|
|
Transgenic RNAi in mouse oocytes
Sarnová, Lenka ; Petr, Jaroslav (referee) ; Svoboda, Petr (advisor)
RNA interference (RNAi) is double-stranded RNA (dsRNA)-mediated mRNA degradation. RNAi has been widely used to investigate gene functions. Many methods to induce transient or stable RNAi have been developed. Transient RNAi can be induced by delivering of a long dsRNA or short interfering RNAs (siRNAs). Stable RNAi may be induced by introducing plasmids expressing a long or a short hairpin RNA. Both small and long RNAs have been used to induce transient RNAi in mouse oocytes. Nevertheless, only long hairpin-expressing system has been used to trigger stable RNAi in oocytes. Although, this system appeared to be highly efficient and specific, it has several disadvantages as complicated long inverted repeat cloning or limited possibility to test these vectors in the cell culture. Here, we constructed a short hairpin-expressing vector suitable for transgenic RNAi induction in mouse oocytes. The new vector, pTMP_ZP3_sh, was derived from a lentiviral short hairpin vector selected based on comparative study of different short hairpin-expressing plasmids. The pTMP_ZP3_sh vector was tested by targeting Moloney sarcoma oncogene (Mos) mRNA, which is a common model for RNAi in mouse oocytes. We designed several candidate short hairpin sequences and tested their efficacy. Subsequently, the most efficient one was selected...
|
|
| |
|
|
Gene expression during oocyte-cumulus expansion and preimplantation embryo development in mammals
Němcová, Lucie ; Kaňka, Jiří (advisor) ; Petr, Jaroslav (referee) ; Lopatářová, Miloslava (referee)
CONCLUSIONS Major resultsof this PhD thesiscan be summarizedas follows: The real-time RT-PCR has become a tool for detection and quantifrcation of RNA targetsandis being utilized in geneexpressionstudyin our laboratory. The gene expression proÍile of GDF9, during porcine oocytes, cumulus andgranulosacells fu vitro cultivation and in vivo cumulus oocyte expansion was assessed.The obtained partial sequence was up|oaded in EMBL/GenBanlďDDBJ databaseand gene was localized on pig chromosome2. Conhary to mouse,GDFS mRNA was detectedfor the ťrrsttime also in porcine somaticfollicular cells. During cultivation,thetranscriptgraduallydecreasedin all the samples. we studied signalling pathways involved in IGFI promotion of FSH-induced oocyte-cumulusexpansion.we found that IGFI in combinationwith FSH increased the production of HA. The expression of Has2 mRNA in cumulus cells gradually decreasedduring cultivation' our results suggest that the PIK3/AKT dépendent pathwayis involved in the promotionof FSH stimulatedsynthesisof HA in porcine OCCs. Bovine blastocystsdeveloped from fertilized oocýes under different in vitro or in vivo conditions were comparedin the expressionof threegenes(Bax, L37 and S3o/.The embryosdiffer in the abundanceof Bax transcripl the elevatedlevels were detected in blastocyst completely cultivated...
|
|
|
Effect of the sperm ubiquitination in the early embryonic development in pig
Petelák, Aleš ; Krylov, Vladimír (advisor) ; Petr, Jaroslav (referee)
The intracellular sperm injection (ICSI) technique is a very effective tool for the fertilization research. In the newly established laboratory at the Faculty of Science of the Charles University it was necessary to introduce this method and define the early developmental potential of fertilized oocytes. After fertilization oocytes were incubated to the blastocyst stage with a success comparable with other laboratories (17%). The ubiquitin-proteasome system which plays a major role in a protein degradation within cells is involved in a regulatory mechanism of sperm maturation. It is also responsible for a penetration of a vitelline membrane. In these processes ubiquitin residues are localized extracellulary. High level of sperm ubiquitination correlates with their low quality. Hypotetically it can be expected that the ubiqutination of impaired sperm cells can be used as a negative marker for their recognition and degradation by 26S proteasome complex localized. Experiments in this diploma thesis were designed based on the hypothesis that the executive part of the selective mechanism is the 26S proteasome. Therefore the effect of MG132 peptide inhibition of the 20S proteasome on the pronuclei formation and subsequent early embryonic development after ICSI was studied. Inhibition of 20S proteasome...
|
|
|
Analysis of short Argonaute isoforms from mouse oocytes
Jankele, Radek ; Svoboda, Petr (advisor) ; Petr, Jaroslav (referee)
AnalysisofshortArgonauteisoformsfrommouseoocytes Abstract: Argonaute proteins carrying small RNAs form the conserved core of RNA silencing mechanisms, which repress viruses, mobile genetic elements, and genes in a sequence specific manner. The microRNA (miRNA) pathway is a dominant mammalian RNA silencing mechanism in somatic cells, which post-transcriptionally regulates large fraction of genes and thereby adjusts protein levels. miRNA-guided Argonautes inhibit translation and induce deadenylation of complementary mRNAs, ultimately resulting in their decay. In contrast to RNA interference (RNAi), which employs Argonaute slicer activity to directly cleave perfectly complementary RNAs, an effective miRNA-mediated mRNA repression requires multiple Argonaute-associated protein factors and enzymes. The miRNA pathway has been implicated in many complex biological processes ranging from organogenesis, stress-response to haematopoiesis or cancer. Surprisingly, canonical miRNAs are not essential for oocytes and early embryonic development in mice. Even the most abundant miRNAs present in mouse oocytes are unable to effectively repress target genes. However, RNAi, which shares key enzymes with the miRNA pathway, is highly active in oocytes and early embryos. The cause of miRNA inactivity in mouse oocytes remains...
