254 DIFFERENTIAL TRANSCRIPTION AND CYSTOPLASMIC POLYADENYLATION ELEMENTS IN PORCINE GERMINAL VESICLE AND METAPHASE II OOCYTES

2006 ◽  
Vol 18 (2) ◽  
pp. 234
Author(s):  
S. Korte ◽  
G. Springer ◽  
W. Spollen ◽  
R. Patel ◽  
K. Whitworth ◽  
...  
Keyword(s):  
Expressed Sequence Tag ◽  
Germinal Vesicle ◽  
Differentially Expressed ◽  
Cdna Libraries ◽  
University Of Iowa ◽  
Cytoplasmic Polyadenylation ◽  
University Of Missouri ◽  
Exact Test ◽  
The University

Identification of transcripts produced during porcine oocyte maturation is one of the first steps in defining pathways important to development. Practically, this information will aid in the refinement of in vitro culture processes, allowing for more efficient in vitro embryo production. To this end, mRNA was isolated from 50 in vitro-matured sow metaphase II stage oocytes (Bomed, Inc., Madison, WI, USA) (MIIs), 50 in vitro-matured gilt metaphase II-stage oocytes (MIIg), and 50 gilt germinal vesicle stage oocytes (GVO) by using the Dynabead® system (Dynal, Inc., Lake Success, NY, USA) and amplified by using the SMART system (Clontech, Palo Alto, CA, USA). The PCR products were ligated into a pSport vector and transformed into electro-competent E. coli. Colonies were randomly picked and sequenced at the University of Missouri DNA Core. Sequences were clustered with similar sequences derived from a larger expressed sequence tag (EST) project (http://genome.rnet.missouri.edu/Swine/) by using the tlcluster program developed at the University of Iowa. Following clustering, individual clusters in the cDNA libraries were compared by using Fisher's exact test (P < 0.01) to determine if they were differentially represented. Two sets of comparisons were performed, one between the MIIs and MIIg libraries, and another between the GVO library and the combination of both metaphase II libraries (MII). The number of clusters per number of clones in the library was 966/1668 (GVO), 458/820 (MIIg), and 158/819 (MIIs). There were 15/419 clusters that were different between the MIIs and MIIg libraries, and 26/1269 that were different between the MII and GVO libraries. Potential cytoplasmic polyadenylation elements (CPEs) identified from the literature were found in the GVO and MII libraries by using a custom pattern-matching program. Of the clusters with differential expression, 4/15 (MIIs vs. MIIg) and 7/26 (MII vs. GVO) contained CPEs. Table 1 contains a partial list of differentially expressed genes and the sequence of their cytoplasmic polyadenylation elements. Many genes were found to be differentially expressed in both (MII vs. GVO and MIIs vs. MIIg) comparisons. Collectively, these findings will facilitate the elucidation of important developmental pathways in swine and other animals. Table 1. Comparison of mRNA expression and cytoplasmic polyadenylation elements This work was partially funded by a University of Missouri Life Sciences Mission Enhancement grant and Food for the 21st Century.

scholarly journals Transcriptome analysis ofSchistosoma mansonilarval development using serial analysis of gene expression (SAGE)

Parasitology ◽  
2009 ◽  
Vol 136 (5) ◽  
pp. 469-485 ◽  
Author(s):  
A. S. TAFT ◽  
J. J. VERMEIRE ◽  
J. BERNIER ◽  
S. R. BIRKELAND ◽  
M. J. CIPRIANO ◽  
...  
Keyword(s):  
Gene Expression ◽  
Sequence Data ◽  
Subsequent Development ◽  
Differentially Expressed ◽  
Cdna Libraries ◽  
Genome Wide ◽  
A Genome ◽  
Genome Wide Expression ◽  
Cell Conditioned Medium

