326 MICROINJECTIONS OF SMALL INTERFERING RNA AND COMPLEMENTARY RNA TO ELUCIDATE THE INVOLVEMENT OF ENDOGENOUS PHOSPHOLIPASE C ISOFORMS IN BOVINE OOCYTE ACTIVATION DURING FERTILIZATION

2010 ◽  
Vol 22 (1) ◽  
pp. 319
Author(s):  
B. R. Sessions ◽  
A. H. Bayles ◽  
J. Collier ◽  
K. Perry ◽  
L. S. Whitaker ◽  
...  
Keyword(s):  
Phospholipase C ◽  
Small Interfering Rna ◽  
Calcium Oscillations ◽  
Activation Process ◽  
Mrna Levels ◽  
Post Injection ◽  
Oocyte Activation ◽  
Bovine Oocyte ◽  
Interfering Rna ◽  
Bovine Oocytes

Phospholipase C (PLC) isoforms stimulate the hydrolysis of phosphatidyl inositol (4,5)-bisphosphate (PIP2) to produce diacylglycerol (DAG) and 1,4,5 inositol trisphosphate (IP3), with IP3 regulating the release of calcium (Ca2+) from the endoplasmic reticulum. This release of calcium is essential for oocyte activation, and a sperm-specific PLC isoform, PLCγ;, has been proposed as the primary agent that initiates the activation process. However, the oocyte contains many endogenous PLC isoforms (PLC β, γ, and δ) that could also be involved in regulating or initiating these calcium oscillations downstream of other initiating events. In order to better elucidate the involvement of endogenous PLC isoforms as well as the specific role of the sperm-specific form, small interfering RNA (siRNA) directed against the specific bovine PLC isoforms (PLCζ;, PLCγ1, PLCγ2, PLCδ1, PLCδ3, PLCδ4, PLCβ1, PLCβ3) were microinjected into bovine oocytes, and the subsequent effects on PLC mRNA levels and bovine fertilization were evaluated. Real-time PCR (qPCR) was used to quantify the levels of PLC message present in bovine oocytes at the time of injection (15 h post-maturation) and 6, 10, and 14 h post-injection. The qPCR results indicated a near-complete knockdown of mRNA levels in bovine oocytes 10 h post-injection for the isotypes PLCγ1, PLCγ2, PLCδ3, PLCδ4, PLCβ1, PLCβ3, but only partial knockdown of PLCS 1 mRNA. Oocytes microinjected with PLC siRNA were also fertilized and cultured in vitro according to our standard laboratory procedures (Reed et al. 1996 Theriogenology 45, 439-449). The oocytes microinjected with PLCζ;, PLCδ1, PLCδ3, PLCδ4, PLCβ1, PLCβ3 siRNA resulted in cleavage rates similar to the negative control siRNA, non-injected, and sham-injected treatment groups, whereas bovine oocytes microinjected with PLCγ1 and PLCγ2 siRNA had significantly lower cleavage rates compared with the controls. Additionally, complementary cRNA for each specific PLC isoform was microinjected into bovine oocytes to ascertain each isoform’s ability to induce parthenogenetic activation. Development was observed in oocytes microinjected with a variety of cRNAs, and the activating effects of the cRNA were negligible if the oocytes were microinjected with the corresponding siRNA before microinjection with cRNA. Interestingly, siRNA specific for PLCζ; failed to reduce cleavage when treated bovine oocytes were fertilized. These data illustrate the potential involvement of multiple endogenous PLC isoforms and not just the sperm-specific PLCζ; isoform in bovine oocyte activation during fertilization.

scholarly journals Optimization Procedure for Small Interfering RNA Transfection in a 384-Well Format

2007 ◽  
Vol 12 (4) ◽  
pp. 546-559 ◽  
Author(s):  
Jason Borawski ◽  
Alicia Lindeman ◽  
Frank Buxton ◽  
Mark Labow ◽  
L. Alex Gaither
Keyword(s):  
High Throughput Screening ◽  
Mammalian Cells ◽  
Small Interfering Rna ◽  
Dna Analysis ◽  
Lentiviral Vector ◽  
Mrna Levels ◽  
Sirna Transfection ◽  
Rna Transfection ◽  
Well Assay ◽  
Interfering Rna

