scholarly journals 216 IDENTIFICATION OF DIFFERENTIALLY EXPRESSED GENES IN BOVINE EMBRYOS CULTURED IN VIVO OR IN VITRO

2005 ◽  
Vol 17 (2) ◽  
pp. 259 ◽  
Author(s):  
D. Corcoran ◽  
T. Fair ◽  
D. Rizos ◽  
G.W. Smith ◽  
P.M. Coussens ◽  
...  
Keyword(s):  
In Vitro Culture ◽  
Differentially Expressed Genes ◽  
Intracellular Signalling ◽  
Differentially Expressed ◽  
Regulation Of Transcription ◽  
Gene Transcript ◽  
Bovine Embryo ◽  
Total Rna

The post-fertilization culture environment of the bovine embryo is known to influence the quality of the resulting blastocyst, manifested in terms of morphology, cryotolerance, and the relative gene transcript abundance of several candidate genes. This may have consequences for the pregnancy rate following embryo transfer. The objective of the current study was to take a broader approach toward identifying differentially expressed genes in bovine blastocysts derived from either in vivo or in vitro culture. Presumptive zygotes, produced by in vitro maturation and fertilization, were randomly assigned to one of two groups and cultured for 6 days, either in vitro in SOF medium, or in vivo in the ewe oviduct following transfer by mid-ventral laparotomy. Blastocysts were recovered from both systems on Day 7 after insemination, snap frozen in liquid nitrogen, and stored at −80°C. Total RNA was extracted from 50 blastocysts for each culture group from each of four replicates using the PicoPureTM RNA Isolation Kit (ARCTURUS, Mountain View, CA 94043, USA). The RiboAmpTM RNA Amplification Kit (ARCTURUS) was used to linearly amplify the mRNA fraction of total RNA using double-stranded cDNA as template in a T7 RNA polymerase-catalyzed amplification. Samples from both culture environments were differentially labelled using N-hydroxysuccinimide (NHS)-activated fluorescent Cy3 or Cy5 dyes (Amersham Pharmacia Ltd., Piscataway, NJ, USA) and were hybridized onto a cDNA microarray. Each microarray contained 3888 total spots, with 932 bovine EST clone inserts developed from a normalized bovine total leukocyte (BOTL) cDNA library and an additional 459 amplicons representing additional genes including cytokines, receptors, signal transduction molecules, transcription and growth factors, enzymes, cell cycle regulators, and cellular components. Data were normalized and an expression ratio calculated between the two groups. This was compared to 1 within each of the 4 replicates by Student's t-test. Microarray analysis identified 15 gene transcripts that were differentially expressed P ≤ 0.05) between blastocysts produced in vivo or in vitro. Among these, four genes involved in transcription (nuclear receptor co-repressor 1, zinc finger protein 22, CCR4-NOT transcription complex, DOT1) and two genes involved in intracellular signalling (proteasome 26S subunit non-ATPase 13 and guanine nucleotide binding protein) had a higher mRNA expression level in blastocysts produced in vivo compared to those produced in vitro. In addition, Connexin 43, a gene involved in gap junction formation, was down regulated following in vitro culture which is consistent with our previous studies. Among the genes up-regulated following in vitro culture were WNT2B wingless-type MMTV integration site, CD103 integrin, and tumor necrosis factor superfamily member 8. In conclusion, we have identified previously uncharacterized, differentially expressed genes involved in cell communication, intracellular signalling, and regulation of transcription in bovine blastocysts cultured in vivo or in vitro. This work was supported by Science Foundation Ireland under Grant No. 02/IN1/B78.

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.