SUMMARYInfection of the snail,Biomphalaria glabrata, by the free-swimming miracidial stage of the human blood fluke,Schistosoma mansoni, and its subsequent development to the parasitic sporocyst stage is critical to establishment of viable infections and continued human transmission. We performed a genome-wide expression analysis of theS. mansonimiracidia and developing sporocyst using Long Serial Analysis of Gene Expression (LongSAGE). Five cDNA libraries were constructed from miracidia andin vitrocultured 6- and 20-day-old sporocysts maintained in sporocyst medium (SM) or in SM conditioned by previous cultivation with cells of theB. glabrataembryonic (Bge) cell line. We generated 21 440 SAGE tags and mapped 13 381 to theS. mansonigene predictions (v4.0e) either by estimating theoretical 3′ UTR lengths or using existing 3′ EST sequence data. Overall, 432 transcripts were found to be differentially expressed amongst all 5 libraries. In total, 172 tags were differentially expressed between miracidia and 6-day conditioned sporocysts and 152 were differentially expressed between miracidia and 6-day unconditioned sporocysts. In addition, 53 and 45 tags, respectively, were differentially expressed in 6-day and 20-day cultured sporocysts, due to the effects of exposure to Bge cell-conditioned medium.

Aptamers against viral proteins as a tool to answer biological questions

2019 ◽  
Author(s):  
◽  
Carolina Camargo
Keyword(s):  
Lentiviral Vector ◽  
Rna Aptamers ◽  
Target Cells ◽  
University Of Missouri ◽  
Practical Applications ◽  
Intracellular Expression ◽  
The University ◽  
Further Development ◽  
Viral Surface

[ACCESS RESTRICTED TO THE UNIVERSITY OF MISSOURI AT REQUEST OF AUTHOR.] There are two experimental chapters in this dissertation in which the fundamental questions center around aptamers and viruses and how these two concepts interlace. The first experimental chapter (chapter two) seeks to utilize previously characterized aptamers against HIV-1 Reverse Transcriptase (RT) that will be delivered by a lentiviral vector and which intracellular expression from different human promoters was evaluated. The goal of this study was to identify important elements of vector design that will impact transgene expression in target cells. This study was based on the hypothesis that intracellular expression of RNA aptamers delivered by a lentiviral vector could offer a platform to enable adequate aptamer expression that would translate into viral suppression. And the second experimental chapter (chapter four) describes an in vitro 2'FY-RNA selection against Filoviral glycoproteins and outlines three different strategies that were followed to achieve selection of specific aptamers. Aptamers described in this chapter were able to recognize Ebolavirus glycoprotein ectodomain as well as in its native conformation displayed on the viral surface. Taking the observations obtained in this dissertation, aptamer technology could be expanded into further development for practical applications.

Advances in aptamer evolution and engineering

2016 ◽  
Author(s):  
◽  
Khalid Kamal Alam
Keyword(s):  
High Throughput Sequencing ◽  
Target Selection ◽  
Three Dimensional ◽  
Rna Aptamers ◽  
Hiv Reverse Transcriptase ◽  
University Of Missouri ◽  
Selection Approach ◽  
And Function ◽  
The University

[ACCESS RESTRICTED TO THE UNIVERSITY OF MISSOURI AT AUTHOR'S REQUEST.] Aptamers are single-stranded nucleic acids that fold into unique three-dimensional shapes that allow them to bind with high affinity and specificity to targets of interest. They are selected through the process of in vitro evolution, wherein large libraries of randomized sequence are iteratively partitioned and amplified to enrich for high-fitness, functional molecules. Selected libraries are sequenced and individual aptamers are characterized for their structure and function. Aptamers have found use as research tools, diagnostics, and therapeutics and in the control of biological systems. The work described herein presents several advancements to the selection and application of aptamers. I first describe an aptamer bioinformatics platform, FASTAptamer, which performs the primary sequence tasks common to all combinatorial selection techniques. I then describe a poly-target selection approach that leverages high-throughput sequencing, the aptamer bioinformatics platform, and parallel selections against a family of related targets to identify the first RNA aptamers capable of potent broad-spectrum inhibition of HIV reverse transcriptase. Finally, this work describes the engineering and in vitro validation of a bifurcated aptamer, Split-Broccoli, for direct visualization of RNA:RNA processes.

