scholarly journals Sensitivity of bovine blastocyst gene expression patterns to culture environments assessed by differential display RT-PCR

Reproduction ◽  
2001 ◽  
Vol 122 (5) ◽  
pp. 687-693
Author(s):  
D. Natale
Keyword(s):  
Gene Expression ◽  
Differential Display ◽  
Expression Patterns ◽  
Gene Expression Patterns ◽  
Rt Pcr ◽  
Bovine Blastocyst

scholarly journals Sensitivity of bovine blastocyst gene expression patterns to culture environments assessed by differential display RT-PCR

Reproduction ◽  
2001 ◽  
pp. 687-693 ◽  
Author(s):  
DR Natale ◽  
PA De Sousa ◽  
ME Westhusin ◽  
AJ Watson
Keyword(s):  
Gene Expression ◽  
Differential Display ◽  
Culture Media ◽  
Expression Patterns ◽  
Molecular Techniques ◽  
Preimplantation Embryos ◽  
Gene Expression Patterns ◽  
Bovine Embryos ◽  
Rt Pcr ◽  
Defined Culture

The use of culture media to support the development of preimplantation embryos to the blastocyst stage is often associated with detrimental effects on normal development. These effects have been uncovered largely by investigating the phenotypic abnormalities displayed by fetuses and newborns derived from cultured preimplantation embryos. Research to understand the impact of culture on the embryonic developmental programme has focused on embryo metabolism, gene expression and genomic imprinting. We have used differential display RT-PCR to examine culture influences on global transcript pools in bovine embryos. Others have examined culture influences on candidate "marker genes" in cultured murine, ovine and bovine embryos. These studies have demonstrated that culture conditions influence the amount of marker gene transcripts and downregulate or induce the expression of novel genes during early development. Optimized defined culture media maintain embryonic gene expression patterns closely resembling those displayed by embryos derived in vivo. Preimplantation mammalian embryos display an impressive capacity to respond to the pressures that suboptimal culture environments place upon them. However, this plasticity operates within a defined range of tolerances. Continued research using molecular techniques will lead to increased understanding of developmental mechanisms causing culture-related phenotypic abnormalities in post-implantation embryos.

Differential Expression of Genes Associated with Toll-Like Receptor-4 (TLR4) Signal Transduction Pathway in Lipopolysaccharide (LPS)-Activated Cord Blood (CB) vs. Adult Peripheral Blood (APB) Monocyte (Mo)-Derived Dendritic Cells (DC).

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 3444-3444
Author(s):  
Hong Jiang ◽  
Mary Brigid Bradley ◽  
Carmella van de Ven ◽  
Prakash Satwani ◽  
Laxmi Baxi ◽  
...  
Keyword(s):  
Gene Expression ◽  
Signal Transduction ◽  
Protein Expression ◽  
Expression Patterns ◽  
Signal Transduction Pathway ◽  
Gene Expression Patterns ◽  
Transduction Pathway ◽  
Rt Pcr ◽  
Cell Surface Molecule ◽  
Expression Of Genes

