...synthetic vectors and cultured mammalian cells. Of course, these mammalian vectors must be compatible with...discernable effect on mammalian cells. In this blog post we will discuss how mammalian plasmids differ from...supplement in mammalian cell culture to suppress bacterial growth, and is not appropriate for mammalian selection...Although plasmids do not naturally exist in mammals, scientists can still reap the benefits of plasmid-based...ORI) will not allow for plasmid replication in mammalian cells, for example, and a toxin that kills bacteria...for transfected cells. Before getting into the mammalian plasmid components, it may be useful to describe...introducing genetic material (such as plasmids) into mammalian cells, a process called transfection. Transfection...

...each clone in case of contamination. We do not normally use antibiotics. Lastly, and perhaps obviously... The Challenges of Cell Culture Plasmids 101: Mammalian Vectors Resources on Addgene.org Check Out Our...Plasmids Find Mammalian Expression Vectors ... mammalian cells... mammalian cell clones...

...yellow) - 8.0 (red)). Blood pH normally lies around 7.35-7.4 so mammalian cells are sensitive to pH. Anything...Cell Culture Cloning Mammalian Cells with the Agarose Method Plasmids 101: Mammalian Vectors Resources ....org Find mammalian plasmids for your research Find viral vectors for infecting mammalian cells ...flowing in. The vent for the in-flowing air is normally visible and you should always ensure nothing is...cross-contamination can be identified. Even if some mammalian cells lines have similar morphologies, being familiar...

...because of a small, avoidable mistake. Or worse, when you're trapped in a pattern of small errors that ... when you’re in the lab and you're sweating the small stuff? Identifying the Issue You can’t fix an experiment...your prep an investment in accuracy.  Bench setup Small adjustments in the physical organization of your...reduce errors as you work, and the possibilities for small, helpful changes are endless.    Fig. 2... Just like any other experimental approach, use small, incremental changes to understand what works and...

...can help you make an informed decision. Mammalian cells Mammalian cells are a great option for viral production...you. Table 1: Mammalian versus insect cells for viral production Consideration Mammalian cells Insect...multiple different cell types, including plants, mammals, and insects. In this post, we will compare two...two of the most widely used systems, mammalian and insect cells. Whether you’re new to in-house virus production...production, particularly for labs that already do mammalian cell culture. HEK-293T cells are the most common... AAV, and adenoviruses can all be produced in mammalian cells. However, it is much more common to make...capacity of 8–10 kb. Time Viral production in mammalian cells is relatively quick. After transfection,...

...Born to a family in Malawi that pushed science education, Kingdom Kwapata grew up to be one of the best...among 100,000 eligible to attend the University of Malawi where he focused on agricultural engineering through... Kingdom on opportunities to go into science in Malawi 4:36-5:46 : How Kingdom ended up working in the...University 9:36-10:29 : Kingdom's transition back to Malawi and Lilongwe University of Agriculture and Natural...difficulties surrouding and need for biotech research in Malawi 11:41-14:53 : Kingdom's current projects producing...projects - HIV inhibitors, biofuels, challenges in Malawi, and more 23:45-28:07 : How Seeding Labs is helping... malawi...

... design and optimize. Today we’ll cover the normalization and loading controls needed for relative quantification...In relative quantification, the POI bands are normalized to the loading control: the intensity of each...resulting fraction is reported. Figure 2: Normalization with loading controls. Under each lane, the ...the loading controls are equally strong, so the normalized value simply reports which POI band is stronger...is weaker in condition 1 than condition 2, the normalized values for the two conditions are equal. Created...Created with BioRender.com. This kind of normalization is a nearly ubiquitous tool, but proceed with...your western blot, and depending too much on normalization can amplify other errors in experimental design...

