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We narrowed to 720 results for: cel

Showing: 241 - 260 of 720 results

  1. What's New in CRISPR - March 2020

    Type
    Blog Post
    Published
    March 10, 2020, 1:15 p.m.
    ...for studying the bacterial cell cycle, asymmetric cell division, and cellular differentiation, but it takes...crescentus Multiplexed gRNA for human pluripotent stem cells CRISPR-based DNA demethylation Optogenetic repressors...complex remains in the cytoplasm. Read the paper in Cellular and Molecular Bioengineering Find the optogenetic...Figure 1: Serial dilutions of CRISPRi in Caulobacter cells for ctrA gene expression using various dCas9. Image...lab repressed ctrA, a gene encoding an essential cell cycle regulator. The lab expressed both dCas9 and...piggyBac transposon system for human pluripotent stem cells Lindy Barrett’s lab developed new fluorescently ... of multiplexed gRNAs in human pluripotent stem cells. Previously, CRISPRi or CRISPRa depended on continued...

  2. Viral Vectors 101: The Retroviral Lifecycle

    Type
    Blog Post
    Published
    July 27, 2023, 1:15 p.m.
    ...synthesized inside of what is called the “target cell” (or host cell) by the viral protein reverse transcriptase...reverse-transcribed DNA into the host cell’s genome and then use cell machinery for transcription and translation...1971). Noninfectious ARK mouse embryo cell lines in which each cell has the capacity to be activated to ..., interested in transferring genes to mammalian cells (i.e. transferring DNA), yet retroviruses are RNA-containing...separate set of instructions in the same producer cells as the virus. Because it is made at the same time...viral genes. It is derived from the host/producer cell membrane, and acquired as an envelope during exocytotic...particles do once they come in contact with target cells (Fig. 3).     Figure 3: The life cycle of...

  3. DNA Purification Without a Kit

    Type
    Blog Post
    Published
    Feb. 11, 2020, 2:15 p.m.
    ...broken out of cells with a solution of salts, detergent, and ball bearings. Then a cellulose dipstick is... kits and typically follow the same principles: cells are lysed, RNAs and proteins are removed to leave... could be digested with restriction enzymes.  Cellulose nucleic acid purification Starting material: plant... plant, microbe, blood, or mammalian cells or tissue Product: total nucleic acids While glass or silica...silica are commonly used to bind and purify DNA, cellulose also efficiently binds, or at least entraps nucleic...and RNA from plants, microbes, blood or mammalian cells in 30 seconds or less without using a pipette.  ... absorb the nucleic acids. Containinants bind cellulose less tightly than nucleic acids, so placing the...

  4. Minigenomes - a Safe Way to Study Dangerous Viruses Like the Ebola Virus

    Type
    Blog Post
    Published
    March 17, 2016, 2:30 p.m.
    ...choose cell lines that are highly transfectable.  The amounts of each plasmid transfected into cells are ... polymerase gene or using cells that stably express T7 (e.g. BSR T7/5 cells) (1). We hope that minigenomes...capable of being replicated and transcribed in the cell when the appropriate viral proteins are provided... RNA-dependent RNA polymerase into the infected cells. The EBOV polymerase replicates the viral genome...on separate plasmids and are co-transfected into cells alongside a plasmid encoding the minigenome. These...transcribed (and the viral mRNAs are translated) by cellular machinery. NP encapsidates the negative sense,...polyadenylate the mRNA. These mRNAs are translated by cellular host machinery (as viral mRNAs would be in the...

  5. Plasmids 101: Stringent Regulation of Replication

    Type
    Blog Post
    Published
    June 20, 2024, 1:15 p.m.
    ...replicating too quickly can slow cell growth or even kill the host cell. For stringently controlled plasmids...All plasmids rely on their host cell's replication machinery in order to replicate—but not always to ...be synchronized with the replication of the host cell's chromosomal DNA.  Plasmids are said to be under... stringent control when they depend on the host cell’s initiation proteins to start their own replication... occurs at the appropriate time—for example, the cell must have enough nutrients available to complete...the amount of active DnaA available is one of a cell’s best tools to regulate DNA replication. DnaA negatively...energy source, ATP binding is one more way for the cell to control DNA replication. A variety of other proteins...