High-throughput screening of RNAi libraries has become an essential part of functional analysis in academic and industrial settings. The transition of a cell-based RNAi assay into a 384-well format requires several optimization steps to ensure the phenotype being screened is appropriately measured and that the signal-to-background ratio is above a certain quantifiable threshold. Methods currently used to assess small interfering RNA (siRNA) efficacy after transfection, including quantitative PCR or branch DNA analysis, face several technical limitations preventing the accurate measurement of mRNA levels in a 384-well format. To overcome these difficulties, the authors developed an approach using a viral-based transfection system that measures siRNA efficacy in a standardized 384-well assay. This method allows measurement of siRNA activity in a phenotypically neutral manner by quantifying the knockdown of an exogenous luciferase gene delivered by a lentiviral vector. In this assay, the efficacy of a luciferase siRNA is compared to a negative control siRNA across many distinct assay parameters including cell type, cell number, lipid type, lipid volume, time of the assay, and concentration of siRNA. Once the siRNA transfection is optimized as a 384-well luciferase knockdown, the biologically relevant phenotypic analysis can proceed using the best siRNA transfection conditions. This approach provides a key technology for 384-well assay development when direct measurement of mRNA knockdown is not possible. It also allows for direct comparison of siRNA activity across cell lines from almost any mammalian species. Defining optimal conditions for siRNA delivery into mammalian cells will greatly increase the speed and quality of large-scale siRNA screening campaigns. ( Journal of Biomolecular Screening 2007:546-559)

276 ACTIVATION OF ROUND SPERMATID-INJECTED OOCYTES USING PHOSPHOLIPASE C ZETA IN PIGS

2013 ◽  
Vol 25 (1) ◽  
pp. 286
Author(s):  
J. Ito ◽  
E. Yuhara ◽  
A. Nakamura ◽  
N. Kashiwazaki
Keyword(s):  
Phospholipase C ◽  
Mammalian Species ◽  
Treated Group ◽  
Round Spermatid ◽  
Calcium Oscillations ◽  
Oocyte Activation ◽  
Domestic Species ◽  
Electrical Pulses ◽  
Group 2 ◽  
Pronuclear Formation

In several mammalian species, the generation of offspring by round spermatid injection has been reported. However, in domestic species, including pigs, no one has reported success to date. One of the reasons is that round spermatid-injected oocytes require artificial stimuli for oocyte activation, but the developmental ability of the oocytes is low in pigs, suggesting that a more optimal activation protocol is needed. During fertilization, a sperm-derived factor induces repetitive increases in intracellular calcium, known as calcium oscillations. It is now acknowledged that phospholipase C zeta (PLCζ) has an essential role in inducing calcium oscillations, not only in mammals, but also in several other vertebrates. Therefore, if PLCζ is used as a stimulus for oocyte activation, the efficiency of oocyte activation can be improved. Recently, we found that equine PLCζ (ePLCζ) has higher activity than those of other mammalian species to be studied. In the present study, we examined whether injection of ePLCζ complementary RNA (cRNA) improves the activation of round spermatid-injected oocytes in pigs. First, we examined whether ePLCζ is expressed in round spermatids. Porcine round spermatids were isolated from adult testes, and immunostaining using anti-PLCζ antibody was carried out. The PLCζ was localised at the head and tail in mature sperm, and a part of the round spermatid was also stained. Next, we evaluated the developmental ability of round spermatid-injected oocytes activated by different protocols (electrical pulses v. injection of ePLCζ cRNA). The cytoplasts were then injected with round spermatids. One hour later, the oocytes were divided into two groups. In group 1, the oocytes were activated by a direct current pulse (150 V mm–1 and 60 µs). In group 2, the oocytes were injected with ePLCζ cRNA as follows: the reagent (0.1 µg µL–1) was diluted in injection buffer [100 mM KCl and 10 mM HEPES (pH = 7.0)], loaded into glass micropipettes by aspiration, and delivered to the ooplasm by pneumatic pressure (Narishige, Tokyo, Japan). Each oocyte received 3 to 10 pL (1 to 3% of the total volume of the oocyte). After the stimulations, oocytes were cultured in PZM-5 under 38.5°C in a humidified incubator (95% air, 5% CO2). In the ePLCζ-injected group, rates of pronuclear formation (n = 22/32, 68.8%) and blastocysts (n = 2/43, 4.7%) were higher than those in the electrical pulse-treated group (n = 9/41, 22%; and n = 0/51, 0%, respectively; P < 0.05). In conclusion, our data suggest that injection of PLCζ is effective for activation of round spermatid-injected oocytes in pigs.

