...Standard AAV vectors are generated with a producer cell line like the widely used 293T. During vector...Casey Maguire and colleagues from Massachusetts General Hospital and Harvard Medical School took a closer... antibodies. This is particularly useful for therapeutic applications in vivo where endogenous anti-AAV...anti-AAV antibodies often neutralize the therapeutic efficacy of AAV vectors. How are exosome AAVs produced...exosomes or stabilize their structure or their interaction with AAVs (Schiller et al., 2018). Exosomal vesicles...interest special contexts, such as the delivery of therapeutic proteins to the brain which requires crossing...cells using exosome-associated AAV." Molecular Therapy 25.2 (2017): 379-391. PubMed PMID: 28082074. PubMed...

...and easily generate zebrafish transgenic lines to study neurobiology and neuorodgenerative diseases. The...Gateway-Compatible Toolbox is based on the original Tol2kit generated by the Chi-Bin Chien lab (Kwan et al., 2007) and... specific neuronal cell types involved in neurodegenerative diseases, the Cole Lab selected promoters ...The impact of KLF4 N-terminal variants on iPSC generation Deriving iPSCs (induced pluripotent stem cells...reprogramming somatic cells. A common method for generating iPSCs to express the transcription factors from...Polycistronic cassettes that contained KLF4s had overall diminished KLF4 expression levels; this altered...mScarlet. Bindels et al used a combination of deliberate and random mutagenesis to isolate the best RFP...

...Promoters control the binding of RNA polymerase and transcription factors. Since the promoter region ...protein, the repressor protein can bind to the operator. The bound repressor then prevents transcription... repressor (TetR) can bind to the tetracycline operator sequences (TetO), preventing transcription. In...have been engineered and improved. Takara’s “2nd generation Tet-Off vectors (Tet-Advanced)” are the most ...VP16. cTA binds to promoters containing putative operator sequences (CuO) (linked in groups of 6), allowing...the same manner, with LexA binding to the lexA operator (lexAop). Fusing the LexA DNA-binding domain (...refined the Q system and introduced the second generation QF2 activators. These activators solve the problem...

...piggyBac™ transposase or hyperactive transposase mutants [7], it is generally not as efficient as Cre ...How does the piggyBac™ system make it easier to generate transgenic cell lines? One of the major uses of... that have negative impacts on fitness. One consideration when making a transgenic cell line with the ...nucleases (CRISPR/Cas9, TALENs, and ZFNs). However, several challenges still remain, such as making gene edits...completely seamless (i.e., so no other genetic alterations have occurred) and isolating the specific cells...the piggyBac™ system, these challenges can be considerably reduced. The piggyBac™ transposon system can...and M. H. Wilson, “piggyBac-ing models and new therapeutic strategies.,” Trends Biotechnol., vol. 33, no...

...ion-channels and pumps in developmental biology, regeneration studies, and cancer biology. I was first introduced...patterning. Based upon trends in studies over the past several years, it seems that the more polarized (negative...  Additionally, researchers have reprogrammed regenerating tissues without using genome engineering. Planarian... briefly inhibited in the entire worm during regeneration, the worm creates two heads. Subsequent cutting...transcriptional or translational level. However, several genetic tools have been developed to address these...exciting emerging field, with many implications for regenerative medicine, evolution of body plans, synthetic...the advancement of research in birth defects, regeneration, medicine, and synthetic bioengineering. References...

...(tracrRNA) which interacts with the Cas9 protein. These RNA molecules are vulnerable to attack by both...effectively serving their function of guiding Cas9 to generate a targeted break. These advances in gRNA technology...foreign RNA. To protect the RNA from degradation, several simple methods have been employed which modify ...modification is best? Studies suggest coupling several of them together, for example phosphorothioates...more efficient on a budget is to simply swap out several of the ribonucleotides for deoxynucleotides. A ...A partially DNA gRNA is surprisingly well-tolerated by the Cas9 protein and can increase on-target efficiency...photocleavable gRNAs. Photocleavable guides are generated by introduction of a single photocleavable 2-nitrobenzyl...

