... trafficking of these biosensors.   Figure 1: Schematic of iGluSnFR3 (left). GluSnFR3 in pMinDisplay... from Abhi Aggarwal.   Brandán Pedre Pérez: Redox biosensors Brandán Pedre Pérez is a postdoc at... enable multiplexed imaging of neurotransmitter, calcium, and membrane voltage,” says Tian. Figure...Congratulations to the five winners! Abhi Aggarwal Brandán Pedre Pérez Elizabeth Hubbard Haoyi Qiu He Tian...hippocampal cultured neurons, imaged 14 days post-transfection, excited at 505 nm (right). Scalebar represents...hydropersulfide-propagating enzymes 3-mercaptopyruvate sulfurtransferase (MPST) which catalyzes the desulfuration of...an enzyme-bound hydropersulfide that is then transferred to thiol-containing acceptors. These hydropersulfides...

... Science Find the SEND plasmids    Figure 1: SEND combines an endogenous Gag homolog, cargo RNA...Cas9 fused to the reverse transcriptase (RT), which induces two double-strand breaks and deletes the intervening...They further develop a version of SEND for gene transfer with pseudotyped viral particles, expanding options...enzyme from the Liu lab to target opposite DNA strands and specify a large intervening deletion. Not only...deletions and introduce epitope tags and RNA transcription start sites. The lab further demonstrated that...uses paired pegRNAs targeting complementary DNA strands to program large genomic deletions with or without...PEDAR is capable of correcting larger genomic rearrangements as demonstrated in a mouse model of tyrosinemia...

...for RaPID is an RNA motif of interest. See figure 1 below for details on the key components of RaPID. ...and IREB2, which post-transcriptionally regulate iron metabolism pathway transcripts. By using RaPID, Ramanathan... include both scrambled RNA motifs and cells transfected with BirA* protein alone (i.e. no RNA bait). ...changes in RC2H1 protein expression and post-transcriptional regulation of SM1v1-containing RNAs could play...often trick host proteins into binding their untranslated regions (UTRs) to aid in the viral life cycle...IRE) RNA motif of the ferritin light chain RNA transcript. IRE motifs are bound by IRE binding proteins...

...best tips beyond the basics to get you started. 1) Be visible and open to connections This is a site... Also, many people on LinkedIn don’t appreciate random requests without a personal message and they may...trolls. People don’t like to connect with complete strangers as it borders on my first rule of networking--...a useful way to build or use a network. Total strangers can’t really help or refer you. Do not ask people... lot about requesting connections to complete strangers for informational interviews. Many have had success...considered intrusive and it’s still connecting with a stranger so is highly discouraged. You should be using ...

...manipulation, there are two main decisions to make: 1. Which modification? and 2. Which viral vector should... linked to a certain behavior. It is a very old branch of science, dating back several hundred years, ...genes. A popular method uses Cre recombinase on transgenic flox animals to introduce modifications at the...Timing: High expression levels of the viral vector trangenes typically occur two weeks after injection. Therefore... after several weeks which may also depend on transgene or promoter choice. Behaviors you can study using...Stoica L, Ahmed SS, Gao G, Sena-Esteves M. Gene transfer to the CNS using recombinant adeno-associated ...

...of interest and the metal ions.      Fig. 1: A sample containing a polyhistidine tagged protein...and several contaminating proteins (blue, green, orange) flows through the Ni2+-containing resin. The protein...polyhistidine containing contaminating protein (orange). A low imidazole wash buffer disrupts the weak...polyhistidine-metal ion interaction is not disrupted by denaturants (Hochuli, 1988). In some cases, proteins can ...tag and then refolded by gradual removal of the denaturant. Not all proteins will refold once denatured,...

...incorporated at the termini of the crRNA molecule (usually 1-3 of the first 3 bps are modified) for optimal stability... 3’ end. This repeat region associates with the trans-activating crRNA (tracrRNA) which interacts with... important cellular RNA molecules with post translational modifications. One of the most common of these...targeting experiments allows you to visualize transfection efficiency and even track localization in cellular...

