... What kind of control? On, off, tunable, and/or inducible? How long do you need control? Will temporary...temporary control (a few days) be enough, or do you want a permanent control mechanism? Where is the control...transcription instead of introducing a DNA double strand break. Transient introduction of CRISPRi or inducible...vector! On/off control all the time If your experimental system requires tunable control - or maybe you...price of tunable control These systems can be easily implemented for exogenously introduced proteins, as ...prior to introduction of the construct into cells. However, if you would like tunable control of an endogenously...forever? You don’t need to resort to the force to control protein expression in mammalian cells: easy to ...

...viruses: gamma-retrovirus, lentivirus, adenovirus, and adeno-associated virus. Gamma-retrovirus Retroviruses...type of gamma-retrovirus is Murine Leukemia virus (MLV). Naturally isolated gamma-retroviruses have been ...Lentivirus is a type of retrovirus and shares many common features with gamma-retrovirus including genome architecture... nucleus as gamma-retroviruses do. Fun fact: Since lentivirus and gamma-retrovirus are so closely related...expression (Robinson, 1982). To add insult to injury, retroviruses generally have moderate to high immunogenicity...immune response in the host. So why use a gamma-retrovirus in the lab? Well, they are essentially evolutionarily... types with a high efficiency. However, gamma-retrovirus infection is restricted to dividing cells only...

...A common means to achieve ligand control is to fuse a mutant estrogen receptor (ER) ligand binding domain...PSD95-mcherry) under the control of Cre recombinase. After in-utero electroporation, this plasmid mix results...tracking for instance) can be controlled with FLP-FRT. For further temporal control, Tamoxifen inducible Cre...system, but it only gives you a single level of control without modification - either Cre is there or it...for site specific (and often cell type specific) control of DNA recombination and gene expression can be...addition, a variety of means to spatiotemporally control FLP and Cre expression have been developed. Read...FLP and Cre enable additional levels of genetic control. What is FLP FRT and how does it work? FLP recombinase...

...Include proper controls To ensure accurate data analysis, include a complete set of controls. Ideally, every...will have a negative control that does not express the target and a positive control that does. The negative...of experiments easier and faster to perform. Introduction to flow cytometry Flow cytometry allows users... (3): An optics system A fluidics system An electronics system  Cells are first resuspended in a pressurized...fluorophore or fluorochrome-stained protein will emit a stronger photon than a cell expressing low levels of the... further the shift of a cell from the negative control, the higher the level of expression. A routine ...triton-x, saponin, or tween. Take care when using strong detergents like triton-x and tween as extended ...

...larger amount of vector using a serotype with strong CNS tropism (4). If axonal transport is not desirable...receptor physiology and the effects of metabotropic and ionotropic influences on overall cell function. ... thanks to our guest blogger Katrina Armstrong. Katrina Armstrong is a Neurophysiology Msc Student at ...post was contributed by guest blogger Katrina Armstrong, a Neurophysiology Msc Student at the University...known is that different serotypes have different tropisms but, as you can see, AAVs of each serotype can... asking questions!  Table 1: AAV Serotypes and Tropism Experiment  Detection Method CNS AAV1, AAV2...>AAV8>AAV6>AAV5>AAV1>AAV2 This preferential tropism for different brain regions is largely due the ...

...Moloney murine leukemia virus (MMLV)-derived retroviruses. Retroviral vectors, such as pMXs or pMSCV, infect...delivery methods. Retrovirus has a cloning capacity of ~8 kb, so either a single polycistronic vector or multiple...10 kb) than other retroviral vectors. This makes it possible to use a polycistronic cassette that expresses...Mechanism Efficiency Safety Pros Cons MMLV-derived Retrovirus Integrating High Low Efficient and Stable Genome...Need to sequence cells to verify excision didn’t introduce mutations Adenovirus Non-integrating Low Medium...required for translational use. Viral Vectors Retroviral: Some of the first iPSCs were generated with ... corresponding endogenous pluripotency genes. Retroviral vectors are prone to incomplete silencing of ...

...DNA.  Plasmids are said to be under stringent control when they depend on the host cell’s initiation ...replication independently are said to be under relaxed control, as they only require the host's replication machinery...replication mechanism of plasmids under stringent control, such as pSC101. DNA replication and regulation...round of replication. Initiation is the primary control point of DNA replication in bacteria, so the process... the bacterial chromosome and for stringently controlled plasmids. Binding of IHF (green) redistributes...binding is necessary to initiate replication, controlling the amount of active DnaA available is one of...source, ATP binding is one more way for the cell to control DNA replication. A variety of other proteins also...