|
|
|
Recognition of expressed double-stranded RNAs in mammalian cells
Vaškovičová, Michaela ; Svoboda, Petr (advisor) ; Petr, Jaroslav (referee)
Long double-stranded RNA (dsRNA) is a unique structure formed during viral replication or transcription of repetitive elements. Mammalian cells evolved several mechanisms how to respond to dsRNA. dsRNA can be engaged in one of three pathways: interferon response, RNA editing, and RNA interference (RNAi). RNAi is evolutionary conserved effect of dsRNA, which results in sequence-specific messenger RNA degradation. However, in mammals, RNAi is functional only in mouse oocytes, which express truncated version of Dicer (DicerO ). In somatic cells, dsRNA triggers sequence-independent interferon pathway. The main aim of this Master's thesis was to examine how specific double-stranded RNA-binding proteins (DRBPs) influence distribution of long dsRNA into RNAi and sequence-independent pathways. We used a luciferase-based reporter RNAi assay to monitor sequence-specific and sequence-independent effects of dsRNA co-expressed with selected DRBPs. Our results suggest that none of the tested DRBPs is sufficient to stimulate RNAi in somatic cells. Interestingly, the overexpression of either TARBP2 or PACT suppressed RNAi in cells expressing DicerO . Moreover, microRNA pathway, which employs the same protein factors as RNAi, is not inhibited by TARBP2 or PACT. Therefore, we propose that DRBPs overexpression...
|
|
|
Formation of spatio-temporal molecular gradients in early embryonic development of Xenopus laevis.
Šídová, Monika ; Tlapáková, Tereza (advisor) ; Pěknicová, Jana (referee) ; Petr, Jaroslav (referee)
Clarifying the underlying spatio-temporal mechanisms that determine body pattern is important for detailed understanding of embryonic development. A crucial question of vertebrate embryogenesis remains: when and how are single blastomeres determined for differentiation that subsequently leads to body axes specification and the formation of different tissues and organs? The answer to this question will be beneficial for primary research as well as in the field of applied medicine. The main aim of the presented thesis was to study spatio-temporal molecular gradients of cell fate determinants during early embryonic development. The African clawed frog Xenopus laevis was used as a model organism because of their large size of oocytes and external embryonic development. Due to late activation of embryonic transcription, a crucial mechanism of early blastomeres determination is dependent on asymmetric localization of maternal factors within oocyte and their uneven distribution into single blastomeres during early cell division. Two main localization patterns were identified along the animal-vegetal axis of the mature Xenopus oocyte using qPCR tomography. The localization gradient with preference in either animal or vegetal hemisphere was found for maternal mRNA as well as miRNAs. Moreover, two vegetal...
|
|
|
Sperm acrosomal reactien in selected species of mammals
Frolíková, Michaela ; Stopka, Pavel (advisor) ; Jonáková, Věra (referee) ; Petr, Jaroslav (referee)
Mammalian sperm must undergo the process of capacitation - series of physiological and biochemical modifications prior fertilization. In last stage of capacitation sperm undergoes acrosome reaction (AR). During AR the cell membrane of the sperm fuses with the outer acrosomal membrane and the contents of acrosomal vesicle are released into extracellular space. Sperm which did not undergo AR or sperm missing acrosome at all are unable to fertilize. AR results into dramatical changes in the sperm head. Most of the proteins present in plasmatic and outer acrosomal membrane are reorganized or lost. There are also significant changes in cytoskeletal and intraacrosomal proteins are released to extracellular space uncovering new surface domains. Some sperms undergo AR even without presence of inductor of AR during capacitation in vitro. This event is called spontaneous (accelerated) AR. The latest research indicates that spontaneous AR is natural part of the process of fertilization. Field mice (Apodemus) show high level of promiscuity leading to significant risk of sperm competition. Unique reproduction strategy where the sperms form so-called sperm trains was evolved in field mice. Spontaneous AR is probably enabling the dissociation of sperms from the sperm train. The spontaneous AR rate is dependent on...
|
|
|
The effects of long double-stranded RNA expression in mammalian cells.
Nejepínská, Jana ; Svoboda, Petr (advisor) ; Petr, Jaroslav (referee) ; Štefl, Richard (referee)
Double stranded RNA (dsRNA) is a foreign molecule that arises in the cell either as a by-product of viral replication or it is produced by the intramolecular or intermolecular pairing of complementary RNAs, often originating from repetitive sequences. In mammals, dsRNA can enter one of three pathways: the sequence-specific RNA silencing, the sequence-independent interferon (IFN) response, or editing by adenosine deaminases. The main focus of my PhD project was to comprehensively analyze the effects of the expressed dsRNA in mammals in the context of the whole organism. To follow this aim, we generated a construct expressing dsRNA in a form of an mRNA containing a long perfect hairpin structure. Transgenic mice ubiquitously expressing dsRNA were viable and, in contrast to the previous studies, the IFN response was not activated. In somatic cells, dsRNA was poorly processed into small interfering RNAs, did not cause transcriptional silencing in trans, and underwent low adenosine deamination without the nuclear retention. Consistent results were obtained in human cells transiently transfected with a dsRNA-expressing plasmid. On the other hand, dsRNA expression caused robust RNA interference (RNAi) in oocytes. Thus, we show for the first time that expressed dsRNA, in contrast to many other forms of...
|
|
| |
guest ::
RSS feed.