...antibody for a…less sensitive moment.   Fig 1: Blugene fading into darkness because they accidentally...spilling over" into another channel's detection range. Though you can compensate for this when reading...

...advantage this cycle, consider the advice below:  1. Eligibility check Scrutinize every opportunity prior...increase your competitive edge, they alone, are no guarantee of success. Don’t luck into an internship, prepare...

...sites that were previously unavailable. Figure 1: The PAM sequence that the Cas nuclease recognizes...across a number of different sites. Using a combined random and directed mutagenesis approach, the Anna Cereseto...

...the output of the analysis performed in experiment 1. Use camelCase: In camelCase, each subsequent word...like a wet lab experiment I use the text editor TextWrangler to record the following, which are later printed...

...with each protein of interest.    Figure 1: The direct approach (left) versus the indirect approach...in light green; and proteins are represented in orange. Created with BioRender.com.    How do secondary...

... an entirely new affinity reagent.    Fig. 1: A comparison of an antibody (left) to several alternative...bonds, monobodies can be easily expressed in transfected eukaryotic cells, allowing for them to be used...an excellent option for many applications, and frankly, if ain't broke, don't fix it!  That being said...

.... See figure 1 for an example of how changing transfection ratios can affect transfection efficiency. ...containing structural genes and a transfer plasmid carrying the transfer gene. By dividing the viral elements... structural elements, envelope, and transfer gene are transfected into a packaging cell line, usually .... For some transgenes, transfection optimization simply is not enough to get a sufficient titer; this ...may not translate to the target cell line. To overcome this issue, we recommend using a range of doses...systems such that it no longer requires the viral transactivator, Tat. For more information on the different...requirement for additional DNA can impact the transfection efficiency of the packaging cell line and thereby...

...If you already have an end product in mind, steps 1 and 2 will generally be straightforward. Likewise ...reagents can be delivered via transfection, nucleofection, viral transduction or injection as either protein...cell line that can be transfected at high efficiency. Lentiviral transduction Cas9 and gRNA can be present... present in a single lentiviral transfer vector or separate transfer vectors. May contain reporter gene...inserted into an AAV transfer vector and used to generate AAV particles. Transient or stable expression...Chemical transfection   Lipid mediated Cationic polymers Calcium phosphate Cells Transformed cell lines... lines Transformed cell lines Transformed cell lines Cargo Nucleic acids or protein Nucleic acids Nucleic...

...patients lives. References 1. Ong-Abdullah, Meilina, et al. "Loss of Karma transposon methylation underlies...paternal grandmothers early food supply influenced cardiovascular mortality of the female grandchildren." BMC...histone modifications, which can influence the transcription of genes by RNA polymerase II, or by the addition...methylcytosine in CpG islands makes  genes less transcriptionally active. The role of epigenetics in studying...to play a large role in a variety of diseases, ranging from cancer, to diabetes, to congenital disorders...isolated population in Sweden found that paternal grandfathers who had experienced famine before their adolescence...adolescence passed on a heritable trait to their grandsons that decreased their likelihood of death from ...

...basis of many molecular biology tools! Figure 1: Common functions of natural plasmids. Created with...lateral”) gene transfer. Horizontal gene transfer can occur naturally through transformation (cellular uptake...? Plasmids 101: Transformation, Transduction, Bacterial Conjugation, and Transfection Resources on Addgene.org...environment), transduction (viral infection by bacteriophages), or conjugation (direct transfer from one cell...one popular method scientists use to promote transformation, but it isn’t just for lab specimens — there... evidence that lightning can also promote gene transfer in soil bacteria! (Demanèche et al., 2001) Beneficial...humans only noticed last century! Nevertheless, the transfer of plasmids carrying these genes between bacteria...

... inhibitory synapses in the brain.  Figure 1:Schematic diagram of different FingR constructs and... efficient double stranded templates, rather than the more efficient single-stranded oligodeoxynucleotide...generally very small, and can contain different neurotransmitters. Using genetically encoded fluorescent proteins... them to localize to the synapse and minimize extraneous fluorescence from other regions of the cell. ...synaptic maturation pattern of hippocampal adult-born granule cells over time that were not previously possible... al., 2020). Figure 3: BaF3 cells were transduced with H2B-FT virus and grown in varying concentrations...lentiviral CRISPR library that targets human glycosyltransferase genes and other proteins related to cellular...

