...1983, but wasn’t successfully used for genetic manipulation in mammalian cells until 2005 [1]. Like other...stable cell lines is to transfect, select, and isolate clones. This procedure is cumbersome and inefficient... other genetic alterations have occurred) and isolating the specific cells that have the edit of interest... CRISPR/Cas9 to efficiently perform genetic manipulations in animal models or cells lines, including stem...included in a standard homology directed repair template to facilitate the selection of cells containing... technology using a site-specific nuclease to stimulate homologous recombination and Footprint-Free™ removal...re-cutting. Transposase removal of the cassette later restores the target sequence. This allows researchers...

...Transcription, translation, and prone to degradation – those are the words that describe RNA! Double ...transcription. In an uninfected cell, dsRNA rarely circulates, but upon viral infection, this nucleic acid ... RNAi is designed against), RISC can repress translation and/or degrade target mRNA. These RNA tools ... almost always epigenetically silenced, but dysregulation of these systems can lead to local dsRNA production...source of dsRNA (that doesn’t involve cellular dysregulation) is the mitochondria. These organelles have ...During transcription, RNA is synthesized from a DNA template, inherently generating RNA:DNA intermediates in...which suppress endogenous dsRNA formation are dysregulated, immune responses can mount in the absence of...

...scientists frequently use growth factors or serum to stimulate a pathway of interest, but there can be a lot ...KTR-mRuby2). The ERK KTR is a target for MEK1 phosphorylation, much like endogenous ERK, and its localization...indicator of MEK1 activity. When MEK1 is active, it phosphorylates the ERK KTR and red fluorescence localizes ...MEK1 and ERK are inactive, the ERK KTR isn’t phosphorylated and red fluorescence can be found in the nucleus...as a stimulus. This system also allows precise modulating of a particular arm of the signaling pathway ...the negative feedback on endogenous MEK1/2 phosphorylation by creating a short pulse of psRaf1 activity...nm light (turn off psRaf1). After this, the phosphorylation status of MEK1/2 was tracked at 5 minute intervals...

...revolution in genetic engineering techniques is a speculation of yesteryear which has been realized recently...explained how the four letters of the DNA get translated into the twenty amino acids. Once scientists ...DNA encoded all the information necessary for regulating and propagating life, there was no turning back... molecular biologist was to devise methods to manipulate it. The term genetic engineering itself is attributed...what would happen if these corporations would extrapolate genetic engineering to exploit human beings. ...repaired by homologous recombination with a repair template.  Each zinc finger recognizes a nucleotide triplet...advertisement of the technology on various non-scientific platforms, CRISPR has also transcended into the world of...

.... Eight years later, scientists isolated pink, purple, and blue chromoproteins isolated from reef-building...reach zooxanthellae, the coral’s symbiotic dinoflagellates that are photosynthetic (Salih et al., 2000...experiment worked just by looking at the cells on the plate. For example, if your plasmid contains a gene encoding...directly use in bacteria, where most genetic manipulations start. Anthony Forster’s lab used codon optimization... Chromoprotein expression from bacteria on agar plate. Image: Liljeruhm et al., 2018. For each chromoprotein...exchange. These vectors contain a toxin gene (regulated by a rhamnose-inducible promoter) and a chromoprotein...Dove SG, Takabayashi M, Hoegh-Guldberg O (1995) Isolation and Partial Characterization of the Pink and Blue...

...solution of benzyl alcohol and methyl salicylate which was later modified by others to produce Murray’...major advances in light microscopy, particularly related to the neurosciences.  These developments in microscopy...single gold particles with diameters less than 4nm. Lateral view of a cleared rat pons, medulla and the first...using optical projection tomography (OPT)  (12) and later Hans-Ulrich Dodt’s lab pioneered the use of light...) (6).   The Dodt and Frank Bradke laboratories later developed the 3DISCO (3-dimensional imaging of solvent...tetrahydrofuran-based tissue clearing (13,14). A few years later, Karl Deisseroth’s lab developed CLARITY, which ...relied on the use of transgenic mice in which a subpopulation of neurons are brightly labeled with GFP (e....

