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...genome-wide libraries for CRISPR screening. Sanjana NE, Shalem O, Zhang F. Nat Methods. 2014 Aug;11(8):783...cells were then used to generate a monoclonal cell lines by limiting dilution. The rest of the cells were... you’re doing a screen. I made a few monoclonal lines (Figure 2) and I was surprised at how variable Cas9... Figure 2: Cas9 expression in monoclonal cell lines generated from A549 cells transduced with lentiCas9...results. (Figure 5). The digestion product (Figure 4, lanes 0.3 % 3.0, Figure 5, lane 2) is faint, so comparing...formed from control (GFP gRNA-targeted) BRAF sites (lanes 3, 4) were used as controls. Duplexes were generated...Resources at Addgene.org Generating Stable Cell Lines with Lentivirus Isolating a Monoclonal Cell Population...

...in between a protein and a tag, such as an NLS or NES, enables control over localization. Inserting PhoCl...acrIIA1 Listeria monocytogenes  pRAU168  No  pJH372  No  acrIIA2 Listeria monocytogenes  pRAU171 Partially...  Listeria monocytogenes  pCSW65 N/A (Toxic)   None N/A   acrIIA4 Listeria monocytogenes  pCSW21  Yes...quickly and easily generate zebrafish transgenic lines to study neurobiology and neuorodgenerative diseases...compatible cloning sites for easy cloning of your genes of interest. In order to improve the study of specific...induction of structural plasticity in single dendritic spines. The set of CyRFP constructs available at Addgene... used a combination of deliberate and random mutagenesis to isolate the best RFP. They targeted known ...

...are (i) brightness, (ii) photostability and (iii) aggregation tendency. Brightness Brightness refers ...molecule. A higher brightness increases the detected signal and therefore high brightness is an important...how the brightness is best defined or determined. Here, we will explain how practical brightness is determined...determined and why practical brightness is a better criterion than theoretical brightness for selecting the brightest...protein. Theoretical brightness Two key properties of fluorophores that determine brightness are the extent ...number, the higher the theoretical brightness. The theoretical brightness often appears in tables with fluorescent...and the sample. Practical brightness A protein with a high intrinsic brightness that does not fold well ...

... the Lindberg Lab we often make cell lines that overexpress genes of interest; more recently we have also...CRISPR vectors to generate cell lines with knockouts of specific genes. Many years ago, people in the ...give rise to single clones, and thus many plates must be examined for single clones and then handled. Additionally... Additionally, many cell lines, especially the endocrine cell lines we most commonly work with, require...cell clones with sterile truncated pipet tips. Indeed, an experienced operator can pick 48 clones from...individual cell clones with different expression characteristics from a population of transfected clones. The technique...number of single clones obtained (on occasion we have prepared two 48-well plates of clones) which enables...

...targeting their genes of interest to generate mAID fusions to endogenous genes. Cell lines with the appropriate...targeting a total of 20,121 genes within the human genome (the identity of the genes and target sequences can...continuous expansion of compatible backbones, promoters, and genes available to the community. If you ...identify both core and context-dependent human fitness genes, the laboratory of Jason Moffat at the University...cell models and identified ~4-5-fold more fitness-related genes than have been found in previous screens...uncharacterized genes that are conserved among apicomplexans and important for T. gondii fitness. Sixteen of...enable researchers to screen the entire genome for genes that regulate a wide variety of phenotypes, including...

..., which genes are good candidates for being involved in the studied phenotype. For robustness of a screen...presenter then showed which genes they had generated knockout mice for (all of the genes involved as I recall...differentially expressed genes that are known drug targets, or all differentially expressed genes that code for ... if genes that you asked for were not found in the reference library. It’s not uncommon that genes have...5 guide RNAs (gRNAs) targeting each of the 1000 genes you’d like to investigate in your next CRISPR screen...CRISPR screens targeting a (long or short) list of genes provided by the user. This approach is useful for...investigator generates a cell population where all genes are knocked out at the population level, but each...

...of insertional mutagenesis. Random integration also creates heterogeneous iPSC cell lines, which can complicate...complicate comparisons made between lines. Incomplete silencing of transgenes is a concern as well, and reactivation...reprogramming transgenes and the up-regulation of corresponding endogenous pluripotency genes. Retroviral...to insertional mutagenesis and random integration creates variation between iPSC clones. Retroviruses ...disrupt the expression of tumour suppressor genes and/or oncogenes, potentially leading to the presentation...insertional mutagenesis, the presence of a genetic scar, and incomplete silencing of transgenes. Overall,...Efficient and Stable Genome integration Insertional mutagenesis, transgene reactivation, residual expression ...

