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... one target is labeled and imaged at a time, inactivating the fluorescent secondary antibodies before ...comes in. After you image the first target, you inactivate the fluorophores in the sample, either by chemical...new primary, wash, add secondary, image again, inactivate, and repeat these cycles for all your targets...from. But by labeling sequentially with one, inactivating the fluorophores, then labeling with the other...brain samples from the James Trimmer Lab, using recombinant antibodies with different isotypes. (A) Neocortex...antibodies are commercially available. Pick whichever combination of host and target species, isotype specificity... 2A above, Andrews et al. used the following combination of antibodies:   Target (Rat) Primary antibody...

...piggyBac™ transposon? The piggyBac™ transposon was originally identified in moths in 1983, but wasn’t successfully...or when the gene is constitutively-active or a dominant-negative mutant. Reducing the copy number can ...host machinery for DNA repair by homologous recombination, which results in the incorporation of your ...containing a negative selection marker such as thymidine kinase, a researcher may subsequently kill off any cells...site-specific nuclease to stimulate homologous recombination and Footprint-Free™ removal of the selection...First, instead of using piggyBac™, you could use recombinase technology (Cre/loxP) to remove the donor cassette...[7], it is generally not as efficient as Cre recombinase. However, the use of negative selection largely...

...another biological workhorse, Cre recombinase, which induces recombination at LoxP sites. Placing a lox-STOP-lox...researchers to activate transcription or Cre recombinase activity only in the presence of GFP. These systems... transcription. Thus, T-DDOGs can be used in combination with green-red Cre-lox systems. T-DDOGs can also...T-DDOGs, GFP, and a reporter construct into the mouse retina. GFP expression robustly activated the reporter...Cre-DOG is both robust and specific. When tested in retinal electroporation studies, DsRed reporter expression...system, including the motor cortex, cerebellum, and spinal cord. Cre-DOG is also suitable for optogenetic ...type-specific manipulation with GFP-dependent Cre recombinase.” Nat. Neurosci. 18(9) (2015): 1334-1341. PubMed...

...novel way. Finally, engineering new applications to solve problems will provide originality to your work...on how to submit your work either through seminars or webinars. Sending on your grant application to your... the deadline isn’t going to be much use. 2. Originality is essential for grant success The two most important...be looking for in your research proposal are originality and relevance. When reading through your proposal...our own or other groups, but your work must be original (otherwise why would you be interested in it?)...Frontier Science Program's website. One way to add originality to your work is to collaborate with specialists...your work, or even the successful use of your preliminary data to secure further funding. Make it clear...

... that exterminate entire humanity were the two most popular topics that sparked the imagination of most... CBE but use a ssDNA adenosine deaminase (evolved from a tRNA deaminase enzyme, TadA) to induce a A→G ...hybrids. This book is published 30 years after the seminal work of Gregor Mendel on the genetic inheritance... first ever X-ray diffracted image of the DNA. Finally, in 1953, Franklin along with James Watson and ...Thurman and Jude Law. The movie’s name is itself a combination of the four bases of DNA – A, T, G, and C. In...These breaks can then be repaired by homologous recombination with a repair template.  Each zinc finger recognizes...The cytidine base editors (CBE) use a cytidine deaminase to introduce a C→T change at precise locations...

...communities. Many thanks to our guest blogger Katrina Armstrong. Katrina Armstrong is a Neurophysiology Msc Student...This post was contributed by guest blogger Katrina Armstrong, a Neurophysiology Msc Student at the University...which cell type? Location (3): Traveling from the original location - Axonal Transport in the CNS What tissue... Photoreceptor Cells AAV2, AAV5, AAV8 RPE (Retinal Pigment Epithelium) AAV1, AAV2, AAV4, AAV5, AAV8...your particular experiment! Traveling from the Original Location - Axonal Transport It has been demonstrated...serotonin in movement. Follow her on twitter @katrinaboards.   References 1. Russell, David W. "AAV vectors...and Richard J. Samulski. "Self-complementary recombinant adeno-associated virus (scAAV) vectors promote...