...produced from its serum several weeks later. Created with biorender.com. To generate polyclonal antibodies... response.   Polyclonal antibodies are generated by injecting an animal with an immunogen, then...might be the full-length protein that you want to generate antibodies against, or a unique peptide sequence...Freund’s adjuvant is injected, which stimulates the overall immune response. Once the initial antibody response...designed for experiments where antibodies raised in several different species are present. One common example...antibody for your experiment, you should either order several vials of the same lot of polyclonal antibody, or...experiments to be aware of signal strength. In general, polyclonal antibodies produce a higher level of...

... islandicus CRISPR-Cas system. 5. Phage therapy Phage therapy uses phage to treat bacterial infections...have discovered over 50 anti-CRISPR proteins that interact with CRISPR-Cas systems such as Cascade-Cas3, ... DNA binding Example:  AcrIIA4 blocks Cas9’s interaction with the PAM site (Dong et al., 2017), and AcrIIC3...2017). Inhibit crRNA loadingExample: AcrIIC2 interactions with Cas9 interferes and prevents the correct...bacterial pathogens possess CRISPR-Cas systems, phage therapy might not be as effective. This is where Acr proteins...enough that they could be engineered to “armor” therapeutic phages to protect them from pathogenic bacteria...activity is an important step towards CRISPR-based therapies and for more precise editing in the lab. References...

... top of the ever expanding body of biomedical literature! PubMed PubMed is the classic search engine ...Twitter into a paper-finding-machine. There are four general types of accounts you can follow: Scientists: ...updated. This Twitter list contains >300 Twitter literature bots, including a bot for biosensor papers, microbe...Semantic Scholar Semantic Scholar is an AI-backed literature search developed by the Allen Institute for Artificial...search terms. Key features Semantic Scholar has several ways to focus your search results. A bar graph ...quickly identify papers with greater influence, the average number of citations per year and the number of ...aren’t familiar with the topic.  Meta Meta is a literature discovery platform developed by the Chan Zuckerberg...

... to consider one of several Cas9s that recognize NG PAMs. The Nureki lab generated SpCas9-NG, a rationally...biology, the answer is always it depends, but generally the efficiency of the desired edit is reduced ...name implies (Nishimasu et. al, 2018). xCas9 was generated by the Liu lab through phage-assisted evolution...evolution, specifically with the goal of generating Cas enzymes with alternative PAM requirements (Zhao et. ...through structure-guided engineering of the PAM-interacting domain of Cas9. After multiple rounds of engineering...helps even when it’s not an ideal site: moving several bps away from a desired site does not have the ...most gene targeting experiments. Between SpRY and several of the other Cas variants described, nearly all...

... with single-pot assembly Tracking cell-cell interactions New auxin inducible degron system for C. elegans...    New system developed to track cell-cell interactions By Jennifer Tsang GFP-based Touching Nexus (...Winslow’s lab to label cells after cell-cell interactions. Using G-baToN, “sender” cells express GFP on...anti-GFP nanobody on the cell surface. When the cells interact, GFP is transferred to the receiver cell, and ...cell pairs.   Figure 2: Schematic of the interaction between the sender cell and the receiver cell...with BFP, sender cells expressing surface BFP generated BFP-labeled receiver cells. What’s more, G-baToN...provides versibility in tracking and modulating the interactions between cells.      Tang et al., eLife 2020...

... and use temperature to denature and renature the dsDNA. Upon renaturation, you will generate DNA duplexes...Expression of gRNAs Generating Mouse Models Using CRISPR/Cas9 Resources at Addgene.org Generating Stable Cell...inactivate the gene BRAF (a kinase implicated in several human cancers) in A549 cells (a human lung cancer...express Cas9. In these particular experiments, we generated A549 cells stably expressing Cas9 via transduction...52962-LV, Figure 1). Some cells were then used to generate a monoclonal cell lines by limiting dilution. ...Figure 2: Cas9 expression in monoclonal cell lines generated from A549 cells transduced with lentiCas9-blast...lanes 3, 4) were used as controls. Duplexes were generated from BRAF-edited and GFP-edited amplification ...

