...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...

...antibody’s labeling of the cell lines to the -omics data. The cell lines with higher expression of the ...approaches are commonly used to produce KO cell lines, some of which are commercially available. Various... – you’ll need to ensure that the parental cell lines of your chosen KO line are likely to express the...Correlation with RNA or Proteomic data from multiple cell lines/ types with different expression Flow cytometry ...combination with other approaches. If several cell lines express the target at different levels, it is possible...assessed through its ability to separate the cell lines based on expression of the target. In this case,...should have more antibodies labeling them; the cell lines with lower expression should have less antibodies...

...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...

... you wait for the anesthetized mice to breathe in the liquid. Restraint: anesthesia Location: nose Gauge...Intradermal Anesthesia Between skin layers 26 < 0.05 mL Advanced Intrathecal Anesthesia Spinal cord ...Intratracheal Anesthesia Trachea 20–23 (tubing) < 0.07 mL Advanced Intranasal Anesthesia Nose n/a < 0.05...be less stressful, because the mice are under anesthesia, but they are not recommended for tumorigenic... required. Restraint: mechanical restraint or anesthesia Location: tail vein or retro-orbitally Gauge:... the muscle. Common uses include administering vaccines, pain meds, or other drugs. Identifying the proper...sciatic nerve. Mice likely experience some muscle soreness after injection, like you might after getting ...

...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...

... to insertional mutagenesis (Howe et al., 2008). 2. Transient expression of transgenes/shRNAs If you want.... Insertional mutagenesis combined with acquired somatic mutations causes leukemogenesis following gene...of research purposes, like creating stable cell lines, delivering CRISPR/Cas9 to cells for genome editing...packaging plasmid contains Gag, Pol, Rev, and Tat genes. Specifically for our knowledge here, the Pol gene...transcriptase), and IN (integrase). These viral packaging genes are required for the production of lentiviral particles...portion does not contain any of the viral packaging genes, the natural cycle of the lentiviral vector ends...process and episomal forms of non-integrating transgenes. Created with BioRender.com; adapted from Banasik...

...protein-coding genes thanks to systems like CRISPR/Cas9 that allow them to manipulate full genes and observe...nearby genes and its role in gene expression, though this may still unexpectedly affect nearby genes (Figure...DA, Weissman JS. Fitness effects of CRISPR/Cas9-targeting of long non-coding RNA genes. Nature biotechnology...only 2% of our genome is made of protein-coding genes, and you might be wondering what the rest of our...manipulate and study in comparison to protein-coding genes (Rinn and Chang 2012). Scientists have determined...knock-out, or permanently disable, protein-coding genes by generating insertions or deletions in the DNA...multiple gRNAs poses the risk of disrupting adjacent genes, especially those regulated by or overlapping with...

...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...

...different fly lines. Also, if there is a chance you need to combine multiple transgenes in one fly, having...having the transgenes in different loci allows for genetic recombination so that the transgenes can be inherited...comparisons between lines expressing different variants of YFG because the transgenes are found in the same... same locus across fly lines (i.e. there won’t be positioning effects). When designing experiments where...where you want to express multiple transgenes, remember that you’ll need to integrate them all at different...accomplished with a few matings to balancer chromosome lines but is not necessary with targeted insertions (here...the white gene, which restores eye color to fly lines where the endogenous white gene is knocked out. ...

...bottleneck is that most recurrently mutated cancer genes are rare and most of the individual variants found...as evolutionary conservation, or predicted deleteriousness of the amino acid change based on maps of protein...scale, broadly available collection of mutant cDNA clones corresponding to cancer variants found in genome...genome sequencing studies (as well as reference cDNA clones mapping to well-annotated cellular pathways). It...These include 634 sequence confirmed mutant cDNA clones corresponding to cancer variants derived from analysis...collaborators to aggregate a sequence-confirmed series of clones corresponding to known cellular signaling pathways...Pathway Reference Set. These Gateway-compatible entry clones are the product of many years of work and we’re...

...technology has changed how scientists edit and control genes, but according to the Broad Institute's Silvana ...technical leap from mutating and deactivating a gene or genes to selectively activating them with the CRISPR system...Konermann said. “If you want to activate all 20,000 genes, it is not possible to test each one and make sure...you are really confident about working for most genes and individual targeting modules. Those two things...for the transcriptional activation of endogenous genes and it consists of three components: 1. A nucleolytically...colleagues demonstrated the new system by screening for genes that, when turned on, make melanoma cells resistant...for each transcription start site in 15,977 human genes. The transcriptional repressor (CRISPRi) library...

...combination with different genes. Having CRISPR systems for assessing genes in defined contexts would ...Annotation of genes in immune cells typically involves the creation of germline knockout mice, which ...the constitutive, ubiquitous knockout of single genes in immune cells in vivo (LaFleur et al., 2019a and...knockouts in their immune systems. However, many genes have different functions in different cell types...Mouse Strain C-CHIME Knocks out a pair of genes pXPR_219 Violet-excited GFP (vex) Rosa26-Cas9...; Rosa26-Cas9 S-CHIME Knocks out pairs of genes sequentially pXPR_220 Violet-excited GFP (vex...These systems were benchmarked on positive control genes in CD4+ and CD8+ T cells. To demonstrate the power...

...choose from when looking to activate genes across diverse cell lines.   dCas9-VP64 Description CRISPR activation...loci across Drosophila cell lines (Sajwan et al., 2019). SPH SPH combines components of SAM and SunTag...variant lacking its endonuclease ability, to bind to genes without editing the genome (Qi et al., 2013). To...exhibits modest levels of gene activation, with some genes experiencing around 2-fold activation levels. Although...HS1 and p65). Performance When targeting single genes, SAM consistently shows the highest levels of gene... multiplex gene regulation (activating multiple genes at once), however, SAM exhibits activation levels...design (SAM) or peptide design (SunTag). This streamlines its delivery, making it a common choice for CRISPR...

... own money at this business and started things up for real: bank account, business cards, incorporation...issues about continuing with the business. We ended up applying to a business accelerator freely stating that...how to run a business. SynBio Axlr8r thought color-changing flowers was a great business idea as well,...social media comments were more often along the lines of “I hate GMOs, but what you are doing makes sense...GMOs that result in a new utility, was there a business opportunity out there for us? After about a month...research and another $30,000 in mentorship and business training. The money is a drop in the bucket against...to develop the technology we will need, and the business training to make the most of it. It has also allowed...