...through the use of protein tags and separated from contaminants using a variety of purification methods. Deciding...expression systems are very powerful for high level recombinant protein expression. These systems enable high...cheap and low-tech means of mass expression of recombinant proteins. Many cells from various types of plants...modifications. Extraction and purification of recombinant proteins from plants however can be expensive...biochemicals and proteins through plant roots. Tagging recombinant proteins with a naturally secreted plant peptide...expression system to generate large quantities of recombinant eukaryotic proteins. Although many species of...using S. cerevisiae, researchers often place recombinant proteins under the control of the galactose inducible...

...to the final topic, which is that natural AdVs, replication-competent viruses with a recombinant genome... Virus type 2 (AAV2) – or some unprecedented combination of the three (Servellita et al., 2023). Normally...lack of exposure, a lack of immunity, and then finally a synchronized exposure under these conditions ...virus, we will see that they are very central determinants of how AdVs behave, and in fact, of how adenoviral...from the genomic plasmid can be used to house a recombinant sequence (transgene) instead.    Although the...strong innate and adaptive immune responses that inactivate viral genomes or otherwise clear the virus from...been reported when using vectors, and immune inactivation of the vector can still occur even in the case...

...infect. Finally, the transfer plasmid encoded the desired transgene flanked by HIV-1 long terminal repeats... the original system, the possibility of creating a replication-competent virus via recombination between...encoding Gag and Pol, increasing the number of recombination events necessary to create a replication-competent...replication-competent virus. Second, Tat is eliminated through the addition of a chimeric 5’ LTR containing a Tat-independent...oncogenesis by altering local gene expression. Self-inactivating (SIN) lentiviral vectors contain a deletion ...F, Beuzard Y, Gluckman E, Bushman F, Hacein-Bey-Abina S, Leboulch P. Nature. 2010 Sep 16;467(7313):318...PubMed PMID: 25804479. 9. Development of a self-inactivating lentivirus vector. Miyoshi H, Blömer U, Takahashi...

...contrast, fluorescence imaging has emerged as the dominant light microscopy contrast technique in modern ...molecular or structural specificity to the viewer. Finally, modern microscope designs may further exploit ... light exits the microscope objective evenly illuminating the entire (wide) field of view. Fluorescent...and focused onto a camera for visualization. Illuminating a sample in the opposite direction to fluorescence...fluorescence collection is referred to as “Epi” illumination. Therefore these are sometimes call Epi-fluorescence...sample and raster scanning the point to build up a final image pixel by pixel. The collected fluorescent ...location in the sample. As more than one hole is illuminated at a time, the image can be acquired more quickly...

...activation-induced cytidine deaminase (AID) and an engineered tRNA adenosine deaminase (TadA) to a catalytically...fusing catalytically inactivated RNA-targeting Cas13 (dCas13) with the adenine deaminase domain of ADAR2. ... by evolving the adenine deaminase domain of ADAR2 into a cytidine deaminase. This editor still retains...Cas9/gRNA ribonucleoprotein delivery Cytosine deaminase for RNA editing CRISPRi toolkit for Burkholderia...Find the gRNA and packaging plasmids   Cytosine deaminase for RNA editing Feng Zhang’s lab previously developed...

...multiplexed using tRNAs, within the lentiviral 3’ long terminal repeats (LTR). The 3’ LTR is duplicated during...the lentiviral insert was minimized to reduce recombination and avoid separation of the multiplexed elements...fold improvement in detection efficiency over the original CROPseq. Find CROPseq-multi plasmids here Walton...developed a new prime editor that incorporates La’s N-terminal domain, PE7 (Figure 2).  PE7 shows enhanced editing...-2 research   By Ashley Waldron Ready-to-use recombinant antibodies were recently added to the Addgene...Image from Addgene. These antibodies are recombinant versions of mouse monoclonal antibodies generated...2021). Check out their paper for details on the original antibodies. Find Anti-SARS-CoV-2 Antibodies here...

...homologous recombination efficiency to reduce the number of plants you need to grow to get a recombinant because...solicitation requires funded labs to rapidly disseminate protocols, methods, data, and tools that result...published, but to engage in ongoing, real-time dissemination of information and materials. Many groups publicized...labs all include materials sharing in their dissemination plans and in particular, that they all share...organismal systems described at this meeting were extraordinarily diverse. Plans to develop tools to study their...   Genus Microtus (Zoe Donaldson) Voles have fascinating social behavior. For example, two similar species...because they have only 1,700 genes total). Hydra (Celina Juliano) This is a known stem cell and regeneration...

