...interest: The transgene sequence is flanked by long terminal repeat (LTR) sequences, which facilitate integration...line that stably expresses Gag-Pol and/or Env, eliminating the need to deliver these genes in trans by specific...feature may be useful for the transduction of terminally differentiated (non-dividing) cells. Both lentiviruses...produce additional virus. Are γ-retroviruses self-inactivating (SIN)? They can be. Plasmids that encode SIN... LTR Subcomponents: -U3 -R -U5 LTR; Long terminal repeats; U3-R-U5 regions found on either side ...U5 and the rest of the provirus. 3’ LTR Terminates transcription started by 5' LTR by the addition...written and updated by multiple Addgenies. It was originally published in 2018, in the Viral Vectors 101 eBook...

...efficiency.  Finding recombinant psychrophilic enzymes for the host The expression of recombinant proteins for...conditions decrease the potential for microbial contamination. Additionally, the high activity of these enzymes...well under suboptimal conditions. This means recombinant proteins can aggregate or precipitate out as ...would allow the stable folding and expression of recombinant psychrophilic proteins. As the figure below shows... as engineering and regulating psychrophilic recombinant proteins.  To infinity, and beyond! Keeping ...brainstorm with each other. The event also featured a seminar on “Tools to increase scientific reproducibility...

...With no blue light, the channelrhodopsin remains inactive and neurons don't fire. With blue light, ions ...minimally-invasive, Frances Arnold’s lab and Viviana Gradinaru’s lab used machine learning to design new channelrhodopsins... To overcome these challenges, the Arnold and Gradinaru Labs developed a machine-learning model to help...information about parental channelrhodopsins to create recombinant channelrhodopsin sequences. The model first removed...their success in vivo. Therefore, the Arnold and Gradinaru Labs expressed ChRgers in mice to see if they ...broad transduction of the nervous system which eliminates the need for intracranial injections. When packaged...Bedbrook CN, Yang KK, Robinson JE, Mackey ED, Gradinaru V, Arnold FH (2019) Machine learning-guided channelrhodopsin...

...second day of Antibody, Addgene gave to me…  A recombinant anti-GFP!    🎶On the third day of Antibody, ...fourth day of Antibody, Addgene gave to me…  A recombinant Anti-Arl13b!    🎶On the fifth day of Antibody...sixth day of Antibody, Addgene gave to me… A recombinant Anti-PSD-95!    🎶On the seventh day of Antibody...eighth day of Antibody, Addgene gave to me…  A recombinant Anti-DYKDDDDK!   🎶On the ninth day of Antibody...tenth day of Antibody, Addgene gave to me…  A recombinant Anti-APP!   🎶On the eleventh day of Antibody... of Antibody, Addgene gave to me…  A growing recombinant antibody collection!     ...

...with a terminator. A simple TU could be comprised of just three parts: promoter, gene, and terminator.   ...functional relevance following a set hierarchy. MoClo originally developed from Golden Gate Cloning, in which ...this category also includes promoters, UTRs, terminators, etc. Once selected, individual units can be ...modularity — many parts can be assembled into many combinations. Even with current technologies, it can be extremely... name implies, gene stacking is the use of a combination of multiple genes to achieve a desired effect...resistance. MoClo is a great tool to deliver many combinations of different candidate genes at once to identify...

... for the production of offspring that have a combination of the mutations engineered into each of their...journey towards producing mice with your desired combination of mutations. Assuming your parental mice were...crosses. In Part 1, we explained how genetic recombination in mice expressing both Cre and LoxP occurs.... Part 1 of this series). In any case, Cre is a dominant phenotype, while you’ll want the floxed gene to... to be homozygous in order for Cre-mediated recombination to affect every copy of your target gene in ... you see in your experiment are a result of recombination at the floxed site, your control should be littermates...29768166. Zickler, Denise, and Nancy Kleckner. "Recombination, pairing, and synapsis of homologs during meiosis...

...vectors and 5 lentiviral Destination vectors. These vectors can induce combinatorial constitutive or inducible...vector from the Gage lab encodes GFP and Cre-recombinase. The Cre-lox system is a technology that can ...can be used to induce site-specific recombination events and is widely used in the field of mouse transgenics...(Plasmid #44246) Expression of a catalytically inactive (dead), human codon-optimized Cas9 under the control...deposited by Dr. Tyler Jacks. It carries CRE recombinase and a puromycin resistance cassette. No time... higher titer of lentiviral particles than the original lentiCRISPR v1. Special note from the Zhang lab...

