...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 well... 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, (for.... 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 different...Matrix, Capsid, and Nucleocapsid components. Pol Precursor protein containing Reverse Transcriptase...

...makes polyclonal antibodies unique and what experiments you should choose them for. How polyclonal antibodies...cross-reactivity. When to use polyclonal antibodies One advantage of polyclonal antibodies is that they are...barrier to entry makes polyclonal antibodies a common reagent in many labs. Polyclonal antibodies also have...When to stay away from polyclonal antibodies The most prevalent issue with polyclonal antibodies is their...from the same species your polyclonal antibody was generated in or a polyclonal isotype control, to account...options to choose from. In this article we’ll cover polyclonal antibodies, one of the many different types of...antibodies are produced Polyclonal antibodies are a heterogeneous mixture of many antibodies that recognize...

... 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...consensus sequence for the addition of a poly(A) tail, the addition of a poly(A) tail on a prokaryotic transcript... termination and polyadenylation Unlike prokaryotes that have a single RNA polymerase for transcription...transcription, eukaryotes have three RNA polymerases (Polymerases I, II, and III), each responsible for transcribing...different types of RNA: Polymerase I is responsible for ribosomal RNA, Polymerase II is responsible for ...

...resistant DNA polymerase from the thermophilic bacterium, Thermos aquaticus (Taq). Taq polymerase is thus a...slightly to Taq polymerase’s ideal temperature (70-75oC). At this temperature Taq polymerase can synthesize...mutagenesis.  Quantitative polymerase chain reaction, qPCR Quantitative polymerase chain reaction (qPCR) also...What is polymerase chain reaction? If you have ever worked in a molecular biology laboratory you have...have likely done a polymerase chain reaction (PCR). PCR is an in vitro method in which a small amount of...Yellowstone National Park.  In addition to Taq DNA polymerase, PCR requires free nucleotides (dNTPs), template... Primers are crucial for this process as DNA polymerases require an existing strand of DNA to add nucleotides...

... science policy, there is often discussion of “big P” and “little p” policy.  “Big P” policy is what you...about where science policy happens.  When do you do science policy? Many science policy training programs... is science policy?”  I have been asked this many times throughout my short science policy career and ...importantly, how, of a science policy career.  Who does science policy? I’ve intentionally started with... many science policy professionals that entered the field from a more traditional policy background, learning...for science policy folks is on Twitter- start an account and start engaging with the #scipol hashtag. Often...to start. What is science policy? THE IMPOSSIBLE QUESTION. Science policy is the work you do to creatively...

...500 bp cassettes containing the RNA pol III promoter, gRNA and pol III terminator. These relatively large...packaged together in a single vector. In addition, the pol III promoter is relatively weak, and low expression...contain internal transcriptional elements that enhance pol III transcription, potentially increasing the levels...different biological systems. Size is also a factor; the pol lll promoter and terminator need be encoded only ... tRNA-gRNA takes up half the space of a standard pol III-gRNA, which will facilitate CRISPR/Cas9 multiplexing...separated by 28 base Csy4 recognition sites from a polycistronic transcript. Although this method shortens construct...requires coexpression of Csy4 with Cas9 and the polycistronic transcript. As nucleases may have off-target...

...gamma-retroviruses use the same genes for packaging (that's gag, pol, and env), the isoforms of these proteins, as well...and a packaging plasmid encoding HIV proteins gag, pol, rev, and tat. 3rd generation systems require an ...and two packaging plasmids, one encoding gag and pol and a second encoding rev.  Secondly, 3rd generation... (3 or 4). Third-generation vectors contain gag, pol, and rev. Second-generation contains those three ...

...placing the gRNA downstream of a plant-specific RNA pol III promoter, such as AtU6, TaU6, OsU6 or OsU3, which...>30 “empty gRNA” backbones which contain a plant pol III promoter and gRNA scaffold sequence and allow...expression is commonly driven by plant-derived RNA pol II promoters which regulate expression of longer ... gene expression). Examples of commonly used RNA pol II promoters for Cas9 expression include the ubiquitously...expressed transiently using a standard detergent, Polyethylene Glycol (PEG), although the application of this...

...This post was contributed by Maaike Pols, PhD, a Developmental Editor at F1000Research.  With many changes... below. Thank You to Our Guest Blogger! Maaike Pols is a Developmental Editor for F1000Research, responsible...

