Biomedical subjects

Vladimir Pekarik

Publications and source records attributed to Vladimir Pekarik.

5 recordsLinked to original sources

PubMed2005

Design of shRNAs for RNAi-A lesson from pre-miRNA processing: possible clinical applications.

RNA interference has become the tool of choice to analyse the loss-of-function of individual genes and has been exploited to identify complex regulatory pathways following genomic screening. RNAi has both admirers and detractors, but is undeniably a technique with great potential, which has come a long way in the short time since its discovery. RNAi utilises cellular machinery associated with the processing of naturally occurring micro RNA (miRNAs). Effective use of RNAi requires detailed knowledge of the individual steps and the proteins involved, as well as the similarities and distinctions between miRNA and siRNA pathways. RNAi was originally induced by the introduction of long double stranded RNAs (dsRNAs) into cells in which the RNA was cleaved into short RNAs which effectively interfered with a transcription of cognate mRNA. More recently an introduction of short approximately 22 nucleotide RNA duplexes has become the standard in short-term experiments, but is insufficient for long-term knock-down assays. Long-term expression of siRNAs has been achieved by in vivo transcription from plasmids coding for short hairpin RNAs (shRNAs). The cellular processing of shRNAs shares common features with the biogenesis of naturally occurring miRNA such as cleavage by nuclear RNase Drosha, export from the nucleus, processing by a cytoplasmic RNase Dicer, and incorporation into the RNA-induced silencing complex (RISC). Each step has a crucial influence on the efficiency of RNAi and their consideration should be a part of a standard experimental design. RNAi has moved from a purely experimental technique to the stage of potential clinical applications. The possible use of RNAi in the treatment of spinocerebellar ataxia or amyotrophic lateral sclerosis, with its advantages and pitfalls and possible extensions to other diseases are discussed.

Animals↗

PubMed2005

Sonic hedgehog guides commissural axons along the longitudinal axis of the spinal cord.

Dorsal commissural axons in the developing spinal cord cross the floor plate, then turn rostrally and grow along the longitudinal axis, close to the floor plate. We used a subtractive hybridization approach to identify guidance cues responsible for the rostral turn in chicken embryos. One of the candidates was the morphogen Sonic hedgehog (Shh). Silencing of the gene SHH (which encodes Shh) by in ovo RNAi during commissural axon navigation demonstrated a repulsive role in post-commissural axon guidance. This effect of Shh was not mediated by Patched (Ptc) and Smoothened (Smo), the receptors that mediate effects of Shh in morphogenesis and commissural axon growth toward the floor plate. Rather, functional in vivo studies showed that the repulsive effect of Shh on postcommissural axons was mediated by Hedgehog interacting protein (Hip).

Age Factors↗

PubMed2005

The survival of neural precursor cell grafts is influenced by in vitro expansion.

Embryonic neural precursor cells (ENPs) provide a potential alternative for transplantation in neurodegenerative diseases, as they can be expanded in culture, avoiding many of the practical obstacles that limit the application of transplanting primary neurones. However, grafts of ENPs into animal models show variable survival and limited differentiation into neurones. The effect of expansion time on their ability to survive and differentiate may be an important factor in this and has not been examined directly. In these experiments, murine and human ENPs were expanded for short (4 weeks) and long (20 weeks) periods before transplantation into the adult rat striatum. Whereas grafts of both short- and long-term expanded human ENPs survived for 4 weeks following transplantation, by 20 weeks all long-term expanded grafts had disappeared. Murine ENPs behaved similarly: only grafts of short-term expanded ENPs survived at 12 weeks following transplantation. RT-PCR analysis of ENP cultures after 4 and 20 weeks of expansion demonstrated changes in expression of a number of different groups of genes. We conclude that long-term expansion of ENPs profoundly impairs their ability to survive long-term after transplantation into the adult brain. This has implications for the potential use of these cells for neural transplantation strategies.

Animals↗

PubMed2002

Screening for gene function in chicken embryo using RNAi and electroporation.

In the postgenomic era the elucidation of the physiological function of genes has become the rate-limiting step in the quest to understand the development and function of living organisms. Gene functions cannot be determined by high-throughput methods but require analysis in the context of the entire organism. This is particularly true in the developing vertebrate nervous system. Because of its easy accessibility in the egg, the chicken embryo has been the model of choice for developmental in vivo studies. However, its usefulness has been hampered by a lack of methods for genetic manipulation. Here we describe an approach that could compensate for this disadvantage. By combining gene silencing by dsRNA (through RNA interference, RNAi) with in ovo electroporation, we developed an efficient method to induce loss of gene function in vivo during the development of the chicken CNS. This method opens new possibilities for studying gene function not only by gain-of-function but also by loss-of-function approaches and therefore represents a new tool for functional genomics.

Animals↗

PubMed2002

Analysis of the prion protein in primates reveals a new polymorphism in codon 226 (Y226F).

Bovine spongiform encephalopathy has been epizootic in cows for the last two decades, and most probably causes variant Creutzfeldt-Jakob disease in humans. A thorough understanding of prion pathogenesis relies on suitable animal models. Modeling the transmission of BSE to primates is a crucial public health priority, necessary for determining the tissue distribution of the agent and for devising therapies. Susceptibility of humans to BSE is partly determined by polymorphism within the gene encoding the cellular prion protein, Prnp, a fact that must be taken into account in primate studies. However, no information is available on Prnp polymorphisms in primates. We have sequenced the Prnp open reading frames of 30 non-consanguineous Rhesus macaques. All macaques were homozygous for methionine at codon 129, which is polymorphic in humans and seems to modulate prion susceptibility. However, we identified a novel polymorphism in macaque Prnp, localized on codon 226 (Y226F). A modulatory effect of this polymorphism on the development of prion disease is possible because codon 226 is close to the suggested binding side of the factor X, which has been invoked as a determinant of the prion species barrier.

Amyloid↗