scholarly journals Expression of RONIN and NANOG-associated proteins in goat parthenogenetic embryos

2017 ◽  
Vol 11 (2) ◽  
pp. 145 ◽  
Author(s):  
Marcelo Tigre Moura ◽  
Pamela Ramos-Deus ◽  
José Carlos Ferreira-Silva ◽  
Priscila Germany Corrêa Silva ◽  
Ludymila Furtado Cantanhêde ◽  
...  
Keyword(s):  
Transcription Factors ◽  
Expression Patterns ◽  
Preimplantation Embryos ◽  
Rt Pcr ◽  
Cleavage Stage ◽  
Specific Expression ◽  
Early Embryos ◽  
Associated Proteins ◽  
Gene Transcripts

The expression of a subset of transcription factors is enriched in early preimplantation embryos, which contributes to their cellular plasticity. RONIN, NANOG and its associated proteins are PluripotencyAssociated Transcription Factors (PATF) that control relevant downstream pathways in pluripotent stem cells, but their activity in early embryos remained less understood. The work was aimed to determine the expression of RONIN and four NANOG-associated PATFs in goat preimplantation embryos. Goat embryos were produced in vitro by parthenogenetic activation. Gene transcripts of cleavage-stage embryos were investigated by reverse transcriptase-polymerase chain reaction (RT-PCR), while blastocysts were analyzed by both RTPCR and quantitative RT-PCR (RT-qPCR) assays. Gene transcripts of ZFP281, NAC1, and NR0B1 were detected in cleavage-stage embryos, while RONIN and OCT4 were not found expressed. Detection in blastocysts by RT-PCR confirmed the activity of NR0B1, RONIN, and OCT4. Moreover, all five PATF were detected in blastocysts by RT-qPCR (ZFP281, NAC1, RONIN, OCT4, and NR0B1). In conclusion, RONIN and NANOG-associated proteins are active during goat parthenogenetic preimplantation development and hold stage-specific expression patterns.

scholarly journals 236 SPATIAL GENE EXPRESSION PATTERNS OF Dnmt1, Dnmt3a, AND Hdac2 IN PREIMPLANTATION EMBRYOS

2005 ◽  
Vol 17 (2) ◽  
pp. 268
Author(s):  
C. Wrenzycki ◽  
D. Herrmann ◽  
A. Lucas-Hahn ◽  
K. Korsawe ◽  
E. Lemme ◽  
...  
Keyword(s):  
Dna Methylation ◽  
Relative Abundance ◽  
Expression Patterns ◽  
Dna Methyltransferases ◽  
Histone Deacetylation ◽  
Preimplantation Embryos ◽  
Differential Staining ◽  
Rt Pcr

In mammals, DNA methylation and the modification of histones account for the major epigenetic alterations. Usually DNA methylation is associated with transcriptional silencing, which is catalyzed by two important classes of DNA methyltransferases. DNA methyltransferase 1 (Dnmt1), a maintenance enzyme, methylates hemi-methylated DNA after DNA replication. Dnmt3a and Dnmt3b are required for de novo methylation in vivo and for establishing new DNA methylation marks during development. In addition to DNA methylation, post-translational modifications of the amino termini of core histones are thought to affect the expression or repression of transcription. Histone deacetylation catalyzsed by histone deacetylases (Hdac) often results in transcriptional repression. Perturbated epigenetic reprogramming of the genome is a likely cause of developmental abnormalities and epigenetic diseases introduced by assisted reproduction technologies. The objective of the present study was to determine the relative abundance of Dnmt1, Dnmt3a, and Hdac2 transcripts in ICM and TE cells of pre-implantation bovine embryos of different origin. Embryos were generated with standard protocols of in vitro production (IVP) and parthenogenetic activation using SOF medium supplemented with BSA (SOF d7/d8, Parth d7/d8) or TCM medium supplemented with estrus cow serum (TCM d7/d8; Wrenzycki et al. 2001 Hum. Reprod. 16, 893–901; Wrenzycki et al. 2002 Biol. Reprod. 66, 127–134). Expanded blastocysts were collected at Days 7 and Day 8 (d7/d8) of culture (Day 0 = IVF). In vivo-generated blastocysts collected from superovulated animals were included as controls (Wrenzycki et al. 1996 J. Reprod. Fert. 108, 17–24). A highly sensitive RT-PCR assay (Wrenzycki et al. 2003 Biol. Reprod. 68, 2073–2080) was used to determine the relative abundance (RA) of gene transcripts in isolated ICM and TE cells. The allocation of ICM and TE cells was analyzed using a differential staining technique (Eckert and Niemann 1998 Mol. Hum. Reprod. 4, 957–965) to calculate the RA of each transcript on a per cell basis. RT-PCR assays were repeated at least six times. Significant differences in the RA of Dnmt1 (In vivo, TCM d7, Parth d7/d8, SOF d8), Dnmt3a (In vivo, SOF d7), and Hdac2 (TCM d7) transcripts were detected between ICM and TE cells. No differences were detected for all transcripts in ICM cells of embryos collected at either Day 7 or Day 8. In TE cells, the RA of Dnmt1 transcripts was significantly reduced or increased in embryos generated in SOF or TCM medium at Day 7, respectively, and the RA of Hdac2 transcripts was significantly higher in embryos generated in TCM medium at Day 8. These results suggest that in vitro culture alters the spatial expression pattern of genes related to epigenetic modifications in the cell lineages critical for a normal embryonic and fetal development. This work was supported by the Deutsche Forschungsgemeinschaft (DFG Ni 256/18-1).

