...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...

...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...

...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...

...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. ...

...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...

...transgene but also that of adjacent cellular genes. Q7: Which genes are deleted or modified to result in replication...1]; and 2) the potential for oncogenesis through insertional mutagenesis. These risks are largely based...lentiviruses and gamma-retroviruses use the same genes for packaging (that's gag, pol, and env), the isoforms...specifically for transient expression (find some useful backbones here) or from viral transduction. While not ideal...cell line is used; several common laboratory cell lines including 293 were immortalized with adenoviral ...3rd-generation transfer vectors apart? A3: Addgene defines 2nd- or 3rd-generation transfer vectors based on...expression of lentiviral vectors in producer cell lines. Identifying less toxic envelope proteins or envelope...

... identified ricin toxicity genes. The screen also discovered several genes previously not associated with...-throughput systematic approach for identifying genes involved in a biological process. These screens ...per gene and targets all ~20,500 protein-coding genes. Unique, non-overlapping sites in the genome were... pooled library via a lentiviral vector to cell lines that either stably expressed Cas9 or that had been...toxicity screen since essential and non-essential genes for both pathways had previously been identified...identifying >88% of previously identified essential genes while previous Cas9 and shRNA library screens only...only identified  60% of the essential genes. The ricin toxicity screen had a 10% false discovery rate ...

...multiple genes with Gal4/UAS system One can also use Gal4 to drive expression of multiple genes under the... Drosophila geneticist’s main workhorse to turn genes on or off. Gal4 is a transcriptional activator that...machinery to the site to induce gene expression. Thus, genes and siRNA encoded downstream of the UAS sequence...promoter, and Bloomington’s Stock Center has fly lines expressing Gal4 under a variety of promoters resulting...quickly mate your UAS-YFG line with a variety of Gal4 lines and study gene expression in many different tissue...sequences. For example, if you discovered five new genes and wanted to express them within a specific neuronal...dosage effects. For example, overexpression of two genes by one Gal4 driver will produce roughly half as ...

...disadvantages of luminescence versus fluorescence.  Table 1: Properties of Luminescence versus Fluorescence... the emission of photons. The most familiar bioluminescent organism for most people is the firefly (Photinus...surprisingly it is also the most commonly used bioluminescent reporter. This beetle emits a yellow-green ...as substrates. Production of photons by this bioluminescent reporter occurs slower than fluorescent-based...the different mechanisms to produce photons, chemiluminescent reporters are generally less bright than fluorescent...Fluorescence   Luminescence Fluorescence Source of Emitted Photons Chemical reaction High-energy photons...Photinus pyralis) is the most frequently used bioluminescent reporter, other luciferases have been identified...

... They share about 70% of their genes with humans and 85% of human genes associated with a disease have...Zuber, Johannes, et al. "Mouse models of human AML accurately predict chemotherapy response." Genes & development...researchers around the globe. These organisms share many genes with humans, are easily maintained in the lab, and...manipulated with tools like CRISPR to make transgenic lines. The fruit fly (Drosophila melanogaster) Another... of Drosophila research. Morgan discovered that genes were found within chromosomes using the fruit fly... as it is estimated that 75% of disease causing genes in humans have a functional homolog in the fly (...packaged into chromosomes and about 23% of yeast genes have a counterpart in humans (Liu et al., 2017) ...

...carrying genes for bacterial toxins like colicin typically also carry the necessary resistance genes to help...cell to another). Bacteria need special fertility genes to be capable of conjugation, and these are encoded...organisms to thrive in competitive environments. The genes encoding these functions form the basis of many ... Resistance (R) plasmids: microbial defense Genes allowing bacteria to survive toxins are the clearest...Nevertheless, the transfer of plasmids carrying these genes between bacteria can also have serious implications...: microbial infection Virulence plasmids encode genes that allow the host to infect other organisms. If...difference is virulence plasmids! This category includes genes that help bacteria invade hosts, including attaching...

...replaced S. pyogenes Cas9 in the ABE complex with PAM-variant Cas9 proteins: the S. pyogenes NG-Cas9 (PAM...While editing single genes is very useful, sometimes, you need to modify multiple genes to generate a cellular...adenine base editors with improved editing in cell lines Nicole Gaudelli, the Associate Director and Head...ABE8 constructs using a catalytically “dead” S. pyogenes Cas9 (dC9-ABE8.x-m/d). The dC9-ABE8.x-m/d constructs...adenine base editors The most commonly used S. pyogenes Cas9 requires the target site in the genomic DNA...(“N” can be any nucleotide base, followed by 2 guanines). This limits the use of ABEs as DNA sites that...In addition to stem cells the authors targeted 6 genes in primary human T cells using 8 selected ABE8s ...

...from Streptococcus pyogenes.   Figure 2:  Crystal structure of S. pyogenes Cas9 in the apo state... we can see why: two arginines make base-specific hydrogen bonds to the guanines, but the N (a thymine...    Figure 3: Crystal structure of S. pyogenes Cas9 bound to guide RNA (PDB ID 4ZT0), shown opaque...the protein, spanning both lobes. Stem loop 1, nestled between the REC and CTD domains, is required for...protein portion of the CTD, ready to check for PAM sequencines. This helps explain why Cas9 can’t locate PAM...sequence isn’t in place yet.     Figure 4: S. pyogenes Cas9 in the apo state (PDB ID 4CMP, top) compared... residues in the CTD can get cozy with DNA. S. pyogenes Cas9 recognizes the PAM sequence 5’-NGG-3’, where...