...neural connectivity (Nassi et al. 2015). The recombinant viruses typically used in neuroscience research...transmitted transsynaptically). This is because the recombinant forms of these viruses typically used in research...G receptor is restricted to presynaptic nerve terminals (Lafon 2005). Therefore, once RABV is released... it is only taken up by the presynaptic nerve terminals of adjacent neurons (i.e., it is taken up by projection... neural tracing. Using these technologies in combination with molecular tools (e.g., optogenetic or chemogenetic...characteristics of a G-deficient rabies virus recombinant: an in vitro and in vivo study. J Gen Virol. ...Osakada F, Callaway EM. Design and generation of recombinant rabies virus vectors. Nat Protoc. 2013;8:1583...

...Miyawaki, Griesbeck, Heim, & Tsien, 1999), monitoring kinase activity (Ni, Titov, & Zhang, 2006), quantifying...tRNA suppression competes with natural chain termination at stop codons, expression levels of these tagged...acceptors, then biarsenicals are a good option. Originally developed by Roger Tsien and co-workers (Griffin...compounds such as British anti-Lewisite (which was originally developed to treat heavy metal intoxication in...insert your protein of interest, resulting in a C-terminal fusion of a tetracysteine tag. In addition, since...so don’t be afraid to try novel, unpublished combinations of labeling strategies. FRET experimental design...Roger Y. Tsien. "Specific covalent labeling of recombinant protein molecules inside live cells." Science281.5374...

...including "CLAP", combining an N-terminal CLIPf tag and a C-terminal SNAPf tag; see Figure 2), the authors...al., 2024). Their optimized strategy uses new combinations of PE protein components (Figure 1), an altered...negative-going GEVI that can be used in different illumination regimes with fast activation kinetics and high...://doi.org/10.1016/j.cell.2024.01.028. New recombinant antibodies for neurodegeneration research   By...-MAPT [AT8] and Anti-APP [6E10-A5]. Both are recombinant mouse monoclonal antibodies targeting the human...Immunohistochemistry with Addgene's Anti-APP [6E10-A5] recombinant antibody in human postmortem brain samples. Results...

...guests, Melina Fan, Kenneth Fan, and Benjie Chen! Melina Fan is a co-founder of Addgene. Melina received... podcast, we sit down with Addgene’s Founders - Melina Fan, Ken Fan, and Benjie Chen. We learn what it...University, where she studied diabetes and metabolism. Melina has worked at Genetics Institute in Cambridge, ... led business efforts at Addgene as the Chief Financial Officer and Chief Operating Officer. Prior to ...

...three codons in eGFP to eliminate fluorescence. This created three inactivated eGFP reporters- eGFP L202...reporter that monitors APOBEC (a family of cytidine deaminase proteins) and Cas9-mediated editing in real time...downstream constitutively active eGFP. To create the inactive mCherry, the lab introduced a 43 base-pair insertion...conversion to GFP after being targeted by a cytidine deaminase base DNA editor. Specifically the BFP mutant (... Figure 3: Targeting pEF-BFP with a cytidine deaminase base editor results in shift in emission spectra...resistance markers, and other enzymes such as Cre-recombinase drastically expanding the base-editing reporter...

...Originally published Aug 30, 2018 and updated April 16, 2020. Sensitive and specific nucleic acid detection... diagnose the presence of SARS-CoV-2 RNA, but inadequate access to reagents and equipment has become a... from a sample is first amplified using RPA (recombinase polymerase amplification) or reverse transcriptase...detection by Cas13. This amplification step in combination with the ssRNA reporter enables SHERLOCK to detect..., Cox DBT, Shmakov S, Makarova KS, Semenova E, Minakhin L, Severinov K, Regev A, Lander ES, Koonin EV,...2018) CRISPR-Cas12a target binding unleashes indiscriminate single-stranded DNase activity. Science 360...Makarova KS, Wolf YI, Gootenberg JS, Semenova E, Minakhin L, Joung J, Konermann S, Severinov K, Zhang F,...

