Expression optimization
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The oviduct provides an optimal environment for the final preparation, transport, and survival of gametes, the fertilization process, and early embryonic development. Most of the studies on reproduction are based on in vitro cell culture models because of the cell's accessibility. It creates opportunities to explore the complexity of directly linked processes between cells. Previous studies showed a significant expression of genes responsible for cell differentiation, maturation, and development during long-term porcine oviduct epithelial cells (POECs) in vitro culture. This study aimed at establishing the transcriptomic profile and comprehensive characteristics of porcine oviduct epithelial cell in vitro cultures, to compare changes in gene expression over time and deliver information about the expression pattern of genes highlighted in specific GO groups. The oviduct cells were collected after 7, 15, and 30 days of in vitro cultivation. The transcriptomic profile of gene expression was compared to the control group (cells collected after the first day). The expression of COL1A2 and LOX was enhanced, while FGFBP1, SERPINB2, and OVGP1 were downregulated at all selected intervals of cell culture in comparison to the 24-h control (p-value < 0.05). Adding new detailed information to the reproductive biology field about the diversified transcriptome profile in POECs may create new future possibilities in infertility treatments, including assisted reproductive technique (ART) programmes, and may be a valuable tool to investigate the potential role of oviduct cells in post-ovulation events.
- MeSH
- buněčné kultury metody MeSH
- epitelové buňky * metabolismus cytologie MeSH
- kultivované buňky MeSH
- prasata MeSH
- regulace genové exprese MeSH
- stanovení celkové genové exprese MeSH
- transkriptom * MeSH
- vejcovody u zvířat metabolismus cytologie MeSH
- vejcovody metabolismus cytologie MeSH
- zvířata MeSH
- Check Tag
- ženské pohlaví MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
Lipases are industrially important enzymes having vast applications in various fields. Cloning and expression of lipase enzyme-encoding genes in suitable host lead to their widespread use in different fields. The present study represents the first attempt towards the expression of the synthetic lipase gene in Pseudomonas aeruginosa. An alkalophilic lipase gene (GenBank accession number: NP_388152) from Bacillus subtilis was synthetically designed and introduced in the pJN105 vector and subsequently cloned in Pseudomonas aeruginosa SDK-6. Agarose gel electrophoresis confirmed the transformation of SDK-6, exhibiting a band difference of ~ 700 bp between native and recombinant pJN105. Further amplification of cloned lipase gene was confirmed using PCR amplification with Lip 1 and Lip 2 primers respectively, followed by restriction analysis. Approximately 15-fold increase in lipase production was observed in recombinant Pseudomonas as compared to the native strain. One factor at a time (OFAT) analysis revealed L-arabinose, inoculum size (0.5%; v/v), and agitation (120 rpm) as significant factors affecting the over-expression of lipase enzyme. Optimization of enzyme induction conditions by central composite design (CCD) led to 1.60-fold increase in the production of lipase at 0.65% (w/v) inducer concentration, OD600-1.075 before induction and 35 °C post induction temperature with overall lipase production of 50.50 IU/mL. Statistical validation of observed value via ANOVA showed an F-value of 138.70 at p < 0.01 with R2 of 0.9921.
- MeSH
- arabinosa metabolismus MeSH
- Bacillus subtilis * genetika enzymologie MeSH
- bakteriální proteiny genetika metabolismus MeSH
- exprese genu MeSH
- genetické vektory genetika MeSH
- klonování DNA MeSH
- lipasa * genetika metabolismus MeSH
- Pseudomonas aeruginosa * genetika enzymologie MeSH
- rekombinantní proteiny * genetika metabolismus MeSH
- Publikační typ
- časopisecké články MeSH
Background and Objectives Two-dimensional SDS PAGE (sodium dodecyl sulfate polyacrylamidegel electrophoresis) coupled with mass spectrometry is still a mainstream approach to analysing multiple protein expression levels. The requirement for some sophisticated devices and the lack of quantitative measurements for low-abundant proteins (e.g. cytokines) greatly limit its broad application. Cytokines present in the pg/ml levels in non-stimulated biological samples are traditionally detected by ELISA. We used a cytokine antibody array, a highly sensitive protein chip, for simultaneous detection of multiple cytokine expression levels in rat sarcoma lysates and serum samples . Material and methods We present here an optimized protocol for preparation and handling of tumour tissue lysates in protein chip detection. The sarcoma samples were processed at low temperatures to prevent cytokine degradation. Tumour cryosections (8?10 mm) were used for extraction of cytokines. The addition of NaN3 destroyed a high endogenous peroxidase activity, which may interfere with protein chip assay and decrease the signal/noise ratio. The data for the protein matrix effect from sandwich ELISA can also affect the protein chip detection. The optimal dilution of samples must be found to prevent pitfalls due to the non-optimal signal-to-noise ratio. This also enables recovery of low amounts of cytokines from difficult samples. Results We report optimized procedures for extraction, sample handling, inhibition of endogenous peroxidase activity and prevention of the protein matrix effect in serum and tumour lysates by detection of cytokine expression using the cytokine antibody array protein chip.
