...Activation 95 10 2 1 Denaturation 94 0.5 2 40 Annealing/Extension 60 1 2 40 Enzyme Deactivation 98 10 2 1 Hold ...copies of RPP30 in diploid cells, thus the reason for multiplying by 2. $$V = 2*{copies\ RRE \over copies...diluted 2-fold serially, the concentration of RRE positive droplets should decrease by a factor of 2 across...each well. Detach cells by incubating with 200 µL TrypLE for 1–2 min. Resuspend cells in 500 µL DMEM complete...Lentivirus is generally considered biosafety level 2+. Please ensure that you are in compliance with your...channel pipette 200–1000 µL single channel pipette 2–50 µL multichannel pipette 20–200 µL multichannel ... DG8 cartridge into the cartridge holder. Using a 2–50 µL multichannel pipet, load 20 µL of the reaction...

...156–159. https://doi.org/10.1016/0003-2697(87)90021-2 Chomczynski P, Sacchi N (2006) The single-step method...are commonly found in cells, and without something to block RNase activity upon cell lysis, RNA is degraded...RNases such as RNaseZAP. When harvesting tissues, cells, plants, fungi, or bacteria, keep samples cold and...before RNases had a chance to break down RNA upon cell lysis.  In the late 1970’s, Chirgwin and colleagues...keeping your samples cold until homogenization and cell lysis. This can be challenging depending on your...developed ways to “stabilize” RNA within tissues before cell lysis. These products, namely RNAlater®  from Thermo...solutions that work by inhibiting RNase activity within cells or tissues- they don’t actually chemically stabilize...

...Antibody Fragments. Antibodies (Basel). 2019 Apr 9;8(2):28. doi: 10.3390/antib8020028. PMID: 31544834; PMCID...in transfected eukaryotic cells, allowing for them to be used as intracellular inhibitors.  Aptamers are...the kids say) affinity reagents deserved to be celebrated, there are many other amazing affinity reagents...small enough to be expressed in bacterial and other cell systems. Since the majority of these alternative...the constant region. Nonspecific binding, caused cell receptors for the constant region, is also reduced..., peptides, amino acids, drugs, metal ions, and cells. They can be used either with or as fluorophores... may sound. Validated antibodies are still an excellent option for many applications, and frankly, if ...

...beginning Let’s take a quick flash back to 2004. “Shrek 2” was the most popular movie in theaters, the Razr ...amazing years in front of us. So in our last celebratory moment, we wanted to walk through some of our...Facebook and Gmail, were launched. And Melina Fan, a cell biology graduate student, was frustrated by the ...to receive the Massachusetts Nonprofit Network Excellence Award for our electronic MTA system. We also ...now our Sr. Director of Production) pie-fully celebrating Addgene's 10 year anniversary.   Expanding...

... solution can be stored at 4 °C for up to 2 months. After 2 months, discard the tube and thaw a new working...μg total DNA to μg PEI ratios of 1:1, 1:2, 1:3 and 1:6. The 1:2 and 1:3 total DNA:PEI μg ratios provided... transfect cells with a fluorescent plasmid using a variety of ratios. Check the cells 1-2 days after ...Transfect packaging cells Day 2 (am): 18 h post-transfection. Remove media, replace with fresh media. ...Biological Safety Cabinet 0.5–10 µL single channel pipette 2–20 µL single channel pipette 20–200 µL single channel... 200–1000 µL single channel pipette Ice bucket CO 2 incubator Pipet controller Hazardous waste container..., 5% CO 2 for ~20 h. Prepare a mixture of the three transfection plasmids into OptiPro SFM: Reagent Amount...