... difficult to eradicate. You can suppress the growth of mycoplasma but cannot eradicate it completely....nutrient-rich and also the best source for mycoplasma proliferation. Plus, it is added to the media after autoclaving...being contamination free. The aerosols that are generated (while talking or pipetting) during working in...leading causes of contamination. These aerosols generated will enter the culture medium during subculturing... contact with a nutrient source, they start proliferating shortly. Once they are present in the laboratory...are used which trigger cellular mechanisms that generate luminescence. This can then be detected by a luminometer...important to prevent contamination rather than eradicating it later. Don’t be scared when mycoplasma infects...

...AAAS Science and Technology Policy Fellow at the Veterans Health Administration. “But, seriously, what even...Science and Technology Policy Fellow (STPF) at the Veterans Health Administration, I could never get one straight...these changes to policy could shift how society interacts with certain scientific areas. Whether you’re ... or even run for office yourself! Plus, at the federal or state level some professional societies host...taken through your own personal lens.  For me, the Veterans Health Administration was the perfect position...example proposals and consider applying for their accelerator for some hands-on guidance.   Check out my webinar...fellowship is that you are often vetted for your general scientific expertise and then once selected, you...

... by Single-stranded Virus Next-generation Sequencing. Molecular Therapy - Nucleic Acids 4:e260 . https...our viral service to continue our mission of accelerating scientific research. To make sure that adeno-associated...more rigorous quality control, including next generation sequencing (NGS) of the viral genome to confirm... the virus itself. While these impurities are generally considered innocuous for research-grade vectors...likelihood of recombinase-independent expression. Next-generation sequencing (NGS) is a powerful tool that can ...Vivo Persistence after Vector Delivery. Molecular Therapy 12:744–753 . https://doi.org/10.1016/j.ymthe.2005.06.003...Mueller I, Mitsopoulos A, Fan M (2020) A Novel Next-Generation Sequencing and Analysis Platform to Assess the...

...SaCas9 with an NNGRRN PAM by mutagenizing the PAM-interacting domain. However, these alternative PAM sequences...often more complex than NGG, resulting in lower overall genome frequency. Despite the progress we’ve made...evolution Hu et al. used phage-assisted evolution to generate and select for Cas9 mutants with PAM flexibility...catalytically dead SpCas9 (dCas9) fused to the bacterial polymerase subunit ω. This construct is designed to activate...another 72 hours of phage-assisted evolution, generating xCas9 2.0-2.6. This second round of evolution... Cas9 variants with activity on non-NGG PAMs, generating xCas9 3.0-3.13. To test xCas9 3.0-3.13, Hu et...transcription. Sites containing NG, GAA, or GAT PAMs averaged 56-91% transcriptional activation compared to ...

...proteins that interact. Indeed, FRET is often used to determine if two proteins interact. Oligomerization...10hours. Temperature: FPs maturation times and fluorescent intensity can be affected by the temperature. For...filter that will pass blue light to the detector/camera, then BFPs are of no use to you. When using more...Oligomerization: The first generations of FPs were prone to oligomerize. This may affect the biological function...parameters (e.g. excitation light intensity, pH or temperature). Photostability: Fluorescent molecules gets ...parameters (e.g. excitation light intensity, pH, or temperature). pH Stability: This parameter is important if...Helpful Websites & Resources: FP guide at Addgene Interactive Visualization of Fluorescent Protein Properties...

...organisms or organism/gene interactions with the host. This scenario allows for several potential CRISPR applications... functions in S. pneumoniae and can be used to generate multiple mutations simultaneously (Jiang et al... with CRISPR to create a system for scarless, iterative genome engineering (Jiang et al., 2015). pCas ... or a dCas9 fused to the omega subunit of RNA polymerase, the lab   successfully repressed or activated...Noting that the more common spCas9 system has generally low efficiencies or can be proteotoxic when used... orthologs to identify a more efficient, well tolerated, Cas enzyme. They identified the Cas9 orthologue...problem than repression, scientists have deposited several plasmid tools that can be used in this way. As ...

