scholarly journals Microsurgical nuclear transfer by intraooplasmic karyoplast injection as an alternative embryo reconstruction method in somatic cloning of pigs and other mammal species; application value of the method and its technical advantages: a review

2011 ◽  
Vol 50 (No. 6) ◽  
pp. 235-242 ◽  
Author(s):  
M. Samiec ◽  
M. Skrzyszowska
Keyword(s):  
Molecular Mechanisms ◽  
Genetic Material ◽  
Injection System ◽  
Total Efficiency ◽  
Reconstruction Method ◽  
Cell Nuclei ◽  
Mammal Species ◽  
Cloning Technique ◽  
Significant Stage ◽  
Chromatin Rearrangement

The introduction of cell nuclei into enucleated recipient cells, beyond enucleation, is the most significant stage of somatic cloning procedure. Microsurgical transfer of somatic nuclei can be an alternative method of clonal nuclear-cytoplasmic hybrid reconstruction towards cell fusion induced in the electric field, not only from the aspect of molecular mechanisms of nuclear chromatin rearrangement, advantageously influencing epigenetic reprogramming and structural remodelling of exogenous genetic material but also because in recent studies on pig cloning it was proved that the effectiveness of piezo-driven microinjection of ear-derived fibroblast karyoplasts measured by the percentage of oocytes preserving vitality after the cell nuclei transplantation operation did not differ significantly from the survival rate (viability) of clonal cybrids reconstituted by an electrofusion method. An intraooplasmic injection system of karyoplasts prepared from cells at G0/G1 or G2/M stages of cell cycle could also increase considerably the total efficiency of somatic cloning technique in pigs and in other mammal species.  

scholarly journals Molecular conditions of the cell nucleus remodelling/reprogramming process and nuclear-transferred embryo development in the intraooplasmic karyoplast injection technique: a review

2011 ◽  
Vol 50 (No. 5) ◽  
pp. 185-195 ◽  
Author(s):  
M. Samiec ◽  
M. Skrzyszowska
Keyword(s):  
Molecular Mechanisms ◽  
Genetic Material ◽  
Injection System ◽  
Injection Technique ◽  
Total Efficiency ◽  
Cell Nuclei ◽  
Mammal Species ◽  
Cloning Technique ◽  
Significant Stage ◽  
Chromatin Rearrangement

The introduction of cell nuclei into enucleated recipient cells, beyond enucleation, is the most significant stage of somatic cloning procedure. Microsurgical transfer of somatic nuclei can be an alternative method of clonal nuclear-cytoplasmic hybrid reconstruction towards cell fusion induced in the electric field, not only from the aspect of molecular mechanisms of nuclear chromatin rearrangement, advantageously influencing epigenetic reprogramming and structural remodelling of exogenous genetic material, but also because it was proved in recent studies on pig cloning that the effectiveness of piezo-driven microinjection of ear-derived fibroblast karyoplasts measured by the percentage of oocytes preserving vitality after cell nuclei transplantation operation did not differ significantly from the survival rate (viability) of clonal cybrids reconstituted by an electrofusion method. The intraooplasmic injection system of karyoplasts prepared from cells at G0/G1 or G2/M stages of cell cycle could also increase considerably the total efficiency of somatic cloning technique in pigs and other mammal species.    

Microglia extracellular vesicles: focus on molecular composition and biological function

2021 ◽  
Vol 49 (4) ◽  
pp. 1779-1790 ◽  
Author(s):  
Lorenzo Ceccarelli ◽  
Chiara Giacomelli ◽  
Laura Marchetti ◽  
Claudia Martini
Keyword(s):  
Extracellular Vesicles ◽  
Molecular Mechanisms ◽  
Biological Function ◽  
Biological Effects ◽  
Genetic Material ◽  
Cell Communication ◽  
Molecular Composition ◽  
Cargo Proteins ◽  
A Cell ◽  
The Central Nervous System

Extracellular vesicles (EVs) are a heterogeneous family of cell-derived lipid bounded vesicles comprising exosomes and microvesicles. They are potentially produced by all types of cells and are used as a cell-to-cell communication method that allows protein, lipid, and genetic material exchange. Microglia cells produce a large number of EVs both in resting and activated conditions, in the latter case changing their production and related biological effects. Several actions of microglia in the central nervous system are ascribed to EVs, but the molecular mechanisms by which each effect occurs are still largely unknown. Conflicting functions have been ascribed to microglia-derived EVs starting from the neuronal support and ending with the propagation of inflammation and neurodegeneration, confirming the crucial role of these organelles in tuning brain homeostasis. Despite the increasing number of studies reported on microglia-EVs, there is also a lot of fragmentation in the knowledge on the mechanism at the basis of their production and modification of their cargo. In this review, a collection of literature data about the surface and cargo proteins and lipids as well as the miRNA content of EVs produced by microglial cells has been reported. A special highlight was given to the works in which the EV molecular composition is linked to a precise biological function.