...phosphate monobasic monohydrate (NaH 2 PO 4 ∙H 2 O), pH 7.0 138 g NaH 2 PO 4 ∙H 2 O 1 L deionized water Adjust...sodium phosphate dibasic (NaH 2 PO 4 ), pH 7.0 142 g of NaH 2 PO 4 ∙H 2 O 1 L deionized water Adjust pH...monobasic monohydrate (NaH 2 PO 4 ∙H 2 O) 610 mL of sterile sodium phosphate dibasic (NaH 2 PO 4 ) Store up to...recombinant antibody. Section 2: Buffer exchange Choose Option 1 or Option 2 based on the concentration ...antibodies. Workflow Timeline Day 1: Purify antibody Day 2 or later: Buffer exchange Equipment Class II, Type...conical tubes NanoDrop spectrophotometer 37 °C, 5% CO 2 incubator with shaking platform set to 120 rpm 37 ... channel pipette 20–200 µL single channel pipette 2–20 µL single channel pipette Reagents Aspirating pipette...

...localizations. Figure 2: Split-wrmScarlet labeling proteins with different subcellular locations. Image from...determine cell cycle length by examining the ratio of blue to red cells. Cells undergoing rapid cell cycles...engineered C. elegans cell lines to express wrmScarlet1-10 unattached to any other cellular protein. They used...’s difficult to observe the cell cycle in vivo, particularly if the cells are migratory or in deep tissues... BaF3 cells were transduced with H2B-FT virus and grown in varying concentrations of IL-3. Cells grown...proportion of blue fluorescence, indicating cell proliferation. BaF3 cells maintained in media without IL-3 show...H2B-FT consistently predicted the cell cycle speed across these different cell types. In one example, H2B-FT...

... already have an end product in mind, steps 1 and 2 will generally be straightforward. Likewise for step...Calcium phosphate Cells Transformed cell lines Transformed cell lines Transformed cell lines Cargo Nucleic...identify and enrich positive cells or a selection marker to generate stable cell lines. Transient or stable...and enrich positive cells. Likely contains a selection marker to generate stable cell lines. Packaging and...wide variety of mammalian cell lines. Useful for difficult to transfect cell types and can be used in ...protein are degraded within the cell.   Delivery methods for mammalian cell lines As mentioned above, the...components into your target cells. Delivery of these components into mammalian cell lines is quite broad and...

... Cure for HIV/AIDS. Int J Mol Sci. 2023 Jan 13;24(2):1563. doi: 10.3390/ijms24021563 Resources on Addgene.org...Cas9 in Intestinal Stem Cell Organoids of Cystic Fibrosis Patients." Cell Stem Cell. 13, 653-658 (2013). ...patient’s own cells that have been modified ex vivo, previously edited healthy donor cells can be delivered...Genetic Disease in Mouse Via Use of CRISPR/Cas9." Cell Stem Cell. 13, 659-662 (2013). Gerald Schwank et al. ...on editing animal models of disease and cultured cells. For example, in 2013, Jinsong Li and colleagues...applied CRISPR to disease correction in adult stem cells isolated from two patients with cystic fibrosis ...delivery directly to the patient. Patient-derived cells can alternatively be harvested, edited ex vivo, ...

... tabular HTML formatted output. Surprisingly only 2 tools provide FASTA output. A few notable tools allow...Nature biotechnology(2014). PubMed PMID: 24880488. 2. Hsu, Patrick D., et al. "DNA targeting specificity...the research community. I’ve likened it to the celebration surrounding the piñata. From the perspective ...

...C-terminus of human codon-optimized  cryptochrome 2 (CRY2) from Arabidopsis thaliana. When exposed to ...illumination of neighboring cells evokes action potentials (APs) in patched cells expressing wild type CoChR...techniques for live-cell imaging of the dynamic changes in biochemical activity within the cell. Their Fluorescence...distribution of clusters without accumulation in subcellular compartments such as nuclear speckles. Furthermore...OptoSTIM1), caused two-fold faster changes in intracellular Ca2+ levels than the original version containing...CRY2clust should prove a useful tool to further probe cellular function. The CRY2clust Plasmid can be found here...28646204. soCoChR: A novel opsin that enables single-cell optogenetics Article contributed by Meghan Rego ...