... also confines channelrhodopsin activation to a small volume of brain tissue (~1 mm3). Current channelrhodopsins... To make channelrhodopsin-based optogenetics minimally-invasive, Frances Arnold’s lab and Viviana Gradinaru...channelrhodopsins paired with a systemic AAV allow for minimally-invasive optogenetics While ChRgers performed ...in mice to see if they were suited for in vivo minimally invasive optogenetics. Both experiments delivered...paired with PHP.eB’s systemic delivery enables minimally-invasive gene delivery and optogenetic excitation...learning-guided channelrhodopsin engineering enables minimally invasive optogenetics. Nat Methods 16:1176–1184...

...replicated many of the properties once exclusive to small-molecules: red-shifted spectra, ion sensitivity,...improve the photon budget. Of course, “reverting” to small molecule dyes seems daunting—no one wants to spend...that reacts specifically and irreversibly with a small substrate ligand motif attached to a fluorophore...These modified antibody fragments bind and enhance small molecule fluorogens (Figure 1e). Stains – These..., no genetically encoded tag is required as the small-molecule binding motif targets endogenous protein...the system. A particularly desirable property for small-molecule labeling strategies is fluorogenicity, ...flurry of activity and the majority of the classic small molecule fluorophores, such as rhodamines (Figure...

...post we’ll provide an overview of some CRISPR mammalian expression systems, the typical applications for...what CRISPR components you will need to deliver. Minimally, one or more sgRNAs and Cas9 are required for ...those CRISPR components into your target cells.  Mammalian CRISPR expression systems Each model system has...Expression System Components of System Application Mammalian expression vector Promoter driving Cas9 expression...or stable expression of Cas9 and/or gRNA in a mammalian cell line that can be transfected at high efficiency...expression of Cas9 and/or gRNA in a wide variety of mammalian cell lines. Useful for difficult to transfect ...degraded within the cell.   Delivery methods for mammalian cell lines As mentioned above, the expression ...

... them 17-26% smaller than other Cas13s and a whopping 33% smaller than Cas9! Their small size makes then...specific editor in mammalian cells Not all Cas13d proteins are functional in mammalian cells, but Konermann... easy to package in low-capacity vectors! These small, but mighty type VI-D enzymes are the latest tools... does not occur when Cas13d is expressed in a mammalian system. Since Cas13d is functionally similar to...compared CasRx to multiple RNA regulating methods: small hairpin RNA interference, dCas9-mediated transcriptional...

...CRISPR article "Generation of Genomic Deletions in Mammalian Cell Lines via CRISPR/Cas9" from the Journal of...of CRISPR/Cas9 to create genomic deletions in mammalian cell lines. Below Bauer and Canver discuss the...genomic deletions We were inspired to produce intrachromosomal deletions based on the experiments of Kim ...References: 1. Lee HJ, Kim E, Kim JS. Targeted chromosomal deletions in human cells using zinc finger nucleases...palindromic repeats (CRISPR)/Cas9 nuclease system in mammalian cells. J.Biol.Chem. 2014 Aug 1;289(31):21312-21324... mammalian cell...

...to the XFP in the construct, dBrainbow includes small epitopes fused to each XFP. Secondary antibodies...signal. Imaging endogenous fluorescence in live animals only permits visualization of two colors. Similarly...protein microinjection or crossing of Zebrabow animals to an inducible Cre line. The Schier group developed...cell populations using the Confetti mouse For mammalian systems, the Clevers lab developed the Confetti...expression in the Ubow mouse Another murine reporter animal, the Ubow mouse (Ghigo et al., 2013), was developed...RFP are not present at the same frequencies. The small population size of CFP++ cells permitted researchers...

...virus- a non-integrating RNA virus Episomal vectors- extrachromosomally maintained DNA plasmids that express...Reprogramming efficiency of PBMCs with episomal vectors Episomal vectors are based on the Epstein-Barr ... pCXLE episomal vector system generated by the group is an optimized mixture of three episomal plasmids...expression from the other episomal plasmids. When used on PBMCs, the pCXLE episomal vector system increases...reprogramming purposes. In this post, I’ll describe an episomal vector system that increases reprogramming efficiency...thereby increasing the costs of the procedure. Episomal reprogramming is an ideal and cost-effective method...encoding reprogramming factors behave as separate extrachromosomal elements in the nucleus of the target cell...