...host's replication machinery for elongation and termination. These types of plasmids tend to be high copy...synthesizes a new DNA strand at each replication fork. Termination sequences present in the chromosomal DNA ensure... although ATP does get hydrolyzed over time, inactivating the protein binding it (Grimwade et al., 2018...replication and balance population growth against sustainability. Plasmid DNA replication and regulation Plasmids...which replication of the plasmid proceeds (and terminates) similarly to the bacterial chromosome. Since.... Thanks, stringent regulation! This post was originally written by Jason Niehaus in December 2015 and...Leonard, A. C. (2018). Origin recognition is the predominant role for DnaA-ATP in initiation of chromosome...

...nature can inform us on how to create better recombinant vectors. In this blog post, we’ll define plasmid...harness those features to create broad host range recombinant vectors.  A plasmid’s host range is determined...phylogenetic groups (Jain and Srivastava 2013).  Recombinant vectors that are made from broad host range plasmids...host-independent   Building broad host range recombinant vectors Replicons, which consist of a plasmid...broad host range plasmids can be used to create recombinant vectors that can replicate in different bacteria...to incorporating broad host range replicons, recombinant vectors can be modified further to increase the...cloning sites (Keen 1988).  Find broad host range recombinant vectors at Addgene!   References and resources...

...Addgene. Anti-V5 [IPI-SV5-Pk1] is a recombinant version of the original V5 tag mouse monoclonal antibody,...different antibody-based applications.  IPI’s recombinant version is a fusion of mouse variable and rabbit...with the multiple myeloma drug mezigdomide and ubiquitination substrate Ikaros (IKZF1ZF2) (B). Image adapted...structure-guided drug design and studies of protein ubiquitination and degradation.   Find the CRBNmidi plasmid...4 protein, mScarlet3 was 50% brighter than the original worm-optimized wrmScarlet. mScarlet-I3, while ...104 times higher than traditional homologous recombination methods). By using mCherry for selection of ...

..., Bjorck 1984). Protein A, Protein G, or the recombinant Protein A/G are conjugated to agarose or magnetic...their primary antibody. To circumvent this, a recombinant protein, Protein A/G, was developed, which can...primary antibody and serum immunoglobulins indiscriminately, causing competition for binding sites. In...antibody will not co-elute with the target protein, eliminating the risk of the primary antibody interfering ...antibodies conjugated within the pores are often inaccessible to their targets. Magnetic beads are solid spheres...need to be verified using alternative methods.  Finally, IP can also be used to study proteins that interact...on Addgene.org: Find plasmids encoding recombinant antibodies at Addgene  ...

...not. The lack of an exemption for postdocs in the final ruling was, for a lot of us, frankly quite surprising...which leads to many complications, including the inability of some institutions to know exactly how many ...of 10.4%. The Icahn School of Medicine at Mount Sinai in New York is raising the minimum from $43,692 ... funding climate combined with the lack of a sustainable research enterprise, there is no easy way for...cheap trainee workforce has clearly not been a sustainable approach to biomedical research in the U.S. and...we must make real changes to make the system sustainable. For years, there have been recommendations that... they are necessary to produce a healthy and sustainable research ecosystem and that we cannot ignore ...

... requires an N-terminal consensus sequence (MG-) and prenylation requires a C-terminal consensus sequence... filter sets on a fluorescence microscope, and finally (iv) good toleration in fusion proteins. Since ... introducing two mutations (F64L, S65T) in the original jellyfish GFP (AvGFP) to create enhanced GFP (... loop-structure (Querido and Chartrand, 2008). Finally, a nuclease-deficient Cas9 can bind loci in the...GFP is fused to these sequences. When N- and C-terminal fusions are not tolerated the alternative is to... subunit fusions that do not tolerate N- and C-terminal fusions (Adjobo-Hermans, 2011). Structural information...

...markers and homologous recombination As an alternative, endogenous homologous recombination enzymes can also...enzymes and those that use endogenous homologous recombination. Modifying bacterial genomes using exogenous...exchange requires two sequential homologous recombination events, but because these events are rare, each... ability to grow on sucrose, perhaps by sacB inactivation.  Bacterial toxins for counter-selection In ...mix. Introduction of the strong transcriptional terminator BBa_B1002 (from IGEM) upstream of the MCS to ...genetic engineering in bacteria using homologous recombination." Current protocols in molecular biology 106.1...