...profiles (Dwight, 2011).  Reference Genes  Choosing a set of reference genes deserves more attention than it...want to ensure that your reference genes are suitable for your cell lines and/or tissue types.  Please do...reference genes, plural. Your data will be much improved if you move to using multiple reference genes whenever... multiplex your reference genes to save on plate space. For many cell lines and tissue types, appropriate...and most machines have a protocol that automatically performs the normalization step.  Machines Most general-use...researcher defines log-linear phase Liu and Saint No Raw Data Reference and target genes can have...previously-generated validation data, be aware that different machines, samples, and treatments may affect the assay....

... epitope tagging of a large cohort of genes (e.g. tagging genes representing a protein super family). ...generation of clonal knock-in lines that we now use for IP and ChIP assays. Tag 96 genes at a time using Custom...knock-in for 96 different genes in a single go (Figure 3). We were able to tag >30 genes/plate in the first ...Derive clonal lines by single-cell deposition in 96-well plates: Characterize clonal lines by ICC and PCR...designed for ~250 genes, we have had about 30% tagging success, although many of these genes were expressed...be used at high throughput because numerous cell lines can be generated, each with a different tagged protein...ChIP assays. Creating epitope-tagged clonal cell lines remains a challenge as it relies on inefficient ...

...community – particularly ones that help address ongoing problems, like cells lines. A 2014 study in Science...145,000 cell lines. Cellosaurus Cellosaurus seeks to identify and describe all cell lines used in research...and dog cell lines is included when available, and can be submitted via email for cell lines profiled in...Cellosaurus also identifies problematic cell lines: ones either suspected to be contaminate or easily ...as commonly misidentified lines. Browsing this feature to know if your lines may be especially vulnerable...to help you identify proximal lines you care about. If you have cell lines without a certified origin (...Science found that approximately one-third of cell lines used in scientific research are misidentified! Rigor...

...70% of human genes have a counterpart in zebrafish. Understanding the function of these genes in fish will...Additional mutagenesis projects have since been initiated and today thousands of mutations in genes affecting...zebrafish Random mutagenesis screens provided a major toehold into understanding how genes control biology..., making them ideal for studying vertebrate embryogenesis. To illustrate some of the reasons that zebrafish... is to examine the consequences of manipulating genes. One reason for the success of zebrafish as a model...studies, in which, for example, the activity of all genes in response to an experimental treatment can be ...This can reveal previously unknown functions for genes, and lays the groundwork for understanding the gene...

...reaction is not fluorescence! Chemiluminescence, which includes all bioluminescence, is the production of light.... Table 1: Properties of luminescence versus fluorescence   Luminescence Fluorescence Source of ...needed — just the luminescent substrate. This approach preserves the advantages of luminescent assays (low ...For most people, the most familiar source of bioluminescence is the firefly. Along with charming nighttime...releasing light. Created with BioRender.com. Bioluminescent reporters require a chemical substrate (and...a laser in fluorescence assays), which means luminescence assays have lower background levels and improved...luciferases Although firefly luciferase was the first bioluminescent reporter to be cloned and remains popular, ...

...   Saving clones for later culture: At this point, you will want to hang onto your clones for later use...start cultures of your positive clones. If you want to store your clones longer term, just streak the colonies...liquid cultures with the clones you pick and only mini-prep the positive ones. Regardless of which method...larger sized products for positive clones (+) compared to negative clones. Finally, orientation-specific ...identifying a few positive clones, the last step is to mini-prep these clones and submit the plasmids for...type of primer pair is also great for screening clones created with the same backbone but that contain... and back-bone specific primers. Find Empty Backbones at Addgene Each approach has advantages and drawbacks...

...recombinant clones are generated.  A simple way to do this is to express lambda red genes from a plasmid...the red genes needed for recombineering, but also leads to expression of the int and xis genes which are...generation Expression of lambda red recombination genes. Electroporation of substrate DNA and outgrowth ...bacteria. Selection and confirmation of recombinant clones.    Figure 2: Overview of using Lambda...selectable DNA fragments, such as antibiotic resistance genes, as well as non-selectable DNA fragments, such as...activation Expression of Lambda Red recombination genes There are four ways to express the lambda red recombineering...exist that stably express lambda red recombineering genes due to integration of a defective lambda red phage...

