scholarly journals Identification of the Role of SOX2 During Early Embryogenesis in Pigs

2020 ◽  
Author(s):  
Mingyun Lee ◽  
Jong-Nam Oh ◽  
Seung-Hun Kim ◽  
Kwang-Hwan Choi ◽  
Dong-Kyung Lee ◽  
...  
Keyword(s):  
Late Stage ◽  
Developmental Process ◽  
Early Stage ◽  
Expression Patterns ◽  
Cell Number ◽  
Early Embryogenesis ◽  
Human Embryos ◽  
Pcr Analysis ◽  
Lineage Specification ◽  
Model Animal

Abstract BackgroundThe lineage specification of mammalian embryos during preimplantation development has been studied for a long time but is still unclear. To understand the developmental process, many studies have examined lineage markers and mechanisms focusing on mouse embryos, but there are differences from human embryos. Pigs have been studied extensively in the field of disease model animals and xenotransplantation because of their physiological similarity with humans. Therefore, it is necessary to analyze gene expression patterns and lineage specification markers during early embryogenesis in pigs, a model animal similar to humans.ResultsAnalysis of the expression pattern of the core pluripotent factors (OCT4, SOX2 and NANOG) of preimplantation porcine embryos showed that SOX2 was only expressed in some cells from the early stage, so SOX2 was selected as an ICM inducible factor candidate. Next, transcript and protein expression patterns were estimated at the early stage (Day 5) and late stage (Day 7) of blastocysts injected with the CRISPR Cas9 system selected through gRNA validation. An ICC assay revealed that the expression of ICM-related genes (SOX2, NANOG and SOX17), except OCT4, was suppressed, and the total cell number was also decreased. Likewise, according to real-time PCR analysis, pluripotency-related genes (NANOG, SOX17 and SMAD7), excluding OCT4, and proliferation-related genes (KDM8 and DDB1) were decreased in SOX2-targeted blastocysts, which showed more differences in late-stage blastocyst than in early-stage blastocyst. Last, in SOX2-overexpressing embryos, the total blastocyst cell number was greatly increased, but the ICM/TE ratio decreased.ConclusionsTaken together, our results demonstrated SOX2 is essential for ICM formation and cell proliferation in porcine early stage embryogenesis. These findings will help to elucidate gene regulation related to lineage specification during porcine early development.

scholarly journals MircroRNA Profiles of Early Rice Inflorescence Revealed a Specific miRNA5506 Regulating Development of Floral Organs and Female Megagametophyte in Rice

2021 ◽  
Vol 22 (12) ◽  
pp. 6610
Author(s):  
Zhixiong Chen ◽  
Yajing Li ◽  
Peigang Li ◽  
Xiaojie Huang ◽  
Mingxin Chen ◽  
...  
Keyword(s):  
Developmental Process ◽  
Early Stage ◽  
Expression Patterns ◽  
Embryo Sac ◽  
Floral Organ ◽  
Inflorescence Development ◽  
Floral Organs ◽  
Transgenic Lines ◽  
Mirnas Expression ◽  
Female Gametophyte Development

The developmental process of inflorescence and gametophytes is vital for sexual reproduction in rice. Multiple genes and conserved miRNAs have been characterized to regulate the process. The changes of miRNAs expression during the early development of rice inflorescence remain unknown. In this study, the analysis of miRNAs profiles in the early stage of rice inflorescence development identified 671 miRNAs, including 67 known and 44 novel differentially expressed miRNAs (DEMs). Six distinct clusters of miRNAs expression patterns were detected, and Cluster 5 comprised 110 DEMs, including unconserved, rice-specific osa-miR5506. Overexpression of osa-miR5506 caused pleiotropic abnormalities, including over- or under-developed palea, various numbers of floral organs and spikelet indeterminacy. In addition, the defects of ovaries development were frequently characterized by multiple megasporocytes, ovule-free ovary, megasporocyte degenerated and embryo sac degenerated in the transgenic lines. osa-miR5506 targeted REM transcription factor LOC_Os03g11370. Summarily, these results demonstrated that rice-specific osa-miR5506 plays an essential role in the regulation of floral organ number, spikelet determinacy and female gametophyte development in rice.

