Analysis of LINE-1 Retrotransposition at the Single Nucleus Level | Protocol
After generating stable clones expressing retrotransposition-competent L-1, or vector backbone, grow HepG2 cells stably ... Then aspirate the medium from the cell pellet and use DPBS to wash the cells. Aspirate all but 200 microliters of DPBS and use ... And incubate for 90 minutes to arrest cells at metaphase.. Use 10 milliliters of 1X Dulbeccos PBS, or DPBS, to wash the cells ... Next, transfer the cells to a 15 milliliter tube and centrifuge at 1, 000 Gs and four degrees Celsius for two minutes. ...
A β-glucuronidase (GUS) Based Cell Death Assay | Protocol (Translated to Russian)
Takayama, S. Cloning and functional analysis of BAG-1: a novel Bcl-2-binding protein with anti-cell death activity. Cell. 80 (2 ... Kang, C. H. AtBAG6, a novel calmodulin-binding protein, induces programmed cell death in yeast and plants. Cell Death Differ. ... Kabbage, M., Ek-Ramos, M., Dickman, M. A β-glucuronidase (GUS) Based Cell Death Assay. J. Vis. Exp. (51), e2680, doi:10.3791/ ... Kabbage, M., Ek-Ramos, M., Dickman, M. A β-glucuronidase (GUS) Based Cell Death Assay. J. Vis. Exp. (51), e2680, doi:10.3791/ ...
Cell-Free Protein Synthesis from Exonuclease-Deficient Cellular Extracts Utilizing Linear DNA Templates | Protocol
Presented here is a protocol for the preparation and buffer calibration of cell extracts from exonuclease V knockout strains of ... Preparing plasmid DNA depends on many time-consuming steps during cloning and large amounts of DNA isolation. On the other hand ... Transfer the cells to chilled and pre-weighed 50 mL tubes.. *Spin down the cells at 2,000 x g for 8 min at 4 °C, and then ... Resuspend the cell pellets in 800 mL of chilled S30A+DTT.. NOTE: The volume of S30A+DTT is 5x less than the original cell ...
Reproductive Cloning and Somatic Cell Nuclear Transfer | Biology | JoVE
O clone resultante tem o DNA cromossómico idêntico ao animal original. Mas o DNA mitocondrial é frequentemente diferente já que ... Embora o DNA cromossómico do clone seja o mesmo do dador do núcleo, ele pode ter DNA mitocondrial diferente, uma vez que as ... Embora os clones possam ser produzidos dividindo um embrião inicial-semelhante ao que acontece naturalmente com gémeos ... Ayala, Francisco J. "Cloning Humans? Biological, Ethical, and Social Considerations." Proceedings of the National Academy of ...
Catalytically Perfect Enzymes | Cell Biology | JoVE
42.8: Cloning of Dolly the Sheep. Chapter 43: Embryonic and Induced Pluripotent Stem Cells ... Substrates must diffuse within the cell to interact with their enzymes.. The reaction rates of catalytically perfect enzymes ... Perfect enzymes not only catalyze highly efficient reactions but can also help protect cells from harmful reaction ...
COP Coated Vesicles | Cell Biology | JoVE
42.8: Cloning of Dolly the Sheep. Chapter 43: Embryonic and Induced Pluripotent Stem Cells ... Coated vesicles are transport vesicles that bud off from specialized regions of the cell membrane. Specific coat proteins cover ... Membrane-enclosed structures called vesicles transport proteins and lipids across the cell. The vesicles derive their cargo ... Lodish, H., & Zipursky, S. L. (2001). Molecular cell biology. Biochem Mol Biol Educ, 29, 126-133. ...
De-differentiation and Induced Pluripotent Stem Cells | Molecular Biology | JoVE
Stem cells are undifferentiated cells that divide and produce different types of cells. Ordinarily, cells that have ... The cells can now divide, and ideally, be directed to produce a specific cell type in culture. The new cells could then be ... These cells are also pluripotent like embryonic stem cells-able to produce all cell types-and are therefore called induced ... Induced pluripotent stem cells, iPSCs, are mature, differentiated cells, such as skin cells, that are reprogrammed in the ...
Chromatin Modification in iPS Cells | Cell Biology | JoVE
42.8: Cloning of Dolly the Sheep. Chapter 43: Embryonic and Induced Pluripotent Stem Cells ... Stem cells international, 2016, 3162363.. *Sha K, Boyer LA. The chromatin signature of pluripotent cells. 2009 May 31. In: ... Scientists experimentally induce chromatin remodeling to enhance the conversion of cells into pluripotent stem cells. Chromatin ... and chromatin remodeling complexes to somatic cells to aid reprogramming into pluripotent stem (iPS) cells. ...
Condensins and Condensation of Chromosomes | Cell Biology | JoVE
42.8: Cloning of Dolly the Sheep. Chapter 43: Embryonic and Induced Pluripotent Stem Cells ... these protein complexes are integral to the cell cycle and cell survival. ... A fundamental part of life is a cells ability to replicate its genome and divide; these processes occur over two main phases ... By the time the cell reaches metaphase, the sister chromatids are only loosely connected along the arms but are still tightly ...