scholarly journals Overexpression of the Oral Mucosa-Specific microRNA-31 Promotes Skin Wound Closure

2019 ◽  
Vol 20 (15) ◽  
pp. 3679 ◽  
Author(s):  
Lin Chen ◽  
Alyne Simões ◽  
Zujian Chen ◽  
Yan Zhao ◽  
Xinming Wu ◽  
...  
Keyword(s):  
Wound Healing ◽  
Oral Mucosa ◽  
Wound Closure ◽  
Expression Patterns ◽  
Skin Wound ◽  
Skin Wound Healing ◽  
Specific Expression ◽  
Keratinocyte Proliferation

Wounds within the oral mucosa are known to heal more rapidly than skin wounds. Recent studies suggest that differences in the microRNAome profiles may underlie the exceptional healing that occurs in oral mucosa. Here, we test whether skin wound-healing can be accelerating by increasing the levels of oral mucosa-specific microRNAs. A panel of 57 differentially expressed high expresser microRNAs were identified based on our previously published miR-seq dataset of paired skin and oral mucosal wound-healing [Sci. Rep. (2019) 9:7160]. These microRNAs were further grouped into 5 clusters based on their expression patterns, and their differential expression was confirmed by TaqMan-based quantification of LCM-captured epithelial cells from the wound edges. Of these 5 clusters, Cluster IV (consisting of 8 microRNAs, including miR-31) is most intriguing due to its tissue-specific expression pattern and temporal changes during wound-healing. The in vitro functional assays show that ectopic transfection of miR-31 consistently enhanced keratinocyte proliferation and migration. In vivo, miR-31 mimic treatment led to a statistically significant acceleration of wound closure. Our results demonstrate that wound-healing can be enhanced in skin through the overexpression of microRNAs that are highly expressed in the privileged healing response of the oral mucosa.

scholarly journals Single cell RNA-seq reveals genes vital to in vitro fertilized embryos and parthenotes in pigs

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Zhi-Qiang Du ◽  
Hao Liang ◽  
Xiao-Man Liu ◽  
Yun-Hua Liu ◽  
Chonglong Wang ◽  
...  
Keyword(s):  
Embryo Development ◽  
Gene Networks ◽  
Regulatory Networks ◽  
Rna Binding ◽  
Expression Patterns ◽  
Early Embryo ◽  
Specific Factor ◽  
Early Embryos ◽  
Genome Activation

AbstractSuccessful early embryo development requires the correct reprogramming and configuration of gene networks by the timely and faithful execution of zygotic genome activation (ZGA). However, the regulatory principle of molecular elements and circuits fundamental to embryo development remains largely obscure. Here, we profiled the transcriptomes of single zygotes and blastomeres, obtained from in vitro fertilized (IVF) or parthenogenetically activated (PA) porcine early embryos (1- to 8-cell), focusing on the gene expression dynamics and regulatory networks associated with maternal-to-zygote transition (MZT) (mainly maternal RNA clearance and ZGA). We found that minor and major ZGAs occur at 1-cell and 4-cell stages for both IVF and PA embryos, respectively. Maternal RNAs gradually decay from 1- to 8-cell embryos. Top abundantly expressed genes (CDV3, PCNA, CDR1, YWHAE, DNMT1, IGF2BP3, ARMC1, BTG4, UHRF2 and gametocyte-specific factor 1-like) in both IVF and PA early embryos identified are of vital roles for embryo development. Differentially expressed genes within IVF groups are different from that within PA groups, indicating bi-parental and maternal-only embryos have specific sets of mRNAs distinctly decayed and activated. Pathways enriched from DEGs showed that RNA associated pathways (RNA binding, processing, transport and degradation) could be important. Moreover, mitochondrial RNAs are found to be actively transcribed, showing dynamic expression patterns, and for DNA/H3K4 methylation and transcription factors as well. Taken together, our findings provide an important resource to investigate further the epigenetic and genome regulation of MZT events in early embryos of pigs.