scholarly journals Comparative transcriptome analysis of inbred lines and contrasting hybrids reveals overdominance mediate early biomass vigor in hybrid cotton

2020 ◽  
Author(s):  
Kashif Shahzad ◽  
Xuexian Zhang ◽  
Liping Guo ◽  
Tingxiang Qi ◽  
Huini Tang ◽  
...  
Keyword(s):  
Circadian Rhythm ◽  
Transcriptome Analysis ◽  
Molecular Mechanisms ◽  
Early Stage ◽  
Expression Patterns ◽  
Pcr Analysis ◽  
Genetic Constitution ◽  
Comparative Transcriptome ◽  
Comparative Transcriptome Analysis ◽  
Extracellular Region

Abstract Background: Heterosis breeding is the most useful method for yield increase around the globe. Heterosis is an intriguing process to develop superior offspring to either parent in the desired character. The biomass vigor produced during seedling emergence stage has a direct influence on yield heterosis in plants. Unfortunately, the genetic basis of early biomass vigor in cotton is poorly understood. Results: Three stable performing F1 hybrids varying in yield heterosis named as high, medium and low hybrids with their inbred parents were used in this study. Phenotypically, these hybrids established noticeable biomass heterosis during the early stage of seedling growth in the field. Transcriptome analysis of root and leaf revealed that hybrids showed many differentially expressed genes (DEGs) relative to their parents, while the comparison of inbred parents showed limited number of DEGs indicating similarity in their genetic constitution. Further analysis indicated expression patterns of most DEGs were overdominant in both tissues of hybrids. According to GO results, functions of overdominance genes in leaf were enriched for chloroplast, membrane, and protein binding, whereas functions of overdominance genes in root were enriched for plasma membrane, extracellular region, and responses to stress. We found several genes of circadian rhythm pathway related to LATE ELONGATED HYPOCOTYL (LHY) showed downregulated overdominant expressions in both tissues of hybrids. In addition to circadian rhythm, several leaf genes related to Aux/IAA regulation, and many root genes involved in peroxidase activity also showed overdominant expressions in hybrids. Twelve genes involved in circadian rhythm plant were selected to perform qRT-PCR analysis to confirm the accuracy of RNA-seq results. Conclusions: Through genome-wide comparative transcriptome analysis, we strongly predict that overdominance at gene expression level plays a pivotal role in early biomass vigor of hybrids. The combinational contribution of circadian rhythm and other metabolic process may control vigorous growth in hybrids. Our result provides an important foundation for dissecting molecular mechanisms of biomass vigor in hybrid cotton.

scholarly journals Involvement of Clostridium botulinum ATCC 3502 Sigma Factor K in Early-Stage Sporulation

2012 ◽  
Vol 78 (13) ◽  
pp. 4590-4596 ◽  
Author(s):  
David G. Kirk ◽  
Elias Dahlsten ◽  
Zhen Zhang ◽  
Hannu Korkeala ◽  
Miia Lindström
Keyword(s):  
Late Stage ◽  
Sigma Factor ◽  
Parent Strain ◽  
Clostridium Botulinum ◽  
Early Stage ◽  
Gene Knockout ◽  
Model Organism ◽  
Stage Iv ◽  
Pcr Analysis ◽  
Content Type