...dimerization-dependent protein interactions. FEBS letters, 591 2, 415-424. PubMed PMID: 28032891. This is a different... expressed in cells via plasmids or by the creation of stable cell lines and these cells are then grown... complex. Not performed in a native cell context. For AP-MS, cells are lysed before the bait protein is...and interacting proteins in mammalian cells. The Journal of cell biology. PubMed PMID: 22412018. PubMed...against PPIs that aren't stable outside the native cell context. The Split-BioID method Enter Split-BioID...tagging of proteins in both BioID or Split-BioID, cells are lysed, protein extracted, and labeled protein...proteins that interact with the bait in a native cell contact. Other PPI methods may require protein interactions...

...divided by the off-target activity. Figure 2: Specificity is the measure of off-target activity...CRISPR-Cas12b for mammalian genome engineering." Cell discovery 4.1 (2018): 63. PubMed PMID: 30510770....

...still depolarize neurons at lower MOIs. Figure 2: ChRgers are delivered by intravenous injection into... channels in living cells. Multiple properties of a channelrhodopsin, such as cell localization, kinetics...

...main decisions to make: 1. Which modification? and 2. Which viral vector should be used? Modifying gene...Different AAV serotypes have preferences for specific cell types. For example, AAV1 and AAV9 serotypes have...most cases with VSV-G that generally mediates broad cell tropism and has a high percentage of neuronal targeting...

...method 2): Method 1 Method 2 $$T = {N*F*D\over V_T}$$ Where: T = Titer, TU/mL N = Number of cells transduced...Workflow Timeline Day 0: Seed 293T cells Day 1: Transduce cells Day 2 (am): Remove media, replace with ...,000 cells into 14 mL of DMEM complete. Mix well by pipetting or inverting. Aliquot 2 mL of cell suspension...fluorescent cells Equipment Class II, Type A2 Biological Safety Cabinet 0.5–10 µL single channel pipette 2–20 ...Lentivirus is generally considered biosafety level 2+. Please ensure that you are in compliance with your... 200–1000 µL single channel pipette Ice bucket CO 2 incubator Pipet controller Hazardous waste container...added to 150,000 cells resulted in 25% fluorescent cells, then the titer is: (150,000 cells x 0.25 fluorescent...

...Components." Cell (2016). PubMed PMID: 27160350. Find Plasmids from the publication at Addgene 2. Green, ...circuits to be deployed out of the laboratory in a cell-free and biosafe format that was stable for up to...possible and submitted our manuscript to the journal Cell just six weeks later. The Zika publication timeline...of Toronto. His research is focused on developing cell-free synthetic biology applications for human health...de-novo-designed regulators of gene expression." Cell 159.4 (2014): 925-939. PubMed PMID: 25417166. PubMed...Keith, et al. "Paper-based synthetic gene networks." Cell 159.4 (2014): 940-954. PubMed PMID: 25417167. PubMed...

...protein from cells or tissue samples. Equipment Microcentrifuge 0.5–10 µL single channel pipette 2–20 µL single...transfer, block, incubate with primary antibody Day 2: Incubate with secondary antibody Video Watch this...Heat block Mini gel tank chamber Power supply iBlot 2 Gel Transfer Device Roller Spatula Platform shaker...running buffer Prestained protein ladder Ethanol iBlot 2 PVDF Mini Stack, Thermo Fisher IB24002 20X TBS Tween...immediately or store at -80 °C until ready to use. Section 2: Determine the total protein concentration and prepare...in deionized water. To prepare 20% ethanol, dilute 2 mL of ethanol into 8 mL of deionized water and mix...the transfer sandwich as follows: Unseal the iBlot 2 PVDF Mini transfer stack. Set the Top Stack to one...

...unc- 119 + selection." MicroPublication 1 (2018): 2–3. Wang, Han, et al. "cGAL, a temperature-robust GAL4...currently doing this using two approaches: single-cell RNA-sequencing to identify neuronal class and deep...WDDD aims to acquire quantitative data on all the cell divisions of the C. elegans embryo when all essential...Wang et al., 2018).” With the split system, only cells that have both halves of cGAL can drive expression...