Abstract LPS activates immature DC via TLR4 and induces maturation of DC for initiating antigen presenting activity (Medzhitov; Nat Rev Immunol 2001). We have previously demonstrated decreased gene expression and protein production of IL-12, IL-15, IL-18 in activated CB MNC and decreased DC MLR (Lee/Cairo, Blood 1996; Qian/Cairo, Blood 1997; Wu/Cairo, Blood 100:3668 p51b 2002). Recently, we have identified differential gene expression patterns including differential immunoregulatory and chemokine genes in LPS-CB vs APB Mo by microarray (Jiang/Cairo, J. Immunol 2004). Since the myeloid lineage DC is derived from Mo, we sought to determine in LPS activated CB vs. APB DC, differential expressed genes that associate with TLR4-mediated signaling pathway. Briefly, Mo were purified from fresh CB or APB and cultured for 7 days with GM-CSF & IL-4 [immature DC (iDC)] and LPS [mature DC (mDC)]. Aliquots from iDC and mDC were analyzed for DC immunophenotype, morphology and DC allogeneic antigen activity. mRNA was isolated, reverse transcripted to cDNA, labeled & hybridized to oligonucleotides (Affymetrix, U133A). Data was analyzed by MAS 5.0 (Affymetrix) and GeneSpring 5.0 software (Silicon Genetics). Several genes were analyzed by RT-PCR (One-Step SuperScript, Invitrogen) and protein expression was analyzed by Western Blot (Bio-Rad). Inverted microscopy demonstrated DC mature morphology at day 8 and flow cytometry demonstrated decreased CD14 and increased CD83 expression in CB & APB mDC. We also demonstrated significant increase in the allogeneic stimulatory effects on CD4+ T cells in APB vs. CB mDC. The microarray analysis demonstrated a significant decreased gene expression of TLR4 [3 fold (F)] and CD14 (2.1 F) (p<0.05) in CB vs APB-DC. We further identified LPS significantly induced increased expression of TLR4 downstream signaling molecular genes such as MAPKKK, NF-kB and TANK in APB compared to CB mDC (3–8 F) (p<0.05). There were also significant amplifications of a variety of other gene categories in LPS activated APB vs CB mDC (p<0.05) including cell surface molecule CD80 (3.7F) and IL-2Ra (5.3 F), cytokine IL-23 (3.5F) & IL-12 (13 F), signal transduction STAT1 (3.4F) & IRF-7 (7.7 F), and immunoregulatory TNFSF10 (12F) & ISG20 (39F). Gene expression of NF-kB1, TRAF1 & IRF-7 by RT-PCR demonstrated an increased expression in LPS-APB vs CB mDC and were compatible with microarray. Moreover, Western analysis of IRF-7 demonstrated increased protein expression in LPS-APB vs CB mDC. In summary, we have identified decreased gene expression patterns in LPS-CB vs APB DC, especially those in the TLR4 signal transduction pathway (MAP3K, TRAF, TANK & NF-kB), and suggest these differentially expressed genes may enhance the activation of TLR4 pathway in LPS-APB vs CB DC, resulting in differential regulation of CB vs APB DC antigen presentation capacities. Furthermore, these decreased expressed genes in other molecular categories (e.g.IL-23, IFNg, IL6, CD80, STAT1, IRF-7, SOCS3) in LPS-CB vs APB DC may be partially responsible for differential innate and adaptive immune function of CB vs APB. Moreover, the differential regulated expression of genes may in part help to explain reduced incidence of severe aGVHD, delay in immune reconstitution and/or increased infectious mortality following HLA disparate UCBT.

Sexually dimorphic gene expression patterns during gonadal differentiation in olive flounder, Paralichthys olivaceus

2015 ◽  
Vol 65 (3-4) ◽  
pp. 193-207 ◽  
Author(s):  
Aiyun Wen ◽  
Feng You ◽  
Peng Sun ◽  
Jun Li ◽  
Dongdong Xu ◽  
...  
Keyword(s):  
Gene Expression ◽  
Transcription Factor ◽  
Synergistic Effects ◽  
Paralichthys Olivaceus ◽  
Expression Patterns ◽  
Olive Flounder ◽  
Gene Expression Patterns ◽  
Gonadal Differentiation ◽  
Rt Pcr ◽  
Related Transcription Factor

The present study aims to elucidate the different expression patterns and possible roles of Doublesex and Mab-3-related transcription factor 1 (dmrt1), dmrt4, SRY-related transcription factor 9 (sox9) and cytochrome P450 aromatase 19a (cyp19a) during gonadal differentiation in olive flounder, Paralichthys olivaceus. We first analyzed the gene expression patterns in tissues using RT-PCR, which indicated dmrt1, sox9 and cyp19a were sex-related genes with sexual dimorphic expression. The quantitative expression changes of these three genes together with dmrt4 during gonadal differentiation were further examined using real-time RT-PCR. The results showed that dmrt1 was scarcely expressed in the primitive gonad and during following periods of gonadal differentiation. Its expression increased rapidly in the differentiating testis. Dmrt4 was strongly expressed in primitive gonads and much less expressed during following periods of gonadal differentiation. Its expression became strong in differentiating testes. While sox9 was highly expressed in the primitive gonad, it was expressed with fluctuations during following periods of gonadal differentiation. Cyp19a started expressing in primitive gonads, and its expression quantity fluctuated during latter periods of gonadal differentiation, but was strongly expressed in the early stage of differentiating ovaries. Results of in situ hybridization showed that dmrt4 and sox9 transcripts were both mainly localized in spermatocytes and our results suggested these four sex-related genes might be involved in gonadal differentiation through their synergistic effects in flounder.

scholarly journals 363. Characteristics of Envelope and Nuclear Gene Expression Patterns in Asymptomatic SARS-CoV-2 Patients: A Single Center Retrospective Observational Study

2021 ◽  
Vol 8 (Supplement_1) ◽  
pp. S284-S284
Author(s):  
Keiko Mizuno ◽  
Takayuki Komatu ◽  
Daisuke Usuda ◽  
Shiori Hocchi ◽  
Karin Ashizawa ◽  
...  
Keyword(s):  
Gene Expression ◽  
Statistical Significance ◽  
Expression Patterns ◽  
Gene Expression Pattern ◽  
Nuclear Gene ◽  
Categorical Variables ◽  
N Gene ◽  
Gene Expression Patterns ◽  
Rt Pcr ◽  
Asymptomatic Patients