...wondered what goes into ‘making virus’ or what gag and pol stand for, we demystify all components of viral vectors...needed for viral packaging, assembly, and display. Pol, the last component, contains the reverse transcriptase...The 5’ LTR and 3’ LTR, respectively, act as RNA polymerase II promoter and terminator regions and are slightly...replication, with E2B being the primary viral polymerase. E4/E1A activate transcription of other relevant...

...the Didier Trono lab that expresses the HIV gag, pol, rev, and tet genes. It can be used with 2nd or 3rd...plasmid from the Trono lab that contains the gag and pol genes. It also requires pRSV-Rev and the envelope...

...toolbox of processing factors, including polymerases (Pol μ and Pol λ), nucleases (Artemis), and structure-specific...

...separate plasmid (envelope plasmid), and the polymerase (Pol) for replication and Gag structural protein...line with those particles. Since the Env, Gag, and Pol genes are not encoded in the transfer plasmid, the... 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...with your specific organization/department/lab’s policies – basically, you’ll need to get trained in each...

...class of components, such as your CRISPR enzyme or Pol 2 promoter. The options available in each class, ...an overhang specific to the type of module it is (Pol 2 protomer, Cas protein, ect...). Whatever individual...

...Image from Mangeot et al., 2019.   MLV GAG-POL polyprotein Nanoblades are formed when viral structural... protease, supplied as part of the MLV Gag-Pol polyprotein, cleaves the proteolytic site connecting the...incubating Nanoblade particles with polybrene, a cationic polymer. Polybrene has long been used to increase...

...production: Gag (structural precursor protein), Pol (polymerase), Tat (viral transactivator for transcriptional...plasmids: one encoding Rev and one encoding Gag and Pol, increasing the number of recombination events necessary... L, Bernaudin F, Girot R, Dorazio R, Mulder GJ, Polack A, Bank A, Soulier J, Larghero J, Kabbara N, Dalle...

...plasmid contains Gag, Pol, Rev, and Tat genes. Specifically for our knowledge here, the Pol gene encodes three...

...necessary for the production of a viral particle (Gag, Pol, Tat, Rev and Env) and the transfer plasmid encoding...this vector requires only three HIV-1 genes (gag, pol, and rev) for production. Third generation lentiviral... you will find will recommend using a polycation, like polybrene (protocol) or DEAE dextran (protocol)...

...replication. They contain two genes: gag and pol. The pol polyprotein encodes the reverse transcriptase and ...reading frames (ORFs) which often terminate with a poly(A). ORF2 encodes endonuclease and reverse transcriptase...

...When Publishing a Scientific Manuscript by Maaike Pols, PhD, a Developmental Editor at F1000Research ...

...incorporated into HIV-1 particles independently of the Gag/Pol precursor protein. The EMBO Journal, 16(16), 5113...viral vector particles are made while missing the polymerase gene may seem like a mystery. But it’s simple... M., & Bishop, J. M. (1973). RNA-directed DNA polymerase of Rous sarcoma virus: Initiation of synthesis...

...scaffold) in a pol-III (OsU3) promoter driven expression system. We use a single polycistronic tRNA-gRNA (...inosine), which is treated as ‘G’ by cellular polymerases. Subsequently, an original genomic A-T base pair...

...civilization in general (1). Science and politics? Science is an apolitical process for seeking knowledge. The...inclusion of science to inform policy (17). The facts surrounding a matter or policy should have more weight ...science rallies scientists to political action, and inspires politicians and citizens to include science... again. No part of this process is inherently political so why is there a march in Washington, D.C., the... the seat of the United States government? Politics and science are intrinsically linked: governments ...when we drop them. Scientific realities limit what policy can do and, in this way, science has always been...been related to government and, consequently, politics. More concretely, scientists gather information ...

... own RNA-dependent RNA polymerase into the infected cells. The EBOV polymerase replicates the viral genome...under control of a T7 RNA polymerase promoter sequence. Thus, T7 RNA polymerase also needs to be present...gene start) and a transcription termination/polyadenylation signal sequence (gene end). A minigenome is...replication and reporter gene transcription by the EBOV polymerase (reviewed in 2, 5). The minigenome is capable...nucleoprotein, NP. NP associates with the viral polymerase, which is formed by a complex of two proteins...sense, single-stranded RNA minigenome. The viral polymerase complex then recognizes the encapsidated minigenome... order to replicate the minigenome, the viral polymerase initiates RNA synthesis at the 3’ end of the ...