Genomic expression profiles in cumulus cells derived from germinal vesicle and MII mouse oocytes

2016 ◽  
Vol 28 (11) ◽  
pp. 1798 ◽  
Author(s):  
Li Shao ◽  
Ri-Cheng Chian ◽  
Yixin Xu ◽  
Zhengjie Yan ◽  
Yihui Zhang ◽  
...  
Keyword(s):  
Expression Profiles ◽  
Expression Patterns ◽  
Germinal Vesicle ◽  
Cumulus Cells ◽  
Developmental Competence ◽  
Differentially Expressed ◽  
Genomic Expression ◽  
Oocyte Competence ◽  
Before And After

Cumulus cells (CCs) are distinct from other granulosa cells and the mutual communication between CCs and oocytes is essential for the establishment of oocyte competence. In the present study we assessed genomic expression profiles in mouse CCs before and after oocyte maturation in vitro. Microarray analysis revealed significant changes in gene expression in CCs between the germinal vesicle (GV) and metaphase II (MII) stages, with 2615 upregulated and 2808 downregulated genes. Genes related to epidermal growth factor, extracellular matrix (Ptgs2, Ereg, Tnfaip6 and Efemp1), mitochondrial metabolism (Fdx1 and Aifm2), gap junctions and the cell cycle (Gja1, Gja4, Ccnd2, Ccna2 and Ccnb2) were highlighted as being differentially expressed between the two development stages. Real-time polymerase chain reaction confirmed the validity and reproducibility of the results for the selected differentially expressed genes. Similar expression patterns were identified by western blot analysis for some functional proteins, including EFEMP1, FDX1, GJA1 and CCND2, followed by immunofluorescence localisation. These genes may be potential biomarkers for oocyte developmental competence following fertilisation and will be investigated further in future studies.

177 GENE EXPRESSION PROFILES OF IN VITRO- AND IN VIVO-DERIVED BOVINE EMBRYOS

2011 ◽  
Vol 23 (1) ◽  
pp. 190
Author(s):  
D. Aktoprakligil Aksu ◽  
C. Agca ◽  
S. Aksu ◽  
T. Akkoc ◽  
A. Tas Caputcu ◽  
...  
Keyword(s):  
Gene Expression ◽  
Functional Annotation ◽  
Differentially Expressed ◽  
Annotation Tool ◽  
Bovine Embryos ◽  
University Of Missouri ◽  
Total Rna ◽  
David Functional Annotation Tool

Microarray technology is one of the most powerful tools for gene expression profiling in animal sciences. The objectives of this study were to determine the effect of vitrification on gene expression in in vitro- and in vivo-derived bovine embryos, and to identify differential mRNA expression patterns between embryos produced by in vivo v. in vitro conditions. Three pools of in vivo- and in vitro-derived blastocyst-stage embryos were used for microarray analysis. Total RNA was isolated using the PicoPure RNA Isolation Kit (Arcturus Bioscience, Mountain View, CA). Bovine ovarian tissue total RNA was used as the reference. Total RNA samples were amplified using an Ovation® Pico WTA System (NuGEN Technologies, San Carlos, CA). The bovine 16 846-member microarrays spotted with 70-mer oligonucleotides were purchased from the Bovine Genomics Laboratory, University of Missouri. Amplified cDNA samples were labeled with Alexa Fluor 647 and 546 dyes (Molecular Probes, Eugene, OR), respectively. Combined, labeled samples were dried and resuspended in hybridization buffer containing 50% formamide (vol/vol), 5× SSC, and 0.1% sodium dodecyl sulfate (wt/vol). After denaturation and cooling, cDNA was applied onto a microarray slide. Microarrays were hybridized overnight at 42°C. Following hybridization, the slides were washed with different stringency buffers and water. After drying by centrifugation, the arrays were scanned on a GenePix 4000B scanner (Axon Instruments, Union City, CA). GenePix Pro4.1 software was used for griding and analysis of spot intensities. Good-quality spots were analyzed using the GeneSpring 7.3 software (Agilent Technologies, Inc., CA, Santa Clara, CA). The data were normalized per spot and per array by Lowess normalization. When comparing two treatments, the Welch t-test with Benjamini and Hochberg multiple testing correction was performed to determine the differentially expressed genes between embryo groups. Microarray experiments were performed in 3 biological and 2 technical replicates for all embryo samples. Differentially expressed genes between all embryo groups were identified. The DAVID Functional Annotation Tool was used to analyze the genes that were differentially expressed. The DAVID Functional Annotation Tool determined the co-occurrence probability and provided gene-GO term enrichment analysis to highlight the most relevant GO terms associated with a given gene list. Differentially expressed Kyoto Encyclopedia of Genes and Genomes pathways are as follows: Ribosome, oxidative phosphorylation, spliceosome, and oocyte meiosis were significantly upregulated in the fresh embryos, whereas sphingolipid and purine metabolism was the upregulated in the vitrified in vitro-derived embryos. Gene expression was very similar between fresh and vitrified in vivo-derived, as opposed to in vitro-derived, embryos. This study was funded by the TUBITAK (Project no. KAMAG107G027) and startup funds to Yuksel Agca at the University of Missouri.