176 Localization and Quantitative Expression of Phospholipase C Zeta in Equine Sperm Using Commercial Antibodies

2018 ◽  
Vol 30 (1) ◽  
pp. 228
Author(s):  
R. A. Gonzalez-Castro ◽  
J. K. Graham ◽  
E. M. Carnevale
Keyword(s):  
Flow Cytometry ◽  
Phospholipase C ◽  
Fluorescence Intensity ◽  
Molecular Probes ◽  
Calcium Oscillations ◽  
Oocyte Activation ◽  
Flow Cytometric ◽  
Live Sperm ◽  
Commercial Antibodies ◽  
Flow Cytometric Evaluation

Fertilization failure in vivo and in vitro (intracytoplasmic sperm injection, ICSI) can be caused by the inability of sperm to elicit intracellular calcium oscillations and to induce oocyte activation. Phospholipase C zeta (PLCz) is sperm-associated protein that can induce oocyte activation. Male infertility has been associated with PLCz deficiency in various species, although this has not been studied in the stallion. We hypothesised that the location and amount of PLCz on sperm varies among stallions. The aim of this study was to validate commercial antibodies (Ab) to detect PLCz on stallion sperm, and then to use these Ab to quantify the amount of PLCz, using flow cytometry, with the long-term goal of correlating PLCz on sperm with stallion fertility. Frozen-thawed sperm were analysed (20 stallions in 3 replicates) using 2 commercial Ab (anti-mouse PLCz M163 and anti-human PLCz H50, Santa Cruz Biotechnology, TX, USA). Western blot and immunofluorescence microscopy were used to validate Ab binding. For microscopy, sperm DNA was counterstained with 1 µg mL−1 Hoechst 33258. For flow cytometry, samples were incubated with Live Dead Fixable Far Red Stain Kit (Molecular Probes, Eugene, OR, USA), fixed, permeabilized, incubated overnight with primary Ab, and labelled with conjugated secondary Ab (anti-rabbit IgG Alexa Fluor 488, Molecular Probes). Green and far red mean fluorescence intensity (MFI) were measured for 20,000 cells per sample. Results are presented as mean ± SEM. Wilcoxon test, Spearman rank correlation, and linear regression were performed for analyses. Immunoblot analyses for both commercial Ab identified an immunoreactive band of ~70 kDa in sperm heads, tails, and whole sperm; β-tubulin was used as loading control and for normalization. Microscopy revealed PLCz in the acrosomal and post-acrosomal regions, connecting piece, midpiece, and tail. Post-acrosomal localization was the pattern most frequently observed (55%), followed by acrosomal plus post-acrosomal regions (25%). The PLCz labelling was observed on >85% of midpiece and tail regions, independent of Ab used. Flow cytometric evaluation revealed that percentage of live sperm was 47 ± 2%. Similar fluorescence intensity was exhibited for both Ab (M163 and H50) with a wide range of values among stallions [M163, mean 30.7 ± 1.9 × 103 (range, 8.8-82.2 × 103); H50: 25.5 ± 3.2 × 103 (7.3-55.0 × 103)]. The percentage of live sperm within a sample was not associated with Ab MFI. However, when samples were gated for live/dead cells, live sperm exhibited higher (P < 0.001) MFI than dead sperm for M163 (42.6 ± 6.0 v. 30.6 ± 3.9 × 103) and H50 (38.4 ± 4.7 v. 25.6 ± 3.7 × 103). There was a strong and positive correlation between M163 and H50 MFI for total sperm and live sperm (total: r = 0.81, P < 0.001; live: r = 0.71; P < 0.001). In conclusion, 2 anti-PLCz commercial antibodies detected equine PLCz, and the PLCz was localised on the sperm as described. Flow cytometric evaluation showed that stallions have different quantities of PLCz on their sperm, and this may provide a mean to determine if PLCz on stallion sperm is associated with fertility.

