... 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...

...Knockout Screening in Human Cells. Shalem O, Sanjana NE, Hartenian E, Shi X, Scott DA, Mikkelson T, Heckl...and easily manipulate specific genes. But what if you want to study genes all across the genome? Two new...screen for genes involved in bacterial toxin resistance, identifying 4 previously unknown genes.  “With ...library, which they call GeCKO, targets 18,080 human genes with 64,751 unique guide sequences to enable both...including Feng Zhang used the GeCKO library to identify genes essential for cell viability in cancer and pluripotent...In a melanoma model, they also went in search of genes involved in resistance to the cancer drug vemurafenib...guide RNAs (gRNAs) covering a total of 7,114 human genes. Unlike the GeCKO library, each library has been...

...expressing nLightG or nLightR before/after application of NE (10 μM) and corresponding pixel-wise ΔF/F0 heatmaps... expression of the indicators over white dashed lines. Scale bars, 10 μm (HEK293T), 20 μm (neurons). Figure...options for multiplexing in experiments, including ones that study alpha2-AR. Selectivity Norepinephrine...

...Feb;10(4):337-47. PubMed PMID: 12595892. 4. Sanjana NE, Shalem O, Zhang F. Improved vectors and genome-wide...virus can be used to generate Cas9-expressing cell lines, which can then be used both for screens (using ...

...Gootenberg JS, Abudayyeh OO, Platt RJ, Brigham MD, Sanjana NE, Zhang F (2017) Genome-scale CRISPR-Cas9 knockout...available reference sequence or plasmids containing genes with many modifications. We look for point mutations...

...specific genes using dCas9-VP64 activator fragments (dCas9(C)-FKBP-2xNLS-VP64 and dCas9(N)-FRB-NES). This...FKBP rapamycin binding domain of mTor (Cas9(N)-FRB-NES) resulting in a rapamycin-inducible Cas9 for genome...editing. Without rapamycin treatment, the Cas9(N)-FRB-NES fragment is actively shuttled out of the nucleus ...sequence. Treatment with rapamycin induces Cas9(N)-FRB-NES and Cas9(C)-FKBP-2xNLS dimerization and net influx...of downstream genes. Nihongaki et al describe the targeted activation of endogenous genes and detail the... promoter-less Gateway® entry clones to be used with other entry clones encoding the ORFs of interest:...neuron-like” phenotype when targeting genes involved in neurogenesis, which had proven challenging using...

...beta-hydroxylase (DBH, magenta) to label norepinephrine (NE) expressing neurons and GFP (green) to label neurons...’m willing to bet the answer would be along the lines of “we have a lot”. In fact, scientists often cite..., C. M., Leib, R. D., Cirolia, G., Thomas, D., Stamnes, S., Holt, K., Sinn, P., May, A. P., & Paulk, N...availability upon request differ across scientific disciplines. Scientific Data, 8(1), 1–11. https://doi.org...

...genome-wide libraries for CRISPR screening. Sanjana NE, Shalem O, Zhang F. Nat Methods. 2014 Aug;11(8):783...2nd-generation lentiviral system (Figure 2). The HIV genes that do remain are very important for viral production...make stable transgene-expressing or knockout cell lines. Many CRISPR plasmids are designed for lentiviral...integrate into the genome, they could promote oncogenesis by altering local gene expression. Self-inactivating...LTR that prevents aberrant activation of nearby genes. These safer vectors have become standard in gene...

...Gootenberg JS, Abudayyeh OO, Platt RJ, Brigham MD, Sanjana NE, Zhang F. Genome-scale CRISPR-Cas9 knockout and transcriptional...

...genome-wide libraries for CRISPR screening. Sanjana NE, Shalem O, Zhang F. Nat Methods. 2014 Aug;11(8):783...cells were then used to generate a monoclonal cell lines by limiting dilution. The rest of the cells were... you’re doing a screen. I made a few monoclonal lines (Figure 2) and I was surprised at how variable Cas9...results. (Figure 5). The digestion product (Figure 4, lanes 0.3 % 3.0, Figure 5, lane 2) is faint, so comparing...Resources at Addgene.org Generating Stable Cell Lines with Lentivirus Isolating a Monoclonal Cell Population...

...doi.org/10.1089/hum.2019.277 Lavin TK, Jin L, Lea NE, Wickersham IR (2020) Monosynaptic Tracing Success...used different scrambled sequences to flank the transgenes (non-homologous pairs) (Figure 2.). They found...

...in between a protein and a tag, such as an NLS or NES, enables control over localization. Inserting PhoCl...acrIIA1 Listeria monocytogenes  pRAU168  No  pJH372  No  acrIIA2 Listeria monocytogenes  pRAU171 Partially...  Listeria monocytogenes  pCSW65 N/A (Toxic)   None N/A   acrIIA4 Listeria monocytogenes  pCSW21  Yes...quickly and easily generate zebrafish transgenic lines to study neurobiology and neuorodgenerative diseases...compatible cloning sites for easy cloning of your genes of interest. In order to improve the study of specific...induction of structural plasticity in single dendritic spines. The set of CyRFP constructs available at Addgene... used a combination of deliberate and random mutagenesis to isolate the best RFP. They targeted known ...

...are (i) brightness, (ii) photostability and (iii) aggregation tendency. Brightness Brightness refers ...molecule. A higher brightness increases the detected signal and therefore high brightness is an important...how the brightness is best defined or determined. Here, we will explain how practical brightness is determined...determined and why practical brightness is a better criterion than theoretical brightness for selecting the brightest...protein. Theoretical brightness Two key properties of fluorophores that determine brightness are the extent ...number, the higher the theoretical brightness. The theoretical brightness often appears in tables with fluorescent...and the sample. Practical brightness A protein with a high intrinsic brightness that does not fold well ...

... the Lindberg Lab we often make cell lines that overexpress genes of interest; more recently we have also...CRISPR vectors to generate cell lines with knockouts of specific genes. Many years ago, people in the ...give rise to single clones, and thus many plates must be examined for single clones and then handled. Additionally... Additionally, many cell lines, especially the endocrine cell lines we most commonly work with, require...cell clones with sterile truncated pipet tips. Indeed, an experienced operator can pick 48 clones from...individual cell clones with different expression characteristics from a population of transfected clones. The technique...number of single clones obtained (on occasion we have prepared two 48-well plates of clones) which enables...

...targeting their genes of interest to generate mAID fusions to endogenous genes. Cell lines with the appropriate...targeting a total of 20,121 genes within the human genome (the identity of the genes and target sequences can...continuous expansion of compatible backbones, promoters, and genes available to the community. If you ...identify both core and context-dependent human fitness genes, the laboratory of Jason Moffat at the University...cell models and identified ~4-5-fold more fitness-related genes than have been found in previous screens...uncharacterized genes that are conserved among apicomplexans and important for T. gondii fitness. Sixteen of...enable researchers to screen the entire genome for genes that regulate a wide variety of phenotypes, including...