... transformation. There are many motivations to generate transgenic Arabidopsis, from studying transcriptional... floral drip or dip transformation method. The general steps for this method include: Cloning and transforming...appropriate expression vector is the first step in generating transgenic Arabidopsis. There are a bunch of ...vectors and can be used for promoter swapping and generating N and C terminally tagged fusion proteins with...plants you are transforming into will be your T0 generation. These plants will be ready to transform when...will also increase the number of inflorescences overall, which may improve transformation efficiency. If...such a construct will be silenced in subsequent generations. You will know if you obtained a transformant...

...enhanced the ability of scientists to make genomic alterations, bringing about a revolution in genome engineering...engineering applications of CRISPR including: generating single or multi-gene knockouts in a wide variety...editing using homology directed repair (HDR), and generating large genomic deletions. What are the advantages...and specificity is a concern, such as knockout generation or homologous recombination.  However, experiments...step in this type of CRISPR experiment is the generation of the RNP complex.  While one option is to purchase...in vitro transcribed from ssDNA, which can be generated by commercial vendors such as IDT. These two components...electroporation (A in the figure above), which generates pores in the cell membrane, allowing for entry...

...neurons throughout the whole brain. Neurons of the peripheral nervous system (PNS), which connect the heart...mutant AAV capsid (cap) expressing plasmids is generated. Diversity is introduced into the cap plasmid ...was neurons and astrocytes. Why screen for IV deliverable AAVs? Typically AAVs are directly injected into...transduction efficiency of neurons were measured. On average, AAV.PHP.eB and AAV.PHP.S had 1.5- to 2-fold higher...They also transduced a higher number of neurons overall (GFP+ cells that also stained for a neuronal marker...labeling of neurons, because it doesn’t require the generation of a transgenic mouse. 2. Cell-type-specific...translational applications (mechanistic studies and gene therapy treatments for neurological diseases). The Gradinaru...

...technology and its advantages? Fujii: In the last several years, my lab has been working on development of...specific genomic regions while retaining molecular interactions. For this purpose, we developed the insertional... regions. I believe that enChIP will markedly accelerate research of regulation mechanisms of genome functions...some of its potential applications? Fujii: We generated a series of retroviral vectors expressing tagged...biological systems. We are currently developing several additional systems of enChIP to make the technology...to journals. In addition, it is my pleasure to interact with my colleagues in meetings of scientific societies... Rhine River. In the US, I very much enjoyed interactions with many excellent colleagues at NYU and visiting...

...implantation and generation of transgenic mice. Purification: GFP can be used as a general epitope tag for...biosensor or cell marker, studying protein-protein interactions, visualizing promoter activity, and much more...fluorescent activity; very little truncation is tolerated, however, point mutations are acceptable. GFP'...mutations have since been introduced to GFP and new iterations of fluorophores are constantly being engineered...energy transfer (FRET): This is used to study the interactions between two proteins or between two domains ... has also been used to study protein-protein interactions. In this case, two portions of EGFP are fused... their activity and visualize the effects of alterations to this activity. Learn more about optogenetics...

..., the legacy of each government and generation is in the veracity of its work. Alina Chan is a postdoctoral...these mistakes quickly. However, many scientists operate in a hyper-competitive environment where getting...speak out in unprecedented force during the 2015 federal election.   Even as we marched in solidarity with... Washington, DC, USA 38.9072°N 77.0369°W – Mark Lucera I am a passionate scientist.  I care about my ...People asked, and we talked.  That's a win. Mark Lucera is a PhD trained scientist at the University of...Postdoctoral Association.  Follow him on twitter at @Mark_Lucera. Ann Arbor, MI, USA 42.2808°N 83.7430°W – Alexandra...Aurora, CO where she studies the development and regeneration of mammalian taste buds. Erin is also the President...