scholarly journals EST analysis of cDNA libraries from the entomopathogenic fungus Beauveria (Cordyceps) bassiana. I. Evidence for stage-specific gene expression in aerial conidia, in vitro blastospores and submerged conidia

Microbiology ◽  
2006 ◽  
Vol 152 (9) ◽  
pp. 2843-2854 ◽  
Author(s):  
Eun-Min Cho ◽  
Li Liu ◽  
William Farmerie ◽  
Nemat O. Keyhani
Keyword(s):  
Gene Expression ◽  
Entomopathogenic Fungus ◽  
Expressed Sequence Tag ◽  
Biological Control Agent ◽  
Expression Profiles ◽  
Cdna Libraries ◽  
Specific Gene ◽  
Cell Type ◽  
Aerial Conidia

The entomopathogenic fungus Beauveria (Cordyceps) bassiana holds much promise as a pest biological control agent. B. bassiana produces at least three in vitro single cell infectious propagules, including aerial conidia, vegetative cells termed blastospores and submerged conidia, that display different morphological, biochemical and virulence properties. Populations of aerial conidia, blastospores and submerged conidia were produced on agar plates, rich liquid broth cultures and under conditions of nutrient limitation in submerged cultures, respectively. cDNA libraries were generated from mRNA isolated from each B. bassiana cell type and ∼2500 5′ end sequences were determined from each library. Sequences derived from aerial conidia clustered into 284 contigs and 963 singlets, with those derived from blastospores and submerged conidia forming 327 contigs with 788 singlets, and 303 contigs and 1079 contigs, respectively. Almost half (40–45 %) of the sequences in each library displayed either no significant similarity (e value >10−4) or similarity to hypothetical proteins found in the NCBI database. The expressed sequence tag dataset also included sequences representing a significant portion of proteins in cellular metabolism, information storage and processing, transport and cell processes, including cell division and posttranslational modifications. Transcripts encoding a diverse array of pathogenicity-related genes, including proteases, lipases, esterases, phosphatases and enzymes producing toxic secondary metabolites, were also identified. Comparative analysis between the libraries identified 2416 unique sequences, of which 20–30 % were unique to each library, and only ∼6 % of the sequences were shared between all three libraries. The unique and divergent representation of the B. bassiana transcriptome in the cDNA libraries from each cell type suggests robust differential gene expression profiles in response to environmental conditions.

Investigating the role of arginine 21 in the structure and function of human [alpha]a-crystallin

2018 ◽  
Author(s):  
◽  
Ashutosh Shripad Phadte
Keyword(s):  
Functional Characterization ◽  
Lens Fiber Cell ◽  
University Of Missouri ◽  
Mutant Proteins ◽  
Plausible Mechanism ◽  
And Function ◽  
The University

[ACCESS RESTRICTED TO THE UNIVERSITY OF MISSOURI AT AUTHOR'S REQUEST.] Cataractogenesis in the eye lens occurs as a result of protein aggregation. Of the multiple mutations in [alpha]A-crystallins associated with the development of congenital hereditary cataract, three identified mutations target R21 within the N- terminal domain of the protein. On structural and functional characterization of a recently identified mutant of [alpha]A-crystallin, [alpha]A-R21Q, we revealed the contribution of R21 in dictating the interaction of [alpha]A-crystallin with other proteins. [alpha]A-R21Q showed and enhanced chaperone-like function, and increased binding to lens fiber cell membranes. Transduction of mutant proteins in ARPE-19 cells prevented their apoptosis in the presence of oxidative stress, suggesting a role for R21 in modulating the anti-apoptotic function of [alpha]A-crystallin. In addition, the R21Q point mutation rescued the chaperone-like activity of [alpha]A-G98R crystallin as well as palliated [alpha]A-G98R mediated cytotoxicity otherwise observed in transduction experiments. Although another mutation, R157Q rescued the chaperone-like activity of [alpha]A-G98R, the double mutant exhibited a loss of its cytoprotective function. The results therefore implicate an important role of R21 in regulating the functional aspect of [alpha]A-crystallin. [alpha]A-crystallin derived peptides have been shown to prevent non-specific aggregation of unfolding proteins in vitro. We show that the [alpha]A-crystallin derived mini-chaperone (mini-[alpha]A) mediated stabilization of self-aggregating [alpha]A-G98R crystallin and bovine [subscript]-crystallin occurs via compensation of lost surface charge. The observation therefore suggests a plausible mechanism of action of [alpha]A-crystallin derived peptides of therapeutic interest.

