scholarly journals Oxygen-dependent modulation of erythropoietin mRNA levels in isolated rat kidneys studied by RNase protection.

1990 ◽  
Vol 172 (2) ◽  
pp. 657-660 ◽  
Author(s):  
P J Ratcliffe ◽  
R W Jones ◽  
R E Phillips ◽  
L G Nicholls ◽  
J I Bell
Keyword(s):  
High Amplitude ◽  
Mrna Levels ◽  
Chain Reaction ◽  
Rnase Protection ◽  
Perfusate Flow ◽  
Polymerase Chain ◽  
Perfusate Flow Rate ◽  
Isolated Kidneys ◽  
Rat Kidneys

Using oligonucleotide primers complementary to conserved regions in the mouse erythropoietin (Epo) gene, a portion of the rat Epo gene was amplified by the polymerase chain reaction to produce a probe suitable for assay of rat Epo mRNA by RNAse protection. The assay, which has sufficient sensitivity to measure to Epo mRNA in unstimulated rat kidneys, was used to demonstrate high amplitude in vitro modulation of Epo mRNA levels in response to changes in perfusate flow rate and oxygen tension in isolated kidneys, thus providing clear evidence that all the necessary events linking changes in oxygen delivery to the modulation of Epo mRNA levels can occur intrarenally.

scholarly journals In vivo and in vitro regulation of erythropoietin mRNA: measurement by competitive polymerase chain reaction

Blood ◽  
1993 ◽  
Vol 81 (3) ◽  
pp. 617-623 ◽  
Author(s):  
J Fandrey ◽  
HF Bunn
Keyword(s):  
Polymerase Chain Reaction ◽  
Hepatoma Cell ◽  
Hepatoma Cell Line ◽  
Mrna Levels ◽  
Chain Reaction ◽  
Human Hepatoma Cell ◽  
Competitive Polymerase Chain Reaction ◽  
Polymerase Chain

Abstract The regulation of erythropoietin (Epo) production was investigated by competitive polymerase chain reaction, a highly sensitive and accurate means of measuring Epo mRNA levels. Co-amplification of the test sample with added mutant Epo cDNA template corrects for variability in the efficiency of amplification. Epo mRNA levels were determined in tissues of normal rats and in animals with varying degrees of anemia. Reduction of the hematocrit level from 0.40 to 0.15–0.20 resulted in a 300-fold increase in kidney Epo mRNA, which comprised 80% of the total Epo mRNA versus 20% from the liver. In contrast, very low levels detected in lung and spleen were not significantly increased by anemia. The human hepatoma cell line, Hep3B, secretes high levels of Epo in response to hypoxia. This regulation is, to a large extent, transcriptional. When Hep3B cells were incubated in the presence of decreasing O2 tension from 160 to 7 mm Hg, there was a monotonic increase in Epo mRNA to 50 to 100 times the normoxic level. Hyperoxia did not suppress basal expression. When cells were incubated at a PO2 of 7 mm Hg, induction of Epo mRNA was first noted at 30 minutes and was maximal at 5 to 6 hours. After Epo mRNA was boosted by a 4-hour hypoxic incubation, cells were then exposed to normoxia, which shut off further transcription of the Epo gene. The decay of Epo mRNA levels closely followed first order kinetics with a half-life of 2 hours, an effective measurement of message stability.

scholarly journals In vivo and in vitro regulation of erythropoietin mRNA: measurement by competitive polymerase chain reaction

Blood ◽  
1993 ◽  
Vol 81 (3) ◽  
pp. 617-623 ◽  
Author(s):  
J Fandrey ◽  
HF Bunn
Keyword(s):  
Polymerase Chain Reaction ◽  
Hepatoma Cell ◽  
Hepatoma Cell Line ◽  
Mrna Levels ◽  
Chain Reaction ◽  
Human Hepatoma Cell ◽  
Competitive Polymerase Chain Reaction ◽  
Polymerase Chain