Cytokinesis, RhoA and Contractile Ring | Cell Biology | JoVE
42.8: Cloning of Dolly the Sheep. Chapter 43: Embryonic and Induced Pluripotent Stem Cells ... The final step of the cell cycle that divides a cell into two daughter cells is called cytokinesis. Cytokinesis begins after ... The contraction of the ring continues until it pinches off two new cells. The ring is degraded once the new cells are formed. ... The midbody ring then carries out the abscission of the parent cell, resulting in the formation of two daughter cells. ...
Positive Regulators of Cell Cycle: Cyclins and Cdks | Biology | JoVE
To consistently produce healthy cells, the cell cycle-the process that generates daughter cells-must be precisely regulated. ... "Immunomodulatory Roles of Cell Cycle Regulators." Frontiers in Cell and Developmental Biology 7 (2019). [Source] ... At these checkpoints, positive and negative regulators promote or inhibit a cells continuation through the cell cycle. ... To regulate the cell cycle, cyclins must be bound to a Cyclin-dependent kinase (CDK)-a type of enzyme that attaches a phosphate ...
Protocols and Video Articles Authored by Yayoi Obata
Separation of Follicular Cells and Oocytes in Ovarian Follicles of Zebrafish ... Identification of Genes Aberrantly Expressed in Mouse Embryonic Stem Cell-cloned Blastocysts Biology of Reproduction. Apr, 2008 ... Separation of Follicular Cells and Oocytes in Ovarian Follicles of Zebrafish. Wenyi Wang1, Tao Kang1, Lin Bai1, Wei Hu2, Yayoi ... Development of Fertile Mouse Oocytes from Mitotic Germ Cells in Vitro Nature Protocols. Sep, 2017 , Pubmed ID: 28796235 ...
Bacterial Inner-membrane Display for Screening a Library of Antibody Fragments | Protocol
Figure 3. E.coli cells and spheroplasts. (A) E. coli cells are cylindrical in shape. (B) After spheroplasting using EDTA and ... Insert the DNA library into the pIMD plasmid (Figure 2) using standard molecular cloning methods23.. NOTE: This plasmid ... Add 1 ml of a cell lysis detergent per 0.2 g of cell pellet. Resuspend by pipetting and gently rotate for 20 min to lyse the ... MC4100 E. coli cells. Coli Genetic Stock Center. 6152. Cells need to be chemically competent or electrocompetent, depending on ...
Co-expression of Multiple Chimeric Fluorescent Fusion Proteins in an Efficient Way in Plants | Protocol
To prepare tobacco BY-2 suspension cells for bombardment, filter-collect 30 milliliters of 3-day cultured BY-2 cells onto a ... To begin, design the primers for molecular cloning of DNA fragments as described in the text protocol. Amplify DNA fragments ... As a negative control, little autofluorescent signal is observed in tobacco BY-2 cells and arabidopsis root and root hair cells ... Keep the bombarded cells and plants in the dark in the plant growth chamber for six to 72 hours prior to observation of ...
Saccharomyces cerevisiae (Yeast) as a Model Organism | Biology I | JoVE
... which grows in size throughout the cell cycle until cytokinesis. Unlike typical eukaryotic cell division, the two cells are not ... This allows molecular cloning to be performed in E. coli, say to incorporate the gene for green fluorescent protein from ... Saccharomyces Cerevisiae Bakers Yeast Model Organism Genetics Cell Division Cell Death Biological Research Biomedical Research ... The cell cycle is a series of cellular events that includes the proper replication and segregation of nuclear DNA before a cell ...
A Portal Vein Injection Model to Study Liver Metastasis of Breast Cancer | Protocol
A surgical procedure was developed to deliver mammary tumor cells to the murine liver via portal vein injection. This model ... Now pipette the tumor cells up and down several times to re-suspend the cell solution, and load a 25 microliter removable ... Advanced Animal Model of Colorectal Metastasis in Liver: Imaging Techniques and Properties of Metastatic Clones… ... Using mouse memory tumor cells tagged with GFP, tumor cell dispersal throughout the liver is apparent at 90 minutes post ...
Subcutaneous Administration of Muscarinic Antagonists and Triple-Immunostaining of the Levator Auris Longus Muscle in Mice |...
Witzemann, V., Brenner, H. R., Sakmann, B. Neural factors regulate AChR subunit mRNAs at rat neuromuscular synapses. J Cell ... Dorje, F. Antagonist binding profiles of five cloned human muscarinic receptor subtypes. J Pharmacol Exp Ther. 256, 727-733 ( ... 3Shriners Hospitals Pediatric Research Center and Department of Anatomy and Cell Biology, Temple University School of Medicine ...
Combining X-Ray Crystallography with Small Angle X-Ray Scattering to Model Unstructured Regions of Nsa1 from S. Cerevisiae |...
This method describes the cloning, expression, and purification of recombinant Nsa1 for structural determination by X-ray... ... Culture the cells, induce Nsa1 expression, and harvest the cells.. Resuspend the cells in 25 milliliters of lysis buffer, pre- ... This method describes the cloning, expression, and purification of recombinant Nsa1 for structural determination by X-ray ... Sonicate the cells at four degrees Celsius for seven minutes, with a cycle of two seconds on, two seconds off. Then, centrifuge ...
