Microinjection of in vitro transcribed RNA and antisense oligonucleotides in mouse oocytes and early embryos to study the gain- and loss-of-function of genes

1996 ◽  
Vol 6 (2) ◽  
pp. 191-199
Author(s):  
Ismail Kola ◽  
Sony Heru Sumarsono
Keyword(s):  
Antisense Oligonucleotides ◽  
Loss Of Function ◽  
Mouse Oocytes ◽  
Early Embryos ◽  
Gain And Loss

DNA methylation pattern in mouse oocytes and their in vitro fertilized early embryos: effect of oocyte vitrification

Zygote ◽  
2012 ◽  
Vol 22 (2) ◽  
pp. 138-145 ◽  
Author(s):  
Ying Liang ◽  
Xiang-Wei Fu ◽  
Jun-Jie Li ◽  
Dian-Shuai Yuan ◽  
Shi-En Zhu
Keyword(s):  
Dna Methylation ◽  
Fluorescein Isothiocyanate ◽  
Methylation Pattern ◽  
Oocyte Vitrification ◽  
Developmental Potential ◽  
Mouse Oocytes ◽  
Early Embryos ◽  
Vitrification Solution ◽  
Early Mouse

SummaryThis study was conducted to investigate the pattern of DNA methylation in vitrified–thawed mouse oocytes and their in vitro fertilized early embryos. Firstly, mouse oocytes at metaphase II (MII) stage of meiosis were allocated randomly into three groups: (1) untreated (control); (2) exposed to vitrification solution without being plunged into liquid nitrogen (toxicity); or (3) vitrified by open-pulled straw (OPS) method (vitrification). Oocytes from all three groups were fertilized subsequently in vitro. The level of DNA methylation in the MII oocytes and their early embryos was then examined by immunofluorescence using an anti-5-methylcytosine (anti-5-MeC) monoclonal antibody and fluorescein isothiocyanate (FITC)-conjugated goat anti-mouse IgG. Developmental rates to 2-cell embryos (62.28%) and blastocysts (43.68%) of the vitrified–thawed oocytes were lower (P < 0.01) than those of fresh oocytes (81.47%, 61.99%) and vitrification solution treated (79.20%, 60.04%) oocytes. DNA methylation (as reflected by 5-MeC fluorescence intensity) in the vitrification group was less (P < 0.01) for MII oocyte and 2- to 8-cell stages compared with that in the control and toxicity groups. Accordingly, a reduction in global genomic methylation due to vitrification of MII oocytes may result in compromised in vitro developmental potential in early mouse embryos.

scholarly journals MiR-21-3p regulation FGFR1/FGF21/PPARγ pathway induces atrial fibrosis by targeting FGFR1 in diabetes

2020 ◽  
Author(s):  
Jian-an Pan ◽  
Hao Lin ◽  
Jian-ying Yu ◽  
Hui-li Zhang ◽  
Jun-feng Zhang ◽  
...  
Keyword(s):  
Growth Factor Receptor ◽  
Loss Of Function ◽  
Atrial Fibrosis ◽  
Diabetic Model ◽  
Direct Target ◽  
Gain And Loss ◽  
Masson Staining