196 EMBRYO BIOPSY TRANSCRIPTOMICS: A POTENTIAL TOOL TO IDENTIFY TRANSCRIPTS DIRECTLY RELATED TO THE ABILITY OF THE EMBRYO TO INDUCE PREGNANCY AFTER TRANSFER

2009 ◽  
Vol 21 (1) ◽  
pp. 196
Author(s):  
D. Tesfaye ◽  
N. Ghanem ◽  
F. Rings ◽  
E. Tholen ◽  
C. Phatsara ◽  
...  
Keyword(s):  
Gene Expression ◽  
Pregnancy Outcome ◽  
Expression Profile ◽  
Expression Profiles ◽  
Transcript Abundance ◽  
Differentially Expressed ◽  
Embryo Biopsy ◽  
Bovine Embryo

The incidence of pregnancy loss due to embryonic mortality in cattle is one of the major causes of reproductive failure. The early embryonic loss can be due to problems with the embryo itself, the uterine environment, or interactions between the embryo and the uterus. So, this study was conducted to investigate the gene expression profile of bovine embryo biopsies produced in vivo and in vitro that resulted in different pregnancy outcomes. For this, biopsies representing 30 to 40% of the intact in vitro and in vivo blastocysts were taken, and 60 to 70% part was allowed to re-expand prior to transfer to recipients. Based on the pregnancy outcome after transfer, biopsies (n = 10 per pool) were grouped into 3 distinct phenotypes: those that resulted in no pregnancy, those that resulted in resorption, and those that resulted in successful pregnancy and subsequent calf delivery. A bovine cDNA microarray with 2000 clones was used to analyze the gene expression profiles of 3 replicates from each embryo biopsy group. Array data analysis revealed a total of 50 and 52 genes to be differentially expressed between biopsies derived from in vivo blastocysts that resulted in no pregnancy v. calf delivery and resorption v. calf delivery, respectively. Similarly, a total of 52 and 58 transcripts were differentially expressed between biopsies derived from in vitro-produced blastocysts that resulted in no pregnancy v. calf delivery and resorption v. calf delivery, respectively. Quantitative real-time PCR has confirmed the expression profile of 6 selected candidate genes. A distinct set of genes were found to be commonly expressed between in vitro- and in vivo-derived blastocyst biopsies, which ended up with the same pregnancy outcome. Biopsies, which ended up with calf delivery, were found to be enriched with transcripts involved in nucleosome assembly (KRT8), translation (RPLPO), electron transport (COX-2), and placenta specific (PLAC8). On the other hand, transcripts regulating immune response (TNFa), response to stress (HSPD1), and cell adhesion (CD9) were up-regulated in embryos that resulted in no pregnancy or resorption. Differences in transcript abundance of some genes have been seen between biopsies derived from in vitro and in vivo blastocysts. Biopsies from in vivo-derived blastocysts and that ended up with resorption were found to be enriched with transcripts regulating calcium-binding protein (S100A10, S100A14). Transcription factor-related transcripts (CDX2, HOXB7) were up-regulated in vitro-derived blastocyst biopsies that resulted in no pregnancy. In conclusion, the results evidenced that embryos derived from either in vitro or in vivo have more similarities than differences in their transcript abundance with respect to the ability in initiating pregnancy.

6 EXPRESSION PROFILING OF SINGLE BOVINE EMBRYOS REVEALS SIGNIFICANT EFFECTS OF IN VITRO MATURATION, FERTILIZATION AND CULTURE

2006 ◽  
Vol 18 (2) ◽  
pp. 111
Author(s):  
S. L. Smith ◽  
L.-Y. Sung ◽  
R. Page ◽  
B. Henderson ◽  
F. Du ◽  
...  
Keyword(s):  
Gene Expression ◽  
Differentially Expressed Genes ◽  
Oocyte Maturation ◽  
Expression Profiling ◽  
Bovine Serum ◽  
In Vitro Maturation ◽  
Differentially Expressed ◽  
In Vitro Oocyte Maturation