  6. Hot Plasmids Spring 2024

    Type
    Blog Post
    Published
    May 23, 2024, 1:15 p.m.
    ...with enrichment, single-cell sequencing, and optical pooled screens in human cells. Addgene offers vectors...reverse transcriptase enzymes, inhibition of host cell proteins, and modifications to the pegRNA. The Britt...shows enhanced editing efficiencies in a number of cell types at different genomic loci — in some cases,... editing efficiencies at different loci in U2OS cells. Image adapted from Yan et al. 2024 under a CC-BY..., target distinct epitopes on the SARS-CoV-2 nucelocapsid protein, one of the virus’s most abundant proteins...western blots (Figure 3).      Figure 3: HeLa cells transduced with SARS-CoV-2 nucleocapsid protein ...desirable than having many closely-packed labeled cells. Plus, Voltron2’s fluorescence is tunable by changing...

  7. Neuronal labeling with Spaghetti Monster

    Type
    Blog Post
    Published
    Aug. 14, 2018, 12:42 p.m.
    ...of neuronal sub-cellular structures GFP is limited in its ability to label subcellular structures in the...ability to interact with protein partners within the cell. However, antibodies can have low affinity for these... generally bright, stable and well tolerated by cells. They can be used for protein localization, isolation...trigger protein aggregation which is toxic for the cells and affects the labeling of small structures like...team, an ideal probe should combine the solubility, cell tolerance, and optional fluorescence of FPs with...used to amplify the direct smFP signal within sub-cellular structures. For instance, smFPs have been used...example, N-cadherin (cadherin-2) is a postsynaptic cell adhesion protein that plays a critical role in neural...

  8. Sonic Hedgehog? Sleeping Beauty? Learn About the Genes Behind Addgene’s Conference Room Names

    Type
    Blog Post
    Published
    April 2, 2019, 1:44 p.m.
    ... are filled with early mitotic cells as well as necrotic or dead cells (Jiang and White-Cooper, 2003)....Ivics et al 1997. Spaghetti Squash, a regulator of cellular contraction You may think that a gene called "...the fly actomyosin cytoskeleton that regulates cellular contraction. This network is composed of actin...interact with each other to generate forces that cause cells and muscles to contract depending on the context...context. Contraction is critical for processes such as cell movement/motility, development, cytokinesis, and...scientists who discovered comr thought the sperm cells in mutant flies looked like a “whole bunch of cookie...are part of a genetic cascade that determines the cell fate of the dorsal mesoderm in developing Drosophila...

  9. Antibody Neutralization Response Against Pseudoviruses Expressing SARS-CoV-2 Spike Protein Variants

    Type
    Blog Post
    Published
    June 22, 2021, 1:15 p.m.
    ...expressing target cells. Any neutralized pseudoviruses would be unable to get into the target cells and this can...disease severity and survival. Cell 184:476-488.e11. https://doi.org/10.1016/j.cell.2020.12.015 Garcia-Beltran...vaccine-induced humoral immunity. Cell 184:2372-2383.e9. https://doi.org/10.1016/j.cell.2021.03.013 Additional resources...vaccines and allows the virus to gain entry into human cells via human ACE2 receptors. In their assay, the pseudoviruses...result was similar to studies that used live virus (Cele et al., 2021). “We weren't completely shocked [with...quick to deposit their plasmids at Addgene to accelerate ongoing COVID-19 research. “I hope that by sharing...Balazs.     References and Resources References Cele S, Gazy I, Jackson L, Hwa S-H, Tegally H, Lustig...

  10. Donations from Addgene to Yield Answers for Rare Disease Researchers

    Type
    Blog Post
    Published
    Sept. 15, 2015, 2:30 p.m.
    ...transformation of healthy cells into melanoma cells. They’ll combine induced pluripotent stem cell (iPS) technology...gene appears to play a role in many cell processes involving cell membrane dynamics. When they learned...: This year, the BeHEARD (Helping Empower and Accelerate Research Discoveries) Award attracted submissions...still no treatment available. Aguilar is a basic cell biologist. He got interested in Lowe Syndrome initially...the gene, it piqued their interest. They obtained cells from patients with the condition to reveal some ...explore the effect of various OCRL1 mutations on cell behavior in greater detail. The goal is not just...now plan to use CRISPR-Cas9 technology to create cell lines and/or mouse models carrying different SOX10...