Abstract Background: A relationship between the abundance of epicardial adipose tissue (EAT) and the risk of atrial fibrosis and atrial fibrillation (AF) in diabetes mellitus (DM) has been reported. And previous studies have shown that MicroRNA-21 (miR-21) is a regulatory factor in atrial fibrosis and AF. The aim of this study was to examine the role of different subtypes of miR-21 in EAT browning and atrial fibrosis under hyperglycemia conditions.Methods: In vivo, C57BL/6 wild type (WT) and miR-21 knockout (KO) mice were used to establish the diabetic model by intraperitoneal injection of streptozotocin (STZ). In vitro, the EAT adipocytes from miR-21 KO mice were cultured and transfected with miR-21-3p mimic or miR-21-5p mimic and co-cultured with atrial fibroblasts in both HG or LG conditions. The browning of EAT and the fibrosis of fibroblasts were assessed by western blotting, immunofluorescence, Masson staining, and ELISA. The gain- and loss-of-function experiments were used to identified fibroblast growth factor receptor 1 (FGFR1) as the target gene of miR-21-3p.Results: In patients with DM and/or AF, serum hsa-miR-21-3p, instead of hsa-miR-21-5p, was significantly up-regulated. And miR-21 KO clearly ameliorated the atrial fibrosis in the diabetic mice. miR-21-3p as a key regulator that controls EAT browning and participates in atrial fibrosis under hyperglycemia conditions. Moreover, our gain- and loss-of-function experiments showed that FGFR1, as a direct target of miR-21-3p identified a regulatory pathway in EAT adipocytes. Conclusions: MiR-21-3p regulated EAT browning and participated the process of hyperglycemia-induced atrial fibrosis by targeting FGFR1.

Pelle kinase is activated by autophosphorylation during Toll signaling in Drosophila

Development ◽  
2002 ◽  
Vol 129 (8) ◽  
pp. 1925-1933 ◽  
Author(s):  
Baohe Shen ◽  
James L. Manley
Keyword(s):  
Dependent Manner ◽  
Loss Of Function ◽  
Concentration Dependent Manner ◽  
Downstream Target ◽  
Toll Signaling ◽  
Early Embryos ◽  
High Concentrations ◽  
L2 Cells

The Drosophila Pelle kinase plays a key role in the evolutionarily conserved Toll signaling pathway, but the mechanism responsible for its activation has been unknown. We present in vivo and in vitro evidence establishing an important role for concentration-dependent autophosphorylation in the signaling process. We first show that Pelle phosphorylation can be detected transiently in early embryos, concomitant with activation of signaling. Importantly, Pelle phosphorylation is enhanced in a gain-of-function Toll mutant (Toll10b), but decreased by loss-of-function Toll alleles. Next we found that Pelle is phosphorylated in transfected Schneider L2 cells in a concentration-dependent manner such that significant modification is observed only at high Pelle concentrations, which coincide with levels required for phosphorylation and activation of the downstream target, Dorsal. Pelle phosphorylation is also enhanced in L2 cells co-expressing Toll10b, and is dependent on Pelle kinase activity. In vitro kinase assays revealed that recombinant, autophosphorylated Pelle is far more active than unphosphorylated Pelle. Importantly, unphosphorylated Pelle becomes autophosphorylated, and activated, by incubation at high concentrations. We discuss these results in the context of Toll-like receptor mediated signaling in both flies and mammals.

Hydrogen peroxide levels in mouse oocytes and early cleavage stage embryos developed in vitro or in vivo

Development ◽  
1990 ◽  
Vol 109 (2) ◽  
pp. 501-507 ◽  
Author(s):  
M.H. Nasr-Esfahani ◽  
J.R. Aitken ◽  
M.H. Johnson
Keyword(s):  
Reproductive Tract ◽  
Female Reproductive Tract ◽  
Cell Stage ◽  
Mouse Oocytes ◽  
Early Embryos ◽  
Individual Mouse ◽  
The Relationship ◽  
Developmental Block

We describe a fluorimetric method for measuring the level of H2O2 in individual mouse oocytes and early embryos. Levels of H2O2 are low but detectable in unfertilized oocytes recovered freshly from the female reproductive tract. The levels in early cleaving embryos (1-cell to 8-cell stages) immediately after recovery from the female tract seem to be slightly higher the later the stage examined. However, when embryos are cultured in vitro from the 1-cell or early 2-cell stage, H2O2 levels rise when the embryos reach the mid-2-cell stage and remain elevated until they enter the early 4-cell stage. No equivalent elevation of H2O2 is seen during the transition from 1-cell to 2-cell or from 4-cell to 8-cell stages. Embryos that are able to develop successfully in vitro, as well as those that show a developmental block at the 2-cell stage on culture in vitro, both show this rise in H2O2 levels after in vitro culture. The relationship between the rise in H2O2 and the ‘2-cell block’ to development is discussed.

