• MeSH
• genetika MeSH
• RNA MeSH

• Konspekt
• Obecná genetika. Obecná cytogenetika. Evoluce
• NLK Obory
• genetika, lékařská genetika

Východisko. U pacientů s chronickou myeloidní leukémií jsme detekovali zvýšenou hladinu exprese genu PCNA, který by se potenciálně mohl podílet na rozvoji onemocnění. Cílem této práce bylo nalézt vhodné podmínky pro transfekci molekul PCNA-siRNA do buněčných linií K562 a MOLM-7 s up-regulovaným PCNA a zvýšenou expresi tlumit. Metody a výsledky. Klíčovými parametry úspěšného vnášení siRNA do buněk jsou typ a množství transfekčního činidla, koncentrace buněk a vnášené siRNA nebo doba inkubace transfekovaných buněk před analýzou exprese. Byla testována tato činidla: ExGene 500 (Fermentas), Metafectene (Biontex), Oligofectamine (Qiagen) a siPORT Amine (Ambion). Účinnost transfekce siRNA značených fluoresceinem byla sledována pomocí fluorescenční mikroskopie. Míra potlačení genové exprese na úrovni mRNA byla stanovována pomocí RT-PCR v reálném čase a na proteinové úrovni metodou western blotů. Jako nejvhodnější byl pro další pokusy vybrán Oligofectamine, pomocí kterého bylo dosaženo 70% poklesu hladiny mRNA genu PCNA. Dále byla zvolena 50 nM koncentrace siRNA, 1x106 buněk/ml a množství Oligofectaminu 4 µl/1ml transfekovaných buněk. Nejvhodnější doba kultivace buněk po transfekci byla 48 hodin. Závěry. Na základě výsledků této práce byl navržen protokol pro vnášení siRNA do buněčných linií K562 a MOLM-7. Tuto techniku lze přenést i na další geny potenciálně přispívající k CML a sledovat dopad siRNA-inhibice genů na expresní profil takto ovlivněných buněk. siRNA proti některým z over-exprimovaných genů by navíc mohly být v budoucnu využity pro genovou terapii CML.

Background. Increased expression of PCNA gene was detected in chronic myeloid leukemia (CML) patients in our laboratory. The gene may participate in the disease development. The aim of the study was to develop appropriate conditions for PCNA-siRNA transfection into K562 and MOLM-7 cell lines (which both have the up-regulated PCNA gene) and to silence the increased expression. Methods and Results. Key parameters of successful siRNA delivery into the cells are type and quantity of transfection reagent, cells and siRNA concentration or cultivation time before an expression analysis. Transfection reagents ExGene 500 (Fermentas), Metafectene (Biontex), Oligofectamine (Qiagen) and siPORT Amine (Ambion) were tested. Transfection efficiency was monitored by fluorescence microscopy of fluorescein labeled siRNA. Gene silencing was determined at mRNA level by real-time PCR and at protein level by western blots. As the most suitable reagent was chosen Oligofectamine, which achieved 70% decrease of PCNA mRNA level. Further, 50 nM siRNA concentration, 1x106 cells/ml and amount of Oligofectamine 4 µl per 1 ml of transfected cells were selected. The best cultivation time after siRNA delivery was 48 h. Conclusions. Based on the results of this study, transfection method for siRNA delivery into the K562 and MOLM-7 cell lines was proposed. The procedure can be transferred also on further selected genes potentially involved in CML and afterwards it will be possible to monitor the impact of siRNA-inhibition on expression profile. In the future siRNAs against some over-expressed genes would be used for gene therapy of CML.