...conditions. Expression of an error prone polymerase I (PolI3M) is capable of mutagenesis in E. Coli, but... error prone polymerase to introduce mutations. However, by using nCas9 to direct PolI3m to a particular...Figure 1: EvolvR consists of an error-prone polymerase (PolI) that is fused to a nicking Cas9. A guide ...EvolvR: (1) two additional mutations to PolI3M generated PolI5M which increased EvolvR’s mutagenesis rate... from bacteriophage T7 DNA polymerase increased the processivity of PolI3M, and thereby increasing the... system has two key components: the error prone PolI3M that’s fused to a nicking Cas9 (nCas9). Similar... site of interest. A gRNA is used to direct the PolI3M-nCas9 complex to a DNA site of interest, which ...

...approach policy makers take in abusing climate science skepticism to promote harmful policies that benefit...move slowly, news about possible broad changes to policies seems to break everyday. Many researchers and ...participation in government, evidence to inform policies, a protected place to work, funding for science...medicines, technologies, defense, and governing policies are developed primarily by wishful thinking. The...rains, it was weather befitting of the ominous political climate in which American scientists find themselves...scientists found themselves not too long ago.   From policies that “muzzle” government scientists to the de-...Canada.  Outside the lab, she dabbles in science policy, public outreach, and restorative justice.  Follow...

...Sharing Policy Discussion PLOS’ New Data Policy: Public Access to Data PLOS’ New Data Policy: Part Two...advice about the scope of the policy. End of story? Not quite. The PLOS policy on data access is a formal...clarified what the policy intent was and provided an FAQ list to further explain the policy. The fact that...year when it announced its data access and sharing policy. Since early March, the open access publisher has...the research reported in PLOS publications. The policy was not an overnight revelation, rather it was ...reasonable. PLOS has since clarified their data sharing policy and recently announced that of the 16,000 manuscripts...agencies (e.g. Wellcome Trust, NIH Data Sharing Policies, etc.) and is founded on the principle that scientific...

...purification based on the polyhistidine tag. What is the polyhistidine tag? The polyhistidine tag is a string of...      Fig. 1: A sample containing a polyhistidine tagged protein of interest (purple) and several...to the Ni2+ and is immobilized along with a polyhistidine containing contaminating protein (orange). A...is successfully isolated.   Prior to the polyhistidine tag, alternative fusion tags were used, such...interfering with protein function. In contrast, the polyhistidine tag is very small and rarely affects protein...function (Bornhorst 2000).  One downside of polyhistidine tag-based purification, however, is the propensity... containing Co2+, have a lower affinity for polyhistidine than more commonly used substrates such as Ni2...

...you need a high-fidelity polymerase. There are many high fidelty polymerases on the market; you need one...a DNA polymerase that produces blunt-ended PCR products (e.g. Phusion, Pfu, and Vent polymerases). Note... PCR followed by Restriction Fragment Length Polymorphism (RFLP) analysis (right). Introduction of restriction...amplified fairly easily by simply following the polymerase manufacturers’ PCR protocol. Be sure to adjust...plasmid template for site directed mutagenesis.  Polymerase To ensure that you don’t introduce undesired ... one with 5’->3’ polymerase activity (for amplification), 3’->5’ exonuclease activity (increases amplification...Note that certain polymerases such as Taq generate A-overhangs (a feature used in TA-cloning). Such non-complementary...

...      The RNA polymerase(s) RNA is transcribed from DNA using an RNA polymerase (RNAP). In bacteria...bacteriophage Constitutive, but requires SP6 RNA polymerase. SP6 polymerase has a high processivity. When used for...describes the combination of the promoter (RNA polymerase binding site) and operators (response elements...promoter region controls the binding of the RNA polymerase and transcription factors, therefore promoters...single enzyme; however, eukaryotes have muliple polymerases which are each responsible for a specific subset... used in expression systems for Drosophila. Polyhedrin General expression mRNA  Strong insect promoter... H1 small RNA expression shRNA From the human polymerase III RNA promoter Constitutive  May have slightly...