ABSTRACTA key survival mechanism ofClostridium botulinum, the notorious neurotoxic food pathogen, is the ability to form heat-resistant spores. While the genetic mechanisms of sporulation are well understood in the model organismBacillus subtilis, nothing is known about these mechanisms inC. botulinum.Using the ClosTron gene-knockout tool,sigK, encoding late-stage (stage IV) sporulation sigma factor K inB. subtilis, was disrupted inC. botulinumATCC 3502 to produce two different mutants with distinct insertion sites and orientations. Both mutants were unable to form spores, and their elongated cell morphology suggested that the sporulation pathway was blocked at an early stage. In contrast,sigK-complemented mutants sporulated successfully. Quantitative real-time PCR analysis ofsigKin the parent strain revealed expression at the late log growth phase in the parent strain. Analysis ofspo0A, encoding the sporulation master switch, in thesigKmutant and the parent showed significantly reduced relative levels ofspo0Aexpression in thesigKmutant compared to the parent strain. Similarly,sigFshowed significantly lower relative transcription levels in thesigKmutant than the parent strain, suggesting that the sporulation pathway was blocked in thesigKmutant at an early stage. We conclude that σKis essential for early-stage sporulation inC. botulinumATCC 3502, rather than being involved in late-stage sporulation, as reported for the sporulation model organismB. subtilis. Understanding the sporulation mechanism ofC. botulinumprovides keys to control the public health risks that the spores of this dangerous pathogen cause through foods.

5 CLONING AND CHARACTERIZATION OF NEWBORN OVARY HOMEOBOX GENE (NOBOX) IN CATTLE

2010 ◽  
Vol 22 (1) ◽  
pp. 160
Author(s):  
S. K. Tripurani ◽  
K. B. Lee ◽  
L. Wang ◽  
G. W. Smith ◽  
J. Yao
Keyword(s):  
Bovine Genome ◽  
Expression Patterns ◽  
Homeobox Gene ◽  
Early Embryogenesis ◽  
Pcr Analysis ◽  
Dna Encoding ◽  
Coding Region ◽  
Rt Pcr ◽  
Cell Stage ◽  
Genome Database

Newborn ovary homeobox (NOBOX) is a homeobox gene that is preferentially expressed in the oocytes and is essential for folliculogenesis in mice. NOBOX knockout mice are infertile, and lack of NOBOX perturbs the expression of many germ-cell-specific genes and microRNAs. Furthermore, mutations in the NOBOX gene associated with premature ovarian failure have been described in humans. However, the temporal and cell-specific expression of NOBOX in bovine oocytes and the potential function of NOBOX in early embryogenesis have not been described previously. The objectives of this study were to clone the complementary (c)DNA encoding for bovine NOBOX, analyze the expression of NOBOX mRNA in bovine tissues including fetal ovaries of different developmental stages, and characterize the temporal expression patterns of NOBOX mRNA during oocyte maturation and early embryogenesis. Based on the sequence of a predicted cDNA for bovine NOBOX, we successfully amplified, using RT-PCR, a cDNA fragment representing the coding region of bovine NOBOX from bovine fetal ovary cDNA. Additional 5′ and 3′ sequences were obtained using rapid amplification of cDNA ends (RACE) procedures. The assembled full-length NOBOX cDNA is 2275 bp with an open reading frame encoding a protein of 500 amino acids with a conserved homeodomain and typical nuclear localization signal. The predicted NOBOX protein shares 61% and 49% amino acid sequence identity with its human and mouse counterparts, respectively. A BLAST search of the bovine genome database at the National Center for Biotechnology Information (NCBI) revealed that the bovine NOBOX gene is located on chromosome 4, spans approximately 5.5 kb, and is encoded by 7 exons. Northern blot analysis revealed an approximately 2.3-kb bovine NOBOX RNA transcript. RT-PCR analysis of RNA samples from a panel of 14 different bovine tissues revealed that expression of NOBOX mRNA is restricted to ovarian samples and can be detected in fetal ovaries harvested as early as 105 days of gestation, when primary follicles start to form. Further RT-PCR analysis using RNA isolated from oocytes and granulosa and cumulus cells of antral follicles indicates that bovine NOBOX is expressed in oocytes but not in other follicular cells. Real-time PCR analysis demonstrated that NOBOX mRNA is abundant in germinal vesicle and metaphase II stage oocytes, as well as from pronuclear to 8-cell stage embryos, but barely detectable in embryos collected at the morula and blastocyst stages, suggesting that NOBOX might be a maternal effect gene. Collectively, our results demonstrate that bovine NOBOX is specifically expressed in oocytes and may play a role in early embryonic development in addition to its known function in folliculogenesis.