scholarly journals Brick Strex: a robust device built of LEGO bricks for mechanical manipulation of cells

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Elina Mäntylä ◽  
Teemu O. Ihalainen
Keyword(s):  
Molecular Mechanisms ◽  
Compressive Force ◽  
Tissue Mechanics ◽  
Cell Junctions ◽  
Physical Factors ◽  
Force Transmission ◽  
Cell Nuclei ◽  
Microscopy Imaging ◽  
Robust Solution ◽  
Mechanical Manipulation

AbstractCellular forces, mechanics and other physical factors are important co-regulators of normal cell and tissue physiology. These cues are often misregulated in diseases such as cancer, where altered tissue mechanics contribute to the disease progression. Furthermore, intercellular tensile and compressive force-related signaling is highlighted in collective cell behavior during development. However, the mechanistic understanding on the role of physical forces in regulation of cellular physiology, including gene expression and signaling, is still lacking. This is partly because studies on the molecular mechanisms of force transmission require easily controllable experimental designs. These approaches should enable both easy mechanical manipulation of cells and, importantly, readouts ranging from microscopy imaging to biochemical assays. To achieve a robust solution for mechanical manipulation of cells, we developed devices built of LEGO bricks allowing manual, motorized and/or cyclic cell stretching and compression studies. By using these devices, we show that $$\upbeta$$ β -catenin responds differentially to epithelial monolayer stretching and lateral compression, either localizing more to the cell nuclei or cell–cell junctions, respectively. In addition, we show that epithelial compression drives cytoplasmic retention and phosphorylation of transcription coregulator YAP1. We provide a complete part listing and video assembly instructions, allowing other researchers to build and use the devices in cellular mechanics-related studies.

scholarly journals Making of fusion genes in cancer: An in-silico study of mechanism of chromosomal translocations

2018 ◽  
Author(s):  
Kiran Lalwani ◽  
Shivani Sheth ◽  
Inayatullah Sheikh ◽  
Afzal Ansari ◽  
Fulesh Kunwar ◽  
...  
Keyword(s):  
Dna Sequences ◽  
In Silico ◽  
Molecular Mechanisms ◽  
Fusion Gene ◽  
Genetic Material ◽  
Chromosomal Translocations ◽  
In Silico Study ◽  
Clinical Consequences ◽  
The Stability ◽  
Genomic Regions

Chromosomal translocations involve exchange of genetic material between non- homologous chromosomes leading to the formation of a fusion gene with altered function. The clinical consequences of non-random and recurrent chromosomal translocations have been so well understood in carcinogenesis that they serve as diagnostic and prognostic markers and also help in therapy decisions, mainly in leukemia and lymphoma. However, the molecular mechanisms underlying these recurrent genetic exchanges are yet to be understood. Various approaches employed include the extent of the vicinity of the partner chromosomes in the nucleus, DNA sequences at the breakpoints, etc. The present study addresses the stability of DNA sequences at the breakpoint regions using in-silico approach in terms of physicochemical properties such as; AT%, flexibility, melting temperature, enthalpy, entropy, stacking energy and free energy. Changes in these properties may lead to instability of DNA which could affect gene expression in particular and genome organization in general. Our study indicates that the fusion sequences are comparatively more unstable and hence, more prone to breakage. Current study along with others could lead to developing a model for predicting breakage prone genomic regions using this novel in-silico approach.

scholarly journals Cell Cycle and DNA Repair Regulation in the Damage Response: Protein Phosphatases Take Over the Reins

2020 ◽  
Vol 21 (2) ◽  
pp. 446 ◽  
Author(s):  
Adrián Campos ◽  
Andrés Clemente-Blanco
Keyword(s):  
Cell Cycle ◽  
Dna Damage ◽  
Cellular Response ◽  
Molecular Mechanisms ◽  
Protein Phosphatases ◽  
Genetic Material ◽  
Protein Composition ◽  
Genotoxic Stress ◽  
Damage Response ◽  
Protein Dephosphorylation

Cells are constantly suffering genotoxic stresses that affect the integrity of our genetic material. Genotoxic insults must be repaired to avoid the loss or inappropriate transmission of the genetic information, a situation that could lead to the appearance of developmental abnormalities and tumorigenesis. To combat this threat, eukaryotic cells have evolved a set of sophisticated molecular mechanisms that are collectively known as the DNA damage response (DDR). This surveillance system controls several aspects of the cellular response, including the detection of lesions, a temporary cell cycle arrest, and the repair of the broken DNA. While the regulation of the DDR by numerous kinases has been well documented over the last decade, the complex roles of protein dephosphorylation have only recently begun to be investigated. Here, we review recent progress in the characterization of DDR-related protein phosphatases during the response to a DNA lesion, focusing mainly on their ability to modulate the DNA damage checkpoint and the repair of the damaged DNA. We also discuss their protein composition and structure, target specificity, and biochemical regulation along the different stages encompassed in the DDR. The compilation of this information will allow us to better comprehend the physiological significance of protein dephosphorylation in the maintenance of genome integrity and cell viability in response to genotoxic stress.