The lung-specific CC10 gene is regulated by transcription factors from the AP-1, octamer, and hepatocyte nuclear factor 3 families

1993 ◽  
Vol 13 (7) ◽  
pp. 3860-3871
Author(s):  
P L Sawaya ◽  
B R Stripp ◽  
J A Whitsett ◽  
D S Luse
Keyword(s):  
Transcription Factors ◽  
Rat Liver ◽  
Transcription Initiation ◽  
Clara Cell ◽  
Clara Cells ◽  
Mobility Shift ◽  
Specific Expression ◽  
Fetal Sheep ◽  
Region I

We have shown that a large fragment (-2339 to +57) from the rat CC10 gene directed lung-specific expression of a reporter construct in transgenic animals. Upon transfection, a smaller fragment (-165 to +57) supported reporter gene expression exclusively in the Clara cell-like NCI-H441 cell line, suggesting that a Clara cell-specific transcriptional element resided on this fragment (B. R. Stripp, P. L. Sawaya, D. S. Luse, K. A. Wikenheiser, S. E. Wert, J. A. Huffman, D. L. Lattier, G. Singh, S. L. Katyal, and J. A. Whitsett, J. Biol. Chem. 267:14703-14712, 1992). The interactions of nuclear proteins with a particular segment of the CC10 promoter which extends from 79 to 128 bp upstream of the CC10 transcription initiation site (CC10 region I) have now been studied. This sequence can stimulate both in vitro transcription in H441 nuclear extract and transient expression of reporter constructs in H441 cells. Electrophoretic mobility shift assays using extracts from H441, HeLa, rat liver, and fetal sheep lung cells were used to demonstrate that members of the AP-1, octamer, and HNF-3 families bind to CC10 region I. Transcription factors from H441 cells which are capable of binding to CC10 region I are either absent in HeLa, rat liver, and fetal sheep lung extracts or enriched in H441 extracts relative to extracts from non-Clara cells.

scholarly journals 256CONSTRUCTION OF A BOVINE CDNA ARRAY TO MONITOR GENE EXPRESSION PROFILES IN BOVINE PREIMPLANTATION EMBRYOS

2004 ◽  
Vol 16 (2) ◽  
pp. 248
Author(s):  
C. Wrenzycki ◽  
T. Brambrink ◽  
D. Herrmann ◽  
J.W. Carnwath ◽  
H. Niemann
Keyword(s):  
Gene Expression ◽  
Gene Expression Profiling ◽  
Expression Profiling ◽  
Expression Profiles ◽  
Cdna Array ◽  
Preimplantation Embryos ◽  
Average Insert Size ◽  
Rt Pcr ◽  
Public Data