Expression and purification of whole and nuclear localization signal (NLS) deleted ORF2 capsid protein of porcine circovirus 2 (PCV2) is demonstrated in the present study. Gene coding for both protein forms were cloned into pDest17 vector and expressed in BL21 (DE3)AI cells and in BL21-CodonPlus (DE3)-RIPL E. coli cells. The later cells were used to overcome difficulties with the heterologous expression of viral proteins in prokaryotic systems. Whole 30 kDa recombinant ORF2 protein was successfully expressed in BL21-CodonPlus (DE3)-RIPL cells only, 3 mg of pure protein was consistently obtained per liter of bacterial culture. NLS deleted ORF2 protein was expressed in both cell types. Resulting proteins reacted with PCV2 positive swine serum in immunofluorescent test and immunoblot.
Adaptation to chronic hypoxia renders the heart more tolerant to ischemia/reperfusion injury. To evaluate changes in gene expression after adaptation to chronic hypoxia by RT-qPCR, it is essential to select suitable reference genes. In a chronically hypoxic rat model, no specific reference genes have been identified in the myocardium. This study aimed to select the best reference genes in the left (LV) and right (RV) ventricles of chronically hypoxic and normoxic rats. Sprague-Dawley rats were adapted to continuous normobaric hypoxia (CNH; 12% O2 or 10% O2) for 3 weeks. The expression levels of candidate genes were assessed by RT-qPCR. The stability of genes was evaluated by NormFinder, geNorm and BestKeeper algorithms. The best five reference genes in the LV were Top1, Nupl2, Rplp1, Ywhaz, Hprt1 for the milder CNH and Top1, Ywhaz, Sdha, Nupl2, Tomm22 for the stronger CNH. In the RV, the top five genes were Hprt1, Nupl2, Gapdh, Top1, Rplp1 for the milder CNH and Tomm22, Gapdh, Hprt1, Nupl2, Top1 for the stronger CNH. This study provides validation of reference genes in LV and RV of CNH rats and shows that suitable reference genes differ in the two ventricles and depend on experimental protocol.
- MeSH
- chronická nemoc MeSH
- hypoxie genetika MeSH
- myokard metabolismus patologie MeSH
- potkani Sprague-Dawley MeSH
- referenční standardy MeSH
- regulace genové exprese * MeSH
- srdeční komory metabolismus patologie MeSH
- zvířata MeSH
- Check Tag
- mužské pohlaví MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
Detailed understanding of the mechanisms employed in transfer of drugs across the placenta is essential for optimization of pharmacotherapy during pregnancy. Disclosure of drug efflux transporters as an "active component" of the placental barrier has brought new important insights into the field of transplacental pharmacokinetics. P-glycoprotein (P-gp, MDR1) is the first discovered and so far the best characterized of drug efflux transporters, whose role in the regulation of drug disposition to the fetus has been extensively studied. Expression of P-gp in the placental trophoblast layer was confirmed at the mRNA and protein levels in all phases of pregnancy, and several in vitro and in vivo studies demonstrated functional activity of the transporter in materno-fetal drug transport. P-gp is able to actively pump drugs and other xenobiotics from trophoblast cells back to the maternal circulation, providing thus protection to the fetus. This review summarizes the current knowledge on the expression, localization and function of P-gp in the placenta. In addition, we include the latest data concerning transcriptional regulation of placental P-gp expression and polymorphisms of the MDR1 gene. Clinical significance of placental P-gp and its future perspectives for pharmacotherapy during pregnancy are also discussed.
