...post has provided some insight on these differences in viral production and will help you decide which is.... In this post, we will compare two of the most widely used systems, mammalian and insect cells. Whether...cells are also easy to cultivate, fast-growing, and widely available. The viruses Lentiviruses, AAV, and adenoviruses...lentivirus production). The packaging plasmids are widely available, so you’ll only have to worry about cloning..., they are easy to cultivate, fast-growing, and widely available. Unlike mammalian cells, they do not ... (about three to four days), this second round provides the added benefit of producing a stock P1 virus... your viral supernatant. Baculovirus replicates inside insect cells, and its lifecycle ends in cell lysis...

...important factor to consider is your desired library coverage, since this will help decide what transformation...and how will you ensure this?). You should also consider if there are any features of the library backbone...difficult to transform.    Figure 1: Factors to consider when creating a pooled library amplification protocol...amplification protocol is the Chong Zhang E. coli Genome-wide Inhibition Library. DNA and cell amounts After ... work! Selecting your media Lastly, you need to decide between growing your library in liquid media or...similar in size and composition as examples. Table 1 provides an overview of pooled library protocols you can...DOCX, 15 KB) CRISPR Activation Human Genome-wide CRISPRa-v2 Libraries 104,540–209,080 20 bp ...

...here to help with that! Four important factors to consider for any experiment are: Speed Cost Difficulty...For cloning in particular, another important consideration is: The number of inserts ...as some methods...versatile Type IIS restriction enzymes. These cut outside their restriction sites allowing you to easily ...little as 30 minutes! Alternatively, you might consider Gibson cloning. In this technique, you first perform...order determined by their homology. You might also consider FastCloning, first described by Chaokun Li and...Finally, TOPO cloning is another good option to consider when time is of the essence. This method requires...-generated insert. When mixed, topoisomerase I provides both the endonuclease and ligase function while...

.... Amp is a semisynthetic derivative of the more widely known antibiotic, penicillin, and is a beta-lactam...buys you a little more time, since bacteria won’t divide immediately after it has been heat-shocked or electrically... it by producing a lot of beta-lactamase. Pros: Widely available, cost-effective, timesaving for transformations...Expensive.   Kanamycin Kanamycin, or kan, is an aminoglycoside which inhibits protein synthesis. The kan resistance...The NPTII gene confers resistance to several aminoglycosides, including G418 which is commonly used for ...recovery period than amp or carb (60 minutes). Pros: Widely available, cost-effective, confers mammalian resistance... with all bacteria used in research, it isn’t a widely popular selection agent. Pros: Stable (spectinomycin...

... Addgene's Lentiviral Guide Most popular lentiviral plasmids γ-Retrovirus Guide References Cullen, B....retroviruses have been developed into some of the most widely used tools in molecular biology. “Retroviruses”...immune system directly). It may seem odder when you consider that out of the 6 x 106 virus-like particles that...DNA called a “provirus.” This DNA is synthesized inside of what is called the “target cell” (or host cell...reverse transcriptase are packaged (Wu et al., 1997) inside of the capsid . This is useful for viral reproduction... particle, some of the RT protein gets packaged inside of the vector particle, along with its RT-deficient...clock to the 6 o’clock positions at the left-hand side. These steps are common between natural infection...

... of regulation of genome functions, we need to identify molecules associated with specific genomic regions... genomic regions can be easily isolated for identification of associated proteins, RNAs, and other genomic... retroviral vectors expressing tagged dCas9 and guide RNA. The retroviral enChIP system enables us to ...system will allow researchers to use enChIP in a wide variety of biological systems. We are currently ...addition, we have recently developed the oligoribonucleotide (ORN) interference-PCR (ORNi-PCR) method ...shipping. Addgene realizes the One-Stop shopping for a wide range of plasmids. The merit of depositing plasmids...community is a kind of duty for researchers when considering the peer-review system of journals and grant ...

... While not ideal, transient expression of viral constructs is a useful tool as it provides a means to ...the specific target cell or tissue needs to be considered. Q2: Can I use lentiviral transfer vectors for... for the HIV Tat protein and consequently are considered safer. Second-generation transfer plasmids MUST...of lentiviral vectors in producer cell lines. Identifying less toxic envelope proteins or envelope proteins...to produce viral particles. The components are divided as follows: Transfer vectors contain minimal cis-acting...those three genes plus tat. The envelope plasmid provides a heterologous envelope for pseudotyping and is...to lower the risk. Biosafety should always be considered with respect to the specific experiments being...

