...assembly methods. A new method, REPLACR-mutagenesis, harnesses the power of bacterial recombineering to...REPLACR-mutagenesis could save busy scientists a lot of time. Let us know how REPLACR-mutagenesis works ...Comparison of REPLACR-mutagenesis with other common methods for site directed mutagenesis   References 1. ...al. (2016). “REPLACR-mutagenesis: a one-step method for site-directed mutagenesis by recombineering.” ...Site-directed mutagenesis (SDM) is one of the key tools researchers use to prove causation in molecular... Plasmids After PCR). Current site directed mutagenesis methods Current SDM methods rely on in vitro ...structure or too much sequence similarity. REPLACR-mutagenesis: Letting bacteria do the work If you’ve conducted...

...Healthcare consulting firms that specialize in business strategy tend to be mostly engaged by pharma and...companies. Medical Affairs groups work on building awareness among physicians and scientists about clinical...collaborative, and financial functions associated with the business. Commercial teams routinely work with consulting... a very short amount of time. Such a decision determines whether the client invests substantial monetary...consultants are usually trained in the life sciences, in business, in healthcare economics, or in some combination... you, you’ll have the opportunity to learn the business side of consulting cases, while feeling like you...healthcare space that can benefit from the blend of business and scientific understanding of successful therapeutics...

...Versatile and Efficient Cloning System for Plant Transgenesis." PLoS ONE. 8(12): e83043 (20 December 2013)...

...yeast gene of interest. These fluorescently tagged genes are expressed under native conditions and allow ...looking at their performance in categories such as brightness, stability, and disruption of the tagged protein...be interested in attaching epitope tags to their genes of interest, allowing for easy capture and detection...

...effects. One-pot saturation mutagenesis The one-pot saturation mutagenesis technique described in Wrenbeck...saturation mutagenesis with the same protocol. What do you think about one pot saturation mutagenesis? How .... “Random Mutagenesis by Error-Prone PCR”. Methods in Molecular Biology In Vitro Mutagenesis Protocols...Efficient, Expansive, User-Defined Mutagenesis.” Ed. D. Dafydd Jones. PLoS ONE 7.12 (2012): e52031. PubMed...the Whitehead lab developed One pot saturation mutagenesis as a quick and easy technique that can be used...library generation prior to one pot saturation mutagenesis Several methods can be used to create mutational... shortcomings compared to one pot saturation mutagenesis. Error-prone PCR: This method amplifies the...

...temperature ranges and make them grow better at suboptimal ones, which could allow even your regular E. coli to ...where we could incorporate cold-tolerance inducing genes within a regular mesophilic host system. This would...folding at low temperatures, since mesophilic chaperones would not function well under suboptimal conditions...engineering a plasmid which contained cold tolerant chaperones (the Cpn 10/60 isolated from Oleispira antarctica...the figure below shows, E. coli expressing the chaperones protein resulted in higher levels of growth compared...

...website navigation paths, the organization and completeness of our information, and the accuracy and relevance...

...bound to modified proteins called histones. We can detect modified histones with “GPS for proteins,” ChIP-... of modified histones, a genomic region that has mono-methylated and acetylated histones, but not tri-...which NANOG-related genes are critical to stem cell survival. Knowing which genes influence the survival...Locates ChIP-seq Proteins (for our example later, histones) ChiRP-seq RNA Hi-C DNA (genome-to-genome ...tri-methylated histones, functions as an enhancer. We can therefore denote enhancers as follows: (mono_methylation...combination of coiled DNA and structural proteins called histones. Chromatin folds into secondary structures (loops...

...coming from.   Acoustic reporter genes (ARGs) Acoustic Reporter Genes (ARGs) are engineered gene clusters... reporters has its drawbacks. Acoustic reporter genes (ARGs) overcome these limitations by using gas vesicle... require injection of substrate to produce bioluminescence. Substrate pharmacokinetics and biodistribution...localization: In vivo imaging with fluorescent or bioluminescent reporters usually only detects the signal of...and high spatial resolution. By piecing together genes from gas vesicle operons from two different bacteria...their ultrasound signal. Using acoustic reporter genes for in vivo imaging Following in vitro characterization...S.P., & Shapiro, M.G. (2018). Acoustic reporter genes for noninvasive imaging of microorganisms in mammalian...

...exploding. Researchers are increasingly able to harness the body’s immune system to fight progressing cancers...this multi-pronged attack. Inflammation and tumorigenesis The conference opened with a very interesting...

...Generation of Genomic Deletions in Mammalian Cell Lines via CRISPR/Cas9" from the Journal of Visualized ...Cas9 to create genomic deletions in mammalian cell lines. Below Bauer and Canver discuss the motivations ... and colleagues using zinc finger nucleases to harness non-homologous end joining repair (NHEJ) [1]. Our...targeted deletions is to produce monoallelic deletion clones. Cells with just a single intact copy at a given...cells can be easily identified. Even in the case of clones with monoallelic deletion, the nondeleted allele...validation, and screening by PCR for biallelic deletion clones:    Like the video? The Orkin and Bauer lab's...enhancer of BCL11A subject to genetic variation determines fetal hemoglobin level. Science 2013 Oct 11;342...