• MeSH
• finanční podpora výzkumu jako téma MeSH
• lidé MeSH
• malá interferující RNA aplikace a dávkování   imunologie   MeSH
• myeloidní leukemie imunologie   MeSH
• nádorové buněčné linie imunologie   účinky léků   MeSH
• proliferační antigen buněčného jádra účinky léků   MeSH
• Check Tag
• lidé MeSH

In plants, posttranscriptional gene silencing (PTGS) is induced by small RNAs (sRNAs) generated from various dsRNA precursors. To assess the impact of dsRNA origin, we compared downregulation of GFP expression triggered by inverted repeat (IR), antisense (AS) and unterminated sense (UT) transcripts transiently expressed from the estradiol-inducible promoter. The use of homogeneously responding tobacco BY-2 cell lines allowed monitoring the onset of silencing and its reversibility. In this system, IR induced the strongest and fastest silencing accompanied by dense DNA methylation. At low induction, silencing in individual cells was binary (either strong or missing), suggesting that a certain threshold sRNA level had to be exceeded. The AS variant specifically showed a deviated sRNA-strand ratio shifted in favor of antisense orientation. In AS lines and weakly induced IR lines, only the silencer DNA was methylated, but the same target GFP sequence was not, showing that DNA methylation accompanying PTGS was influenced both by the level and origin of sRNAs, and possibly also by the epigenetic state of the locus. UT silencing appeared to be the least effective and resembled classical sense PTGS. The best responding UT lines behaved relatively heterogeneously possibly due to complexly arranged T-DNA insertions. Unlike IR and AS variants that fully restored GFP expression upon removal of the inducer, only partial reactivation was observed in some UT lines. Our results pointed out several not yet described phenomena and differences between the long-known silencer variants that may direct further research and affect selection of proper silencer variants for specific applications.

• MeSH
• DNA bakterií MeSH
• dvouvláknová RNA genetika   MeSH
• metylace DNA MeSH
• posttranskripční úpravy RNA * MeSH
• regulace genové exprese u rostlin * MeSH
• reportérové geny MeSH
• RNA interference * MeSH
• RNA rostlin genetika   MeSH
• tabák genetika   MeSH
• umlčovací elementy transkripční MeSH
• umlčování genů * MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

• MeSH
• biogeneze organel MeSH
• malá interferující RNA MeSH
• umlčování genů MeSH
• Publikační typ
• sborníky MeSH

• Konspekt
• Biochemie. Molekulární biologie. Biofyzika
• NLK Obory
• biologie
• cytologie, klinická cytologie
• onkologie

It has been well established that trans-acting small RNAs guide promoter methylation leading to its inactivation and gene silencing at the transcriptional level (TGS). Here we addressed the question of the influence of the locus structure and epigenetic modifications of the target locus on its susceptibility for being paramutated by trans-acting small RNA molecules. Silencing was induced by crossing a 35S promoter silencer locus 271 with two different 35S-driven transgene loci, locus 2 containing a highly expressed single copy gene and locus 1 containing an inverted posttranscriptionally silenced (PTGS) repeat of this gene. Three generations of exposure to RNA signals from the 271 locus were required to complete silencing and methylation of the 35S promoter within locus 2. Segregating methylated locus 2 epialleles were obtained only from the third generation of hybrids, and this methylation was not correlated with silencing. Strikingly, only one generation was required for the PTGS locus 1 to acquire complete TGS and 35S promoter methylation. In this case, paramutated locus 1 epialleles bearing methylated and inactive 35S promoters segregated already from the first generation of hybrids. The results support the hypothesis that PTGS loci containing a palindrome structure and methylation in the coding region are more sensitive to paramutation by small RNAs and exhibit a strong tendency to formation of meiotically transmissible TGS epialleles. These features contrast with a non-methylated single copy transgenic locus that required several generations of contact with RNA silencing molecules to become imprinted in a stable epiallele.

• MeSH
• alely MeSH
• epigeneze genetická MeSH
• genetická transkripce MeSH
• geneticky modifikované rostliny genetika   MeSH
• genomový imprinting MeSH
• malá interferující RNA genetika   MeSH
• metylace DNA MeSH
• promotorové oblasti (genetika) MeSH
• regulace genové exprese u rostlin MeSH
• RNA interference MeSH
• tabák genetika   MeSH
• transgeny genetika   MeSH
• umlčovací elementy transkripční genetika   MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

Here, we describe a protocol to prepare and administer glucan-encapsulated RNAi particles (GeRPs), for specific delivery of siRNA and subsequent gene silencing in Kupffer cells (KCs) in mice. This technology is based on baker's yeast and allows gene manipulation in macrophages in a tissue-specific manner depending on the route of administration and the model that is used. GeRP administered by intravenous injection in mice are delivered to KCs. Therefore, using the GeRP technology to silence genes provides a unique method to study the function of factors expressed by KCs in the regulation of liver function.