scholarly journals Circ-0005105 activates COL11A1 by targeting miR-20a-3p to promote pancreatic ductal adenocarcinoma progression

2021 ◽  
Vol 12 (7) ◽  
Author(s):  
Gang Ma ◽  
Guichen Li ◽  
Wufeng Fan ◽  
Yuanhong Xu ◽  
Shaowei Song ◽  
...  
Keyword(s):  
Pancreatic Ductal Adenocarcinoma ◽  
Cell Lines ◽  
Poor Prognosis ◽  
Cellular Proliferation ◽  
Ductal Adenocarcinoma ◽  
Loss Of Function ◽  
Gain And Loss ◽  
Proliferation And Invasion

AbstractGrowing evidence indicates that circular RNAs (circRNAs) are closely involved in tumorigenesis, but the association between circRNAs and pancreatic ductal adenocarcinoma (PDAC) is far from clear. Here, we focused on the functional investigation of circ-0005105, a newly identified circRNA, in PDAC progression. In the present study, we assessed circ-0005105 expression in PDAC tissues and cell lines with quantitative reverse transcription–polymerase chain reaction (qRT-PCR). The biological functions of circ-0005105 in cellular proliferation and invasion were identified through gain- and loss-of-function experiments in vitro and in vivo. The interaction between circ-0005105 and the microRNA (miR)-20a-3p–COL11A1 (collagen type XI alpha 1) axis was examined using luciferase reporter and RNA immunoprecipitation assays. We found that circ-0005105 expression was upregulated in both PDAC tissues and cell lines. Higher circ-0005105 expression correlated positively with the malignant clinical phenotype and poor prognosis of patients with PDAC. Gain- and loss-of-function analysis showed that circ-0005105 facilitated both in vitro and in vivo cellular proliferation and invasion. Mechanistically, circ-000510 served as a competing endogenous RNA (ceRNA) of miR-20a-3p and indirectly modulated COL11A1 expression, leading to activation of epithelial–mesenchymal transition (EMT). Rescue experiments suggested that the oncogenic activity of circ-0005105 was dependent on the modulation of the miR-20a-3p–COL11A1 axis. More importantly, COL11A1 overexpression was significantly associated with poor prognosis in PDAC, and silencing COL11A1 reduced PDAC cell tumorigenicity and metastasis. Taken together, our findings confirm for the first time that circ-0005105 has critical functions by regulating the miR-20a-3p–COL11A1 axis. In the clinic, circ-0005105 can act as a potential prognostic marker and therapeutic target in PDAC.

Effect of ezrin regulation by sperm-borne miR-183 on the formation of microvilli and early development of bovine embryos.

2020 ◽  
Author(s):  
Zhenzi Zuo ◽  
Xu Liu ◽  
Zheng Wang ◽  
Fang Qiao ◽  
Jingyi Wang ◽  
...  
Keyword(s):  
Nuclear Transfer ◽  
Apoptotic Index ◽  
Developmental Competence ◽  
Bovine Embryos ◽  
Loss Of Function ◽  
Regulatory Relationship ◽  
Bovine Sperm ◽  
Early Embryos ◽  
Non Coding Rnas