37 In vitro maturation and fertilization in white-tailed deer (Odocoileus virginianus) oocytes vitrified with trehalose or sucrose

2020 ◽  
Vol 32 (2) ◽  
pp. 144
Author(s):  
V. A. Rubio-Santillanes ◽  
J. Antillón-Ruiz ◽  
F. A. Rodríguez-Almeida ◽  
S. Romo ◽  
H. Álvarez-Gallardo ◽  
...  
Keyword(s):  
Germinal Vesicle ◽  
Future Research ◽  
Fisher Exact Test ◽  
Oocyte Vitrification ◽  
Exact Test ◽  
Significant Difference ◽  
Production Techniques ◽  
Epidermal Growth ◽  
Humidified Air

White-tailed deer (WTD) invitro embryo production is not well documented, due to the seasonality of the wildlife species and available hunting permits. The objective of the present study was to conduct IVM and IVF using WTD oocytes vitrified with trehalose (TH) or sucrose (SC). A total of 121 immature oocytes were obtained from ovaries (n=18) using the slicing technique from hunter-slaughtered deer, not more than 2h after death. The WTD oocytes were vitrified using a solid surface vitrification technique in two different groups: 1) TH (n=60) and 2) SC (n=61). Oocyte warming (OW) was done using four concentrations (OW1 1 M, OW2 0.5 M, OW3 0.25 M, and OW4 0 M) in the oocytes after thawing. Then, a sample was used to evaluate viability for TH (n=5) and SC (n=5) (MTT stain (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide), 0.5mgmL−1) and nuclear status (NS) for TH (n=4) and SC (n=5) with 4′,6-diamidino-2-phenylindole (1μgmL−1), defining the stage as germinal vesicle (GV), MI, and unable to evaluate (NE). The remaining oocytes (n=88) were used for IVM for 36h in tissue culture medium-199 supplemented with human menopausal hormone (75 UImL−1) and epidermal growth factor (10ngmL−1). The evaluation of NS was defined (GV, MI considered immature and MII matured). The IVF (n=15) took place in Tyrode's albumin lactate pyruvate media supplemented with heparin (10μgmL−1), penicillamine (0.075mgmL−1), hypotaurine (10M), epinephrine (1μM), and bovine serum albumin fraction V (0.4%), with a final semen concentration of 3×106 spermmL−1. Frozen straws of conventional WTD semen were used; the straws were split into 3 parts, with one fraction thawed and capacitated using “swim-up” technique. After 24h of incubation at 38.5°C with 5% CO2, and humidified air, nuclear evaluation was made as fertilized (F), not fertilized (NF), or NE. A Fisher exact test was used (SAS, 9.0 version for Windows), α=0.05. After warming, for the TH group (n=5), viability was 60% and nuclear status (n=4) was 50% GV, 50% MI, and 0% NE. For the SC group (n=5), viability was 40% and nuclear status (n=5) was 60% GV, 20% MI, and 20% NE. After 36h IVM, NS evaluation in the TH group (n=38) was 66% GV, 24% MI, 0% MII, and 10% NE; for the SC group (n=50), 84% GV, 10% MI, 2% MII, and 4% NE, not a statistically significant difference (P&gt;0.05). For IVF after 24h, the NS evaluation in the TH treatment (n=10) was 0% F, 60% NF, and 40% NE versus the SC treatment (n=5) with 20% F, 40% NF, and 40% NE, not a statistically significant difference (P&gt;0.05). A statistically significant difference (P&gt;0.05) was not found between the TH and SC groups with regard to post-thaw viability, IVM, and IVF. Future research with larger numbers of immature or mature oocytes is suggested for further evaluation of both TH and SC for WTD oocyte vitrification for use with invitro-production techniques as a model for other endangered cervid species.