... common chemiluminescent methods for detection (though the enzymes used for chemiluminescence can be conjugated...classes of reporters: chemiluminescence enzymes and fluorescent proteins. Chemiluminescence enzymes are cheap...Direct westerns are simpler and faster than indirect ones and can be used in highly quantitative assays. On...appropriate for the target(s) and transfer method. Membranes The two membrane options are PVDF and nitrocellulose...the transfer: you’ll need to pre-wet most PVDF membranes before the transfer and allow them to dry afterwards...has higher background noise than nitrocellulose membranes. Pro tip! You can visualize proteins on a PVDF...reprobing, unless you buy specialty nitrocellulose membranes specifically developed to allow stripping. And...

...used to generate random mutagenesis libraries or to insert heterologous genes, reporters, or other markers...unprecedentedly-high brightness, bfloGFPa1, and the other with extremely-dim brightness, bfloGFPc1. The plasmids...Updated mini-transposon vector for bacterial mutagenesis or gene targeting Victor de Lorenzo's lab...this vector, called pBAM1, by conducting random mutagenesis in the soil bacterium Pseudomonas putida and ...successfully create GFP fusion proteins with a variety of genes across the genome. Although this tool was published...variety of GFPs with diversity in fluorescence brightness, intensity, excitation and emission spectra. ...-on system (pTRE-Tight-MitoTimer) to show its usefulness in cell culture to report changes in mitochondrial...

...antibiotic resistance genes. Genes that are targets of antibiotics are often essential genes which creates a.... ICEBs1 carries the genes needed for conjugation, but can also carry cargo genes, like Mobile-CRISPRi...essential genes were less represented while bacteria containing guides targeting non-essential genes remained...parts: a plasmid expressing the Tn7 transposition genes (tnsA, tnsB, tnsC, tnsD, and tnsE), and a plasmid...interest following IPTG induction of conjugation genes and bi-parental mating. Following excision from ...components and a plasmid with the Tn7 transposition genes are transferred to a recipient bacteria strain of...colleagues used Mobile-CRISPRi to knockdown individual genes in several bacteria associated with human disease...

...could replace one of the reporter genes above with your gene or genes of interest. Some of the plasmids...more of the genes replaced.  Additionally, 2A peptides could be PCR-cloned between your genes of interest...Co-expression of multiple genes is valuable in many experimental settings. To achieve this, scientists...useful almost anytime you want to express multiple genes in the same cell. Although it is possible to drive...multiple, individual expression cassettes, having the genes expressed from the same cassette is sometimes advantageous... relative expression levels between two or more genes is important. How do multicistronic vectors work...allow for the efficient translation of multiple genes from a single transcript. The two strategies most...

...fluorescent donor for a bioluminescent donor, making BRET probes. Here the bioluminescent donor is unaffected...Fusion Protein, codon-optimized Firefly luciferase genes (e.g., luc2), and NanoLuc® Luciferase are available...deep sea shrimp to a compact powerhouse of bioluminescence, NLuc is 100-fold brighter than our more common...NLuc is bright enough to detect endogenous tagged genes generated through CRISPR/Cas9 knock-in. Finally,...wavelengths should be used. Researchers often use bioluminescence to overcome these issues as traditional luciferases...infeasible for certain experimental setups. The brightness of NLuc has caught the attention of researchers...excites the fluor through a process known as bioluminescent resonance energy transfer (BRET). The fluor...

...that for approximately 40% of the identified genes, the genes were not expressed at all. This unexpected... and germline sequence variation in cancer cell lines took them down unexpected paths. Though this research...researchers do large-scale CRISPR screens in cancer cell lines, looking to uncover genetic dependencies of individual...if they could identify the ancestry of the cell lines (European, East Asian, Indigenous American, Africa...each ancestry group. Since about 90% of the cell lines used in DepMap CRISPR screens are of either European...for 994 of the Cancer Dependency Map cancer cell lines (left). Heatmap indicating the breakdown of ancestry...groups were differentially dependent on certain genes. This didn’t prove to be correct, either. However...