...systems to defend against both DNA and RNA viruses - 2 cas systems regulate at the DNA level and 1 at the...origin of cellular life. His lab has long hypothesized that life started with RNA and a simple cell membrane... circular RNA they have identified resulted in a cell migration defect. They have created a circRNA website...

...: PMC4353587. 2. Stephen, Andrew G., et al. "Dragging ras back in the ring." Cancer cell 25.3 (2014): ...molecular level, at the cellular level there is a universal theme - uncontrolled cellular proliferation. We ...cancerous cells Luckily, there are several safety mechanisms in place to protect us from rogue cells when ...process of programmed cell death, or apoptosis, is one such mechanism. When a cell or its genetic material... directly inhibit the ability of a cell to undergo programmed cell death while simultaneously stimulating...eliminate every cancerous cell. Even so, to pose the greatest threat, cancerous cells still need to successfully...universally-dividing behavior of cancer cells, attacking the cellular machinery necessary for division. This...

... 2008 Dec; 18(6): 617–623. Pubmed PMID 19349161.  2. Deshpande A, Bergami M, Ghanem A, Conzelmann KK, ...it first infects one cell (the “starter cell”). It then replicates within that cell, and nascent RABV cross...connections to the starter cell, RABV was modified to only infect user-defined starter cell types, as well as ...dictate infection of target cells through their interactions with target cell surface receptors (Callaway...Callaway 2008). Based on this, cell type specificity of the starter cell can be achieved by using EnvA-pseudotyped...studied biological processes, like how our stem cells are housed in specific locations and give rise to...Tracers Due to their ability to efficiently enter cells and deliver a variety of cargoes, genetically engineered...

... replication competent virus [usually HIV-1]; and 2) the potential for oncogenesis through insertional...of interest into cells. Unlike gamma-retroviruses that can only infect dividing cells, lentiviruses can...dividing and non-diving cells while retroviruses can only infect dividing cells. When choosing the appropriate...to ensure that the proper cell line is used; several common laboratory cell lines including 293 were immortalized...can infect dividing and non-dividing cells.  Addgene has an extensive collection of lentiviral plasmids...transfecting four different plasmids into the producer cells (two packaging plasmids, an envelope plasmid, and...appropriate gene delivery system, the specific target cell or tissue needs to be considered. Q2: Can I use...

...Figure 2: Inner mitochondrial membranes imaged with structured illumination microscopy in HeLa cells expressing...yielded editing of 80–90% in human cell lines and primary cells. Although cell entry of a Cas9 ribonucleoprotein...post: Peptide-assisted genome editing for primary cells Monomeric StayGold variants AAV.CAP-Mac for systemic...antibodies Peptide-assisted genome editing for primary cells By Susanna Stroik Even with the advances in CRISPR...CRISPR technology over the past decade, primary cells remain difficult to genetically edit due to toxicity...toxicity from electroporation or poor cellular entry with other transfection methods. The Junwei Shi Lab addressed...Genome Editing (PAGE). They fused the Cas enzyme to cell-penetrating peptides and co-incubate it with an ...

...PMID: 27151215. PubMed Central PMCID: PMC4938170. 2. Zuris, John A., et al. "Cationic lipid-mediated delivery...can often be used in cells that are difficult to transfect, such as primary cells. Using RNPs can also...interest. This is commonly performed by transfecting cells with a plasmid, such as PX459, which encodes Cas9...must be considered when using this method:     Cells must be amenable to transfection or viral transduction...protein in complex with a targeting gRNA, to your cells of interest. Cas9 RNPs are capable of cleaving genomic...single or multi-gene knockouts in a wide variety of cell types, gene editing using homology directed repair...difficulties with protein expression that occur in cells where common eukaryotic promoters (such as CMV or...

