...Packaging genes (viral Gag-Pol): Gag is a structural precursor protein, and Pol is a polymerase. Envelope gene...γ-retroviruses use the same gene products for packaging (Gag, Pol, and Env), the isoforms of these proteins, as ...utilizes a packaging cell line that stably expresses Gag-Pol and/or Env, eliminating the need to deliver these...Garry Nolan at Stanford (Swift, 2001) contains Gag-Pol and either an ecotropic envelope, Phoenix-ECO, ...days. Another variant, Phoenix-gp, contains only Gag-Pol and enables additional flexibility in pseudotyping...lentiviruses and standard γ-retroviruses use the Gag, Pol, and Env genes for packaging; however, they are...Addgene blog Viral Vector Elements The Retroviral Life Cycle Popular Retroviral Vectors and Their Use in...

... production (3 or 4). Third-generation vectors contain gag, pol, and rev. Second-generation contains those...ingredients: the lentiviral packaging vector, a transfer vector including the gene of interest, and the...gamma-retroviruses use the same genes for packaging (that's gag, pol, and env), the isoforms of these proteins, as ...3rd-generation transfer vectors apart? A3: Addgene defines 2nd- or 3rd-generation transfer vectors based on whether...between 2nd- and 3rd-generation vectors? What does this mean for transfer vectors? A4: The lentiviral packaging... and a packaging plasmid encoding HIV proteins gag, pol, rev, and tat. 3rd generation systems require ...plasmid, and two packaging plasmids, one encoding gag and pol and a second encoding rev.  Secondly, 3rd generation...

...making virus’ or what gag and pol stand for, we demystify all components of viral vectors here! Need Virus...types, but most vectors are based on adenovirus 5 (Ad5).     Fig. 3 - Viral vector components of...of adenovirus Building viral vectors Addgene has many viral vectors and helper plasmids for all the...Check out Addgene's Viral Service! Types of viral vectors Of the many viruses out there, only a small subset...adeno-associated virus (AAV). The components of these vectors are also split across several plasmids (often three...called helper plasmids. Below, we’ll breakdown the vector components first by virus and then by function....particle to interact with the host. This is the role of Gag proteins: they contain the matrix, capsid, and nucleocapsid...

...the HIV gag, pol, rev, and tet genes. It can be used with 2nd or 3rd generation lentiviral vectors and an...  Find the scrambled shRNA vector. pLKO.1 - TRC cloning vector - This vector from the David Root lab is...plasmid from the Trono lab that contains the gag and pol genes. It also requires pRSV-Rev and the envelope...opening our UK office in 2014, to starting our viral vector service in 2016...we’re excited to help scientists...lentiCRISPR v2 - This 3rd generation lentiviral transfer vector also from the Zhang lab is used to deliver Cas9...your gRNA of interest into mammalian cells. This vector was the top requested plasmid of 2015. What makes... This plasmid from the Zhang lab is a mammalian vector that expresses both SpCas9 and your gRNAs and was...

...plasmid (envelope plasmid), and the polymerase (Pol) for replication and Gag structural protein on yet a third...cell line with those particles. Since the Env, Gag, and Pol genes are not encoded in the transfer plasmid... on the Addgene blog Viral Vectors 101: Types of Viruses Viral Vector Elements Tips for Preventing... contact with than GFP. Similarly, if the viral vector carries an shRNA to a gene essential for cellular...cellular function, this would also be a higher risk vector. While it is unlikely that you, or anyone else ... be disposed of following your lab’s biohazard policies. Administering Virus Many virus experiments involve... accident-prone researchers. Note that safety policies will vary slightly from organization to organization...

...important for viral production: Gag (structural precursor protein), Pol (polymerase), Tat (viral transactivator...Lentiviral vectors are one of the most popular and useful viral vectors in the lab. Advantages of lentivirus...dividing and non-dividing cells. Lentiviral vectors are the vector of choice for many CRISPR applications,...and future) applications of lentiviral vectors! Lentiviral vector history and components In the early ...viral vector systems based on retroviruses like Moloney murine leukemia virus (MMLV). The vectors could...adeno-associated viral vectors can also target non-dividing cells, lentiviral vectors can carry much more... plasmids: one encoding Rev and one encoding Gag and Pol, increasing the number of recombination events...