University of Iceland | Protocols and Video Articles
Propagating and Detecting an Infectious Molecular Clone of Maedi-visna Virus that Expresses Green Fluorescent Protein Stefán R ... collected on cell material scraped from bacterial colonies. The pipeline allows researchers to rapidly organize hundreds to ... Jónsson1, Valgerdur Andrésdóttir1 1Institute for Experimental Pathology, University of Iceland We describe a molecular clone of ...
Generation of In-Frame Gene Deletion Mutants in Pseudomonas aeruginosa and Testing for Virulence Attenuation in a Simple Mouse...
Repeat the centrifugation and re-suspend the cells in PBS to a final concentration of 2.5 times 10 to the ninth colony forming ... Compared to current models, procedures with reproducibility plus clone validation before and after infection can benefit other ... On the morning of injections, thaw the cryovials of bacterial cells at four degrees Celsius for three to four hours. Keep the ... For each strain, aliquot 1.5 milliliters of the cell suspension into a two milliliter tube and prepare the PBS for control ...
A Novel Human Epithelial Enteroid Model of Necrotizing Enterocolitis | Protocol
Induced Differentiation of M Cell-like Cells in Human Stem Cell-derived Ileal Enteroid Monolayers… ... Place a small cloning ring into a coverslip. Pipette the tissue processing gel and enteroid mixture into the cloning ring that ... Induced Differentiation of M Cell-like Cells in Human Stem Cell-derived Ileal Enteroid Monolayers… ... Then, submerge the cloning ring with the solidified mixture into 70%ethanol to prepare for paraffin embedding. Immediately ...
The Terroir Concept Interpreted through Grape Berry Metabolomics and Transcriptomics | Protocol
The choice of one single clone to minimize genetic differences, and the multiple duration of the sampling, allowed ... Formaldehyde-assisted Isolation of Regulatory Elements to Measure Chromatin Accessibility in Mammalian Cells… ... Formaldehyde-assisted Isolation of Regulatory Elements to Measure Chromatin Accessibility in Mammalian Cells… ...
Methanol Independent Expression by Pichia Pastoris Employing De-repression Technologies | Protocol
Measure the cell density of the overnight culture diluted at a one to twenty ratio in cultivation medium in a spectrophotometer ... This method can help in the pre selection of chromosomes clones by Pichia pastoris strains expressing recombinant proteins ... Take samples every 24 hours to monitor the cell density and the protein expression by the assay of choice. At the end of the ... Electric Cell-Substrate Sensing for Real-Time Evaluation of Metal-Organic Framework Toxicological Profiles… ...
Initiation of Translation and Initiation Factors | Molecular Biology | JoVE
Before the preinitiation complex binds the mRNA, to make sure that a correctly processed mRNA is translated, the cell uses ... Other cells also frequently use this to produce multiple proteins from the same mRNA molecule. ... Molecular Biology of the Cell. (2016), Pgs 347-348.. *Pestova, Tatyana V., Victoria G. Kolupaeva, Ivan B. Lomakin, Evgeny V. ...
Protein Complexes and Protein-Protein Interactions | Molecular Biology | JoVE
10.1: Cell-Specific Gene Expression. 30. 10.2: Regulation of Expression Occurs at Multiple Steps ...
pH: Acidity and Basicity of Aqueous Solutions | Molecular Biology | JoVE
Chapter 2: Biochemistry of the Cell Back To Chapter 2.10: pH TABLE OF. CONTENTS ...
Reporter Genes: lacZ, Luc, GFP, RFP | Molecular Biology | JoVE
The recombinant plasmid is transfected into a mammalian cell, and its expression shows that the cell is successfully ... The cells expressing the Luc gene produce luciferase enzymes that catalyze a reaction with luciferin to produce light. The lacZ ... Since the gene of interest and the reporter gene have the same cis-regulatory sequence, they are expressed in the same cells ... This is done by placing a reporter gene downstream of the promoter in a plasmid and then introducing it into the mammalian cell ...
Euakryotic Compartmentalization and Microenvironments | Molecular Biology | JoVE
Eukaryotic cells contain numerous internal compartments. Membrane bound organelles allowing for very specific functions to ... One of the distinguishing features of eukaryotic cells is that they contain membrane-bound organelles, such as the nucleus and ... For example, lysosomes in the animal cells maintain an acidic environment compared to the surrounding cytosol. This helps the ... The genetic material of eukaryotic cells is compartmentalized within the nucleus, surrounded by a double membrane called the ...
Copy Number Variations (CNV) and Single Nucleotide Polymorphism (SNP) | Molecular Biology | JoVE
For example, in patients with sickle cell anemia, a single base substitution from adenine to thymine in a specific locus of the ...
Dihybrid Crosses and Independent Assortment | Molecular Biology | JoVE
10.1: Cell Specific Gene Expression. 30. 10.2: Regulation of Expression Occurs at Multiple Steps ...