... to try to determine whether simpler and maybe smaller Cas systems could exist in nature. To do this,... Institute for uncharacterized genes that were proximal to a CRISPR array and the universal CRISPR integrase...cas4, and a new gene cas14. cas14 encodes for a small Cas protein (40-70 kDa), which is half the size ...

...pAquaN1 Cyan Mammalian Expresses mammalian optimized Aquamarine mCerulean N1 Cyan Mammalian Express a ...compartments pCEP4CyPet-MAMM Cyan Mammalian Expresses mammalian optimized CyPet pCyPet-His Cyan Bacterial...pCEP4YPet-MAMM Yellow Mammalian Expresses mammalian optimized YPet pYPet-His Yellow Bacterial Expresses YPet...Yellow Mammalian Express a gene of interest fused to the C-terminus of SYFP2 Clover Green Mammalian Expresses...N-terminus of monomeric Cerulean mCerulean C1 Cyan Mammalian Express a gene of interest fused to the C-terminus...C-terminus of monomeric Cerulean mTurquoise2 Cyan Mammalian Constructs to target mTurquoise2 to various subcellular...Expresses CyPet with C-terminal His tag SCFP3A Cyan Mammalian Express a gene of interest fused to the C-terminus...

... yeast two-hybrid assay are not optimal for studying native mammalian protein interactions, whereas others...is needed to assess its function in mammalian cells or other animal models, but this technique has laid...p2A-ferritin-p2A-mCherry Mammalian Expression 74308 pcDNA3.0-Magneto2.0-p2A-mCherry Mammalian Expression 74334...sheared genomic libraries are cloned downstream of a minimal promoter sequence. Should one of these random stretches...it will activate its own transcription from the minimal promoter and its strength can be determined from...RAS Pathway Clone Collection 2.0 Ras proteins are small GTPases involved in cell signaling pathways that...been difficult to target therapeutically due in no small part to the central role Ras proteins play in cell...

...expression in neuronal cells. U6 small RNA expression shRNA From the human U6 small nuclear promoter Constitutive...expression) you need to use an RNAP II promoter, whereas small RNAs (such as shRNA) are transcribed from the RNAP...Similarly, the various eukaryotic cell types (mammalian, yeast, plants, etc) require unique promoters ...leakiness" of transcription may occur. This is normally not a big issue for cells, but it may confound...listing some of the most common bacterial and mammalian promoters. These lists is by no means exhaustive...considerations CMV General expression mRNA Strong mammalian expression promoter from the human cytomegalovirus... types. EF1a General expression mRNA Strong mammalian expression from human elongation factor 1 alpha...

... by the heat. Isolating male and female flies Differentiating male and female flies is essential for setting...of their abdomens, males have black coloration whereas females do not. Also, males have sex combs on their...is important to isolate females from males while they are still virgins. Females begin mating ~6 hours ...trying to get males to recombine two mutations onto the same chromosome. You should use females for that. ...their forelegs which are black, bristly hairs. Females do not have sex combs. To make genetic crosses ... bodies. This is the ideal stage to select for females because you can be certain that they are too young...fourth chromosomes, you are a very unlucky person. Animals are diploid except for their germ cells (sets of...

...screening 768 F1 animals). Recovering rare editing events in a sea of unedited animals struck me as a “...from 11-100% of F1 animals carrying precise knock-ins, and homozygous knock-in animals can be obtained in... in eight days. The only animals on a plate, other than the parental animal, are rescued progeny, which...for any selective marker to remain in the edited animal. Development of CRISPR/Cas9 editing strategies...must be removed, which can be done by isolating animals with particular visible phenotypes. These approaches...conditional-lethal mutation to identify edits with minimal screening effort. Selecting for repair of a lethal...unedited “haystack”, facilitating recovery of edited animals. Figure 1: In the background are 504 restriction...