scholarly journals Flower Color Diversity Revealed by Differential Expression of Flavonoid Biosynthetic Genes in Sacred Lotus

2016 ◽  
Vol 141 (6) ◽  
pp. 573-582 ◽  
Author(s):  
Yanjie Wang ◽  
Yeqing Chen ◽  
Man Yuan ◽  
Zeyun Xue ◽  
Qijiang Jin ◽  
...  
Keyword(s):  
Anthocyanin Biosynthesis ◽  
Early Stage ◽  
Quantitative Polymerase Chain Reaction ◽  
Expression Patterns ◽  
Flower Color ◽  
Flavonoid Biosynthesis ◽  
Pcr Analysis ◽  
Cdna Clones ◽  
Biosynthetic Genes ◽  
Sacred Lotus

Sacred lotus (Nelumbo nucifera) is an important aquatic ornamental plant which contains several diverse flower colors, but the underlying mechanisms of its flower coloration remain unclear. In this study, seven complementary DNA (cDNA) clones of genes involved in flavonoid biosynthesis, including chalcone synthase (CHS), chalcone isomerase (CHI), flavanone 3-hydroxylase (F3H), flavonoid 3′-hydroxylase (F3′H), flavonoid 3′,5′-hydroxylase (F3′5′H), dihydroflavonol 4-reductase (DFR), and anthocyanidin synthase (ANS), were isolated and characterized. Moreover, expression patterns of these seven genes and pigment profiles were investigated across four N. nucifera cultivars with different flower colors: Zhongguohongbeijing [ZGH (red)], Xinghuafen [XHF (pink)], Molingqiuse [MLQS (yellow)], and Zhufengcuiying [ZFCY (white)]. Real-time quantitative polymerase chain reaction (qRT-PCR) analysis showed that during flower development, transcripts of early biosynthetic genes (NnCHS, NnCHI, and NnF3H) were abundant at the early stage; noticeably, highest expression of NnCHI in MLQS probably induced abundant anthoxanthin synthesis and displayed yellow. Expression of late biosynthetic genes, especially NnDFR and NnANS, was generally consistent with change patterns of anthocyanins in ZGH and XHF, but NnF3′H was barely detectable in the pink cultivars. Meanwhile, negligible expression of NnDFR and NnANS was detected in MLQS and ZFCY, respectively, which blocked their colored anthocyanin biosynthesis. Spatial expression analysis revealed that most flavonoid biosynthetic genes were highly expressed in floral tissues, rather than leaves. These results suggest that in N. nucifera cultivars with different flower colors, flavonoid biosynthesis is differentially regulated by the expression of these flavonoid biosynthetic genes, among which, NnCHI, NnF3′H, NnDFR, and NnANS are supposed to be critical for pigment accumulation, and therefore, affect different flower coloration.

42 Disruption of endogenous SOX2 during porcine embryo development using the CRIPSR/Cas9 system

2021 ◽  
Vol 33 (2) ◽  
pp. 128
Author(s):  
M. Lee ◽  
J.-N. Oh ◽  
D.-K. Lee ◽  
K.-H. Choi ◽  
S.-H. Kim ◽  
...  
Keyword(s):  
Fluorescent Protein ◽  
Developmental Process ◽  
Inner Cell Mass ◽  
Cell Mass ◽  
Value Added ◽  
Pcr Analysis ◽  
Lineage Specification ◽  
Cell Stage ◽  
Porcine Embryo ◽  
Green Fluorescent