Abstract Background: Mature sperm contain both coding and non-coding RNAs, which can be delivered into an oocyte with the sperm at fertilization. Accumulating evidence shows that these sperm-borne RNAs play crucial roles in epigenetic reprogramming, remodeling, embryonic development, and offspring phenotype. MiR-183 is highly expressed in bovine sperm, and can be delivered into oocytes during fertilization. Results: Here we used bioinformatics and luciferase assays to show that the ezrin gene, EZR, is one of the targets of miR-183 in early embryos, while gain- and loss-of-function studies demonstrated their regulatory relationship. Scanning electron microscopy showed that the density of microvilli on the surface of somatic cell nuclear transfer (SCNT) embryos was significantly higher than on in vitro fertilized (IVF) embryos and was significantly reduced by injection of miR-183. EZR-siRNA injected into SCNT embryos had a similar effect. This indicated that a deficiency in sperm-borne miRNA-183 might lead to abnormal changes in microvilli by down-regulating ezrin protein. We used bioinformatics to select the proteins that worked in combination with ezrin to regulate microvilli function. Co-IP mass spectrometry and immunofluorescence identified SLC9A3R1 as the protein functioning in synergy with ezrin in early bovine embryos. Gain-of-function studies showed that miR-183 significantly improved developmental competence of SCNT embryo in terms of cleavage (76.63% vs 64.32%, p<0.05), blastocyst formation (43.75% vs 28.26%, p<0.05), apoptotic index (5.21% vs 12.64%, p<0.05), and the trophoblast ratio (32.65% vs 25.58%, p<0.05) in day 7 blastocysts. Conclusions: In conclusion, the present studies indicated that sperm-borne miR-183 might influence the formation of microvilli and embryo development by regulating expression of EZR mRNA.

scholarly journals A novel lncRNA ROPM-mediated lipid metabolism governs breast cancer stem cell properties

2021 ◽  
Vol 14 (1) ◽  
Author(s):  
Shuiqing Liu ◽  
Yan Sun ◽  
Yixuan Hou ◽  
Liping Yang ◽  
Xueying Wan ◽  
...  
Keyword(s):  
Breast Cancer ◽  
Stem Cells ◽  
Cancer Stem Cells ◽  
Phospholipid Metabolism ◽  
Luciferase Reporter ◽  
Breast Cancer Patients ◽  
Loss Of Function ◽  
Gain And Loss

Abstract Background Cancer stem cells (CSCs) are considered as the major cause to tumor initiation, recurrence, metastasis, and drug resistance, driving poor clinical outcomes in patients. Long noncoding RNAs (lncRNAs) have emerged as crucial regulators in cancer development and progression. However, limited lncRNAs involved in CSCs have been reported. Methods The novel lncROPM (a regulator of phospholipid metabolism) in breast CSCs (BCSCs) was identified by microarray and validated by qRT-PCR in BCSCs from breast cancer cells and tissues. The clinical significance of lncROPM was evaluated in two breast cancer cohorts and TANRIC database (TCGA-BRCA, RNAseq data). Gain- and loss-of-function assays were performed to examine the role of lncROPM on BCSCs both in vitro and in vivo. The regulatory mechanism of lncROPM was investigated by bioinformatics, RNA FISH, RNA pull-down, luciferase reporter assay, and actinomycin D treatment. PLA2G16-mediated phospholipid metabolism was determined by UHPLC-QTOFMS system. Cells’ chemosensitivity was assessed by CCK8 assay. Results LncROPM is highly expressed in BCSCs. The enhanced lncROPM exists in clinic breast tumors and other solid tumors and positively correlates with malignant grade/stage and poor prognosis in breast cancer patients. Gain- and loss-of-function studies show that lncROPM is required for the maintenance of BCSCs properties both in vitro and in vivo. Mechanistically, lncROPM regulates PLA2G16 expression by directly binding to 3'-UTR of PLA2G16 to increase the mRNA stability. The increased PLA2G16 significantly promotes phospholipid metabolism and the production of free fatty acid, especially arachidonic acid in BCSCs, thereby activating PI3K/AKT, Wnt/β-catenin, and Hippo/YAP signaling, thus eventually involving in the maintenance of BCSCs stemness. Importantly, lncROPM and PLA2G16 notably contribute to BCSCs chemo-resistance. Administration of BCSCs using clinic therapeutic drugs such as doxorubicin, cisplatin, or tamoxifen combined with Giripladib (an inhibitor of cytoplasmic phospholipase A2) can efficiently eliminate BCSCs and tumorigenesis. Conclusions Our study highlights that lncROPM and its target PLA2G16 play crucial roles in sustaining BCSC properties and may serve as a biomarker for BCSCs or other cancer stem cells. Targeting lncROPM-PLA2G16 signaling axis may be a novel therapeutic strategy for patients with breast cancer.

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