• MeSH
• glukany genetika   MeSH
• játra fyziologie   MeSH
• Kupfferovy buňky fyziologie   MeSH
• makrofágy fyziologie   MeSH
• malá interferující RNA genetika   MeSH
• myši inbrední C57BL MeSH
• myši MeSH
• RNA interference fyziologie   MeSH
• umlčování genů fyziologie   MeSH
• zvířata MeSH
• Check Tag
• mužské pohlaví MeSH
• myši MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

• MeSH
• geny MeSH
• RNA MeSH

• Konspekt
• Obecná genetika. Obecná cytogenetika. Evoluce
• NLK Obory
• genetika, lékařská genetika

BACKGROUND: Antisense gapmer oligonucleotide drugs require delivery and biodistribution enabling technologies to increase in vivo efficacy. An attractive approach is their binding and consequent transport by the endogenous human serum albumin pool as mediated by fatty acid incorporation into the gapmer design. METHODS: The present study investigated the effect of palmitoyl modification and position on albumin-binding, cellular uptake and in vitro gene silencing of gapmers with either a phosphorothioate (PS) or phosphodiester (PO) backbone. RESULTS: Two palmitoyls positioned exclusively at the 5' end, or a single palmitoyl at both the 3' and 5' positions, showed similar binding to human serum albumin as demonstrated by a gel-shift assay. Decreased cellular uptake determined by flow cytometry (27% compared to nonpalmitoyl gapmers) was observed for palmitoylated Cy5.5 labelled gapmers. However, HER3 (human epidermal growth factor receptor 3) gene silencing was exhibited by the palmitoylated gapmers with transfection agent in PC-3 and Caco-2 cells (68% and 62%, respectively), which was comparable to nonpalmitoyl gapmers (68% and 82%, respectively). Importantly, PO gapmers with a single palmitoyl positioned at both the 3' and 5' positions showed high silencing efficiencies (68% and 66% in PC-3 and Caco-2 cells, respectively) similar to those of PS nonpalmitoylated gapmers (67% and 66% in PC-3 and Caco-2 cells, respectively) in the absence of a transfection agent. CONCLUSIONS: The present study defines phosphodiester gapmer design criteria exhibiting high gene silencing activity and albumin binding that may be utilized with potentially less in vivo toxicity that can be associated with phosphorothioate gapmer designs.

• MeSH
• albuminy metabolismus   MeSH
• antisense oligonukleotidy chemie   genetika   metabolismus   MeSH
• lidé MeSH
• lipoylace MeSH
• molekulární struktura MeSH
• nádorové buněčné linie MeSH
• receptor erbB-3 genetika   MeSH
• techniky in vitro MeSH
• transfekce MeSH
• umlčování genů * MeSH
• vazba proteinů MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

• MeSH
• elektroporace metody   MeSH
• finanční podpora výzkumu jako téma MeSH
• genetická terapie metody   MeSH
• geny p53 genetika   MeSH
• malá interferující RNA genetika   MeSH
• nádory genetika   terapie   MeSH
• regulace genové exprese imunologie   MeSH
• RNA interference MeSH

Despite intensive research efforts and development of numerous new anticancer drugs and treatment strategies over the past decades, there has been only very limited improvement in overall patient survival and in effective treatment options for pancreatic cancer. Current chemotherapy improves survival in terms of months and death rates in pancreatic cancer patients are almost equivalent to incidence rates. It is imperative to develop new therapeutic approaches. Among them, gene silencing shows promise of effectiveness in both tumor cells and stromal cells by inhibiting tumor-promoting genes. This review summarizes potential targets for gene silencing in both pancreatic cancer cells and abundant stromal cells focusing on non-viral delivery systems for small RNAs and discusses the potential immunological implications. The review concludes with the importance of multifactorial therapy of pancreatic cancer.

• MeSH
• antitumorózní látky * terapeutické užití   MeSH
• buňky stromatu MeSH
• lidé MeSH
• nádory slinivky břišní * farmakoterapie   terapie   MeSH
• umlčování genů MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• přehledy MeSH