scholarly journals Defective sperm head decondensation undermines the success of ICSI in the bovine

Reproduction ◽  
2017 ◽  
Vol 154 (3) ◽  
pp. 307-318 ◽  
Author(s):  
Luis Águila ◽  
Ricardo Felmer ◽  
María Elena Arias ◽  
Felipe Navarrete ◽  
David Martin-Hidalgo ◽  
...  
Keyword(s):  
Calcium Oscillations ◽  
Sperm Head ◽  
Oocyte Activation ◽  
Bovine Sperm ◽  
Bovine Oocytes ◽  
Mouse Eggs ◽  
Do So ◽  
Nuclear Decondensation

The efficiency of intracytoplasmic sperm injection (ICSI) in the bovine is low compared to other species. It is unknown whether defective oocyte activation and/or sperm head decondensation limit the success of this technique in this species. To elucidate where the main obstacle lies, we used homologous and heterologous ICSI and parthenogenetic activation procedures. We also evaluated whetherin vitromaturation negatively impacted the early stages of activation after ICSI. Here we showed that injected bovine sperm are resistant to nuclear decondensation by bovine oocytes and this is only partly overcome by exogenous activation. Remarkably, when we used heterologous ICSI,in vivo-matured mouse eggs were capable of mounting calcium oscillations and displaying normal PN formation following injection of bovine sperm, althoughin vitro-matured mouse oocytes were unable to do so. Together, our data demonstrate that bovine sperm are especially resistant to nuclear decondensation byin vitro-matured oocytes and this deficiency cannot be simply overcome by exogenous activation protocols, even by inducing physiological calcium oscillations. Therefore, the inability of a suboptimal ooplasmic environment to induce sperm head decondensation limits the success of ICSI in the bovine. Studies aimed to improve the cytoplasmic milieu ofin vitro-matured oocytes and to replicate the molecular changes associated within vivocapacitation and acrosome reaction will deepen our understanding of the mechanism of fertilization and improve the success of ICSI in this species.

scholarly journals Role of UBC9 in the Regulation of the Adipogenic Program in 3T3-L1 Adipocytes

Endocrinology ◽  
2010 ◽  
Vol 151 (11) ◽  
pp. 5255-5266 ◽  
Author(s):  
Angelo Cignarelli ◽  
Mariangela Melchiorre ◽  
Alessandro Peschechera ◽  
Antonella Conserva ◽  
Lucia Adelaide Renna ◽  
...  
Keyword(s):  
Transcription Factors ◽  
Small Interfering Rna ◽  
Protein Modification ◽  
Covalent Attachment ◽  
Mrna Levels ◽  
Protein Levels ◽  
Mrna Induction ◽  
Induction Of Differentiation ◽  
Interfering Rna

The small ubiquitin-like modifier-conjugating enzyme UBC9, involved in protein modification through covalent attachment of small ubiquitin-like modifier and other less defined mechanisms, has emerged as a key regulator of cell proliferation and differentiation. To explore the role of UBC9 in adipocyte differentiation, the UBC9 protein levels were examined in differentiating 3T3-L1 cells. UBC9 mRNA and protein levels were increased 2.5-fold at d 2 and then gradually declined to basal levels at d 8 of differentiation. In addition, UBC9 was expressed predominantly in the nucleus of preadipocytes but shifted to cytoplasmic compartments after d 4, after induction of differentiation. UBC9 knockdown was then achieved in differentiating 3T3-L1 preadipocytes using a specific small interfering RNA. Oil-Red-O staining demonstrated accumulation of large triglyceride droplets in approximately 90% of control cells, whereas lipid droplets were smaller and evident in only 30% of cells treated with the UBC9-specific small interfering RNA. CCAAT/enhancer-binding protein (C/EBP)-δ, peroxisome proliferator-activated receptor-γ, and C/EBPα mRNA levels were increased severalfold 2–6 d after induction of differentiation in control cells, whereas the expression of these transcription factors was significantly lower in the presence of UBC9 gene silencing. Adenovirus-mediated overexpression of a catalytically inactive mutant UBC9 protein in 3T3-L1 cells resulted in no changes in expression of adipogenic transcription factors and conversion to mature adipocytes as compared with control. In conclusion, UBC9 appears to play an important role in adipogenesis. The temporal profile of UBC9 induction and its ability to affect C/EBPδ mRNA induction support a role for this protein during early adipogenesis.

