scholarly journals Rapid and easy-to-use ES cell manipulation device with a small groove near culturing wells

2020 ◽  
Vol 13 (1) ◽  
Author(s):  
Shun-ichi Funano ◽  
Daisuke Tone ◽  
Hideki Ukai ◽  
Hiroki R. Ueda ◽  
Yo Tanaka
Keyword(s):  
Genetically Modified ◽  
Embryonic Stem ◽  
Operation Time ◽  
Cell Manipulation ◽  
Knock Out ◽  
Training Time ◽  
New Device ◽  
Phenotype Analysis ◽  
Genetically Modified Mice ◽  
Hydrophilic Treatment

Abstract Objective Production of genetically modified mice including Knock-out (KO) or Knock-in (KI) mice is necessary for organism-level phenotype analysis. Embryonic stem cell (ESC)-based technologies can produce many genetically modified mice with less time without crossing. However, a complicated manual operation is required to increase the number of ESC colonies. Here, the objective of this study was to design and demonstrate a new device to easily find colonies and carry them to microwells. Results We developed a polydimethylsiloxane-based device for easy manipulation and isolation of ESC colonies. By introducing ESC colonies into the groove placed near culturing microwells, users can easily find, pick up and carry ESC colonies to microwells. By hydrophilic treatment using bovine serum albumin, 2-μL droplets including colonies reached the microwell bottom. Operation time using this device was shortened for both beginners (2.3-fold) and experts (1.5-fold) compared to the conventional colony picking operation. Isolated ESC colonies were confirmed to have maintained pluripotency. This device is expected to promote research by shortening the isolation procedure for ESC colonies or other large cells (e.g. eggs or embryos) and shortening training time for beginners as a simple sorter.

scholarly journals Genetically Modified Rabbits for Cardiovascular Research

2021 ◽  
Vol 12 ◽  
Author(s):  
Jianglin Fan ◽  
Yanli Wang ◽  
Y. Eugene Chen
Keyword(s):  
Molecular Mechanisms ◽  
Genetically Modified ◽  
Embryonic Stem ◽  
Cardiovascular Research ◽  
Knock Out ◽  
Experimental Animals ◽  
Transgenic Rabbits ◽  
Long Time ◽  
Human Atherosclerosis ◽  
Genome Information

Rabbits are one of the most used experimental animals for investigating the mechanisms of human cardiovascular disease and lipid metabolism because they are phylogenetically closer to human than rodents (mice and rats). Cholesterol-fed wild-type rabbits were first used to study human atherosclerosis more than 100 years ago and are still playing an important role in cardiovascular research. Furthermore, transgenic rabbits generated by pronuclear microinjection provided another means to investigate many gene functions associated with human disease. Because of the lack of both rabbit embryonic stem cells and the genome information, for a long time, it has been a dream for scientists to obtain knockout rabbits generated by homologous recombination-based genomic manipulation as in mice. This obstacle has greatly hampered using genetically modified rabbits to disclose the molecular mechanisms of many human diseases. The advent of genome editing technologies has dramatically extended the applications of experimental animals including rabbits. In this review, we will update genetically modified rabbits, including transgenic, knock-out, and knock-in rabbits during the past decades regarding their use in cardiovascular research and point out the perspectives in future.

scholarly journals Generation of Genetically Modified Mice by Oocyte Injection of Androgenetic Haploid Embryonic Stem Cells

Cell ◽  
2012 ◽  
Vol 149 (3) ◽  
pp. 605-617 ◽  
Author(s):  
Hui Yang ◽  
Linyu Shi ◽  
Bang-An Wang ◽  
Dan Liang ◽  
Cuiqing Zhong ◽  
...  
Keyword(s):  
Stem Cells ◽  
Embryonic Stem Cells ◽  
Genetically Modified ◽  
Embryonic Stem ◽  
Genetically Modified Mice ◽  
Androgenetic Haploid

scholarly journals Robotic Precisely Oocyte Blind Enucleation Method

2021 ◽  
Vol 11 (4) ◽  
pp. 1850
Author(s):  
Xiangfei Zhao ◽  
Maosheng Cui ◽  
Yidi Zhang ◽  
Yaowei Liu ◽  
Xin Zhao
Keyword(s):  
Success Rate ◽  
Nuclear Transfer ◽  
Operation Time ◽  
Volume Control ◽  
Translation Control ◽  
Cleavage Rate ◽  
Training Time ◽  
Manual Method ◽  
Control Oocyte ◽  
Key Techniques

Oocyte enucleation is a critical procedure for somatic cell nuclear transfer. Yet, the main steps of oocyte enucleation are still manually operated, which presents several drawbacks such as low precision, high repetition error, and long training time for operators. For improving the operation efficiency and success rate, a robotic precise oocyte blind enucleation method is presented in this paper. The proposed method involves the following key techniques: oocyte translation control, oocyte immobilization and penetration control, and enucleation volume control based on the adaptive slide mode. Compared with the manual blind enucleation method, the proposed robotic blind enucleation method reduced the operation time by 44.5% (manual method: 62 s vs. proposed method: 34.4 s), increased the accuracy of enucleation by 83.1% (manual method: 30.7 vs. proposed method: 5.2), increased the success rate from 80% to 93.3%, and increased the cleavage rate from 41.7% to 63.3%.