... let’s say you identified gene target X for BRCA-deficient breast cancer and identified several candidate... of two approaches. The first route involves identifying a gene/pathway to develop a drug for (aka a druggable...to their target, or their direct target can be identified if it isn’t known. If a strong candidate emerges...put into them in those later stages. Target identification Not all diseases are fully characterized or...simple pathology. In these cases, preliminary identification of potential targets by CRISPR can accelerate... activate, or knockout many genes at a time to identify ‘hits’ - genes that affect disease progression...off target effects, both of which make it a non-ideal drug. If a drug selectively kills the wildtype line...

...need to be inside of a gene’s open reading frame, in fact, they can be activated a considerable distance... gene expression are all factors to consider. Once you’ve decided on the type of trap, there are many...world, approximately 50% of predicted genes have unidentified roles as well (Vanni, et al). Is it true that...genes are truly dispensable or are the current identification methods missing something? Here we will review...review some time-tested gene identification systems that may have a leg up on their modern counterparts...just yet. Gene and promoter traps unambiguously identify the gene that they are monitoring as they are ...distance away from a gene which can make identification of the gene itself difficult. The best trap depends ...

...we can confirm the identity of the gene and check for any mutations. Once we identify the gene, we can ...talked about how our new Snapgene generated maps provide improved feature detection with an easy to use ...SnapGene for plasmid quality control are: To identify important plasmid features To align NGS sequencing...experiments for further plasmid verification Identifying plasmid features While our quality control process...in Snapgene’s extensive database. Snapgene also provides easy visualization of open reading frames, which...similar features and it allows you to more quickly identify the same feature in other plasmids. Aligning ...Aligning sequences with Snapgene    Once we’ve identified the features within a plasmid, we next verify that...

...UnLOCKING Cas13 (C2c2) was first identified in 2016 by the Zhang lab as an RNA guided RNase (Abudayyeh et al 2016...-related single nucleotide polymorphisms (SNPs) from human saliva. SHERLOCKv2 provides a method to detect... expensive, time-consuming, and complex to use outside of standard laboratories. In the case of the COVID... 2016). Cas13 can be guided by a single CRISPR RNA (crRNA) to cleave ssRNA or mRNA. It also exhibits a...based on DETECTR technology with the mission to “provide a CRISPR-based platform on which an infinite number...2016) C2c2 is a single-component programmable RNA-guided RNA-targeting CRISPR effector. Science 353:aaf5573... Koonin EV, Zhang F (2015) Cpf1 Is a Single RNA-Guided Endonuclease of a Class 2 CRISPR-Cas System. Cell...

...searched for this unique sequence of DNA and both identified lymphocyte antigen 6 complex (LY6A) as a receptor...library of AAV9 mutants that had a small randomized peptide loop inserted in the AAV9 capsid. This mutant library...then screened in vivo in C57BL/6J mice strains to identify variants that reached the CNS following IV injection...hematopoietic stem cells. Its ligand(s) has not been identified. While LY6A or other Ly6 gene family members ...susceptibility. The Deverman and Wilson Labs independently identified LY6A as a receptor for PHP.B by looking for a...total, they tested 13 strains, with seven strains identified as permissive and six as non-permissive to PHP.B...the PHP.B permissive phenotype of mouse strains beside C57BL/6J and BALB/cJ, and the effects of overexpressed...

...range, identify a few of the features that confer broad host range in plasmids in nature, and provide tips... meliloti, Rhodobacter sphaeroides, R. capsulatus, Rhodopseudomonas spheroides, Salmonella spp., Serratia...equally. Only a handful of plasmids have been identified as broad host range plasmids. The properties ...are not fully understood, but scientists have identified some of the features that confer broad host range...metabolic load (Meyer 2009). Figure 1: Idealized examples of a narrow host range plasmid with only...Porphyromonas gingivalis, Proteus mirabilis, Providencia spp., Pseudomonas spp., Rhizobium leguminosarum... M. xanthus, Proteus rettgeri, P. mirabilis, Providencia stuartii, Pseudomonas spp., Rhizobium leguminosarum...