Cattle and sheep embryos transferred after in vitro production are often afflicted by large offspring syndrome (LOS), which has been correlated with the presence of serum and/or cell co-culture. Previous research indicates that post-fertilization culture affects blastocyst quality and gene expression, and in vitro oocyte maturation and fertilization impact developmental competence. To dissect the effects of in vitro maturation, fertilization, and culture, we compared the expression profiles of single bovine blastocysts generated by: (1) in vitro maturation, fertilization and culture (IVF, n = 15); (2) in vivo maturation, in vivo fertilization, and in vitro culture (IVD, n = 14); and (3) in vivo maturation, fertilization, and development (AI, n = 14). For in vitro culture, the embryos were cultured for 2 days in CR1aa medium with bovine serum albumin (BSA) and then transferred to CR1aa with 10% fetal bovine serum (FBS) with cumulus cells until Day 7, at which time the embryos were vitrified. IVD zygotes were surgically collected from two superovulated Holstein donor cows 24 h post-insemination and cultured in the same system. To conduct expression profiling, total RNA was isolated from individual thawed embryos. The RNA was subjected to three rounds of amplification utilizing a previously adapted and validated T7 linear amplification protocol. Amplified RNA from each embryo and from a standard reference was indirectly labeled with Cy3 or Cy5 by dye swap and hybridized to a custom bovine cDNA microarray containing ~6300 unique genes. After Loess normalization, an ANOVA model (GeneSpring 6.1 and SAS 9.0) was used to identify differentially expressed genes. The P-values were adjusted for multiple comparisons using the false discovery rate approach, and a e2-fold differential criterion was applied. A subset of the differentially expressed genes was verified by real-time RT-PCR. The blastocyst rates for IVF and IVD embryos were 37% and 75%, respectively. There were 305, 365, and 200 genes differentially expressed between the AI and IVD, the IVF and IVD, and the AI and IVF comparisons, respectively. Interestingly, 44 differentially expressed genes were identified between the AI embryos and both the IVF and the IVD embryos, making these potential candidates for LOS. There were 61 genes differentially expressed between the IVF embryos and the AI and IVD embryos. The Gene Ontology categories 'RNA processing' and 'RNA binding' were over-represented among the genes that were down-regulated in the IVF embryos, indicating an effect of in vitro oocyte maturation/fertilization on embryonic gene expression. This work was supported by USDA grants to X.Y., H.A.L., and X.C.T.

235 PROGESTERONE AFFECTS THE MESSENGER RNA EXPRESSION OF IN VITRO-PRODUCED BOVINE EMBRYOS

2013 ◽  
Vol 25 (1) ◽  
pp. 265
Author(s):  
K. Knauer ◽  
H. Stinshoff ◽  
S. Wilkening ◽  
C. Wrenzycki
Keyword(s):  
In Vitro Culture ◽  
Messenger Rna ◽  
Blastocyst Stage ◽  
Developmental Competence ◽  
Morphological Development ◽  
Bovine Embryo ◽  
Development Rates ◽  
Vitro Model

It is known that the progesterone (P4) provided by the corpus luteum is essential for the maintenance of pregnancy. It has been suggested that supplying external P4 in vivo is beneficial to the establishment and upkeep of pregnancy. The aim of the present study was to assess the effects of supplementation with different concentrations of P4 on either of 2 days of in vitro culture (IVC) on early bovine embryo development in an in vitro model. A total of 5073 cumulus–oocyte complexes were matured and fertilized in vitro. Before culture, they were collected in groups of 30 and allocated to 1 of 9 groups. The groups were supplemented with 10, 20, or 100 ng of P4 on Days 4 or 5 of IVC (IVF = Day 0). Alcohol (ETOH) was used as the solvent, so 8 µL of ETOH was used per supplementation. Therefore, two additional groups were supplemented with only ETOH on Day 4 or 5 of IVC. The presumptive zygotes allocated to group 9 were not supplemented. A culture system without oil overlay was used to prevent the lipophilic P4 from moving into the oil. Embryo cleavage and development rates were determined solely on Day 8 of IVC. Single expanded blastocysts were stored at –80°C for RT-qPCR. Subsequently, the relative amounts of six developmentally important gene transcripts (IGF1R, SLC2A1, HSD3B1, IFNT, PGRMC1, and PGRMC2) were analysed in single embryos of all groups. Statistical analysis was performed using one-way and two-way ANOVA, and the level of significance was set at P ≤ 0.05. Cleavage and development rates did not differ among groups (see Table 1). The relative abundance of IGF1R, SLC2A1, PGRMC1, and PGRMC2 was not affected by either the concentration or the timing of P4 supplementation. Nevertheless, there was a statistically significant interaction between the day of treatment and the concentration used for the expression of HSD3B1 mRNA. When 20 ng of P4 was added on Day 5 of IVC, significantly more HSD3B1 transcripts were detected than if 10 ng, 100 ng, or ETOH alone was added. The expression of IFNT was not affected by the day of supplementation, only by the concentration used. Thus, supplementation with 20 ng of P4 resulted in a significantly higher level of transcripts than when 10 ng or ETOH was supplemented. The results indicate that the amount of P4 present during early embryonic development and the timing of its presence had an impact on molecular developmental competence. However, no effects concerning morphological development up to the blastocyst stage could be detected. Table 1.Cleavage and development rates (± SEM) of embryos supplemented with 10, 20, or 100 ng on Day 4 or 5 of in vitro culture (P ≥ 0.05) The financial support of the FBF e.V. is acknowledged.