scholarly journals NETWORKS FROM GENE EXPRESSION TIME SERIES: CHARACTERIZATION OF CORRELATION PATTERNS

2007 ◽  
Vol 17 (07) ◽  
pp. 2477-2483 ◽  
Author(s):  
D. REMONDINI ◽  
N. NERETTI ◽  
C. FRANCESCHI ◽  
P. TIERI ◽  
J. M. SEDIVY ◽  
...  
Keyword(s):  
Gene Expression ◽  
Time Series ◽  
Large Scale ◽  
Molecular Mechanisms ◽  
Single Gene ◽  
Biological Information ◽  
Reconstruction Method ◽  
Gene Expression Time Series ◽  
Expression Time

We address the problem of finding large-scale functional and structural relationships between genes, given a time series of gene expression data, namely mRNA concentration values measured from genetically engineered rat fibroblasts cell lines responding to conditional cMyc proto-oncogene activation. We show how it is possible to retrieve suitable information about molecular mechanisms governing the cell response to conditional perturbations. This task is complex because typical high-throughput genomics experiments are performed with high number of probesets (103–104 genes) and a limited number of observations (< 102 time points). In this paper, we develop a deepest analysis of our previous work [Remondini et al., 2005] in which we characterized some of the main features of a gene-gene interaction network reconstructed from temporal correlation of gene expression time series. One first advancement is based on the comparison of the reconstructed network with networks obtained from randomly generated data, in order to characterize which features retrieve real biological information, and which are instead due to the characteristics of the network reconstruction method. The second and perhaps more relevant advancement is the characterization of the global change in co-expression pattern following cMyc activation as compared to a basal unperturbed state. We propose an analogy with a physical system in a critical state close to a phase transition (e.g. Potts ferromagnet), since the cell responds to the stimulus with high susceptibility, such that a single gene activation propagates to almost the entire genome. Our result is relative to temporal properties of gene network dynamics, and there are experimental evidence that this can be related to spatial properties regarding the global organization of chromatine structure [Knoepfler et al., 2006].

scholarly journals Nucleosome–nucleosome interactions via histone tails and linker DNA regulate nuclear rigidity

2017 ◽  
Vol 28 (11) ◽  
pp. 1580-1589 ◽  
Author(s):  
Yuta Shimamoto ◽  
Sachiko Tamura ◽  
Hiroshi Masumoto ◽  
Kazuhiro Maeshima
Keyword(s):  
Molecular Mechanisms ◽  
Mechanical Response ◽  
Nuclear Architecture ◽  
Living Cells ◽  
Genome Integrity ◽  
Biological Processes ◽  
Cell Nuclei ◽  
Histone Tails ◽  
Insight Into

Cells, as well as the nuclei inside them, experience significant mechanical stress in diverse biological processes, including contraction, migration, and adhesion. The structural stability of nuclei must therefore be maintained in order to protect genome integrity. Despite extensive knowledge on nuclear architecture and components, however, the underlying physical and molecular mechanisms remain largely unknown. We address this by subjecting isolated human cell nuclei to microneedle-based quantitative micromanipulation with a series of biochemical perturbations of the chromatin. We find that the mechanical rigidity of nuclei depends on the continuity of the nucleosomal fiber and interactions between nucleosomes. Disrupting these chromatin features by varying cation concentration, acetylating histone tails, or digesting linker DNA results in loss of nuclear rigidity. In contrast, the levels of key chromatin assembly factors, including cohesin, condensin II, and CTCF, and a major nuclear envelope protein, lamin, are unaffected. Together with in situ evidence using living cells and a simple mechanical model, our findings reveal a chromatin-based regulation of the nuclear mechanical response and provide insight into the significance of local and global chromatin structures, such as those associated with interdigitated or melted nucleosomal fibers.