- MeSH
- financování organizované MeSH
- geny MDR MeSH
- jednonukleotidový polymorfismus MeSH
- lidé MeSH
- maternofetální výměna látek MeSH
- messenger RNA metabolismus MeSH
- P-glykoprotein genetika metabolismus MeSH
- placenta cytologie metabolismus MeSH
- těhotenství MeSH
- vývojová regulace genové exprese MeSH
- zvířata MeSH
- Check Tag
- lidé MeSH
- těhotenství MeSH
- ženské pohlaví MeSH
- zvířata MeSH
- Publikační typ
- přehledy MeSH
Although the mechanism of action of leukemic oncogene Wilms' tumor gene 1 (WT1) remains unclear, WT1 has already been used in monitoring of patients with acute myeloid leukemia (AML) and it is being tested for immunotherapy. More detailed understanding of the role of WT1 in leukemia may improve its utilization. At least 36 isoforms may be produced. Four major variants denoted as -5/-KTS, -5/+KTS, +5/-KTS and +5/+KTS are produced by combining splicing of exon 5 and KTS sequence. In this study, we report applicability of newly developed real-time RT PCRs enabling for the first time full quantification of the four major WT1 splicing variants. Following careful optimization and testing of quantification reliability of four assays, we analyzed 34 samples of patients with AML and 12 samples of patients with chronic myeloid leukemia (CML) at the time of diagnosis. Analyses of five more CML patients provided insight into WT1 variants expression kinetics. We found predominance of +5/+KTS in both diagnoses. Comparison of WT1 variant expression in AML and CML patients' groups differing in response to therapy suggested possible importance of particular WT1 variant levels as markers of further disease course.
- MeSH
- akutní myeloidní leukemie diagnóza metabolismus terapie MeSH
- alternativní sestřih MeSH
- buňky K562 MeSH
- chronická myeloidní leukemie diagnóza metabolismus terapie MeSH
- dospělí MeSH
- lidé středního věku MeSH
- lidé MeSH
- mladiství MeSH
- polymerázová řetězová reakce s reverzní transkripcí MeSH
- proteiny WT1 biosyntéza MeSH
- regulace genové exprese u leukemie MeSH
- Check Tag
- dospělí MeSH
- lidé středního věku MeSH
- lidé MeSH
- mladiství MeSH
- mužské pohlaví MeSH
- ženské pohlaví MeSH
- Publikační typ
- časopisecké články MeSH
- klinické zkoušky MeSH
- práce podpořená grantem MeSH
- srovnávací studie MeSH
Formalin-fixed, paraffin-embedded (FFPE) tissue is the most common tissue specimen available after microscopic examination. Molecular methods, such as polymerase chain reaction (PCR) and gene expression examination, serve as a source of diagnostic and prognostic information but require high-quality RNA. However, the increasing application of RNA extracted from FFPE tissue frequently results in very small and degraded quantities of nucleic acid. This study targets gene expression analysis from FFPE specimens using real-time quantitative PCR. The whole protocol consists of several steps, that is, RNA extraction and its quality control, reverse transcription, and fluorescence detection during real-time quantitative PCR. We compared several methods in each step, chose the most effective, and with that combination we successfully examined 95% (62 from 65) FFPE samples for our genes of interest. We reached the best results with RNA isolation by using a commercial kit, carefully interpreted UV spectrophotometric values, and meticulously chose reverse transcriptase and TaqMan fluorescence detection. Our protocol improves the utility of FFPE tissue for molecular profiling studies.
- MeSH
- fixativa farmakologie MeSH
- formaldehyd farmakologie MeSH
- lidé MeSH
- odběr biologického vzorku metody MeSH
- patologie metody MeSH
- polymerázová řetězová reakce metody MeSH
- RNA genetika izolace a purifikace MeSH
- stanovení celkové genové exprese metody MeSH
- uchovávání tkání MeSH
- zalévání tkání do parafínu MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- hodnotící studie MeSH
- práce podpořená grantem MeSH