... – a mechanical device that could be used to bend 1/8th inch steel wire into a backbone structure of a...this surface”….where the complex is docked on the membrane of the endoplasmic reticulum. And graduate students...reported cleavage site on the complementary DNA strand. And this is why we love working with researchers...”. Jacob computationally added the non-template strand to the Jinek structure so that the resulting physical...protein must have in order to scan along the double-stranded DNA, recognize a PAM sequence, allow the complementary...pairing between the guide RNA and the target DNA strand, and then activate two different endonuclease active...structure captures the displaced non-target DNA strand and shows the HNH nuclease domain swung around ...

...article, they investigated three Casɸ orthologs, Casɸ-1, Casɸ-2, and Casɸ-3. This system is notable for several...including excision or insertion, inversion, translocation or cassette exchange. By utilizing recombinases...delivery constraints and scientists seeking a wider range of targetable genomic sequences could benefit from...spatially and temporally target single cells. Now, Francois St-Pierre’s lab developed a new form of FACS-based...-based Dual-fluorescent DSB Repair) is a double-strand break repair assay for mammalian cells. It's based...deposited by Leopold Parts. This library delivers 2 randomly paired guides per construct and allows a reduced...

... little signal detected along the neurite (Figure 1.) Moreover, the group was able to demonstrate that...expression in E. coli. This operon is under the transcriptional control of a strong constitutive promoter (... required enzymes, including a glutathione S-transferase homolog from a Janthinobacterium sp. Moreover... protein kinase A (PKA) activity at the plasma membrane in living cells using a construct in which TagRFP-T...used in quenching, FRET (Förster resonance energy transfer), and FLIM (fluorescence lifetime imaging microscopy...

...technologies (Bradbury & Plückthun, 2015a).   Figure 1: (Top) Polyclonal antibodies are produced in animals...chain and light chain for the antibody and when translated into protein will assemble into a fully functional...synthesize the genes that encode those amino acids (Tran et al., 2016). Each antibody would then be tested...P, Frenzel A, Wong J, Koh XY, Eng H-Y, Lane D, Lefranc M-P, Clark M, Dübel S (2018) When monoclonal antibodies...:633–644 . https://doi.org/10.1083/jcb.201409074 Tran NH, Rahman MZ, He L, Xin L, Shan B, Li M (2016) ...

...your finished constructs at Addgene!). References 1. Li, Chaokun, et al. "FastCloning: a highly simplified...would at least be guaranteed a successful result? While there are never any guarantees, some cloning techniques...Resources” section at the end of the post! Speed: Your grant deadline is approaching or your advisor is demanding...your insert, DpnI digest away the templates, and transform the PCR products into E. coli. Ligases within ...

...most comprehensive for searching. Timesaving Tip #1: If you know the species that your sequencing result...blastn), Standard Protein BLAST (blastp), and Translated BLAST (blastx). NCBI has a terrific getting-started...gaps in the nucleotide sequence, you can run a Translated BLAST (blastx) search to check for expected ORFs..., due to the additional searches involved in translating the nucleotide sequence into all six possible...choose for your sequencing result and can easily translate it, we recommend using blastp over blastx. The...

...SpCas9 base editors with editing windows as small as 1-2 nucleotides! To reduce off-target effects associated...Lower on-target activity than BE3; editing window ~1-2 nt  87438  pET42b-HF-BE3  HF-Cas9 (NGG)  Very ...scientists to quickly and easily generate zebrafish transgenic lines to study neurobiology and neuorodgenerative...pluripotent stem cells) through the expression of transcription factors OCT3/4, SOX2, KLF4, and c-MYC is the... method for generating iPSCs to express the transcription factors from a polycistronic cassette. However...comparable experimental results. Using PiggyBac transposons, the Woltjen Lab compared different variants ...reprogramming discrepancies. The polycistronic piggyBac transposon vectors tested in this work are available at ...