Array technology is a widely used tool for gene expression profiling, providing the possibility to monitor expression levels of an unlimited number of genes in various biological systems including preimplantation embryos. The objective of the present study was to develop and validate a bovine cDNA array and to compare expression profiles of embryos derived from different origins. A bovine blastocyst cDNA library was generated. Poly(A+)RNA was extracted from in vitro-produced embryos using a Dynabead mRNA purification kit. First-strand synthesis was performed with SacIT21 primer followed by randomly primed second-strand synthesis with a DOP primer mix (Roche) and a global PCR with 35 cycles using SacIT21 and DOP primers. Complementary DNA fragments from 300 to 1500bp were extracted from the gel and normalized via reassoziation and hydroxyapatite chromatography. Resulting cDNAs were digested with SacI and XhoI, ligated into a pBKs vector, and transfected into competent bacteria (Stratagene). After blue/white colony selection, plasmids were extracted and the inserts were subjected to PCR using vector specific primers. Average insert size was determined by size idenfication on agarose gels stained with ethidium bromide. After purification via precipitation and denaturation, 192 cDNA probes were double-spotted onto a nylon membrane and bound to the membrane by UV cross linking. Amplified RNA (aRNA) probes from pools of three or single blastocysts were generated as described recently (Brambrink et al., 2002 BioTechniques, 33, 3–9) and hybridized to the membranes. Expression profiles of in vitro-produced blastocysts cultured in either SOF plus BSA or TCM plus serum were compared with those of diploid parthenogenetic ones generated by chemical activation. Thirty-three probes have been sequenced and, after comparison with public data bases, 26 were identified as cDNAs or genes. Twelve out of 192 (6%) seem to be differentially expressed within the three groups;; 7/12 (58%) were down-regulated, 3/12 (25%) were up-regulated in SOF-derived embryos, and 2/12 (20%) were up-regulated in parthenogenetic blastocysts compared to their in vitro-generated counterparts. Three of these genes involved in calcium signaling (calmodulin, calreticulin) and regulation of actin cytoskeleton (destrin) were validated by semi-quantitative RT-PCR (Wrenzycki et al., 2001 Biol. Reprod. 65, 309–317) employing poly(A+) RNA from a single blastocyst as starting material. No differences were detected in the relative abundance of the analysed gene transcripts within the different groups. These findings were confirmed employing the aRNA used for hybridization in RT-PCR and showed a good representativity of the selected transcripts. Results indicate that it is possible to construct a homologous cDNA array which could be used for gene expression profiling of bovine preimplantation embryos. Supported by the Deutsche Forschungsgemeinschaft (DFG Ni 256/18-1).

scholarly journals Development of cDNA normalization system and preliminary transcription analysis of KCS genes in apple tissues

2011 ◽  
Vol 59 (3) ◽  
pp. 9-12 ◽  
Author(s):  
Zsolt Albert ◽  
Cs. Deák ◽  
A. Miskó ◽  
M. Tóth ◽  
I. Papp
Keyword(s):  
Gene Expression ◽  
Expression System ◽  
Expression Patterns ◽  
Housekeeping Genes ◽  
Apple Fruit ◽  
Rt Pcr ◽  
Specific Primers ◽  
Specific Expression ◽  
Transcription Analysis ◽  
Tissue Specific

Wax production is an important aspect of apple (Malus domestica Borkh.) fruit development from both theoretical and practical point of views. The complex molecular mechanism that controls wax biosynthesis is still widely unknown but many studies focused on this topic. We aimed to develop further the experimental framework of these efforts with a description of an improved reference genes expression system. Results in the literature show that similarities exist among the expression of some housekeeping genes of different plant species. Based on these considerations and on gene expression data from Arabidopsis thaliana, some genes in apple were assigned for analysis. EST sequences of apple were used to design specific primers for RT-PCR experiments. Isolation of intact RNA from different apple tissues and performing RT-PCR reaction were also key point in obtaining expression patterns. To monitor DNA contamination of the RNA samples, specific primers were used that amplify intron-containing sequences from the cDNA. We found that actin primers can be used for the detection of intron containing genomic DNA, and tubulin primers are good internal controls in RT-PCR experiments. We were able to make a difference between tissue-specific and tissue-independent gene-expression, furthermore we found tissue specific differences between the expression patterns of candidate genes, that are potentially involved in wax-biosynthesis. Our results show that KCS1 and KCS4 are overexpressed in the skin tissue, this could mean that these genes have skin-specific expression in apple fruit.

scholarly journals AtHB7/12 Regulate Root Growth in Response to Aluminum Stress

2020 ◽  
Vol 21 (11) ◽  
pp. 4080
Author(s):  
Yang Liu ◽  
Jiameng Xu ◽  
Siyi Guo ◽  
Xianzheng Yuan ◽  
Shan Zhao ◽  
...  
Keyword(s):  
Cell Wall ◽  
Transcription Factors ◽  
Root Growth ◽  
Crop Production ◽  
Expression Patterns ◽  
Acid Soils ◽  
Limiting Factor ◽  
Aluminum Stress ◽  
Al Stress

