... and the complexity of effectively deploying them in the lab somewhat mirrors the complexity of understanding.... Addgene’s Viral Vector Service Besides being complex to use, viral vectors are also difficult both to..., and jGCaMP8 viral preps distributed per year compared to other AAVs preps from 2016–2023. B) Inset showing...

...virus as self-complementary AAV (scAAV). However, scAAV vectors have limited size compared to single-strand...-multi (Walton et al. 2024). This platform is compatible with enrichment, single-cell sequencing, and ...virus’s most abundant proteins and a critical component for viral genome packaging. Addgene recommends...

...Host range depends on whether the host has the components to recognize the ori and allow for replication...organisms. pBBR1-based plasmids and plasmids in incompatibility groups N, P, Q, and W are known to have replicons...list of the bacterial species these plasmids are compatible with.  Table 1: Non-exhaustive list of common... and Srivastava 2013. Family Replicon Compatible with Shown to work in IncN pCU1 Gram-negative...

...meets two conditions:  1) The sequence is unique compared to the rest of the genome.  2) The target is present...found. If you’re lucky enough to find a SpCas9 compatible PAM at your site of interest, that is the most...expanding number of Cas9s with looser PAM requirements compared to SpCas9. xCas9 has a PAM requirement of NG, ... proteins. The Synergistic Activation Mediator complex, generally the strongest activator, consists of...

...interior target sites. In a second step, the two compatible target sites are cleaved out, leaving behind ...a vector with the inverted DNA flanked by non-compatible target sites (Schnütgen et al. 2003). Flex vectors...within neuronal networks in living animals. To accomplish such cell specific manipulations in vivo, FLEx...knockout. In this experiment it is possible to compare neuronal morphology and synaptic connectivity before...

...can be difficult to construct and deliver when compared to gRNAs. Additionaly, cDNAs cannot be used to...Housden BE, Perrimon N, Collins JJ, Church G (2016) Comparison of Cas9 activators in multiple species. Nat Methods...transcriptional activation by an engineered CRISPR-Cas9 complex. Nature 517:583–588 . https://doi.org/10.1038/nature14136...Kadoch C (2018) Interrogation of Mammalian Protein Complex Structure, Function, and Membership Using Genome-Scale...

...endogenous homologous recombination enzymes can also accomplish the desired genomic modification. This technique... which results in a merodiploid. This can be accomplished by selecting for an antibiotic resistance marker...scarless modification of bacterial genomes. It can accomplish large deletions, insertions, and even precise...PMC4513841. Latynski US, Valentovich LN. "DNA tuner: a computer program for the construction of polylinker sequences...

... proteins in living cells and organisms. Both components of the chimera are encoded by DNA. Since researchers...When both orientations are available, a direct comparison of these fusion proteins may reveal which of ...the fusion protein can be verified and it can be compared to what is expected based on immunofluorescence...interactions) can be challenging. If a fusion can complement a knock-down or knock-out of the corresponding...

...to the popularity of GFP including (i) fast and complete maturation to functional, fluorescent protein ...diameter of about 2.4 nm (Hink et al., 2000). The complete beta-barrel is necessary for its fluorescence ...genetically encoded tag and exogenously added components. For a review see Lotze et al. (2016). To date...is used to excite GFP, may also excite several components that are naturally present in cells or media....

... (Sandomenico et al., 2020). Figure 1: Comparison between the IgG antibody and scFv.   What...their lack of an Fc region. Disadvantages scFVs:  Compared to antibodies, scFvs tend to have lower affinities...intensity of a tagged protein. SunTag has two components: 1) a protein of interest that’s tagged with ....0000000000000145  Gąciarz A, Ruddock LW (2017) Complementarity determining regions and frameworks contribute...

...the production of lentiviral vectors that are competent for one round of infection (AKA transduction in...progeny are produced (Figure 1).  Figure 1: Comparison of the life cycle of a lentiviral vector with...double-stranded DNA transgene is incorporated into a large complex of both viral and cellular proteins and trafficked...titering IDLVs include detecting vector particle components or performing quantitative PCR titration of vector...

...method for knockouts is Tracking of Indels by Decomposition (TIDE) (Brinkman et al., NAR). This technique...trace files of WT and edited cell lines. Then decomposition analysis is performed yielding a graph of edits...This method requires a set of control cells to compare the sequencing reads from your edited sample to...assessment of genome editing by sequence trace decomposition. (2014) Nucleic Acids Research Volume 42, Issue...

...lacY1 proA2 rpsL20 xyl-5 leu mtl-1 Stbl4   Electrocompetent cells for cloning and storage of unstable ...XL10 Gold Tetracycline and Chloramphenicol High competency cloning and propagation of large plasmids, ligated...tables are by no means exhaustive! For a more comprehensive list and additional information, visit OpenWetWare's...of Bacterial Expression Systems. Check out our companion post describing protein expression strains, where...

...helps all viewers, especially if you have some complex patterns with several overlapping targets to interpret...you add the secondary antibody, it should get completely washed away and leave no signal. If you see lots...samples where you label only one target in each. Compare these individually-labeled samples with the results...deep phenotyping and spatial analysis of cells in complex tissues. Proceedings of the National Academy of...

...certain Cas enzymes are less compatible with the bulkiness that accompanies mispairing of bases. To reduce...unnecessary for many applications, it is the only comprehensive method available. Targeted sequencing approaches...must be quantified slightly differently. For a comprehensive approach, RNA-seq would be an appropriate method...

...could avoid potential off target artifacts by comparing the phenotypes of multiple gRNAs. Targeting different...lanes 0.3 % 3.0, Figure 5, lane 2) is faint, so comparing it to the undigested control (Figure 4, lane 0...instead of agarose gives much sharper bands. I compared two MOIs of gRNA transduction to see if that had...testament to how far genome engineering has come. For comparison, in 2007 it took about 3 months to knockout a...

...your data by Nov. 12 to compete. The top ten participants will be invited to compete in our speed round data.... Bobae An will host a Q&A session after the competition, where you can get all your burning AAV questions...

...In complex metazoans, rapid cell division and large scale cell mobility are essential processes during... required for a growing organism to make the complicated transition from a clump of cells to a fully differentiated...organism. Studying cell mobility within these complex environments has never been easy and, for many ...labelling of cells using cre-lox is advantageous when compared to photoactivable or photoconvertible proteins...

...straightforward to learn and relatively cheap (compared to many other technologies and techniques in the...experiment work? What are the various tools and components necessary to run it? And, most importantly, what...writing. Derek is also a pianist and an aspiring composer. View his blog drsimonsaysscience.org or follow...applying optogenetic tools derived from direct comparative analysis of microbial opsins. Nature methods....

...fragments, allowing ordered assembly of multiple components into a destination vector. The first step in ...Oligonucleotides specifying the gRNA seed sequence should be compatible with type IIS enzyme BfuAI. Gateway cloning is...supply it with another plasmid.   Figure 3: Comparison of Multiplex Strategies including Standard PolIII-gRNA...Cas9-containing binary vectors. The system is compatible with both monocot and dicot plants. Liu Lab Golden...