... and whether you are inoculating from a frozen stock or a colony from an LB plate. In my hands, a 5 mL...mL starter culture inoculated from a colony on a plate will reach an optimal density in 18-24 hours. I...Arabidopsis, from studying transcriptional and translational dynamics of genes and proteins in living plants... seeds from the pool you will be selecting from later. The Arabidopsis transformation process The process... T1 plants from each transformation. I like to isolate a minimum of five. You have no control over how..., check out Harrison et al., 2006. Tips: Don’t plate seeds too densely during selection: this will increase...difficult. Don’t leave your plants on selective plates for too long: even resistant plants will be affected...

...tumors, which can lead to new cancer targets and related biomarkers . Data from such screens are often uploaded...Unexpected results Their results showed 96 strongly correlated genetic dependencies that were clearly associated... they looked for a biological explanation, a relationship between the genetic dependencies they identified... ancestries. While there were some exciting relationships identified, 48% were found to be artificial,...bias into their experiments instead of a true correlation.   It turned out there was indeed a bias, and...diversity of genetic variation across the human population. Rather, it primarily reflects European ancestry... to guides in the Broad’s Genomic Perturbation Platform Avana library (used by the Cancer Dependency Map...

...their resistance genes throughout the bacterial population. But what if we could specifically target only...successfully resensitized a beta-lactam resistant population to the antibiotic, despite this resistance gene...plasmid-based sequences (red). Treating a mixed population, represented here by cells of different colors...given genomic or plasmid sequence from a mixed population without affecting the other bacteria. Targeting...small pools of antibiotic resistant cells could repopulate the environment after treatment. The phagemid...antibiotics can be used to target the sensitive population. Yosef et al.’s proof-of-concept work targeted...infected by the lysogenic phage, rendering the population sensitive to antibiotics. Created with BioRender.com...

...transcription and epigenetic regulation. To elucidate molecular mechanisms of regulation of genome functions, ...mechanisms of genome functions such as epigenetic regulation and transcription by using locus-specific chromatin...manner. To achieve this goal, it is necessary to isolate specific genomic regions while retaining molecular... step of knocking-in. In 2013, we showed that isolation of a single copy genomic locus is feasible by ...enChIP, specific genomic regions can be easily isolated for identification of associated proteins, RNAs...that enChIP will markedly accelerate research of regulation mechanisms of genome functions in the hope of... of the reasons why we have only three biology-related Nobel prizes, out of which only one was done in...

...hedgehog signalling pathway, which is a key regulator of all bilateral animal development. SHH plays a key role...Cancer Institute! Sonic Hedgehog, an important regulator of embryonic development The largest conference...publication, Ivics et al 1997. Spaghetti Squash, a regulator of cellular contraction You may think that a gene...component of the fly actomyosin cytoskeleton that regulates cellular contraction. This network is composed...vessels). Spaghetti squash (Sqh) is the Drosophila Regulatory Light Chain (RMLC) of nonmuscle myosin II (Karess... Jordan and Karess, 1997), which contains the regulatory regions that are necessary for myosin activity...Roger Karess. "Myosin light chain–activating phosphorylation sites are required for oogenesis in Drosophila...

...identification system for the isolation of base-edited cell populations. To develop this method the lab... affecting protein translation of different reporter proteins. Protein translation requires the start ...edited cells without the need for single cell isolation and downstream sequencing (Standage-Beier et al...and Brafman lab showed that GFP positive cells isolated with TREE have a significantly higher frequency...base-editor (C>T conversion) they could induce the translation of any detectable protein, not just fluorescent...base-editing reporter. As the initiation of protein translation at the start codon ATG is universal, the Dow ...Dow lab successfully used GO to induce the translation of an array of different reporters including mScarlet-I...

... protein is up or downregulated, examine a protein’s stability or post-translational modifications, or...aureus. Its isolation by affinity chromatography and its use as an immunosorbent for isolation of immunoglobulins...immobilized antibodies, or immunoglobulins, to isolate a specific protein out of a complex mix. Using ...suitable for all applications, such as protein isolation from serum. In this case, Protein A and Protein... assay, an antibody against the tag is used to isolate the protein of interest. The wide availability ...experiments using a tagged protein may not be translatable to the endogenous system and will need to be...modification. Similarly, RNA IP (RIP) can be used to isolate proteins that bind to RNA, though the details of...