246 INDUCTION OF MEIOTIC ARREST IN IMMATURE FELINE OOCYTES WITH ROSCOVITINE AND DIBUTYRYL CYCLIC-AMP

2009 ◽  
Vol 21 (1) ◽  
pp. 221
Author(s):  
C. B. Hanna ◽  
T. M. Glover ◽  
C. R. Long
Keyword(s):  
Germinal Vesicle ◽  
Cumulus Cells ◽  
Culture Conditions ◽  
Control Culture ◽  
Basic Medium ◽  
Meiotic Arrest ◽  
Chi Square ◽  
Exact Test ◽  
Chromatin Configuration

Successful application of some assisted reproduction technologies in feline species requires the use of competent, meiotically mature ova, which can be difficult to obtain in sufficient quantities. Meiotic arrest of immature feline oocytes would allow the accumulation of oocytes over time to maximize laboratory resources. Two meiosis inhibitors commonly used in other species, roscovitine (ROS) and dbcAMP, were evaluated for the ability to maintain a germinal vesicle (GV) in immature feline oocytes after 24 h of in vitro culture. Feline ovaries were obtained from routine ovariohysterectomies and oocytes with homogenous cytoplasm and at least 2 layers of cumulus cells were selected. All oocytes were cultured in a basic medium modified from Gomez et al. (2000 Reprod. Fertil. Dev.) consisting of TCM-199 with Earle’s salts, 0.3% fraction V bovine serum albumin, 2.0 mm L-glutamine, 1.12 mm L-cysteine, 2.2 mm calcium lactate, 0.36 mm sodium pyruvate, 100 μm cysteamine, 10 ng μL–1 epidermal growth factor, 1 μg mL–1 estradiol, and 50 μg mL–1 gentamicin, with or without meiotic inhibitor. After 24 h of culture, cumulus cells were removed; oocytes were fixed, permeabilized, and stained with 5 μg mL–1 of Hoechst 33342; and chromatin configuration was assessed under ultraviolet fluorescence. In 4 replicates of Experiment 1, oocytes were cultured with either 25 nm ROS, 1 mm dbcAMP, both ROS and dbcAMP (Both), or without inhibitor (Control). A greater proportion of oocytes retained the GV when cultured with Both compared with ROS, dbcAMP, or Control (90.6, 67.8, 25.0, and 14.8%, respectively; chi-square P < 0.05), and ROS alone was superior to dbcAMP or Control. Culture of oocytes in the base medium plus 0.5 IU mL–1 eCG and 1.0 IU mL–1 hCG after arrest showed that pretreatment with Both did not decrease their ability to resume meiosis (87.9 v. 87.0%, respectively). Given the low percentage of oocytes with a retained GV in the presence of 1.0 mm dbcAMP, Experiment 2 evaluated the concentration of dbcAMP required to inhibit meiosis. Oocytes were cultured over 4 replicates in the base media containing 0, 0.1, 0.5, 1.0, 2.0, and 10.0 mm dbcAMP. After 24 h of culture, a greater number of oocytes were arrested at the GV stage when cultured in 10 mM dbcAMP (39.8%) than in 0, 0.1, 1.0, and 2.0 mM dbcAMP (22.0, 20.4, 25.8, and 26.3%, respectively; Fisher’s exact test P < 0.05), but there was no difference from those cultured in 0.5 mm dbcAMP (28.1%). For many species, 1.0 mm dbcAMP is commonly used to inhibit meiosis successfully for 24 h. However, dbcAMP alone did not effectively arrest meiosis, but when combined with ROS, it tended to improve meiotic arrest. Experiment 2 indicates that at least 10.0 mm dbcAMP is required to show a significant effect of meiotic inhibition in feline oocytes. Under these culture conditions, dbcAMP at levels up to 10 mm were not effective at inducing meiotic arrest. Interestingly, ROS and dbcAMP may act synergistically to successfully induce a reversible inhibition of meiosis in a high percentage of feline oocytes.

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