...care (4). For this reason, viral vectors often employ other non-poly(A) transcript-stabilizing and nuclear...terminator will suffice. Many commercial expression vectors use double terminators to reduce unwanted translation...Expression of Transgenes  Delivered by Retroviral Vectors. Journal of Virology. 1999. PubMed PMID: 10074136...constitutive RNA transport element lead to enhanced HIV-1 Gag expression in a context-dependent manner. Nucleic... both polyadenylation and termination. Out of those listed, the SV40 late polyA and rbGlob polyA are thought...any 3’ regulatory elements, such as the polyadenylation or poly(A) signal. While many studies focus on ...purpose and mechanism of polyadenylation vary across cell types, but polyadenylation generally serves to promote...

...three HIV-1 genes (gag, pol, and rev) for production. Third generation lentiviral vectors can be used for...your experiment using the lentiviral vector system. Viral vectors have been increasingly popular in fundamental...primary cells. Amongst the different vectors used, lentiviral vector constructs have proven very useful ...fluorescent sensors. Retroviral vector background Until the development of viral vectors, gene transfer into mammalian...un-transfectable cells, labs use viral vectors and particularly retroviral vectors because they target a broader...necessary for the production of a viral particle (Gag, Pol, Tat, Rev and Env) and the transfer plasmid encoding...attractive choice for a gene transfer vector. Lentiviral vectors Although retroviruses are still in use...

...blog Viral Vectors 101: Viral Vector Elements Viral Vectors 101: Types of Viruses Viral Vectors 101: Viral...would not be great for a gene therapy vector! For viral vectors, only the protein for reverse transcriptase...components of the vector particle, some of the RT protein gets packaged inside of the vector particle, along...and this is where viral vectors and natural viruses diverge. For the viral vector, its job as a tool is ...incorporated into HIV-1 particles independently of the Gag/Pol precursor protein. The EMBO Journal, 16(16), 5113...exactly, that heaps of viral vector particles are made while missing the polymerase gene may seem like a mystery...seem like an unlikely candidate to use as a viral vector, but retroviruses have been developed into some...

...replication. They contain two genes: gag and pol. The pol polyprotein encodes the reverse transcriptase ...in viral vectors, but easily fit in a TE. Transposons are also less likely than viral vectors to induce...transposon’s LTRs, similar to when packaging viral vectors.   Let’s take a closer look at three different ... in human cells at rates similar to retroviral vectors. The hyperactive version of the SB transposase,...efficiency in human cells similar to retroviral vectors. The hyperactive PB transposase (hyPB) is ~10-fold...transfer Transposons are an alternative to viral vector delivery of transgenes, like for iPSC reprogramming... piggyBac, which is a big advantage over viral vectors (~5 kB payload for AAV and ~8 kB payload for lentivirus...

...expression vector which gives close to 100% cloning efficiency and gene expression. The pUCXKT vector ensures...science.aaa3380 MuLE vectors for customized lentivirus production Finding a lentiviral vector backbone with ...custom-cloned vectors in a Multisite Gateway reaction with a desired destination vector expressing a separate...a final lentiviral vector containing up to 5 different elements. Lentiviral vectors constructed with the...MXS-chaining kit contains 5 empty chaining vectors plus a destination vector and a collection of building blocks...complex polycistronic lentiviruses. The MuLE toolbox contains a series of building-block vectors that are.... Additionally, by using an adapted destination vector with a heterologous promoter outside of the Multisite...

...of pExpression vectors) plasmids. The type IIS restriction sites used in these vectors are of low abundance...to produce the constructs you need. Expression vectors within this kit contain a variety of promoters ...insects, and even Xenopus oocytes. The expression vectors additionally contain various combinations of protein...Series Confronted with the problem of creating vectors for predictable protein expression in bacteria,...this so-called bicistronic design (BCD). The 49 vectors in this kit can be used to drive production of ...SITS to create the pCell-Free vectors, Gateway-compatible backbones that enable cell-free expression of ...kit represents a set of 88 clones in a pCellFree vector that enables protein expression in any in vitro...

