...and last updated Apr 6, 2021 by Will Arnold. Introducing NGS in 2017 for all incoming plasmids was a big...Addgene - we had used Sanger sequencing for quality control for over ten years. Now, thanks to a generous donation...quantification of various intermediate steps, a sizing electrophoretic gel, and qPCR quantification of the final libraries...The first step we take when performing quality control of newly deposited plasmids is to align the NGS... by the depositing lab, we call these “quality control (QC) issues.” We then ask the depositing laboratory...

... just label cells. If GFP could be co-opted to control gene expression, he could then selectively manipulate...promoters. Moving to an in vivo system, Tang et al. electroporated T-DDOGs, GFP, and a reporter construct into...TdTomato, whose expression was absent without electroporated GFP. They then tested T-DDOGs with two mouse...robust and specific. When tested in retinal electroporation studies, DsRed reporter expression was induced...

...Their system is based on the AraC protein that controls transcription from the PBAD promoter. “AraC is... promoter. While none of the constructs gave as strong a signal as with the wild-type AraC, it still reached... of interest, under the pBAD promoter, can be controlled with light without the need of any cloning. ...

... lentiviral plasmid to express ubiquitin under control of a CMV promoter. Starting with two search terms...What type of experiment are you planning? (e.g. Retroviral) Species: Specify the source organism for a gene...

...an appropriate control? Many CRISPR screens use non-targeting guides as negative controls. Non-targeting...are well characterized and strategies exist to control for these faults. However, it’s still unclear if...screens and how best to design these screens to controls for flaws. Recently the Bassik Lab at Stanford...site in the genome. This means that while they control for many of the effects of gRNA and Cas9 expression...detecting Cas9 cutting. Safe targeting guides better control for non-specific toxicity than non-targeting guides... a coding region. Targeting these sites should control for the effects of guide expression and dsDNA breaks... the genome, and are commonly used as negative controls for genome-wide CRISPR screens; and 3) safe-targeting...

...systematically study gene function with temporal control and cell-type specificity! The Gal4/UAS system...you mate your fly line containing YFG under the control of the UAS with a fly line expressing Gal4 in your...promoter each time a new hypothesis is tested. Controlling multiple genes with Gal4/UAS system One can also...to drive expression of multiple genes under the control of UAS enhancer sequences. For example, if you ...them individually or simultaneously. Do note for control purposes that there are dosage effects. For example...

...for gene control over a 1,000-fold range. Weissman explained that there are two levels of control in the...technology has changed how scientists edit and control genes, but according to the Broad Institute's Silvana...screening In October, Jonathan Weissman’s lab introduced additional pooled CRISPR libraries, also available...abilities to turn genes on and off,” he said. “Some are strong and some more moderate; there is an allelic series...size of the backbone, transformations require electroporation. References & Further Reading: For more ...Epub 2014 Dec 10. Genome-Scale CRISPR-Mediated Control of Gene Repression and Activation. Gilbert LA, ...

...specificity, or retrograde transport in neurons. (b) Genetic cargo can be customized to control transgene expression... Capsid Published Tropism Model Organism Mouse Strain-Specific Tropism?  Reference AAV-...and astrocytes Mice No (LY6C1)   Hanlon et al., 2019 AAV-BI-28 CNS: neurons, astrocytes, oligodendrocytes...-Specific Tropism?" column. For a complete list of mice strains with LY6A-dependent tropism, see S3 table...derived from AAV9 using iTransduce, shows tropism toward astrocytes and neurons, with greatly increased efficacy... appears to show a broader tropism with a potential bias toward astrocytes (Chuapoco et al., 2023). Additionally...systemic AAVs with endothelial-specific tropism in rodents and broad tropism in primates. Nature Communications...

...developed a new AAV variant, rAAV2-retro, that migrates retrogradely at higher rates than any other AAV... AAV2-retro vectors with a variety of transgene/promoter combinations. Check out our retrograde AAV blog...requires both antero- and retrograde labelling, synaptic restriction as well as controlled monosynaptic and polysynaptic...Selective retrograde transsynaptic transfer of a protein, tetanus toxin, subsequent to its retrograde axonal...bodies to the terminals of their axons; while retrograde tracing outlines neurons in the opposite direction...axons to their cell bodies. Anterograde and retrograde tracing take advantage of existing transport ...myosin, and enzymes for transmitter synthesis. Retrograde transport is used for transport of endocytosed...

... 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...