...target genes. The sequence of the promoter region controls the binding of the RNA polymerase and transcription...promoters is commonly employed in lentiviral and retroviral constructs and we will discuss these in a future... all the time while others are more carefully controlled. Regulated promoters might act only in certain... light. In the cell, promoters themselves are controlled by still other regulatory factors: enhancers,...Additional considerations CMV General expression mRNA Strong mammalian expression promoter from the human cytomegalovirus...some cell types. EF1a General expression mRNA Strong mammalian expression from human elongation factor...promoter hybrids. CAG General expression mRNA Strong hybrid mammalian promoter Constitutive  Contains...

... under the control of the cI857 temperature-sensitive repressor. The temperature-controlled expression...activity in the Co-IP compared of a control Co-IP indicates stronger interaction. The system has been demonstrated...DECEMBER 2016 Restoring yeast prototrophy with plasmids Though auxotrophic markers are used frequently ...complement strains with multiple auxotrophies or using different auxotrophic backgrounds can lead to disparate...their Yeast Prototrophy Kit, a set of plasmids designed to complement unused auxotrophies in Saccharomyces... at a dose and duration sufficient to kill all control cells and surviving “mutant” colonies were deep...GTPases involved in cell signaling pathways that control many cellular processes such as differentiation...

.../gamma-retrovirus Lentiviruses are a class of retrovirus (so all lentiviruses are retroviruses, but not...not all retroviruses are lentiviruses.) This means that lentivirus and gamma-retrovirus share a lot of...as tools in the lab. We will discuss the gamma-retrovirus, lentivirus, adenovirus, and adeno-associated...somatitis virus G glycoprotein (VSVG). VSVG is a broad tropism envelope protein that can also be pseudotyped to...the common components outlined above for gamma-retroviruses. While not required for lentiviral production...   Fig. 1 - Viral vector components of retrovirus  Adeno-associated virus AAV’s elements are...are quite different from gamma-retrovirus, but they perform very similar functions to infiltrate the host...

...Viral Vectors 101: An Introduction to AAV Viral Vectors 101: The AAV Retrograde Serotype Viral Vectors... more than one.  Most AAV serotypes have broad tropism (the ability of a virus to enter a particular cell... cell type), but there are differences in tropism between animal species or even strains of the same species...well in a rat or non-human primate. Likewise, in vitro efficacy doesn’t always translate to in vivo efficacy...remember that some serotypes can be transported retrogradely and/or anterogradely along the axon. Others,...site or circuit can occur. Promoters The promoter controls how the gene of interest will be expressed; it...or inducible.  Ubiquitous promoters  These often-strong promoters offer the most flexibility for expression...

... PCR-introduced mutations can make alternative approaches more favorable if you want to introduce a point...highly versatile technique that can be used to introduce specific nucleotide substitutions (or deletions...approach can be used in conventional cloning (to introduce or remove restriction sites), in mapping of regulatory...substitution of key residues), and in SNP analysis (to introduce naturally occuring SNPs in a plasmid context)....genome editing. Tailored mutations are commonly introduced to endogeneous DNA through homology-directed ... Mutagenesis In brief, point-mutations can be introduced to plasmids using primers (with the desired mutation...green) amplify the plasmid template (blue), and introduce a restriction site “A*” (in addition to the “A...

...plasmids: Luc (intronless; CMV-LUC2CP/ARE and Luc-I (intron-containing; CMV-LUC2CP/intron/ARE). The Luc-I...all-optical electrophysiology A major challenge in the field of all-optical electrophysiology is designing...field with an improved tool for electrophysiology, minus the use of electrodes. The Cohen lab has made the...Luc-I intron is a chimeric β-globin/immunoglobulin intron that has been commonly used in constitutive splicing...an effort to obtain even more precise temporal control of gene knockout or activation, the c-terminal ...). This system provides users greater temporal control over CRISPR/Cas9 mediated genome modification and...Optogenetics is a powerful tool that utilizes light to control and monitor individual living cells in order to...