...Reports 9: https://doi.org/10.1038/s41598-019-41088-2 Magnus C, Lee P, Bonaventura J, Zemla R, Gomez J, ...neuroscience where specific tissues, cell types, or even subcellular regions of a neuron need to be targeted...pathways, giving rise to the same cellular responses. The cellular response set off by a DREADD depends ...disease, neuroscientists need to be able to probe cells and circuits without disrupting normal brain function...it provides a noninvasive method for manipulating cell activity, especially neuronal firing. What is chemogenetics...drugs. Once activated, chemogenetic receptors hijack cell signaling pathways and can lead to such responses...requires successfully introducing receptors to target cells. Targeted and specific delivery becomes especially...

... that bind to a cell’s DNA, telling the Cas machinery where to cut.      Fig. 2: Heatmap indicating...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...

...take care when using this method.      Fig 2:  A, The polyhistidine tag is a string of histidine...This can be pretty complicated given that a single cell has tens of thousands of proteins functioning in...

... adapted to a competitive format. Figure 2: Overview of using Competitive ELISA to quantitatively...immunoassays in DNA damage and instability detection. Cell. Mol. Life Sci. 76, 4689–4704 (2019). https://doi.org...

...Workflow Timeline Day 1: Seed cells Day 2: Transfect cells Day 3-6: Feed cells Day 7: Harvest antibody Equipment...Pro-Tip Do not use cells that are over 30 passages. Section 2: Transfection Check the cell density and viability... (EBNA). Safety Warnings HEK293 cells are considered biosafety level 2. Please ensure that you are in ...compatible with 50 mL conical tubes Automated cell counter 37 °C, 5% CO 2 incubator with shaking platform set to...density of 0.9 x 10 6 cells/mL in a 500 mL vented flask.Incubate in a 37 °C, 5% CO 2 incubator on a shaking ...Pro-Tip Culture should be between 1.5–2 x 10 6 cells/mL with >95% viability to proceed with transfection...250 mg Benzamidine 25 mL Aprotinin saline solution (2 mg/mL) Mix well and sterilize through a 0.2 µm PES...

...promising monoclonal antibody with high specificity. (2) Narrow down the specific epitope that your antibody...For about as long as the disciplines have existed, cell and molecular biologists have been continuously ... peer into the mysterious, microscopic world of cells. The development of antibodies as tools allowed ...monoclonals would help the field explore Myc’s role in cell proliferation and tumorigenesis.  Using two different...specific for human c-myc proto-oncogene product. Mol Cell Biol 5:3610–3616. https://doi.org/10.1128/mcb.5.12.3610... and immunoglobulin heavy chain binding protein. Cell 46:291–300. https://doi.org/10.1016/0092-8674(86...signal prevents secretion of luminal ER proteins. Cell 48:899–907. https://doi.org/10.1016/0092-8674(87...

...system (by measuring the fluorescence intensity); and 2) to visualize the localization of the FP (fused to... localization of that biomolecule inside living cells. FPs classified by the emission color (emission .... Therefore, these can be used as “timers” for cellular processes following their activation. The four...Biosensors: Throwing color lights on molecular and cellular processes” Curr. Protein. Pept. Sci. 9(4):338....proteins and their applications in imaging living cells and tissues”  Physiol. Rev. 90:1103. Wu et. al. ... nuclear localization, and dynamics” Curr. Opin. Cell. Biol. 23:310. ...

...integrates into the trnS-leu2 locus.   Figure 2: Introducing Mobile-CRISPRi into bacteria. Top: A ... acid (DAP), a component of the cell wall. Once in the recipient cell, the CRISPRi DNA (purple) flanked... is propagated during normal DNA replication and cell division. Mobile-CRISPRi does not disrupt gene function...mobilize these plasmids for transfer to recipient cells during a tri-parental mating scheme. Mobile-CRISPRi...

...) 12 (>100) xCas9(3.7)-ABE 69 (1.4) 43 (2) 21 (3) Not examined 16 (>100) Average percent... in mammalian cells Hu et al. compared xCas9 activity with wt SpCas9 in mammalian cells, finding xCas9...NG and GAT PAMs. xCas9 3.7 Activity in Mammalian Cells Construct NGG PAM NGA PAM NGC PAM NGT... percent editing efficiencies (edit frequency in cell population measured with NGS) are shown as percentages...