The regulation of erythropoietin (Epo) production was investigated by competitive polymerase chain reaction, a highly sensitive and accurate means of measuring Epo mRNA levels. Co-amplification of the test sample with added mutant Epo cDNA template corrects for variability in the efficiency of amplification. Epo mRNA levels were determined in tissues of normal rats and in animals with varying degrees of anemia. Reduction of the hematocrit level from 0.40 to 0.15–0.20 resulted in a 300-fold increase in kidney Epo mRNA, which comprised 80% of the total Epo mRNA versus 20% from the liver. In contrast, very low levels detected in lung and spleen were not significantly increased by anemia. The human hepatoma cell line, Hep3B, secretes high levels of Epo in response to hypoxia. This regulation is, to a large extent, transcriptional. When Hep3B cells were incubated in the presence of decreasing O2 tension from 160 to 7 mm Hg, there was a monotonic increase in Epo mRNA to 50 to 100 times the normoxic level. Hyperoxia did not suppress basal expression. When cells were incubated at a PO2 of 7 mm Hg, induction of Epo mRNA was first noted at 30 minutes and was maximal at 5 to 6 hours. After Epo mRNA was boosted by a 4-hour hypoxic incubation, cells were then exposed to normoxia, which shut off further transcription of the Epo gene. The decay of Epo mRNA levels closely followed first order kinetics with a half-life of 2 hours, an effective measurement of message stability.

Application of the polymerase chain reaction to the RNase protection assay for 5-HT1B receptor mRNA levels measurement in rat brain tissues

1999 ◽  
Vol 4 (3) ◽  
pp. 322-328 ◽  
Author(s):  
Catherine Drogou ◽  
Mounir Chennaoui ◽  
Lionel Forestier ◽  
Brigitte Grimaldi ◽  
Gilles Fillion ◽  
...  
Keyword(s):  
Polymerase Chain Reaction ◽  
Rat Brain ◽  
Rnase Protection Assay ◽  
Mrna Levels ◽  
Protection Assay ◽  
Chain Reaction ◽  
Rnase Protection ◽  
Receptor Mrna ◽  
Polymerase Chain ◽  
Brain Tissues

scholarly journals Derivation, characterization, and in vitro cell regeneration of canine white adipose tissue-derived mesenchymal stem cells obtained from a mesenteric region

2021 ◽  
Vol 82 (1) ◽  
Author(s):  
Anirban Mandal ◽  
Ajeet Kumar Jha ◽  
Dew Biswas ◽  
Shyamal Kanti Guha
Keyword(s):  
Stem Cells ◽  
Mesenchymal Stem Cells ◽  
Adipose Tissue ◽  
White Adipose Tissue ◽  
Stromal Vascular Fraction ◽  
Cell Regeneration ◽  
Wound Healing Assay ◽  
Chain Reaction ◽  
Polymerase Chain

Abstract Background The study was conducted to assess the characterization, differentiation, and in vitro cell regeneration potential of canine mesenteric white adipose tissue-derived mesenchymal stem cells (AD-MSCs). The tissue was harvested through surgical incision and digested with collagenase to obtain a stromal vascular fraction. Mesenchymal stem cells isolated from the stromal vascular fraction were characterized through flow cytometry and reverse transcription-polymerase chain reaction. Assessment of cell viability, in vitro cell regeneration, and cell senescence were carried out through MTT assay, wound healing assay, and β-galactosidase assay, respectively. To ascertain the trilineage differentiation potential, MSCs were stained with alizarin red for osteocytes, alcian blue for chondrocytes, and oil o red for adipocytes. In addition, differentiated cells were characterized through a reverse transcription-polymerase chain reaction. Results We observed the elongated, spindle-shaped, and fibroblast-like appearance of cells after 72 h of initial culture. Flow cytometry results showed positive expression for CD44, CD90, and negative expression for CD45 surface markers. Population doubling time was found 18–24 h for up to the fourth passage and 30±0.5 h for the fifth passage. A wound-healing assay was used to determine cell migration rate which was found 136.9 ± 4.7 μm/h. We observed long-term in vitro cell proliferation resulted in MSC senescence. Furthermore, we also found that the isolated cells were capable of differentiating into osteogenic, chondrogenic, and adipogenic lineages. Conclusions Mesenteric white adipose tissue was found to be a potential source for isolation, characterization, and differentiation of MSCs. This study might be helpful for resolving the problems regarding the paucity of information concerning the basic biology of stem cells. The large-scale use of AD-MSCs might be a remedial measure in regenerative medicine.