  11. Plasmids 101: Modular Cloning Applications and Kits

    Type
    Blog Post
    Published
    May 14, 2024, 1:15 p.m.
    ...can be used in bacterial and cell-free expression systems, mammalian cells, yeast, flies, and even plants...transport studies in HEK cells, recombinant protein purification in bacterial cells, and a two-hybrid protein...for synthetic biology applications in bacteria or cell-free systems. The approach is particularly useful...tags to your proteins, then the CIDAR Kit is an excellent option (Iverson et al., 2016). CIDAR offers a ...add-on kit and is compatible with E. coli and/or cell-free systems. Protein expression in yeast MoClo ...plant-specific parts you‘ll need, including sub-cellular localization signals, plant markers, and reporters...of expression and integration needed in mammalian cell types, and comes with a large library of common ...

  12. Transferable Skills Guide: Time Management

    Type
    Blog Post
    Published
    Dec. 5, 2017, 1:52 p.m.
    ...splitting my cells first. Time management through planning I probably wouldn’t have fallen into my cell splitting...for Scientists. Back when I used to do mammalian cell culture, I would often respond to friends asking...there in a few minutes, I just need to split some cells.” Inevitably, this task would take longer than I...since my days as an undergrad working with cancer cell lines. As I started planning out my own experiments.... In my anecdote above, I prioritized splitting cells over meeting up with friends, but didn’t realize...mean in terms of the utility of your tasks. In my cell splitting anecdote, I probably could have grabbed...with friends and come back to the lab to split my cells later. Eating with friends has a somewhat immediate...

  13. Top Requested Lentivirus and AAV of 2016

    Type
    Blog Post
    Published
    Jan. 6, 2017, 3:56 p.m.
    ...expressed in cells that express cre recombinase. Typically, this feature is used to attain cell-specific ...tools so that researchers around the world can accelerate their work. After all, as we like to sayat Addgene...CRISPR applications and can be expressed from your cell line of choice after transduction with this virus...blast virus can be used to generate Cas9-expressing cell lines, which can then be used both for screens (...viral prep to create a single stable Cas9-expressing cell line and then transduce this line with lentiviruses...using lentiCas9-blast to generate Cas9-expressing cells. To ensure that this growth rate effect doesn’t ...activating particular signaling pathways in specific cell types (typically neurons). While all of these viruses...

  14. Stabilized Bacterial Promoters: Constant Gene Expression at any Copy Number

    Type
    Blog Post
    Published
    Sept. 4, 2018, 12:53 p.m.
    ... engineer cells for specific purposes. Unfortunately, plasmid copy numbers vary within cell populations...problems. Rapidly dividing cells initiate genome replication more than once per cell division and thus, genes.... When cells are dividing rapidly, multiple rounds of replication are initiated before the cell has divided...growth rate can all impact plasmid copy number in a cell. One can attempt to eliminate fluctuations due ... pSC101 with copy numbers ranging from 3-100 per cell, a stabilized promoter buffered against the effects... to stationary phase, the number of plasmids per cell increases 4-5 fold (for pUC plasmids) or ~2 fold...fold (for p15a, R6K, and ColE2 plasmids) as cell division slows (Kittleson et al., 2011). Segall-Shapiro...

  15. Technical Design of a Western Blot

    Type
    Blog Post
    Published
    Aug. 22, 2024, 1:15 p.m.
    ...expensive Nitrocellulose or low-fluorescence PVDF membranes Increased fragility (nitrocellulose) or increased... mechanical method before your enzyme lysis. For cell cultures, enzyme lysis is typically sufficient.  ...When choosing your lysis buffer, consider the subcellular location of your protein. Proteins located in...proteins easily available in the cytoplasm or whole-cell lysate. A harsher, SDS-containing lysis buffer, ...Membranes The two membrane options are PVDF and nitrocellulose. PVDF is a sturdy membrane that can be stripped...membrane, it also has higher background noise than nitrocellulose membranes. Pro tip! You can visualize proteins...methanol and place it on a light box after drying. Nitrocellulose, on the other hand, doesn’t require any pre-wetting...