Aluminum (Al) stress is a major limiting factor for plant growth and crop production in acid soils. At present, only a few transcription factors involved in the regulation of Al resistance have been characterized. Here, we used reversed genetic approach through phenotype analysis of overexpressors and mutants to demonstrate that AtHB7 and AtHB12, two HD-Zip I transcription factors, participate in Al resistance. In response to Al stress, AtHB7 and AtHB12 displayed different dynamic expression patterns. Although both AtHB7 and AtHB12 positively regulate root growth in the absence of Al stress, our results showed that AtHB7 antagonizes with AtHB12 to control root growth in response to Al stress. The athb7/12 double mutant displayed a wild-type phenotype under Al stress. Consistently, our physiological analysis showed that AtHB7 and AtHB12 oppositely regulate the capacity of cell wall to bind Al. Yeast two hybrid assays showed that AtHB7 and AtHB12 could form homo-dimers and hetero-dimers in vitro, suggesting the interaction between AtHB7 and AtHB12 in the regulation of root growth. The conclusion was that AtHB7 and AtHB12 oppositely regulate Al resistance by affecting Al accumulation in root cell wall.

scholarly journals EMT-Inducing Transcription Factors, Drivers of Melanoma Phenotype Switching, and Resistance to Treatment

Cancers ◽  
2020 ◽  
Vol 12 (8) ◽  
pp. 2154 ◽  
Author(s):  
Yaqi Tang ◽  
Simon Durand ◽  
Stéphane Dalle ◽  
Julie Caramel
Keyword(s):  
Transcription Factors ◽  
Neural Crest ◽  
Epithelial Mesenchymal Transition ◽  
Expression Patterns ◽  
Specific Expression ◽  
Therapeutic Targeting ◽  
Mesenchymal Transition ◽  
Neural Crest Stem Cell ◽  
Resistance To Treatment ◽  
Phenotype Switching

Transcription factors, extensively described for their role in epithelial–mesenchymal transition (EMT-TFs) in epithelial cells, also display essential functions in the melanocyte lineage. Recent evidence has shown specific expression patterns and functions of these EMT-TFs in neural crest-derived melanoma compared to carcinoma. Herein, we present an update of the specific roles of EMT-TFs in melanocyte differentiation and melanoma progression. As major regulators of phenotype switching between differentiated/proliferative and neural crest stem cell-like/invasive states, these factors appear as major drivers of intra-tumor heterogeneity and resistance to treatment in melanoma, which opens new avenues in terms of therapeutic targeting.

In vitro and in vivo culture effects on mRNA expression of genes involved in metabolism and apoptosis in bovine embryos

2005 ◽  
Vol 17 (8) ◽  
pp. 775 ◽  
Author(s):  
Hiemke M. Knijn ◽  
Christine Wrenzycki ◽  
Peter J. M. Hendriksen ◽  
Peter L. A. M. Vos ◽  
Elly C. Zeinstra ◽  
...  
Keyword(s):  
Glucose Metabolism ◽  
Mrna Expression ◽  
Critical Period ◽  
Glucose Transporter ◽  
Expression Patterns ◽  
Culture Conditions ◽  
Glut 4 ◽  
Gene Transcripts

Bovine blastocysts produced in vitro differ substantially from their in vivo-derived counterparts with regard to glucose metabolism, level of apoptosis and mRNA expression patterns. Maternal embryonic genomic transition is a critical period in which these changes could be induced. The goals of the present study were twofold: (1) to identify the critical period of culture during which the differences in expression of gene transcripts involved in glucose metabolism are induced; and (2) to identify gene transcripts involved in apoptosis that are differentially expressed in in vitro- and in vivo-produced blastocysts. Relative abundances of transcripts for the glucose transporters Glut-1, Glut-3, Glut-4 and Glut-8, and transcripts involved in the apoptotic cascade, including BAX, BCL-XL, XIAP and HSP 70.1, were analysed by a semiquantitative reverse transcription–polymerase chain reaction assay in single blastocysts produced in vitro or in vivo for specific time intervals, that is, before or after maternal embryonic transition. Whether the culture environment was in vitro or in vivo affected the expression of glucose transporter transcripts Glut-3, Glut-4 and Glut-8. However, the critical period during culture responsible for these changes, before or after maternal embryonic transition, could not be determined. With the exception of XIAP, no effects of culture system on the mRNA expression patterns of BAX, BCL-XL and HSP 70.1 could be observed. These data show that expression of XIAP transcripts in expanded blastocysts is affected by in vitro culture. These findings add to the list of bovine genes aberrantly expressed in culture conditions, but do not support the hypothesis that maternal embryonic transition is critical in inducing the aberrations in gene expression patterns studied here.

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