...follows: Figure 2 Using the box tool, draw a box around the entire first gel lane (as in Figure 2). Select Analyze... Example for AR0018 (lane 2 in Figure 1): Sample Peak 1 (contaminant) Peak 2 (contaminant) Peak 3 (HC)...choose Use Equation . Select the Show R 2 checkbox. Pro-Tip The R 2 of the trendline should be between 0.95...Equipment Heat block 1–10 µL single channel pipette 2–20 µL single channel pipette 20–200 µL single channel... Add 5 µL of 4X sample buffer to each sample. Add 2 µL 10X reducing agent to each sample. Spin the sample...bottom part of the gel where dye is visible. Section 2: Staining the Gel Place the gel in a plastic tray .... Figure 1 Recombinant antibody preps should have 2 clear bands at ~50 kDa and ~25 kDa corresponding to...

...for ~2 weeks. All of the cells in the untransduced (negative) control well should be killed and no colonies...with 0.1% crystal violet and counted. Figure 2: A549 cells were transduced with the indicated serial dilutions...Day 2: Replace media with fresh media containing selection reagent Days 3–14: Change media as needed Days...Cabinet 0.5–10 µL single channel pipette 2–20 µL single channel pipette 20–200 µL single channel pipette... 200–1000 µL single channel pipette Ice bucket CO 2 incubator Pipet controller Hazardous waste container...disturb the colonies. Count the colonies for at least 2 of the dilutions. Pro-Tip The higher dilution wells...1,000 cells into each well of a 6-well dish. Prepare a batch of cells as follows: Dilute 7,000 cells into...

...You’ve probably heard that only 2% of our genome is made of protein-coding genes, and you might be wondering...encode proteins. They play important roles in many cellular and regulatory processes, but much of their functionality...screening for functional long noncoding RNAs in human cells by Cas9 targeting of splice sites. Nat Biotechnol...non-coding RNAs with the CRISPR/Cas9 system in human cell lines. Nucleic Acids Research. 2014;43(3):e17. doi...

...mazF gene and a gene for Zeocin resistance (Figure 2). This MazF-ZeoR cassette is flanked by two direct...unstable, meaning that the cells must continually produce antitoxin to avoid cell death. In the case of plasmids...addiction systems” require dividing cells to retain plasmids - if a cell does not inherit plasmid due to ...will be degraded leaving the cell to be killed by the more stable toxin. Cells only survive if they retain...10% of cells while plasmids containing the plasmid addiction system were retained in 75% of cells. They...consist of a toxin which can affect a variety of cellular processes and an antitoxin that suppresses the...designated this fragment as the ccdB region (for “coupled cell division”). The encoded toxin-antitoxin system consists...

... vivo and in vitro applications.   Figure 2: Modular features of DIRECTED particles where the ... between living cells by TransitID. Cell, 186(15), 3307–3324.e30. doi: 10.1016/j.cell.2023.05.044. PMID... efficient prime editors. Cell, 186(18), 3983–4002.e26. doi: 10.1016/j.cell.2023.07.039. PMID: 37657419...lentivirus and MMLV gag, to specific cell types. It can also make CAR-T cell therapy more accessible by simplifying...Optogenetic proximity labeling and TransitID Tracking cellular history with protein ticker tape Teaching an old...called DIRECTED (Delivery to Intended REcipient Cells Through Envelope Design). This modular approach ...viral surface, expanding the range of targetable cell types. DIRECTED is compatible with fusogens from...

...replicate, then purify the DNA.     Figure 2: A pooled library page with protocols boxed in red...transduction of cells, resulting in cell population mutated by the viral pooled library. These cells can then...transduce your cells.  Titering After generating your lentivirus, you’ll want to infect your cells with the ...of a cell being infected by only one viral particle, but it also increases the chances that a cell doesn...adequately represented in the cell population, you’ll need to infect many more cells than the number of plasmids...appropriate packaging and envelope plasmids into HEK cells allows production of lentivirus. Purification of...small aliquot is not enough to transfect producer cells, so your first step will be to amplify the library...