...phenotype. Incubating the phage display library with plate-bound antigen helps select for high affinity scFvs...bacteriophages. This library is incubating with plate-bound antigen to select for scFvs with high affinity...the non-oxidizing cytoplasm of bacteria, scFvs accumulate in insoluble inclusion bodies. While it’s possible... has led to their application for basic and translational research.  Basic research applications of scFvs...generate since it’s genetically encoded.   Translational applications of scFvs: Just like antibodies...scFvs are also used for eye diseases, such as age-related macular degeneration (AMD) since direction injection...cell. When a CAR has an scFv that binds cancer-related antigens, binding of the scFv part of the receptor...

...recipient, the only antibiotic resistant colonies isolated are those where the allelic exchange vector has...that contained one of eight bacterial toxin genes isolated from E. coli or Psuedomonas aeruginosa. These ...the transcriptional activator rhaS. To allow manipulation of bacteria without an endogenous rhamnose regulon...synthetic ribosome binding site (using the online calculator from the Salis group) (Espah Borujeni et al.,...designed with the help of the DNA tuner program) (Latynski and Valentovich 2014). We routinely clone the ...facilitate the cloning of toxic products. To allow manipulation of naturally chloramphenicol-resistant strains...Anirudh S. Channarasappa, and Howard M. Salis. "Translation rate is controlled by coupled trade-offs between...

...with ssDNA repair templates. Their work demonstrates that different repair template strategies activate...using dsDNA templates achieving ~1-10% efficiency. Easi-CRISPR uses a long ssDNA template to increase ...These complementary goals are aimed at improving translation of CRISPR technology, but they would also bring...working with CRISPR than just designing a repair template that contains your desired edit, and I want to...CRISPR updates on our blog to keep current with the latest and greatest! Addgene resources 3 Tips to Improve...Editing in Human Cells Hands-on tips for repair template design from guest bloggers Dominik Paquet and ...

...continuing to copy the template rather than dissociating).  MarathonRT, isolated from Eubacterium rectale...fields of biology and enable the conversion of RNA templates to DNA but suffer from several important shortcomings...structured HCV genome (~9.6 kb of highly structured template!). MarathonRT also faithfully transcribes RNA ...protein fused with GFP, YFP, mCerulean, or other related fluorophores. Find all of the nanobody toolkit ...biotin. The biotin-labeled proteins can then be isolated and analysed. In this work, the authors specifically...discovery of proteins involved in transcriptional regulation and chromatin structure at your locus of choice...technology, which they call C-BERST (dCas9–APEX2 biotinylation at genomic elements by restricted spatial tagging...

...mentor. How do you make the most of this new relationship? Consider adding formality and active goal setting... setting to your mentoring relationships, so that you can reap rewards in the form of reaching career ... career path is a great start to a mentoring relationship, but if you don’t take this past the chatting...meetings and setting the direction in a mentoring relationship falls on the shoulders of the Mentee. A Mentor...progress over the course of an ongoing mentoring relationship (in a book or online). Mentee: Make a list of...secret of accountability To make a mentoring relationship really productive, the most important step you...on setting SMART goals using a Development Plan template. A Development Plan or mentoring journal will ...

...transduced by lentivirus and have been difficult to manipulate genetically. To solve this issue, Sarah Teichmann...disease is driven by atherogenic lipoproteins that accumulate within the vascular wall. ApoB, the main structural...lipoproteins, shuttles lipoproteins through the circulatory systems, but can also lead to the formation of... of ApoB-LPs has been shown to have a strong correlation with cardiovascular disease and indirect measurement...in vivo model to identify drug candidates that modulate ApoB-LP size and concentration, Steven Farber ...that the system is active in E. coli and closely related species, is amenable to use with other bEBP/UAS...and the system may not be adaptable to distantly related species from E. coli. This tool should serve as...

... AAV Targeting and manipulating specific, genetically-defined neuronal populations with intersectional...calcium sensors and GABA sensors) Calcium sensors The latest calcium sensors from Janelia (jGCaMP7) continue...potential, GCaMP7s is approximately twice as bright, relative to baseline fluorescence, as the GCaMP7f variant...useful for imaging signaling activity in large populations of densely-labeled neurons because background...all our biosensors inventory, and check out our latest vectors below: pGP-AAV-syn-jGCaMP7f-WPRE (104488...Optogenetics continues to be a useful method for manipulating specific neurons. We recently packaged a soma-targeted...specificity of influence measurements by reducing photostimulation of the axons and dendrites of non-targeted...