Knockdown of Pathologic Immuoglobulin Light Chain Protein by Small Interfering RNA Molecules.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 4407-4407
Author(s):  
Jonathan E Phipps ◽  
James S Foster ◽  
Daniel P Kestler ◽  
Robert Donnell ◽  
Alan Solomon ◽  
...  
Keyword(s):  
Cell Line ◽  
Plasma Cell ◽  
Light Chain ◽  
Small Interfering Rna ◽  
Mrna Levels ◽  
Light Chain Deposition Disease ◽  
Rna Molecules ◽  
Plasma Cell Dyscrasias ◽  
Rpmi 8226 ◽  
Interfering Rna

Abstract Abstract 4407 Introduction A major contributing factor to the morbidity and mortality of patients with plasma cell dyscrasias (i.e., light chain deposition disease, light chain amyloidosis (AL), etc.) is the overproduction and deposition of immunoglobulin light chain (LC) protein. Accumulation of insoluble fibrillar or amorphous aggregates within vital organs, particularly the kidneys, contributes to progressive loss of function and failure. Recent reports have shown that reducing levels of precursor LC proteins by 50% correlated with improved prognosis in patients with AL. Current therapies to suppress the production of monoclonal free LC rely on conventional or high dose myeloablative chemotherapeutic intervention. These therapies carry inherent risk and may not be appropriate in every situation, thus there is a need to employ alternative or adjuvant therapeutic approaches. To this end, we have investigated the ability of small interfering RNA molecules to decrease production of LC mRNA and protein secretion in both a synthetic and naturally occurring myeloma cell line. We report here that exposure of these lines to small interfering RNA (siRNA) oligonucleotides inhibited LC production in a specific and non-cytotoxic manner. Methods SP2/O mouse myeloma cells were stably transfected with a construct encoding a human λ6 LC protein (Wil) and designated SP2/O-1 for this study. The human λ2 producing cell line RPMI 8226 was purchased from ATCC. For both lines, LC mRNA was sequenced and used to design siRNA molecules targeting the V-, J-, or C-region of the LC. Cells were cultured for 1-3 d with sequence-specific siRNA, sham siRNA, or with media alone after which time LC mRNA levels were assessed by real-time PCR, and cellular or secreted LC protein concentrations were measured using flow cytometry or ELISA, respectively. Results Treatment with siRNA was well tolerated and no significant reduction in cell viability was observed in either the SP2/O-1 or RPMI 8226 cells at any of the timepoints assayed. Exposure of either SP2/O-1 or 8226 cells to siRNAs targeting the LC resulted in reductions of mRNA as compared to sham siRNA-treated controls. Flow cytometric analysis of 8226 cells immunostained with anti-LC antibodies at 48 h post transfection, indicated that ∼63% and 83% of the total cell number contained reduced amounts of cytoplasmic LC following treatment with V-, or C- region-specific siRNA, respectively. The 8226 LC protein was also significantly reduced by 20-60% in culture supernatants over the 72h period in cells exposed to experimental siRNAs as compared with sham-treated cells. Similarly, treating the SP2/O-1 cells with siRNAs led to significant (20-52%; P < 0.05) reductions in secreted LC Wil over 72 h as compared to sham-siRNA treatments. Conclusions We demonstrate here that siRNA provides a viable option for preventing the synthesis and thus secretion of pathologic LC protein using two cell lines, and that this reduction is carried out in a specific and non-cytotoxic manner. These results provide support for the potential use of siRNA to decrease the secretion of pathologic LC proteins in patients diagnosed with plasma cell dyscrasias. Our future goal will be to evaluate the action of siRNAs on LC production in vivo using a murine model of the disease. Disclosures: No relevant conflicts of interest to declare.