scholarly journals Evaluation of CRISPR gene-editing tools in zebrafish

BMC Genomics ◽  
2022 ◽  
Vol 23 (1) ◽  
Author(s):  
José M. Uribe-Salazar ◽  
Gulhan Kaya ◽  
Aadithya Sekar ◽  
KaeChandra Weyenberg ◽  
Cole Ingamells ◽  
...  
Keyword(s):  
Differentially Expressed Genes ◽  
Gene Editing ◽  
Differentially Expressed ◽  
Transient Effects ◽  
Cytoskeleton Organization ◽  
Functional Studies ◽  
Network Analyses ◽  
Molecular Features

Abstract Background Zebrafish have practical features that make them a useful model for higher-throughput tests of gene function using CRISPR/Cas9 editing to create ‘knockout’ models. In particular, the use of G0 mosaic mutants has potential to increase throughput of functional studies significantly but may suffer from transient effects of introducing Cas9 via microinjection. Further, a large number of computational and empirical tools exist to design CRISPR assays but often produce varied predictions across methods leaving uncertainty in choosing an optimal approach for zebrafish studies. Methods To systematically assess accuracy of tool predictions of on- and off-target gene editing, we subjected zebrafish embryos to CRISPR/Cas9 with 50 different guide RNAs (gRNAs) targeting 14 genes. We also investigate potential confounders of G0-based CRISPR screens by assaying control embryos for spurious mutations and altered gene expression. Results We compared our experimental in vivo editing efficiencies in mosaic G0 embryos with those predicted by eight commonly used gRNA design tools and found large discrepancies between methods. Assessing off-target mutations (predicted in silico and in vitro) found that the majority of tested loci had low in vivo frequencies (< 1%). To characterize if commonly used ‘mock’ CRISPR controls (larvae injected with Cas9 enzyme or mRNA with no gRNA) exhibited spurious molecular features that might exacerbate studies of G0 mosaic CRISPR knockout fish, we generated an RNA-seq dataset of various control larvae at 5 days post fertilization. While we found no evidence of spontaneous somatic mutations of injected larvae, we did identify several hundred differentially-expressed genes with high variability between injection types. Network analyses of shared differentially-expressed genes in the ‘mock’ injected larvae implicated a number of key regulators of common metabolic pathways, and gene-ontology analysis revealed connections with response to wounding and cytoskeleton organization, highlighting a potentially lasting effect from the microinjection process that requires further investigation. Conclusion Overall, our results provide a valuable resource for the zebrafish community for the design and execution of CRISPR/Cas9 experiments.

scholarly journals The targeted inhibition of prostate cancer by iron-based nanoparticles based on bioinformatics