...cereblon, leading to rapid target degradation (Figure 1). Plasmids: We generated a series of plasmids that...example, IZKF1 and IZKF3 are cell-type specific transcription factors with important roles in Multiple Myeloma... (Nabet et al., Erb et al., Huang et al.), transcriptional and epigenomic profiling studies (Nabet et ...Featured molecules: dTAG-7 and dTAG-13 Erb et al. Transcription control by the ENL YEATS domain in acute leukemia...

...which you’ve already fluorescently labeled. Figure 1: Using Split-BioID to Identify Protein Protein Interactions...Interactions Difficult to detect weak and/or transient PPIs. Many PPI identification techniques rely ...often prevents the identification of weak and/or transient interacting proteins. No spatiotemporal control...BioID, is also useful for identifying weak and/or transient protein-protein interactions since the biotin ...

...Set a goal for paper-reading, even starting out at 1 paper/week will be useful. Check out the hashtag #365papers...your area. When you are in ‘writing-mode’ for a grant or a scientific manuscript, the reading will likely...

... to the use of multiple antibodies. Figure 1: Overview of using Sandwich ELISA to quantitatively...concentration of any experimental wells in that range of optical densities. If your standard curve does...

...significant progress in clinical trials.     Figure 1: Types of delivery and editing strategies for CRISPR...CRISPR targets — Sickle Cell Disease (SCD) and Transfusion-dependent beta thalassemia (TBT), both of which...clinical trial for SCD in 2022 as well. Hereditary transthyretin amyloidosis (hATTR) hATTR is caused by a variety...

...editing machinery such as CRISPR-Cas. Figure 1: Schematic of targeted gene knock-out (left) and targeted...AdV-mediated transduction of gRNA:Cas9 ribonucleoprotein complexes into transformed and non-transformed cells...(right) are transduced to target cells. Within target cells, Cas9 machinery is transiently expressed and... viruses. Adenoviruses are non-enveloped double-strand DNA viruses with a genome of around 36 kb in size...left), a fragment is deleted from two homologous strands of DNA through error-prone non-homologous end joining...other viral vectors, AdVs can provide a large transgene packaging capacity. For instance, with a packaging...disruption frequencies in the various cell types ranged from 18% to 65% (Maggio et al., 2014). An alternative...

...Figure 1: A schematic illustration of CRISPR/Cas9-based approaches to study ncRNAs. (A) Transcription start...signal downstream of the transcription start site will terminate transcription of most of the ncRNA (Figure... coming, right? Non-coding RNAs (ncRNAs) are transcribed but do not encode proteins. They play important...Using a single guide RNA (sgRNA) to create a double-strand break works well because very small deletions and...sequence can completely disrupt expression of the transcribed ncRNA (Jiang et al. 2014).  Simple knock-out ...lncRNA regulatory elements that are crucial for transcription and downstream function. Rather than attempting...the lncRNA locus, scientists can mutate the transcription start site, splice sites, exons, and/or promoters...

...how the brain operates. References 1. Callaway EM. Transneuronal Circuit Tracing with Neurotropic Viruses...generally are not transmitted to adjacent neurons via synapses (i.e., transmitted transsynaptically). This is ...retrograde transmission (Etessami et al., 2000, Wickersham et al., 2007). Thus, RABV transsynaptic spread ...replicate. Modified Rabies Virus: Converting a transsynaptic neuronal tracer into a monosynaptic (first order...subsequent cycles of viral replication and retrograde transmission occur, spreading the virus through higher and...higher order neural connections. While this transsynaptic spread is useful for neuronal tracing, it has...-dependent TVA to Cre-expressing neurons in a transgenic mouse line. By doing this, RABV expressing the...

...genome engineering and microbiology.     References 1. Chen, Sean, et al. "Highly efficient mouse genome...:     Cells must be amenable to transfection or viral transduction     Appropriate promoters must be...expression     The requirement for Cas9 transcription and translation delays editing What are Cas9-gRNA ... studies in cells that are difficult to transfect or transduce. While using Cas9-gRNA RNPs may require...site of interest. This is commonly performed by transfecting cells with a plasmid, such as PX459, which encodes... often be used in cells that are difficult to transfect, such as primary cells. Using RNPs can also alleviate... are detectable at high levels shortly after transfection, and are quickly cleared from the cell via protein...