...accurately communicating ideas in a content-rich, easy-to-understand set of PowerPoint slides. What are some of...learned as a healthcare consultant and give you an idea of what a career in this field involves, and why...a defined path forward. Communication is key as ideas must be relayed quickly both internally and externally..., where case managers are encouraged to use the wider firm’s resources when their teams are facing a crunch...approach brings to patients, doctors, and insurance providers, and is crucial to how our clients’ products will...you’ll have the opportunity to learn the business side of consulting cases, while feeling like you are ...to get your foot in the door When I was first considering preparing for and transitioning to a career in...

...Any ∼20 nucleotides of DNA can serve as the target for Cas9’s single guide RNA (sgRNA), provided it meets...unique experiment. Here, we will introduce you to the wide array of Cas9s (many available through Addgene’s...Specificity concerns? If off-target effects are a worry, consider a dual-sgRNA system. This system uses a “nicking...little homology to other regions of the genome and consider using a Cas9 variant with higher specificity (...residue of a protein or knocking out a very small peptide. In these cases, you have little flexibility as...specific? Perhaps you need to introduce a single nucleotide modification at a defined amino acid to mimic...requirements, giving it more flexibility. A consideration with this tool is that the desired edit will...

... understanding of CRISPR, and perhaps provide insights and ideas for your own research! Classification... abundant of the two, comprising about 90% of identified CRISPR-Cas in bacteria and nearly 100% in archaea...transposases, by leaving out Cas3. Type I is further subdivided into subtypes A–G. These subtypes have similar...Schematic of a Cascade complex (blue subunits) with a guide RNA (green and orange) bound to the target DNA with...with BioRender.com.   Type III Type III is considered the most complex of the CRISPR types and is thought...et al., 2021). IV-A and IV-B lack what you may consider to be an integral component of CRISPR — a nuclease...engineered Cas enzyme, with adaptations for increased fidelity, activation/repression, high precision edits, ...

...need for target-specific antibodies and have been widely used across species and applications. In this blog... What are epitope tags? Epitope tags are short peptides introduced at the N or C terminus of a protein...purification or detection of the tagged protein. These peptides consist of as little as 8 or as many as several... (rabbit). Myc The Myc tag is a 10 amino acid peptide (EQKLISEEDL) derived from the protooncogene of ...immunofluorescence . However, protein purification is not ideal with Myc, as low pH conditions are required during...these species. HA The HA tag is a 9 amino acid peptide (YPYDVPDYA) derived from human influenza hemagglutinin... chicken) to this tag! GFP  GFP is more than a peptide – it’s a 238 amino acid protein that can also be...

...chance. Luckily, around the time I decided to look for work outside of academia, a job as a ‘Cancer Marketing...next and what options lie ahead of me? Like a considerable number of researchers, I had high hopes of securing...in the business of western blots and cloning, I decided to pack it in. I had just started month 8 of my...my colleagues and what I was researching, but I decided I needed a new challenge. Looking for sales and...yourself, sales and marketing positions like these are widely available, especially for life science researchers...purchased their products, so you have some of the best ideas on how to sell these products to other scientists...marketing knowledge. For most PhDs looking at careers outside of academia, sales and marketing can be great places...

...self-reflection necessary to identify areas that need work or growth. Here are some ideas for making your meetings...do you make the most of this new relationship? Consider adding formality and active goal setting to your...learned, especially noting action items that are identified. Keep a journal to record topics, plans, findings...you can find it in our Mentoring for Scientists Guide.  You can use it to clarify and discuss values. ...Attainable, Relevant, and Time-bound. It is a good idea to take larger goals (e.g. Figure out what I want...items and make progress. To illustrate this point, consider that I try to exercise every morning, but the ...postdoc can mentor a graduate student – you get the idea. There are many advantages to being a Mentor: Learn...

...promoter and the gRNA scaffold are provided with the kit. Oligonucleotides specifying the gRNA seed sequence...promoters. To use this plasmid, you simply order oligonucleotides for your chosen gRNA target sequences and ...uses Type IIS restriction enzymes, which cleave outside of their recognition sequence, creating flanking...Cas9 Golden Gate multiplexing is to clone the oligonucleotides specifying each gRNA target sequence into ...rectangles) are cloned into various plasmids using oligonucleotides. These plasmids contain Type IIS restriction...allows you to express 2-4 gRNAs, with four being the ideal number. First you generate four unique kanamycin-resistant...you to express two gRNAs constructed using oligonucleotide assembly.  Unlike some of the plasmids described...