...are Acoustic Reporter Genes (ARGs)? Fluorescent and bioluminescent reporter genes are widely used to study...Lab - developed Acoustic Reporter Genes (ARGs), which are sets of genes that, when expressed, enable cells... vivo.  But what ultrasound lacked was reporter genes that could produce ultrasound contrast. That is,...gas vesicle-expressing species. The gas vesicle genes from Serratia sp. 39006 produced the highest levels...bARGSer expression, from left to right: colony whiteness on inducer plates, white inclusions in cells with...cell line with strong gas vesicle expression and robustness without chromatin-opening agents. It ended up...being very straightforward to engineer various cell lines from different tissues and mammalian species to ...

...genome-wide libraries for CRISPR screening. Sanjana NE, Shalem O, Zhang F. Nature Methods. 2014 Aug;11(8...Protocols Generating Stable Cell Lines with Lentivirus Generating Stable Cell Lines with Lentivirus You may also...Protocol to generate stable cell lines expressing a gene of interest from an integrated lentiviral vector...This protocol can be used to generate stable cell lines expressing a gene of interest from an integrated...transient transfection approaches, generating cell lines using lentiviral vectors enables long-term protein...293T cells but can be adapted to alternative cell lines. Last Updated: September 9, 2023 Workflow Timeline...needed Day 14–18: Expand and harvest stable cell lines Equipment Class II, Type A2 Biological Safety Cabinet...

...immortalize CDG patient-derived immune cell lines and use these lines to study how immune processes and biological...research on diseases of glycosylation, arginine mutagenesis, and neural development. Paula Videira: Congenital...studying the function of the mouse Nfia, Nfib and Nfix genes in brain development since 2000 and have shown several...

... our paper, “Synthetic recombinase-based state machines in living cells” (1), into a more straight-forward...our engineered devices, recombinase-based state machines for your own experimental needs. For more context...timing of these factors. Recombinase-based state machines (RSMs), which take on a particular DNA-sequence... from different recombinases are overlapping or nested on a register, then the operation of one recombinase...replication), contains large serine recombinase genes downstream of inducible promoters: the BxbI gene...The paper also demonstrates that genetic parts (genes, terminators, and promoters) may be interweaved ...predictable gene regulation programs that specify which genes should be expressed or not expressed in each state...

... fragmented genomes is the key to fitting large genes into AAV First, some history. In 2008, the Auricchio...mini-dystrophin.  3. Hybrid  Overview: The hybrid approach combines the overlapping and trans-splicing methods. The... A (2008) Serotype-dependent packaging of large genes in adeno-associated viral vectors results in effective...Chamberlain K, Riyad JM, Weber T (2016) Expressing Transgenes That Exceed the Packaging Capacity of Adeno-Associated... Auricchio A (2013) Effective delivery of large genes to the retina by dual AAV vectors. EMBO Mol Med ...

...localization signal (bNLS) and a nuclear export signal (NES), both which are phosphorylatable. Prior to phosphorylation... phosphorylation bNLS activity is suppressed and NES activity is enhanced, leading to cytoplasmic translocation...of the phosphorylatable residues and the bNLS and NES sequences, which have previously been characterized.... for more details on the design of the bNLS and NES sequences. Disadvantages of KTRs KTRs also have some...

...repress the expression of genes primarily involved in development. These genes are known to play a role...many contexts within the fly including oogenesis and cytokinesis. Why name this protein after a vegetable..., two genes important for mesoderm differentiation Now, let's turn tinman and bagpipe, two genes important...bagpipe are both homeobox genes that are part of a genetic cascade that determines the cell fate of the dorsal...two homeo box genes that determine cell fates in the dorsal mesoderm of Drosophila." Genes & Development...they can name a gene and many of the interesting ones come from the fly community. Not surprising, this... our repository that you can use to study these genes! Groucho, a transcriptional co-repressor The first...

...2019. PubMed PMID: 30704911. Acoustic reporter genes for noninvasive imaging of microbes in mammalian...through a host organism, the expression of some genes can change depending on the location. Current methods...of microbes rely heavily on fluorescent protein genes. These optical reporters have limited deep-tissue...vivo using ultrasound. These ARG plasmids contain genes for “gas vesicles,” a gas-filled protein structure...demonstrates engineered gas vesicles as the first reporter genes using ultrasound and provide a major benefit to ...active miRNA’s unique to HEK293FT and HeLa cell lines. When the miRNA activity profile for a given cell...segment Understanding the connection between specific genes and an immune response has been a longstanding challenge...