scholarly journals The Yeast Signal Sequence Trap Identifies Secreted Proteins of the Hemibiotrophic Corn Pathogen Colletotrichum graminicola

2008 ◽  
Vol 21 (10) ◽  
pp. 1325-1336 ◽  
Author(s):  
Jorrit-Jan Krijger ◽  
Ralf Horbach ◽  
Michael Behr ◽  
Patrick Schweizer ◽  
Holger B. Deising ◽  
...  
Keyword(s):  
Cell Walls ◽  
Leaf Extract ◽  
Signal Sequence ◽  
Stem Rot ◽  
Colletotrichum Graminicola ◽  
In Planta ◽  
Chain Reaction ◽  
Genes Encoding ◽  
Polymerase Chain

The hemibiotroph Colletotrichum graminicola is the causal agent of stem rot and leaf anthracnose on Zea mays. Following penetration of epidermal cells, the fungus enters a short biotrophic phase, followed by a destructive necrotrophic phase of pathogenesis. During both phases, secreted fungal proteins are supposed to determine progress and success of the infection. To identify genes encoding such proteins, we constructed a yeast signal sequence trap (YSST) cDNA-library from RNA extracted from mycelium grown in vitro on corn cell walls and leaf extract. Of the 103 identified unigenes, 50 showed significant similarities to genes with a reported function, 25 sequences were similar to genes without a known function, and 28 sequences showed no similarity to entries in the databases. Macroarray hybridization and quantitative reverse-transcriptase polymerase chain reaction confirmed that most genes identified by the YSST screen are expressed in planta. Other than some genes that were constantly expressed, a larger set showed peaks of transcript abundances at specific phases of pathogenesis. Another set exhibited biphasic expression with peaks at the biotrophic and necrotrophic phase. Transcript analyses of in vitro-grown cultures revealed that several of the genes identified by the YSST screen were induced by the addition of corn leaf components, indicating that host-derived factors may have mimicked the host milieu.

Εξέλιξη και βελτίωση της τεχνικής βιοψίας βλαστοκύστεων για μοριακές αναλύσεις και εκτίμηση της επιτυχίας εμφύτευσης και εγκυμοσύνης κατά την εξωσωματική γονιμοποίηση σε σχέση με το μοριακό υπόστρωμα

2013 ◽  
Author(s):  
Γεωργία Κόκκαλη
Keyword(s):  
Gene Expression ◽  
Polymerase Chain Reaction ◽  
Clinical Practice ◽  
Expression Profiles ◽  
Chain Reaction ◽  
Trophectoderm Cells ◽  
Monogenic Diseases ◽  
Blastocyst Biopsy ◽  
Polymerase Chain