2020 ◽  
pp. 088532822097524
Author(s):  
Feng Xiao ◽  
Jiayu Liu ◽  
Yongbo Zheng ◽  
Zhen Quan ◽  
Wei Sun ◽  
...  
Keyword(s):  
Prostate Cancer ◽  
Gene Therapy ◽  
Cancer Cells ◽  
Differentially Expressed Genes ◽  
Differentially Expressed ◽  
Urogenital System ◽  
Prostate Cancer Cells ◽  
Iron Based

Prostate cancer is an epithelial malignant tumor of the prostate, and it is one of the malignant tumors with a high incidence of urogenital system in men. The local treatment of prostate cancer is mainly radical resection and radical radiotherapy, but they are not applicable to advanced prostate cancer. Systemic therapy mainly includes targeted therapy and immunotherapy which could cause many complications, and will affect the prognosis and quality of life of patients. It is urgent to find new treatments for prostate cancer. Bioinformatics offers hope for us to find reliable therapeutic targets. Bioinformatics can use the tumor informations in database and analyze them to screen out the best differentially expressed genes. Using the selected differentially expressed genes as targets, a gene interference plasmid was designed, and the constructed plasmid was used for targeted gene therapy. There are some problems about gene therapy that need to be solved, such as how to transfer genes to target cells is also an important challenge. Due to their large molecular weight and hydrophilic nature, they cannot enter cells through passive diffusion mechanisms. Here we synthesized a DNA carrier used surface modified iron based nanoparticles, and used it to load plasmid including ShRNA which can inhibit the expression of oncogene SLC4A4 selected by bioinformatics’ method. After that we use this iron based nanoparticles/plasmid DNA nanocomposite to treat prostate cancer cells in vitro and in vivo. The target gene SLC4A4 we had selected using bioinformatics had a strong effect on the proliferation of prostate cells; Our nanocomposite could inhibit the expression of SLC4A4 effectively, it had strong inhibitory effects on prostate cancer cells both in vivo and in vitro, and can be used as a potential method for prostate cancer treatment.

scholarly journals Genome-Wide Expression Analysis of Burkholderia pseudomallei Infection ina Hamster Model of Acute Melioidosis

2006 ◽  
Vol 74 (10) ◽  
pp. 5465-5476 ◽  
Author(s):  
Apichai Tuanyok ◽  
Marina Tom ◽  
John Dunbar ◽  
Donald E. Woods
Keyword(s):  
Gene Expression ◽  
Differentially Expressed Genes ◽  
Energy Production ◽  
Burkholderia Pseudomallei ◽  
Expression Profiles ◽  
Differentially Expressed ◽  
Bacterial Growth Rate ◽  
Hamster Model

ABSTRACT Burkholderia pseudomallei is the causative agent of melioidosis and represents a potential bioterrorism threat. In the current studies we have examined gene expression in B. pseudomallei in an animal model of acute melioidosis using whole-genome microarrays. Gene expression profiles were generated by comparing transcriptional levels of B. pseudomallei-expressed genes in infected hamster organs including liver, lung, and spleen following intraperitoneal and intranasal routes of infection to those from bacteria grown in vitro. Differentially expressed genes were similar in infected livers irrespective of the route of infection. Reduced expression of a number of housekeeping genes suggested a lower bacterial growth rate during infection. Energy production during growth in vivo involved specific biochemical pathways such as isomerization of 3-phosphoglycerate, catabolism of d-glucosamine and inositol, and biosynthesis of particular amino acids. In addition, the induction of genes known to be involved in oxidative phosphorylation including ubiquinol oxidase, ferredoxin oxidoreductase, and formate dehydrogenase enzymes suggested the use of alternative pathways for energy production, while the expression of genes coding for ATP-synthase and NADH-dehydrogenase enzymes was reduced. Our studies have identified differentially expressed genes which include potential virulence genes such as those for a putative phospholipase C and a putative two-component regulatory system, and they have also provided a better understanding of bacterial metabolism in response to the host environment during acute melioidosis.

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