The lineage specification of the pre-implantation embryo is important to understand the developmental process, but it remains unclear because the expression of lineage-specific genes is distinct among species. Pigs have genetic and physiological traits similar to humans; however, there are differences in gene expression during the pre-implantation stage. To select a candidate gene that affects the formation of the inner cell mass (ICM) in porcine embryo, we conducted preliminary experiments. First, we measured the expression level of candidate genes for lineage specification in parthenogenetic-activated embryos. The expression of pluripotent genes peaked on Day 3 and thereafter decreased gradually. Next, we conducted immunocytochemistry. OCT4 was expressed in all cells in morula and Day 5 blastocyst, but some Day 7 blastocysts expressed OCT4 in both ICM and trophectoderm (TE), whereas others expressed OCT4 only in ICM. NANOG was not observed in the morula stage, whereas SOX2 was located in a restricted area. To examine the effect of SOX2 in ICM formation, we injected plasmid expressing Cas9 and guide (g)RNA using Lipofectamine for efficient transgene expression at the 2-cell stage to increase viability by inducing mosaicism. The expression of enhanced green fluorescent protein (EGFP) contained in the plasmid confirmed that the plasmid was operating normally. In SOX2-knockout (KO) early blastocysts, the numbers of total cells and SOX2- and NANOG-positive cells were greatly decreased, while OCT4 was expressed in most cells. As in early blastocysts, SOX2-KO late blastocysts had fewer cells expressing SOX2, NANOG, and SOX17 than control. To identify the transcriptional consequences of SOX2 reduction, we performed quantitative PCR analysis on non-injected and PX458-gRNA injected blastocysts. Injection of PX458-gRNA resulted in downregulation of NANOG, SOX17, and SMAD7, but not SOX2 and OCT4. Furthermore, proliferation-associated genes were downregulated in injected blastocysts. In conclusion, SOX2-targeted porcine embryos showed blastocoel formation, the inner cell mass formed poorly, and embryos have inefficient cells. Also, the depletion of SOX2 in porcine blastocysts downregulated pluripotent genes and proliferation genes. This work was supported by the BK21 Plus Program, the National Research Foundation of Korea (NRF) grant funded by the Korea government (NRF-2019R1C1C1004514), the Korea Institute of Planning and Evaluation for Technology in Food, Agriculture and Forestry (IPET) through the Development of High Value-Added Food Technology Program funded by the Ministry of Agriculture, Food, and Rural Affairs (MAFRA; 118042-03-3-HD020).

scholarly journals Comparative transcriptome analysis of inbred lines and contrasting hybrids reveals overdominance mediate early biomass vigor in hybrid cotton

2020 ◽  
Author(s):  
Kashif Shahzad ◽  
Xuexian Zhang ◽  
Liping Guo ◽  
Tingxiang Qi ◽  
Huini Tang ◽  
...  
Keyword(s):  
Circadian Rhythm ◽  
Transcriptome Analysis ◽  
Molecular Mechanisms ◽  
Early Stage ◽  
Expression Patterns ◽  
Pcr Analysis ◽  
Genetic Constitution ◽  
Comparative Transcriptome ◽  
Comparative Transcriptome Analysis ◽  
Extracellular Region

Abstract Background: Heterosis breeding is the most useful method for yield increase around the globe. Heterosis is an intriguing process to develop superior offspring to either parent in the desired character. The biomass vigor produced during seedling emergence stage has a direct influence on yield heterosis in plants. Unfortunately, the genetic basis of early biomass vigor in cotton is poorly understood. Results : Three stable performing F 1 hybrids varying in yield heterosis named as high, medium and low hybrids with their inbred parents were used in this study. Phenotypically, these hybrids established noticeable biomass heterosis during the early stage of seedling growth in the field. Transcriptome analysis of root and leaf revealed that hybrids showed many differentially expressed genes (DEGs) relative to their parents, while the comparison of inbred parents showed limited number of DEGs indicating similarity in their genetic constitution. Further analysis indicated expression patterns of most DEGs were overdominant in both tissues of hybrids. According to GO results, functions of overdominance genes in leaf were enriched for chloroplast, membrane, and protein binding, whereas functions of overdominance genes in root were enriched for plasma membrane, extracellular region, and responses to stress. We found several genes of circadian rhythm pathway related to LATE ELONGATED HYPOCOTYL (LHY) showed downregulated overdominant expressions in both tissues of hybrids. In addition to circadian rhythm, several leaf genes related to Aux/IAA regulation, and many root genes involved in peroxidase activity also showed overdominant expressions in hybrids. Twelve genes involved in circadian rhythm plant were selected to perform qRT-PCR analysis to confirm the accuracy of RNA-seq results. Conclusions: Through genome-wide comparative transcriptome analysis, we strongly predict that overdominance at gene expression level plays a pivotal role in early biomass vigor of hybrids. The combinational contribution of circadian rhythm and other metabolic process may control vigorous growth in hybrids. Our result provides an important foundation for dissecting molecular mechanisms of biomass vigor in hybrid cotton.