...concentration gradient and hyperpolarize the cell (Mattis et al., 2012). Bicistronic vectors combine excitation...candidates for your experiments.  Wavelengths Most vectors have an optimal wavelength to use but can be activated...enter the cell with the concentration gradient to depolarize it (Nagel et al., 2003). Inhibitory opsins are...the receptor, when exposed to light, the ligand engages the binding pocket and activates the receptor. ...considering for your experiment, note that you cannot extrapolate results from one cell type to another, as they... A. K., Hegemann, P., & Wiegert, J. S. (2021). BiPOLES is an optogenetic tool developed for bidirectional...

...will describe how to copy a cDNA from one vector into a new vector that is better suited for analyzing the...use a high fidelity polymerase to minimize mutations. The fidelity of the polymerase becomes more important... million bp depending on the polymerase used. No matter which polymerase you use, it is important that.... This gives us a sequence of 5'-TGGCATATCTCGAAGTACTGAGCGGCCGCTAAGCA-3' (30bp with 18bp of homology to...chose for our reverse primer (5’-TGGCATATCTCGAAGTACTGAGCGGCCGCTAAGCA-3’) into this calculator we get a...final Reverse Primer sequence of 5’-TGCTTAGCGGCCGCTCAGTACTTCGAGATATGCCA-3’. Preparing the PCR product for...

...reprogramming discrepancies. The polycistronic piggyBac transposon vectors tested in this work are available...TagRFPt, EGFP, mVenus, mCerulean3, mKOFP2) and empty vectors that have standard cloning sites or gateway compatible...neuronal tools to the expanding gateway compatible vectors currently shared amongst the zebrafish community...for the research community. You can find the AAV vectors for infecting GABA-ergic interneurons here. Dimidschstein...Very low off-target activity; protein purification vector  87439  pCMV-HF-BE3  HF-Cas9 (NGG)  Very low ...low off-target activity; mammalian expression vector Rees H, et al. Nature Communications. 2017. PubMed...editing. The bacterial and mammalian expression vectors for these CRISPR inhibitors are listed in the table...

...restriction enzymes or vector purchases (Table 1). Restriction enzymes and vector purchases can raise the...for the simple insertion of a PCR product into a vector. The time it takes to make a plasmid could increase...plasmid? Figure 2: Survey results from a Twitter poll run in 2023, n=194.   After the great Plasmid...much harder to calculate, in both our 2018 and 2023 poll, most people said it took them more than 22 hours...these rough estimates, it’s clear that community engagement with and use of repositories like Addgene provides...

...domains of the selected scFvs are inserted into a vector encoding the antibody constant domain scaffold ...small size also allows for their delivery by viral vectors like AAV, which could be useful for delivering ...doi.org/10.1038/nbt.1791  Deal CE, Balazs AB (2015) Vectored antibody gene delivery for the prevention or treatment...recombinant antibody. They are ~25 kDa single polypeptides that contain the variable light chain (VL) and...When these molecules, called Bispecific T-cell engagers (BiTE®s), bind CD3 on T cells and a tumor-specific...

...continues to be challenging. Organism specific viral vector systems would also be beneficial for many systems...not just wait for papers to be published, but to engage in ongoing, real-time dissemination of information...No transformation techniques are available yet. Polistes (Amy Toth) These wasps are like the ones that ...Crepidula atrasolea juvenile, light microscope with polarizing filter. Development is direct in this species...actually silence this gene with RNAi to make a polygamous species monogamous. Anolis sagrei (Doug Menke...

...on the Addgene Blog Learn All about Mammalian Vectors Read up on CRISPR Delivery Systems for Mammalian...sharing cells: the black market of cell biology. Engaging in this practice would seem to save time and money...whether the cell lines had isoenzymes and genetic polymorphisms specific to their origins. This technique is...

...experiment, select from a variety of empty sgRNA vectors and design your sgRNA targeting sequence using ... Fig. 2: Prime editor engaging target DNA. The reverse transcriptase polymerizes DNA onto the target strand...