..., 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...

...   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...

...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...

...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...

...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 ...colleagues used Mobile-CRISPRi to knockdown individual genes in several bacteria associated with human disease...the antibiotic trimethoprim. When Enterobacter aerogenes, Klebsiella pneumoniae, and P. aeruginosa (all...

...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...

...directly trafficked to cell membranes. Membrane trafficking of farnesylated derivatives enables labeling...distinct patterns of recombination. In 2007, Joshua Sanes and Jeff Lichtman, both of Harvard’s Department ...Department of Molecular and Cellular Biology, harnessed these attributes of Cre-lox to create the Brainbow mouse...biology towards single-cell resolution imaging. Sanes and Lichtman endeavored to create a map of the brain... deletion, the design of Brainbow-2.1 cleverly combines Cre-mediated deletion and inversion. The Brainbow... prevent constant inversion of the construct. Sanes and Lichtman used pronuclear injection to insert...colors (see table to the right); in various reports, Sanes and Lichtman have observed from 90-160 distinct ...

...play a large role in turning genes ‘off’ and ‘on’, signaling which genes should be ‘read’ by the cellular...with the genes in the compacted region and results in a loss of gene expression. In this way, genes in compacted...the same exact genes, being improperly silenced in both cases. In both diseases, genes from both copies...Epigenetics has recently been hitting the headlines, with sotires like the potential devastation of the...dangerous DNA damage. If the way DNA is wrapped around histones changes, then gene expression can change as well...The more frequently and tightly bound DNA is to histones, the more compacted it becomes. This prevents ...modifications, which can influence the transcription of genes by RNA polymerase II, or by the addition of methyl...

...roles of proto-oncogenes, tumor suppressors, and their protein products. Proto-oncogenes consist of a group...specific genes involved. Downregulation of tumor suppressors and upregulation of proto-oncogenes ­­­can ... group of genes/proteins that, when expressed, normally encourage cell survival, growth and proliferation...of control. One may compare the roles of proto-oncogenes and tumor suppressors to the accelerator and brake... the health of an organism that both classes of genes function normally to maintain balance – encouraging...lining the digestive system, causing discomfort and illness. Further, depending on the specific type of cancer...-Ras, and N-Ras) encoded by a family of proto-oncogenes. Ras proteins are special because they are known...

...mantra being “you can’t trust those whacky cell lines”. This may certainly have been a valid concern for...quality work in cell culture. The problems with cell lines Simply put, the majority of cell line problems come... for authentication and contamination) and cell lines can become cross contaminated and are invariably...Stanley Gartler reported that most of the 18 cell lines he had studied (the majority in those days) were...line. He proved this by examining whether the cell lines had isoenzymes and genetic polymorphisms specific...problem today. It is now known that nearly 500 cell lines suffer from contamination with HeLa being the most...listing all cross contaminated or misidentified cell lines. The solution Fortunately it has never been easier...

...reusing pipettes for multiple cell lines rather than using disposable ones or reusing gloves. The serum is... technique, PCR primers specific to the 16s RNA genes of various commonly infecting mycoplasmas are used...sensitivity but low specificity (Degeling, 2012). Bioluminescence assay kits are available from different biotech...which trigger cellular mechanisms that generate luminescence. This can then be detected by a luminometer.... for cell culturing and maintenance of the cell lines. Providing such an environment for the lab facility...Wear a clean lab coat and mask while handling cell lines. Avoid discussion with other lab members, sneezing...wearing PPE. Photo by Kate Harten DeMaio. Cell lines and reagents Examine your cell line stock for mycoplasma...

...important to know the brightness of individual fluorophores. Table 1 describes the brightness of a few commonly...fluorophore brightness levels, these differences are much more subtle (think of brightness levels on your...there are fixable dye stains that react with free amines on and inside the cells and are thus typically ...en-us/resources/bd-spectrum-viewer.       Brightness Apart from the excitation and emission wavelengths...commonly used fluorophores. A fluorophore's brightness refers to the signal strength of the emitted photons...fluorophores have a stronger signal. Table 1: Degrees of brightness for a few commonly used fluorophores in relation...emea.bd.com/panel-design/en/flow-cytometry-fluochrome-brightness-spillover Excitation Laser Color Very Bright...

...Infection with Agrobacterium rhizogenes can be used to develop stable hairy root lines in tissue culture. This...transformation. Choose good reporter genes There are many great reporter genes available and which one you choose...process. While developing transgenic hairy root lines in tissue cultures takes half a year, and generating...transcription factor candidates and the regulation of genes in the biosynthetic pathway. Why plant scientists...interest in the plant. A common method for silencing genes Virus induced gene silencing (VIGS) is a transient...plant's post-transcriptional machinery to silence genes of interest (Unver and Budak, 2009). This method...in C. roseus. A common method for overexpressing genes In plants like Arabidopsis and tobacco, Agrobacterium...