scholarly journals Recombinant human phospholipase C zeta 1 induces intracellular calcium oscillations and oocyte activation in mouse and human oocytes

2012 ◽  
Vol 27 (6) ◽  
pp. 1768-1780 ◽  
Author(s):  
S.-Y. Yoon ◽  
J. H. Eum ◽  
J. E. Lee ◽  
H. C. Lee ◽  
Y. S. Kim ◽  
...  
Keyword(s):  
Intracellular Calcium ◽  
Phospholipase C ◽  
Calcium Oscillations ◽  
Oocyte Activation ◽  
Human Oocytes

scholarly journals Targeting Protein S Using Small Interfering RNA Is Well Tolerated and Protects Mice with Hemophilia a from Acute Hemarthrosis

Blood ◽  
2020 ◽  
Vol 136 (Supplement 1) ◽  
pp. 20-21 ◽  
Author(s):  
Raja Eladnani Prince ◽  
Ute Schaeper ◽  
Sibylle Dames ◽  
Sara Calzavarini ◽  
Claudia Quarroz ◽  
...  
Keyword(s):  
Gene Expression ◽  
Prothrombin Time ◽  
Small Interfering Rna ◽  
Hemophilia A ◽  
Alpha Angle ◽  
Coagulation Factor ◽  
Protein S ◽  
Mrna Levels ◽  
Spontaneous Bleeding ◽  
Interfering Rna

Introduction & Aim: Hemophilia A (HA) is an X-linked disorder caused by an absence or a reduction of coagulation factor VIII. Patients with HA often suffer from spontaneous bleeding within the musculoskeletal system, such as hemarthrosis. Hemarthrosis is caused by bleeding into joint spaces affecting the synovium, synovial blood vessels as well as cartilage and bone tissues. Current prophylactic treatments are not always effective and hemophilia patients can experience breakthrough bleeds. Recently, we demonstrated that inhibition of protein S (PS), a natural anticoagulant, controls coagulation and constitutes a potential therapeutic target in hemophilia (Blood 2018, 131:1360-1371). Here, we aim to translate our findings using small interfering RNA conjugated to an N-acetylgalactosamine (GalNAc) cluster to target Pros1 gene expression (GalNAc-PS siRNA) in vivo and exclusively in hepatocytes. siRNAs conjugated to a GalNAc cluster bind to asialoglycoprotein receptors expressed predominantly by hepatocytes thereby providing a potentially safe, specific and efficient delivery technology for therapeutic molecules. Methods & Results: Forty-two days after subcutaneous (s.c.) injection of GalNAc-PS siRNA (3mg/kg), wild-type (WT) mice were alive and did not display overt disseminated intravascular coagulation (DIC). In a second study in WT mice, DIC parameters assessed fourteen days after treatment with either 5mg/kg GalNAc-PS siRNA or with vehicle were also comparable between the two groups (platelet count: 578±284 vs 725±186 G/L, p&gt;0.9, n=4-6; prothrombin time: 9.0±0.4 vs 8.9±0.3 seconds, p&gt;0.9, n=4-6; fibrinogen: 1.5±0.5 vs 1.8±0.4 g/L, p&gt;0.85, n=5-6; thrombin anti-thrombin complexes, TAT: 71±63 vs 115±34 μg/L, p&gt;0.9, n=2-4) supporting that GalNAc-PS siRNA treatment can be safe. At the same time, mice treated with GalNAc-PS siRNA displayed lower plasma PS level compared to mice receiving the vehicle (52±12 vs 100±11 %, p&lt;0.001, n=6). In the liver, PS mRNA levels were reduced by 69% compared to mice treated just with the vehicle (31±10 vs 100±24 %, p&lt;0.0001, n=6). Importantly, in a murine model for hemophilia A (F8-/- mice) the intrinsically-activated test using ellagic acid (INTEM) assessed by rotative thromboelastometry (ROTEM®) was improved by treatment with GalNAc-PS siRNA (5mg/kg s.c) as compared to F8-/- mice treated with the vehicle (clotting time: 281±193 vs 802±330 seconds, p&lt;0.01, n=6-11; clot formation time: 109±80 vs 657±466 seconds, p&lt;0.05, n=6-11; alpha angle: 70±13 vs 35±24 mm, p&lt;0.1, n=6-11). To assess if targeting PS using GalNAc-siRNA-PS protects mice from acute hemarthrosis (AH), we applied an AH model to F8-/- mice. Five days after injecting a single dose of 5mg/kg GalNAc-PS s.c., right knees were injured using a 30 gauge-needle and knee diameters were measured 72 hours later. Macroscopically, vehicle treated F8-/- mice developed extensive bleeding in injured knees as compared to GalNAc-siRNA-PS treated mice. Scores for intra-articular bleeding (2.4±0.9 vs 1.0±0.7, p=0.035, n=5-9) and synovial hyperplasia (2.4±0.9 vs 0.6±0.9, p=0.027, n=5-9) were higher in F8-/- mice treated by vehicle than in those treated by GalNAc-PS-siRNA. Moreover, knee joint swelling was reduced in GalNAc-siRNA-PS treated mice compared to those treated by vehicle (0.14±0.15 vs 0.78±0.50 mm, p=0.025, n=7-10). As expected, PS plasma levels were lower in GalNAc-PS siRNA treated mice compared to those which received vehicle (63±9 vs 101±19% of WT PS antigen level, p&lt;0.0001, n=7-13) with no overt DIC (platelets count: 711±149 vs 681±189 G/L, p&gt;0.9, n=7-12; prothrombin time: 8.5±0.3 vs 8.4±0.3 seconds, p&gt;0.9, n=4-13; fibrinogen: 2.7±0.6 vs 3.0±0.8 g/L, p=0.73, n=7-12 and TAT: 33±42 vs 45±66 μg/L, p&gt;0.9, n=6-10). Conclusion: These data provide the first evidence that using a GalNAc-siRNA conjugate to modulate Pros1 gene expression is well tolerated and has the ability to reduce plasma PS level and protect F8-/- mice from AH pointing to PS targeting using GalNAc-siRNA-PS as a new valuable therapeutic approach for hemophilia. Further analysis to understand if the inhibition of PS influences also the inflammatory processes causing the hemophilic arthropathy is ongoing. Disclosures Schaeper: Silence Therapeutics GmbH: Current Employment. Dames:Silence Therapeutics: Current Employment. Eisermann:Silence Therapeutics: Current Employment.