...preps and our lentiviral preps.   Viruses are generated using standard methods that have been optimized...optimized for each specific virus in order to generate high quality preparations. After production, all virus...the University of Pennsylvania Vector Core. In general, titering is performed by the facility that produced...suggests that titers obtained using ddPCR are generally two to three fold higher than those achieved using... us. Full sequencing of the Viral Genome Next-generation sequencing is performed on viral genomes isolated...items) are produced in 293T cells using the 2nd generation psPAX2 and pMD2.G packaging system developed ...lentiviral preparations (“-LVC” catalog items) are generated from lentiviral preparations (described above)...

... developed, though, researchers at HuidaGene Therapeutics guessed that fusing MPG to an ABE would allow...transversion editor AYBEv3. This tool still retained considerable A → G editing activity at most sites, in addition...they co-expressed Polη, a translesion synthesis polymerase known to preferentially incorporate A bases across... N108Q mutation in the deaminase), produced an average A → C product purity of 48%. Like CGBEs, these ...abasic site before filling the empty space. Over several rounds of rational mutagenesis, the team progressed...allow it to recognize thymine (T) or cytosine (C). Several different teams succeeded with this approach around...recognize T and/or C substrates. The HuidaGene Therapeutics team has deposited their editors, called gTBE...

...more stable than the methyltransferase. When a plasmid is lost, the methyltransferase is degraded and/or...stabilized luciferase-expressing plasmids to examine in vivo-induced promoters in the Vibrio cholerae vaccine...ensure that plasmids are passed down through generations (Figure 1). These so called “plasmid addiction...composed of restriction endonucleases and methyltransferases. Restriction endonucleases cleave DNA at ...specific nucleotide sequences. To counter this, methyltransferases add methyl groups to bases at specific nucleotide...stably express antigens in sufficient quantities to generate an immune response. As a proof of concept, they...genes that couple host cell division to plasmid proliferation." Proceedings of the National Academy of Sciences...

...pEcCas/pEcgRNA is superior in several ways: The pEcCas plasmid can be operated at 37 ℃ for faster growth ...plasmid that expresses the S. pyogenes Cas9, a temperature-sensitive replicon for self-curing, an arabinose..., 2) changed the replicon of pCas into a non-temperature-sensitive replicon, and 3) added the sacB gene...containing BsaI restriction enzyme sites at its ends to generate pEcgRNA. The ccdB serves as a counterselection...genes of interest in BL21(DE3) using the newly generated pEcCas/pEcgRNA system.   Advantages of the pEcCas...directly ligated with the annealed 24 nt oligos to generate new pEcgRNA. pEcCas/pEcgRNA can be applied not... the gene dosage of glucose dehydrogenase and generated a strain that could achieve glucose dehydrogenase...

...processes from development to metabolism to fertility. Several zebrafish-specific gene expression databases that...available, new hypotheses about specific genetic interactions arise and tools to deactivate specific genes...way to deactivate genes without spending months generating mutants. Unexpectedly, phenotypes sometimes differ...of gene regulation (El-Brolosy et al., 2019). Generating zebrafish transgenics Creating transgenic organisms...different experimental conditions. Strategies for generating transgenic zebrafish are well established (Felker...cell RNA sequencing approaches (Harland, 2018). Several different methods have been developed, relying ...i.e., redundant) genome, their long three-month generation time and labor-intensive maintenance compared...

... replicates how you’d interact with team members in person, and improves overall communication.  Maintain...project and accelerates the pace at which problems can be addressed and new ideas generated. Furthermore...team in Paris, France. While efforts like these accelerate the progress of research, they can also present...different tasks. But these challenges can be exaggerated when communicating across time zones, digitally...the times that work best for them. This also accelerates the pace of research by circumventing the slower...exchange project at the International Cancer Immunotherapy Conference in Frankfurt, Germany When funding...