Shortcomings of short hairpin RNA-based transgenic RNA interference in mouse oocytes

<p>Abstract</p> <p>Background</p> <p>RNA interference (RNAi) is a powerful approach to study a gene function. Transgenic RNAi is an adaptation of this approach where suppression of a specific gene is achieved by expression of an RNA hairpin from a transgene. In somatic cells, where a long double-stranded RNA (dsRNA) longer than 30 base-pairs can induce a sequence-independent interferon response, short hairpin RNA (shRNA) expression is used to induce RNAi. In contrast, transgenic RNAi in the oocyte routinely employs a long RNA hairpin. Transgenic RNAi based on long hairpin RNA, although robust and successful, is restricted to a few cell types, where long double-stranded RNA does not induce sequence-independent responses. Transgenic RNAi in mouse oocytes based on a shRNA offers several potential advantages, including simple cloning of the transgenic vector and an ability to use the same targeting construct in any cell type.</p> <p>Results</p> <p>Here we report our experience with shRNA-based transgenic RNAi in mouse oocytes. Despite optimal starting conditions for this experiment, we experienced several setbacks, which outweigh potential benefits of the shRNA system. First, obtaining an efficient shRNA is potentially a time-consuming and expensive task. Second, we observed that our transgene, which was based on a common commercial vector, was readily silenced in transgenic animals.</p> <p>Conclusions</p> <p>We conclude that, the long RNA hairpin-based RNAi is more reliable and cost-effective and we recommend it as a method-of-choice when a gene is studied selectively in the oocyte.</p

Molecular Genetic Analysis of 103 Sporadic Colorectal Tumours in Czech Patients

The Czech Republic has one of the highest incidences of colorectal cancer (CRC) in Europe. To evaluate whether sporadic CRCs in Czech patients have specific mutational profiles we analysed somatic genetic changes in known CRC genes (APC, KRAS, TP53, CTNNB1, MUTYH and BRAF, loss of heterozygosity (LOH) at the APC locus, microsatellite instability (MSI), and methylation of the MLH1 promoter) in 103 tumours from 102 individuals. The most frequently mutated gene was APC (68.9% of tumours), followed by KRAS (31.1%), TP53 (27.2%), BRAF (8.7%) and CTNNB1 (1.9%). Heterozygous germline MUTYH mutations in 2 patients were unlikely to contribute to the development of their CRCs. LOH at the APC locus was found in 34.3% of tumours, MSI in 24.3% and MLH1 methylation in 12.7%. Seven tumours (6.9%) were without any changes in the genes tested. The analysis yielded several findings possibly specific for the Czech cohort. Somatic APC mutations did not cluster in the mutation cluster region (MCR). Tumours with MSI but no MLH1 methylation showed earlier onset and more severe mutational profiles compared to MSI tumours with MLH1 methylation. TP53 mutations were predominantly located outside the hot spots, and transitions were underrepresented. Our analysis supports the observation that germline MUTYH mutations are rare in Czech individuals with sporadic CRCs. Our findings suggest the influence of specific ethnic genetic factors and/or lifestyle and dietary habits typical for the Czech population on the development of these cancers

Image-based visual hull of a tennis racket

Researchers are often interested in tracking object movement in sport. This could be useful to identify equipment designs that match the technique of players. Previously, stereo camera systems have been used to track markers attached to striking implements to measure their movement in three dimensions. However, manual selection of markers on the image plane can be time consuming and inaccurate. There is potential however to reduce these drawbacks associated with marker-based analysis by tracking a striking implement using its visual hull. The closest geometric approximation of an object that can be reconstructed using only its silhouette images is its visual hull. Early applications of visual hulls include size and shape estimation of objects such as stones. Recently, subject specific visual hulls constructed from multiple camera views combined with anatomical tracking algorithms have measured human motion through markerless motion capture. However, multiple camera systems are not practical for real play conditions in most sports. The application of visual hulls to measure movements of striking implements used in sport has not yet been explored. A set of calibrated views of a tennis racket were captured and segmented into binary images to obtain silhouettes. The visual hull of a tennis racket was constructed by intersection of the volume of space formed by back-projecting the silhouettes from all input views. This research is the first stage in the development of a system that measures movement of a striking implement in real play conditions by combining its visual hull with footage from a single camera