scholarly journals Tyrosinase Small Interfering RNA Effectively Suppresses Tyrosinase Gene Expression In Vitro and In Vivo

2010 ◽  
Vol 2010 ◽  
pp. 1-6
Author(s):  
Jia Xiu-Hua ◽  
Lin Shao-Chun ◽  
Huang Bing ◽  
Zhu Xiang ◽  
Zhuang Jing ◽  
...  
Keyword(s):  
Small Interfering Rna ◽  
Initial Step ◽  
Tyrosinase Activity ◽  
Stable Gene ◽  
Mrna Levels ◽  
Melanin Content ◽  
Melanin Biosynthesis ◽  
Interfering Rna

Tyrosinase is a bifunctional enzyme which oxidizes the initial step of melanin biosynthesis, that is, conversion of tyrosine to dopa and subsequently dopa to dopaquinone. It is a glycosylated protein and a major regulator of melanogenesis. To date, many approaches have been tried to regulate tyrosinase activity and melanin content. To that end, we screened small interfering RNA sequences for sequence-inhibited tyrosinase expression in B16 cells and in C57BL/6 mice. We analyzed tyrosinase mRNA levels by quantitative real-time PCR and determined tyrosinase activity and melanin content at 24, 48, and 72 hours after transfection. Results showed that siNM_011661_001 was the most efficient small interfering RNA sequence in suppressing tyrosinase mRNA expression, and cells transfected with this sequence showed lower tyrosinase activity. Moreover, intravitreous injection of siNM_011661_001 in C57BL/6 mice induced an efficient and stable gene-specific inhibition of expression at the posttranscriptional level.

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