  16. PRIDICT: Predicting Efficiencies of Prime Editing Guide RNAs

    Type
    Blog Post
    Published
    June 25, 2024, 1:15 p.m.
    ... systems in multiple cell types. Cell, 186, 1–17. https://doi.org/10.1016/j.cell.2023.03.034 Resources...Integrated in Parallel. Cell, 154(4), 914–927. https://doi.org/10.1016/J.CELL.2013.07.018 Anzalone, A....epigenetic manipulation of prime editing. Cell. https://doi.org/10.1016/j.cell.2024.03.020 Mathis, N., Allam, A... balance. Molecular Cell, 81(10), 2216-2230.e10. https://doi.org/10.1016/J.MOLCEL.2021.03.032 Yu, G., ...suited for prediction of very diverse edit types and cell contexts. Figure 1: High-throughput screening...

  17. RNA Interference in Plant Biology: New Tools for an Old Favorite

    Type
    Blog Post
    Published
    Oct. 27, 2020, 1:15 p.m.
    ...genes known to be essential for cell growth. Together, this excellent work highlights the importance of...fluorescence-activated cell sorting (FACS). This is not possible in plants outside of limited, single cell protoplast...exquisitely and robustly self-organize at the cellular and multicellular scale.     References Bezanilla M, Perroud...can be found at Addgene that could serve as an excellent start (#22988, #137884). Both vectors allow the...visually identified subpopulation. For traditional cell culture, enriching your active RNAi subpopulation...where he investigated how plants achieve polarized cell growth in the lab of Luis Vidali. For his next adventure...Screens for Effective Gene Silencing in Plants. Plant Cell 25:1507–1522 . https://doi.org/10.1105/tpc.113.112235...

  18. New Optimized Genome-wide CRISPRko, CRISPRi, and CRISPRa Libraries

    Type
    Blog Post
    Published
    Oct. 4, 2018, 12:44 p.m.
    ...when trying to screen a limited number of cells like primary cells or in vivo. Recently, the labs of David... cultured cells as well as more difficult conditions such as model systems and primary cells. References...with both A375 (melanoma) and AT29 (colon cancer) cells and assessed the libraries performance via the depletion...important criterion for probing a limited number of cells. Get the Brunello CRISPRko sgRNA library as pooled...subject to an increase in dsDNA breaks that can cause cell death. Thus, CRISPR inhibition (CRISPRi) libraries...study screening for vemurafenib resistance in A375 cells. The SAM system (CRISPR/Cas9 Synergistic Activation...CRISPR-mediated control of gene repression and activation." Cell 159.3 (2014): 647-661. PubMed PMID: 25307932. PubMed...

  19. Uncovering Ancestral Bias in CRISPR Research

    Type
    Blog Post
    Published
    Nov. 29, 2022, 2:15 p.m.
    ...ancestry and germline sequence variation in cancer cell lines took them down unexpected paths. Though this...researchers do large-scale CRISPR screens in cancer cell lines, looking to uncover genetic dependencies of...to see if they could identify the ancestry of the cell lines (European, East Asian, Indigenous American...or South Asian) used in the screens based on the cells’ germline genetic variations. They then wanted to...with each ancestry group. Since about 90% of the cell lines used in DepMap CRISPR screens are of either...groups for 994 of the Cancer Dependency Map cancer cell lines (left). Heatmap indicating the breakdown of...CRISPR guides, short pieces of RNA that bind to a cell’s DNA, telling the Cas machinery where to cut.  ...

  20. Cpf1: A New Tool for CRISPR Genome Editing

    Type
    Blog Post
    Published
    Oct. 14, 2015, 2:30 p.m.
    ...orthologs that display cleavage activity in mammalian cells. Like Cas9 nucleases, Cpf1 family members contain...GC-content. After sequencing and searching for cellular RNAs important for Cpf1 function, they found that...the PAM. With this information, they turned to a cell culture system to see if any Cpf1 nucleases would...would exhibit in vivo activity in mammalian cells. From 16 diverse Cpf1 candidates, Zetsche et al. found...particularly helpful for targeting non-dividing cells, which are difficult to modify through homology-... Cpf1 orthologs known to be active in mammalian cells, LbCpf1 and AsCpf1. In both reports, on target editing... (RNPs) failed to induce off-target editing in a cell culture model. The application of Cpf1 to genome...
Showing: 241 - 260 of 720 results