...cells were transfected using 1:1, 1:2, 1:3 and 1:6 µg of pRosetta :µg of PEI. The 1:2 and 1:3 ratios provided...18.9 18.9 1:2 18.9 37.8 1:3 18.9 56.7 1:4 18.9 75.6 1:5 18.9 94.5 1:6 18.9 113.4 Gently add the diluted PEI...possible range of ratios to test: Ratio of DNA:PEI Amount of DNA (μg) Volume of 1 mg/mL PEI (μL) 1:1 18.9 .... Thawed aliquots should be discarded after 1–2 months. 1 mg/mL polyethylenimine, linear MW 25,000 Da ...stable. Dilute 1:3 (µg DNA:µg PEI) in 500 µL total of OptiPro SFM (per 10 cm plate). 56.7 µL of 1 mg/mL PEI...for viral production) Day 1: Transfect Cells Day 2 (am): 18 h post transfection - Remove media, replace ...prepared, transfect cells with a fluorescent plasmid using a variety of ratios. Check the cells 1–2 days ...

... NGT PAM GAA PAM GAT PAM xCas9 3.7 41 (1) 32 (1.6) 13 (2.1) 38 (4.5) 7.2 (5.2) ...xCas9 for transcriptional regulation and base editing Hu et al. also engineered transcriptional activators...activating transcription. Sites containing NG, GAA, or GAT PAMs averaged 56-91% transcriptional activation... to create xCas9, an enzyme recognizing a broad range of PAMs like NG, GAA, and GAT, but also displaying...subunit ω. This construct is designed to activate transcription from the accessory plasmid (AP) encoding phage...could recognize a library PAM, it would induce transcription of gene III, permitting phage propagation and...spectinomycin resistance. xCas9 3.7 showed the broadest PAM range, recognizing NG, NNG, GAT, and CAA PAMs, while xCas9...

...target DNA quickly and accurately.  Figure 1: Steps in PCR reaction   This process of denaturation...separation of the two DNA strands.  Annealing- The temperature is then dropped to a range in which the forward...the primers can anneal to the single stranded DNA template strands. DNA polymerase is also stable at this...template DNA to amplify from and unique single stranded DNA primers that bind upstream (5’) and downstream... process as DNA polymerases require an existing strand of DNA to add nucleotides to.  Using these reagents...reaction:  Denaturation- To amplify DNA, the two strands of the template DNA first have to be separated....used to quantitate RNA levels using reverse transcription-qPCR (RT-qPCR). The first step of this process...

...in restraint. The very first subheading in Chapter 1 asks “What is science?” That’s a very big question... that may have warranted their own chapters, and certainly chapters that could warrant their own books..., and he wisely makes no such attempt. He is transparent about his own biases--many of which will be shared...non-scientists, Zimmer’s observations will likely range from mildly surprising to genuinely shocking. Mainstream...he describes the now infamous tale of Rosalind Franklin and her essential-but-unlauded role in the identification...within this section. The GFP story has its own Franklin in Doug Prasher, who originally isolated the GFP...science produced by the quieter, more reserved Franklins and Prashers will find it increasingly difficult...

...(RBD) of the viral spike protein.   Figure 1. (A) SARS-CoV-2 RBD DMS Library generation and sequencing... lab has created a novel nanocompartment for transporting proteins in yeast by using murine polyomavirus...2A-Synaptophysin-mRuby from Liqun Luo’s lab expresses membrane tethered GFP to enhance axonal labeling and synaptophysin-tagged...expressing GCaMP6f with this promoter. Image calcium transients in PV interneurons with AAV-S5E2-GCaMP6f are ...

...understand these strange results by digging deeply into their data.     Fig. 1: Genomic fraction...Samples are divided into those of African ancestry(orange) and those of other ancestry groups (grey). ...): 250-252. Carlson J, Henn BM, Al-Hindi DR, Ramachandran S. Counter the weaponization of genetics research...