...AAV particle. Finally, the precision of the assay can vary up to two fold for the final titer value, which... of 1:1 (Zeltner et al., 2010). However, for recombinant AAV2, the same study reported a physical-to-infectious...2010), while another reported that only 20% of recombinant AAV2 particles were full, compared to 50% of ...annealing can be affected by the presence of protein contaminants, which is a factor in AAV titration since the... be as important as the relative titer values. Finally, because physical titer does not give an indication...packaging of genomes can limit production of recombinant AAV-2." Gene therapy 6.7 (1999). PubMed PMID:... Lock, Martin, et al. "Characterization of a recombinant adeno-associated virus type 2 Reference Standard...

... at Mount Sinai Hospital, Toronto, Canada where he studies protein-serine/threonine kinases implicated...detect possible errors in assumptions, use of inappropriate statistical methods, omission of some data, ...tools and assumptions to reveal findings that the originator was oblivious to, no matter how much time they...productivity”, so this is unlikely. Usually, the originating scientist and colleagues are privileged in viewing...generators. This practice doesn’t appear to harm the originating researchers, even if they are occasionally beaten...data are likely, at least in part, due to our inadequate cataloguing of how results were obtained and ...multiple, complex panels. PLOS, itself, has not finalized its definition of necessary data requirements ...

...possibility of carrying over contaminants from the plasmid prep process into the final plasmid eluate. Possible...designed with environmental sustainability in mind (Table 2). Table 2: Sustainability features highlighted in...more importantly, the DNA needs to be free of contaminants, such as bacterial endotoxins, that could affect...Possible contaminants can sneak in from almost any step of the process and include things like genomic DNA...by excess material, leading to less DNA in your final eluate. Bead-based kits can be more flexible in ...impact, check out Addgene’s blog post on bringing sustainable practices for the lab. Plastic consumables (including...by more than half 70% recycled materials Sustainable forestry certification Starch-based compostable...

... its destination plasmids. In the destination plasmid, the ccdB gene is flanked by recombination sites...introduce a gene of interest into the destination plasmid, recombination sites must first be added to flank...compounds needed for growth, or auxotrophy, the inability to synthesize a compound needed to grow. Auxotrophy...usually something toxic. This gene is found on the original plasmid and either the insertion of a DNA fragment...flank the gene. Successful recombination and integration of the gene of interest at these sites in the plasmid...

...Minimizing off-target editing ABEs were originally developed from RNA deaminases, using directed evolution to get...DNA-targeting technology with a fused cytidine deaminase to create C→T base edits at a target locus. A ...characterized this RNA-editing activity and developed a deaminase variant with a V106W mutation, which reduced RNA...directed evolution, introducing mutations to the deaminase subunit to produce a series of ABE8s that were...editing. Base editing in the present—and future The final base editor that was administered to KJ was designated...J., Liquori, A. J., Murray, R., Packer, M. S., Rinaldi, C., Slaymaker, I. M., Yen, J., Young, L. E., &...sciadv.aax5717  Richter, M. F., Zhao, K. T., Eton, E., Lapinaite, A., Newby, G. A., Thuronyi, B. W., Wilson, C....

...and practicing my SciComm skills and I had to coordinate with other people for a variety of projects. ...conceptual chunks: prioritization, planning, and procrastination. Time management through prioritiziation Every... more time for that task the next time around. Finally, when prioritizing, you should always be aware ...Susanna’s post Upload Eric’s post and give it a final read Browse list of previous depositors from Boston...Eric before doing anything else. Preventing procrastination Even if you’ve done all the prioritizing and...and planning in the world, procrastination can easily prevent you from making progress on a task. For ...few things I’ve found useful to avoid such procrastination: Remove distractions: Probably the easiest...

...colonies, and screened for the desired modification. Finally, the positive clones are sequenced to confirm the...plasmid. Although it is commonly believed that the final products of the PCR reaction are pieces of double...recent data (see Xia et al., 2015) suggests that the final PCR products are pieces of linear, double stranded...These homologous ends recombine in vivo to give the final circular plasmid product pictured here. B) Screening... of DMSO to the PCR reaction (usually around 3% final concentration). The DMSO reduces secondary structures...using a methylation-dependent enzyme (DpnI) to eliminate the parent plasmid from the PCR products, the ...duplications (slightly larger bands sizes relative to the original template). Due to the small difference in size...