Development of an allele-specific minimal residual disease assay for patients with juvenile myelomonocytic leukemia

Juvenile myelomonocytic leukemia is an aggressive and frequently lethal myeloproliferative disorder of childhood. Somatic mutations in NRAS, KRAS, or PTPN11 occur in 60% of cases. Monitoring disease status is difficult because of the lack of characteristic leukemic blasts at diagnosis. We designed a fluorescently based, allele-specific polymerase chain reaction assay called TaqMAMA to detect the most common RAS or PTPN11 mutations. We analyzed peripheral blood and/or bone marrow of 25 patients for levels of mutant alleles over time. Analysis of pre–hematopoietic stem-cell transplantation, samples revealed a broad distribution of the quantity of the mutant alleles. After hematopoietic stem-cell transplantation, the level of the mutant allele rose rapidly in patients who relapsed and correlated well with falling donor chimerism. Simultaneously analyzed peripheral blood and bone marrow samples demonstrate that blood can be monitored for residual disease. Importantly, these assays provide a sensitive strategy to evaluate molecular responses to new therapeutic strategies

Development of an allele-specific minimal residual disease assay for patients with juvenile myelomonocytic leukemia

Juvenile myelomonocytic leukemia is an aggressive and frequently lethal myeloproliferative disorder of childhood. Somatic mutations in NRAS, KRAS, or PTPN11 occur in 60% of cases. Monitoring disease status is difficult because of the lack of characteristic leukemic blasts at diagnosis. We designed a fluorescently based, allele-specific polymerase chain reaction assay called TaqMAMA to detect the most common RAS or PTPN11 mutations. We analyzed peripheral blood and/or bone marrow of 25 patients for levels of mutant alleles over time. Analysis of pre–hematopoietic stem-cell transplantation, samples revealed a broad distribution of the quantity of the mutant alleles. After hematopoietic stem-cell transplantation, the level of the mutant allele rose rapidly in patients who relapsed and correlated well with falling donor chimerism. Simultaneously analyzed peripheral blood and bone marrow samples demonstrate that blood can be monitored for residual disease. Importantly, these assays provide a sensitive strategy to evaluate molecular responses to new therapeutic strategies

Influence of branching density on the cross-linkability of ethylene-octene copolymers

Ethylene-octene copolymers (EOCs) with two different octene contents (20 and 35 wt%) and the same melt flow index (3 g per 10 min) were cross-linked using various levels (0.3, 0.5 and 0.7 wt%) of dicumyl peroxide at different temperatures. Cross-linking and degradation were analyzed by rubber process analyzer (RPA) within a temperature range of 150-200 °C. The highest s′ max (maximum elastic torque) and the lowest tan(delta) were found for EOC-20 with low-octene content at all cross-linking temperatures. Lower peroxide efficiency was observed in the case of the high-octene copolymer. Increased degradation was observed with increasing cross-linking temperature. High-octene EOC was found to be more vulnerable to degradation. According to dynamic mechanical analysis, the storage modulus (M′) and the glass transition temperature (T g) obtained from the tan(delta) peaks were found to decrease with increasing octene content. The differential scanning calorimetry (DSC) results show that the octene content has an inverse effect on the crystallinity (X) and melting point (T m) - due to the reduction in the average number of consecutive ethylene units. Creep testing at 150 °C confirmed the cross-linkability results obtained by RPA and the gel content analyses. Increased β-scission due to high numbers of tertiary carbon atoms present in the chain has resulted in the poorer cross-linking and inferior properties of high-octene EOC. © 2013 The Society of Polymer Science, Japan (SPSJ) All rights reserved