Generating genetically modified mice using CRISPR/Cas-mediated genome engineering

2014 ◽  
Vol 9 (8) ◽  
pp. 1956-1968 ◽  
Author(s):  
Hui Yang ◽  
Haoyi Wang ◽  
Rudolf Jaenisch
Keyword(s):  
Genome Engineering ◽  
Genetically Modified ◽  
Genetically Modified Mice

scholarly journals FUS driven circCNOT6L biogenesis in mouse and human spermatozoa supports zygote development

2021 ◽  
Author(s):  
Teresa Chioccarelli ◽  
Geppino Falco ◽  
Donato Cappetta ◽  
Antonella De Angelis ◽  
Luca Roberto ◽  
...  
Keyword(s):  
Rna Polymerase Ii ◽  
Rna Binding ◽  
Cannabinoid Receptor ◽  
Rna Binding Proteins ◽  
Embryonic Stem ◽  
Circular Rna ◽  
Physical Interaction ◽  
Knock Out ◽  
Maternal Contribution ◽  
Zygote Development

AbstractCircular RNA (circRNA) biogenesis requires a backsplicing reaction, promoted by inverted repeats in cis-flanking sequences and trans factors, such as RNA-binding proteins (RBPs). Among these, FUS plays a key role. During spermatogenesis and sperm maturation along the epididymis such a molecular mechanism has been poorly explored. With this in mind, we chose circCNOT6L as a study case and wild-type (WT) as well as cannabinoid receptor type-1 knock-out (Cb1−/−) male mice as animal models to analyze backsplicing mechanisms. Our results suggest that spermatozoa (SPZ) have an endogenous skill to circularize mRNAs, choosing FUS as modulator of backsplicing and under CB1 stimulation. A physical interaction between FUS and CNOT6L as well as a cooperation among FUS, RNA Polymerase II (RNApol2) and Quaking (QKI) take place in SPZ. Finally, to gain insight into FUS involvement in circCNOT6L biogenesis, FUS expression was reduced through RNA interference approach. Paternal transmission of FUS and CNOT6L to oocytes during fertilization was then assessed by using murine unfertilized oocytes (NF), one-cell zygotes (F) and murine oocytes undergoing parthenogenetic activation (PA) to exclude a maternal contribution. The role of circCNOT6L as an active regulator of zygote transition toward the 2-cell-like state was suggested using the Embryonic Stem Cell (ESC) system. Intriguingly, human SPZ exactly mirror murine SPZ.

scholarly journals PHF3 regulates neuronal gene expression through the Pol II CTD reader domain SPOC

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Lisa-Marie Appel ◽  
Vedran Franke ◽  
Melania Bruno ◽  
Irina Grishkovskaya ◽  
Aiste Kasiliauskaite ◽  
...  
Keyword(s):  
Rna Polymerase Ii ◽  
Mrna Stability ◽  
Embryonic Stem ◽  
Pol Ii ◽  
Knock Out ◽  
Phd Finger ◽  
Neuronal Gene ◽  
Neuronal Genes ◽  
Knock Out Mouse ◽  
Liquid Liquid Phase Separation

AbstractThe C-terminal domain (CTD) of the largest subunit of RNA polymerase II (Pol II) is a regulatory hub for transcription and RNA processing. Here, we identify PHD-finger protein 3 (PHF3) as a regulator of transcription and mRNA stability that docks onto Pol II CTD through its SPOC domain. We characterize SPOC as a CTD reader domain that preferentially binds two phosphorylated Serine-2 marks in adjacent CTD repeats. PHF3 drives liquid-liquid phase separation of phosphorylated Pol II, colocalizes with Pol II clusters and tracks with Pol II across the length of genes. PHF3 knock-out or SPOC deletion in human cells results in increased Pol II stalling, reduced elongation rate and an increase in mRNA stability, with marked derepression of neuronal genes. Key neuronal genes are aberrantly expressed in Phf3 knock-out mouse embryonic stem cells, resulting in impaired neuronal differentiation. Our data suggest that PHF3 acts as a prominent effector of neuronal gene regulation by bridging transcription with mRNA decay.

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