-mediated RNAi is a powerful gene silencing method, which is useful for gene therapy applications requiring long-term knockdown of gene functions in vitro and in vivo.