...muscular atrophy.   Type of Virus Pros Cons Retrovirus Stable Gene Expression Broad Tropism ...the best one for your experiment. Retro and lenti viruses Retroviruses are a family of viruses that stably...The most well-known retrovirus is HIV. Lentiviruses are a genus of the retroviral family that can infect...which means they do not stimulate a strong immune response when introduced into an organism. This feature ...straightforward across applications, while electroporation of a human, for example, isn’t a reasonable...types of viruses used in mammalian research: 𝛄-retrovirus, lentivirus, adenovirus, and adeno-associated...the need for envelope disassembly. Both lenti and retro viruses can accommodate a cargo up to approximately...

...range (tropism). Pseudotyping is only done on enveloped viruses such as lentivirus, retrovirus, and rabies...et al, 2020). Because retrovirus is closely related to lentivirus, many retroviral envelope proteins have... a few cell types.  If you’re looking for more control in the cell types your lentiviral vector will infect...particles.   Why pseudotype? Lentiviruses and retroviruses typically target immune cells with their wild-type...viruses to infect neurons. Besides altering the host tropism of your virus, there are many other reasons when.... Image from Crawford et al., 2020.   Broad tropism: VSV-G pseudotyping A commonly used envelope protein...stem cells and neurons. However, VSV-G’s broad tropism can be undesired for in vivo studies since there...

...blotting is a loading control. Pro-Tips: 1. It’s all about the MOI. In retrospect I used a way too high...express CAs9 and are included as a control (WT). Vinculin is a loading control. 3. In terms of making the...undigested control. Sometimes the PCR products can look smeared, so having an undigested control can help...those formed from control (GFP gRNA-targeted) BRAF sites (lanes 3, 4) were used as controls. Duplexes were...but that nobody talks about. After months of troubleshooting, those people who did not tell you that essential...Cas9 line that’s “most efficient at editing” to introduce the gRNA, but I just randomly chose one of my ...and BRDN0001146266) ( 76479-LV and 76480-LV) or control GFP gRNA (BRDN0000561167) (80034-LV) were individually...

... application for OptoNBs takes us in vitro: light-controlled affinity purification of unmodified proteins...light-based control of signaling pathways. This is particularly exciting because using light to control signaling...  Option #2 may sound like fiction, but light-controlled protein purification is now a reality thanks ..., and their use for protein purification and controlling cell signaling. Key components of OptoNBs OptoNBs... put AsLOV2 because here it was less likely to destroy the nanobody’s function than if inserted into the...insertion into loops 1 and 6 resulted in light controlled binding, but with opposite effects: insertion...insertion site on light-induced binding.  Light controlled protein purification with OptoNBs The first application...

... when using auxotrophic selection Of course, there are some drawbacks to using auxotrophic markers as ...maintained within the yeast cell. These elements control not only the number of plasmids found in each cell...unstable and may be lost during budding. Yeast Centromere plasmids (YCp): These are considered low copy...incorporate part of an ARS along with part of a centromere sequence (CEN). These vectors replicate as though... The copy number of these vectors can also be controlled if specific regulatable elements are included...yeast Historically, scientists have utilized auxotrophic selection rather than antibiotic selection when...insensitivity of yeast strains to some antibiotics. Auxotrophy is defined as the inability of an organism to...

...possible, include positive and negative controls. Positive controls typically include cell lines or tissues...Gordon J (1979) Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: procedure...Towbin et al., 1979, Burnett et al., 1981).  Introduction to western blotting  In a western, proteins ... Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) is the first step of a western. ...immobilized onto a membrane, usually composed of a nitrocellulose or polyvinylidene difluoride (PVDF) in a step...step called protein transfer. While both nitrocellulose and PVDF are commonly used, PVDF tends to be slightly...validated applications, pay close attention to the controls listed on the vendor’s website. Typically, the...

...www.janelia.org/open-science/voltron-and-positron.  Both Ace2N-mNeon-A122D and Voltron2 are available as Cre-dependent...33714753. Voltron2 voltage sensors By Brian O’Neill New viral vector preps expressing the Voltron2 voltage...performance.      Figure 4: Structure of Voltron or Voltron2 conjugated to a JaneliaFluor dye emitting... SARS-CoV-2 research New viral vector preps of Voltron2 voltage sensors AAV vectors for targeting microglia...membrane protein (Plasmid #141386-LV [+M], negative control) were immunoblotted against Anti-SARS-CoV-2 Nucleocapsid...also immunoblotted against vinculin as a loading control (right panel). Image from Addgene. These antibodies..., are now available! Building on their earlier Voltron indicators (a fusion of Ace2N and HaloTag, Figure...