Identification of Differentially Expressed Genes Using cDNA-AFLP During Six Days In Vitro Tuberization of Potato

Zuriat ◽  
2015 ◽  
Vol 14 (1) ◽  
Author(s):  
Nono Carsono ◽  
Christian Bachem
Keyword(s):  
Gene Expression ◽  
Developmental Process ◽  
Expression Patterns ◽  
Induction Medium ◽  
In Vitro Tuberization ◽  
Storage Organ ◽  
Developmentally Regulated ◽  
Study Gene Expression ◽  
Differential Pattern

Tuberization in potato is a complex developmental process resulting in the differentiation of stolon into the storage organ, tuber. During tuberization, change in gene expression has been known to occur. To study gene expression during tuberization over the time, in vitro tuberization system provides a suitable tool, due to its synchronous in tuber formation. An early six days axillary bud growing on tuber induction medium is a crucial development since a large number of genes change in their expression patterns during this period. In order to identify, isolate and sequencing the genes which displaying differential pattern between tuberizing and non-tuberizing potato explants during six days in vitro tuberization, cDNA-AFLP fingerprint, method for the visualization of gene expression using cDNA as template which is amplified to generate an RNA-fingerprinting, was used in this experiment. Seventeen primer combinations were chosen based on their expression profile from cDNA-AFLP fingerprint. Forty five TDFs (transcript derived fragment), which displayed differential expressions, were obtained. Tuberizing explants had much more TDFs, which developmentally regulated, than those from non tuberizing explants. Seven TDFs were isolated, cloned and then sequenced. One TDF did not find similarity in the current databases. The nucleotide sequence of TDF F showed best similarity to invertase ezymes from the databases. The homology of six TDFs with known sequences is discussed in this paper.

scholarly journals Making the Grade during Pandemic: Early-stage and Late-stage Provisional Institutions

2021 ◽  
pp. 073112142110286
Author(s):  
Alexander B. Kinney ◽  
Nicholas J. Rowland
Keyword(s):  
Late Stage ◽  
Early Stage ◽  
State University ◽  
Institutional Work ◽  
Traditional Grading ◽  
Grading Policy ◽  
Temporary Solution ◽  
Rural State ◽  
Empirical Implications ◽  
Common Understanding

This is an article that draws on the institutional work literature about provisional institutions. To date, nearly every U.S. sector has been impacted by COVID-19. To sustain their core missions, highly institutionalized organizations such as universities have had to rethink foundational structures and policies. Using a historical ethnographic approach to investigate records from faculty senate deliberations at “Rural State University” (RSU), the authors examine the implementation of a temporary grading policy to supplement traditional, qualitative grades spring 2020 during the outbreak. The authors find that RSU implemented a temporary, supplemental grading policy as a provisional institution to momentarily supersede traditional grading as a means to—as soon as possible—return to it. This finding contrasts with the common understanding that provisional institutions operate primarily as a temporary solution to a social problem that leads to more stable and enduring, ostensibly nonprovisional institutions. The temporary grading policy, the authors argue, constitutes a “late-stage” provisional institution and, with this new lens, subsequently characterize the more commonplace understanding of provisional institutions as “early-stage.” This contribution has theoretical implications for studies of institutions and empirical implications for research on shared governance and disruption in higher education.

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