Abstract Background Reverse transcription-polymerase chain reaction (RT-PCR) is used for the diagnosis of COVID-19, caused by SARS-CoV-2. RT-PCR is a method that detects the virus by amplifying two regions of the target viral genome, namely the nuclear (N) and envelope (E) encoding sequences. However, no reports have shown a relationship between the symptoms and the gene expression patterns, especially in asymptomatic patients. Herein, we validated the characteristics of E and N gene expression patterns using RT-PCR on samples obtained from asymptomatic COVID-19-positive patients. Methods In this retrospective cohort study, conducted at Juntendo University Nerima Hospital, Tokyo, Japan, SARS-Cov-2 RT-PCR positive patients whose specimens had been obtained and analyzed by our laboratory technicians from September 1, 2020 to December 31, 2020 were enrolled. For RT-PCR, the LightMix Modular SARS-CoV-2 reagent (TIB MOLBIOL company) was used. After excluding patients who had symptoms, background, demographic, laboratory, and gene expression pattern data were collected from RT-PCR-positive asymptomatic patients. We also investigated patients who met the release criteria of the Center for Disease Control and prevention. Continuous and categorical variables were analyzed, with p&lt; 0.05 set as statistical significance using the student-t test, chi-square test, or Fisher’s exact test, respectively. Results Of 92 RT-PCR-positive asymptomatic patients, 57 comprised the expression E only group (Group E) and 35 comprised the E+N group (Group E+N). Significantly more patients in Group E met the release criteria compared to those in Group E+N [41 (71%) vs 10 (28%), p&lt; 0.001]. Among patients who met the release criteria, those in Group E+N had significantly more immunosuppression [7 (70%) vs 8 (30%), p=0.004]. Moreover, among the patients who underwent RT-PCR screening, no patients in Group E developed symptoms [0 vs 6 (42%), p=0.02]. Conclusion The results of this study suggest that RT-PCR-positive asymptomatic patients can be divided into three patterns: pre-symptomatic, gene E+N-positive patients; post-symptomatic covid-19-recovered patients, regardless of gene E and N expression patterns; and false positive, gene E-positive patients. Disclosures All Authors: No reported disclosures

RT-PCR detection of Candida albicans ALS gene expression in the reconstituted human epithelium (RHE) model of oral candidiasis and in model biofilms

Microbiology ◽  
2004 ◽  
Vol 150 (2) ◽  
pp. 267-275 ◽  
Author(s):  
Clayton B. Green ◽  
Georgina Cheng ◽  
Jyotsna Chandra ◽  
Pranab Mukherjee ◽  
Mahmoud A. Ghannoum ◽  
...  
Keyword(s):  
Gene Expression ◽  
Candida Albicans ◽  
Oral Candidiasis ◽  
Expression Patterns ◽  
Pcr Analysis ◽  
Gene Expression Patterns ◽  
Rt Pcr ◽  
Pcr Assay ◽  
Strain Sc5314 ◽  
Biofilm Development

An RT-PCR assay was developed to analyse expression patterns of genes in the Candida albicans ALS (agglutinin-like sequence) family. Inoculation of a reconstituted human buccal epithelium (RHE) model of mucocutaneous candidiasis with strain SC5314 showed destruction of the epithelial layer by C. albicans and also formation of an upper fungal layer that had characteristics similar to a biofilm. RT-PCR analysis of total RNA samples extracted from C. albicans-inoculated buccal RHE showed that ALS1, ALS2, ALS3, ALS4, ALS5 and ALS9 were consistently detected over time as destruction of the RHE progressed. Detection of transcripts from ALS7, and particularly from ALS6, was more sporadic, but not associated with a strictly temporal pattern. The expression pattern of ALS genes in C. albicans cultures used to inoculate the RHE was similar to that observed in the RHE model, suggesting that contact of C. albicans with buccal RHE does little to alter ALS gene expression. RT-PCR analysis of RNA samples extracted from model denture and catheter biofilms showed similar gene expression patterns to the buccal RHE specimens. Results from the RT-PCR analysis of biofilm RNA specimens were consistent between various C. albicans strains during biofilm development and were comparable to gene expression patterns in planktonic cells. The RT-PCR assay described here will be useful for analysis of human clinical specimens and samples from other disease models. The method will provide further insight into the role of ALS genes and their encoded proteins in the diverse interactions between C. albicans and its host.

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