IntroductionOne of the most difficult aspects in assisted reproductive technology (ART) is the selection of asuitable embryo for transfer to the patient’s uterus, in order to achieve implantation anddevelopment to term. This study was based on the hypothesis that preimplantation embryosmay have different gene expression profiles that characterize their ability to implant in theuterus and develop to a healthy baby at term.The main aim of this study was to investigate molecular markers associated with developmentalcompetence and successful implantation in ART. The primary aim of the study was to developand optimize a blastocyst biopsy method, suitable for application in clinical practice. Thesecondary aim of the study was to investigate the gene expression of beta Human ChorionicGonadotropin (CGβ) in blastocysts and correlate it with their morphology. Previously to thecurrent study, blastocyst biopsy was not implemented in clinical practice and no prior researchon the existence, quantification and standardization of transcripts of CGβ has been performedin blastocysts.MethodologyThe methodology for trophectoderm cell biopsy from blastocysts was developed and optimizedprimary to be a safe technique for the embryo and secondary to ensure biopsy of a sufficientnumber of cells, in order to allow the application of multiple molecular analyses. The blastocystbiopsy method involved three steps: A., opening of a hole in the zona pellucida using lowfrequency laser, B., blastocyst culture to allow trophectoderm cells to herniate from the holeand C., trophectoderm cell dissection of the blastocyst mass by laser ablation.The methodology for the investigation of CGβ gene expression in blastocysts, included RNAisolation, cDNA synthesis, amplification and quantification of CGβ transcripts. Because CGβ isencoded by a cluster of homologous genes (CGβ1, CGβ2, CGβ3, CGβ5, CGβ7, CGβ8),methodology was designed considering the homology between them into groups (A: CGβ1,CGβ2 and B: CGβ3, CGβ5, CGβ7, CGβ8). For group A, real time polymerase chain reaction (RealTime PCR, RT-PCR) was applied and then transcripts were identified using restriction enzymedigestion. For group B, nested polymerase chain reaction (Nested-PCR) was used incombination with polymerase chain reaction temperature decreasing hybridization (Touch-downPCR). Following amplification, the products were sequenced (DNA sequencing) for theiridentification.ResultsThe biopsy technique did not appear to impact on the blastocyst’s ability to reform a blastocoelecavity and continue to grow and hatch from the zona pellucida, as it was shown followingfurther in vitro culture. No blastocyst showed signs of morphological damage at the lightmicroscopic level. Blastocyst biopsy was applied in clinical practice in two steps: A., 49 couples undergoing IVF had a biopsy in 153 blastocysts. The implantation rate per blastocysttransferred was 34.3% and lead to 23 full-term pregnancies (46.9%) with 37 babies born. B.,24 couples undergoing IVF for PGD of monogenic diseases had biopsy in 144 blastocysts. Thediagnosis success rate was 93%, the implantation rate per blastocyst transferred was 40% andlead to 11 full-term pregnancies (50%) with 15 term newborns. Then, a randomized pilot studywas conducted with the aim to evaluate and compare the diagnosis and implantation successrates between patients undergoing blastomere biopsy and blastocyst transfer and those havingtrophectoderm biopsy and blastocyst transfer for the diagnosis of monogenic diseases. Theresults showed that the diagnosis success rate was superior in the blastocyst biopsy group,while implantation and pregnancy rates were not statistically significant between the twogroups.For the study of CGβ expression profiles 45 blastocysts were donated to research, of which 39generated trophectoderm cells cDNA libraries. RT-PCR revealed the presence of CGB3, CGB5,CGB7, CGB8 transcripts in 5 blastocysts. The transcripts CGB5, CGB7, CGB8 were expressed inone hatched and one hatching blastocysts (fair morphology on day 7 post insemination) and thetranscript CGβ3 was expressed in three hatched blastocysts (excellent morphology on day 5/6post insemination). The transcript CGβ1 was identified in one only blastocyst. Four blastocystswere biopsied in order to investigate whether CGβ expression can be detected at the minimallevel of few trophectoderm cells. No transcript was found in trophectoderm cell samples orbiopsied blastocyst proper.DiscussionIn recent years, many new technologies have been introduced in clinical practice of ART.Blastocyst biopsy since its first announcement in 2005, until today, has been adopted andintegrated into the application of preimplantation genetic diagnosis (Kokkali et al., 2005). Asblastocyst biopsy has the advantage of providing adequate number of cells for multipleanalyses, it has been lately used for the PGD for monogenic diseases in combination withhistocompatibility screening (HLA matching) or PGD for monogenic diseases screening forstructural or numerical chromosomal abnormalities. Besides its clinical application, blastocystbiopsy offers great opportunities for research, such as the study for the expression ofpreimplantation genetic profiles for the identification of the single most viable blastocyst amongthe cohort developing in vitro that will enable single blastocyst transfers without a concomitantreduction in pregnancy rates.In this study, we investigated whether the β HCG may be used as a predictive marker ofdevelopmental competence for human embryos. This study showed that CGβ gene expressionwas diverse and heterogeneous between blastocysts. Further studies need to be accomplishedto investigate this further.ConclusionsBlastocyst biopsy was developed and optimized to serve as powerful tool for diagnostics ofhuman diseases or to identify diagnostic markers of competence to develop to term for humanembryos.

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