System for In Vivo Nanoinjection of Tissues

2016 ◽  
Author(s):  
Talmage H. Jones ◽  
Brian D. Jensen
Keyword(s):  
Large Scale ◽  
Genetic Material ◽  
Injection System ◽  
Radius Of Curvature ◽  
Curvature Radius ◽  
Convex Surfaces ◽  
Preliminary Results ◽  
Flexible Designs ◽  
Micrometer Scale

This paper investigates the design of a nano-injection system that can deliver genetic material to cells within live tissue. The approach to creating such a system was to create candidate designs that meet all the requirements for successful in vivo injection and can be fabricated using silicon etching. The designs were tested through large-scale prototyping and through models that describe the systems’ behavior on the micrometer scale. One design consists of an array of lances on a rigid backing. The other design consists of an array of lances grouped in sets of three on a backing that can conform to the shape of the tissue being injected. Each design was prototyped in 3D printed ABS plastic. Preliminary results were qualitative and showed that the rigid and flexible designs performed similarly on mostly flat and irregular surfaces. On convex surfaces with a strong curvature (radius of curvature of about 2 cm), the flexible array gave slightly better results. Final testing gave a quantitative comparison of the two designs’ efficiencies on strongly curved convex surfaces. These results supported the preliminary results that the flexible array is more efficient in reaching points on the tissue than the rigid array is. As the applied force increased, each array performed more efficiently.

scholarly journals Recurrent losses and rapid evolution of the condensin II complex in insectsg

2018 ◽  
Author(s):  
T. King ◽  
C.J. Leonard ◽  
J.C. Cooper ◽  
S. Nguyen ◽  
E. Joyce ◽  
...  
Keyword(s):  
Genome Organization ◽  
Molecular Mechanisms ◽  
Somatic Cells ◽  
Rapid Evolution ◽  
Genetic Material ◽  
Life Forms ◽  
Model Organisms ◽  
Homologous Chromosomes ◽  
Homologous Chromosome Pairing ◽  
Key Aspects

AbstractCondensins play a crucial role in the organization of genetic material by compacting and disentangling chromosomes. The condensin I and condensin II complexes are widely considered to have distinct functions based on studies in a few model organisms, although the specific functions of each complex are yet to be fully understood. The condensin II complex is critical for genome organization in Drosophila, and is a key anti-pairing factor that separates homologous chromosomes in somatic cells. Intriguingly, the Cap-G2 subunit of condensin II is absent in Drosophila melanogaster, and this loss may be related to the high levels of homologous chromosome pairing in somatic cells seen in flies. Here, we find that this Cap-G2 loss predates the origin of Dipterans, and other CapG2 losses have occurred independently in multiple insect lineages. Furthermore, the Cap-H2 and Cap-D3 subunits have also been repeatedly and independently lost in several insect orders, and some taxa lack condensin II-specific subunits entirely. We used Oligopaint DNA-FISH to quantify pairing levels in ten species across seven orders, representing several different configurations of the condensin II complex. We find that all non-Dipteran insects display near-uniform low pairing levels, suggesting that some key aspects of genome organization are robust to condensin II subunit losses. Finally, we observe consistent signatures of positive selection in condensin II subunits across flies and mammals. These findings suggest that these ancient complexes are far more evolutionarily labile than previously suspected, and are at the crossroads of several forms of genomic conflicts. Our results raise fundamental questions about the specific functions of the two condensin complexes and the interplay between them in taxa that have experienced subunit losses, and open the door to further investigations to elucidate the diversity of molecular mechanisms that underlie genome organization across various life forms.

scholarly journals CRISPR-Cas9 mediated nuclear transport and genomic integration of nanostructured genes in human primary cells

2021 ◽  
Author(s):  
Enrique Lin Shiao ◽  
Wolfgang G Pfeifer ◽  
Brian R Shy ◽  
Mohammad Saffari Doost ◽  
Evelyn Chen ◽  
...  
Keyword(s):  
Fluorescent Protein ◽  
Genetic Material ◽  
Specific Cell ◽  
Dna Nanostructures ◽  
Primary Cells ◽  
Cell Nuclei ◽  
Genomic Integration ◽  
Dna Nanostructure ◽  
Human Primary Cells

DNA nanostructures are a promising tool for delivery of a variety of molecular payloads to cells. DNA origami structures, where 1000's of bases are folded into a compact nanostructure, present an attractive approach to package genes; however, effective delivery of genetic material into cell nuclei has remained a critical challenge. Here we describe the use of DNA nanostructures encoding an intact human gene and a fluorescent-protein encoding gene as compact templates for gene integration by CRISPR-mediated homology-directed repair (HDR). Our design includes CRISPR-Cas9 ribonucleoprotein (RNP) binding sites on the DNA nanostructures to increase shuttling of structures into the nucleus. We demonstrate efficient shuttling and genomic integration of DNA nanostructures using transfection and electroporation. These nanostructured templates display lower toxicity and higher insertion efficiency compared to unstructured double-stranded DNA (dsDNA) templates in human primary cells. Furthermore, our study validates virus-like particles (VLPs) as an efficient method of DNA nanostructure delivery, opening the possibility of delivering DNA nanostructures in vivo to specific cell types. Together these results provide new approaches to gene delivery with DNA nanostructures and establish their use as large HDR templates, exploiting both their design features and their ability to encode genetic information. This work also opens a door to translate other DNA nanodevice functions, such as measuring biophysical properties, into cell nuclei.

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