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...hiPSCs for clinical purposes: mRNA transfection- this allows transient expression of the reprogramming factors...cells, thus expressing additional EBNA-1 transiently. The transiently expressed EBNA-1 increases protein expression...insertional mutagenesis, which is the integration of transgenes into the genome that can cause disruption of ...express reprogramming factors To date, the mRNA transfection method has not been used successfully to derive...using PBMCs as the source of somatic cells. This translates to only about 16 hiPSC colonies per 100 ml of...such low efficiency of reprogramming is a major hindrance in using PBMCs as a source of somatic cells for...system thus provides a practical way to generate transgene-free, virus-free clinical grade hiPSCs from easily...

...diderms, have a thinner peptidoglycan membrane and an outer lipid membrane. Common gram negative bacteria include...within their membrane, while the stain is washed from the gram negative bacteria’s thinner membrane. Procedure...destroys the outer lipid membrane, and the thin inner peptidoglycan membrane is unable to retain the stain...expanded on Gram’s discovery, adding a second stain, safranin, as a counterstain to help visualize the bleached...or monoderms, have a single thick peptidoglycan membrane. Common gram positive bacteria include staphylococci...and immediately rinse. Flood the smear with the safranin counterstain for 30 - 60 seconds. Rinse the slide...interaction of CV+ and I- in the thick multilayered membrane of the gram positive bacteria. In a gram negative...

... of the four strands of the cleaved duplex, while the fourth strand (the released strand in Figure 1 below...discovered that single stranded donor DNA: Complementary to the released strand, 127bp in length, With...single or double stranded donor DNA with other design parameters. Example single stranded donor DNA and ...presented in Figure 2. Figure 2: Single-stranded donor DNA interaction with the Cas9-DNA duplex...accessible to anneal with complementary single stranded DNA. We thus wondered if such a structure could... that are an order of magnitude greater than transfection approaches. Moreover, the cost of reagents is...relatively modest, requiring expressed Cas9 protein, transcribed sgRNA, and optional donor DNA. Chris Richardson...

... Retroviruses are single-stranded RNA viruses that use reverse transcriptase to synthesize complementary...for most experimental needs, and can transduce (infect) a wide range of cell types with a high efficiency... such as a heterologous envelope or a post-transcriptional regulatory element can be used for either type...Adeno-associated virus (AAV) is a small, single-stranded DNA parvovirus. AAV can infect humans and some...response as retroviruses. This virus can also transduce dividing and non-dividing cells, making it conducive...issue. Adenovirus Adenovirus has a linear, double stranded DNA genome which is non-enveloped. It can infect...Adenovirus is an effective option if you need high, transient expression of a genetic cargo or have a very large...

...AAV5, AAV8) enable different patterns of transduction and transgene expression across different cell types..., Rumpel S. Analysis of transduction efficiency, tropism and axonal transport of AAV serotypes 1, 2, 5...expressed from your cell line of choice after transduction with this virus. While various Cas9 mutants ...classic Cas9 nuclease, which introduces double stranded DNA breaks, can be expressed from lentiCas9-Blast...single stable Cas9-expressing cell line and then transduce this line with lentiviruses expressing various...experiments and over time (as compared to separate transductions with a single-vector system). Tips for using...is used to attain cell-specific expression in transgenic animals, where cre expression is restricted to...

...SLC6A8 mutations in Creatine Transporter Deficiency Creatine Transporter Deficiency (CTD) is a rare X-... to be transported from the blood and cross the blood brain barrier using creatine transporters. However...However, mutations in the transporter SLC6A8 impede this transport and result in global creatine deficiency...Accelerate Research Discoveries) Challenge provides grants for rare disease research. This year is our fourth... the SLC6A8 gene, also known as the creatine transporter. The Association for Creatine Deficiencies (ACD...GPCRs) detect molecules outside of the cell and translate those signals into cellular responses. For example...consist of many parts that work together on the cell membrane. Germline mutations in one of these parts, the...

... that determine membrane expression and plasma membrane localization of transmembrane proteins, like rhodopsins...capable of broad transduction of the nervous system which eliminates the need for intracranial injections. ... to perform in vivo optogenetic experiments. Intracranial injections of AAV are commonly used to deliver...channelrhodopsin-based optogenetics minimally-invasive, Frances Arnold’s lab and Viviana Gradinaru’s lab used machine...neurons and mouse brain slices collected following intracranial injection of an AAV expressing a ChRger. In ...performed well in a tissue culture dish, that doesn’t guarantee their success in vivo. Therefore, the Arnold and...their cage. Since dopamine is an important neurotransmitter that promotes reward-motivated behavior, if...

...bacterial transformation One protocol that has been automated is high throughput transformation. With our... to do up to 96 transformations in one run compared to doing about 50 transformations in the same amount...easier and more efficient. Here, I’ll discuss transformation, DNA purification, and filling vials with agar...deposits (over 100 deposits per month!), manual transformations were taking a lot of time. Enter the Biomek... We wanted a machine that would improve our transformation protocol and, after looking at a few options... - one pod can pick up 96 tips for easy sample transfer between 96 well plates. The second pod can pick...out your samples. This can be useful if you’re transferring samples from 96 wells plates onto agar q-trays...

... copy. The coding sequence for GFP is transcribed and translated along with the gene, producing both a...exon of the gene to ensure it would be transcribed and translated. For an N-terminally tagged protein, ...destroyed by CRISPR/Cas9 during transfection. Making the double strand break and introducing the tag through...base pairs on each side, in fact. Later, when we transfect the cells, these large regions of homology enable...directed repair (HDR) process following the double strand break CRISPR/Cas9 makes at the target site. The...technique that briefly destabilizes the cell’s outer membrane and allows the components to physically enter ...go to work. Figure 3: Inducing the double strand break and introducing the tag. Created with BioRender.com...

...Interest (GOI), and transfect it into your favorite cell line. In parallel, you transfect cells with another...antigen - Recognition of an immunogen does not guarantee recognition of the target antigen. You want to...Antibody-independent methods may include analysis of transcriptomic or proteomic data from several different tissues...non-specific, background signal, so you’ll need a range of expression strengths to be confident in your ... feasible and (2) large-scale proteomic and transcriptomic datasets already exist.   Example: You want...tissues. You know that there is a wealth of transcriptomic data from this sample type, so you use your...need to be aware of any possible alternative translation start sites or alternative splicing that could...

...selectable, it can be transferred to a different genetic background via P1 transduction. Plasmid Expressing...products: 1) a partially dsDNA duplex with single-stranded 3’ overhangs or 2) if the dsDNA was short enough...enough, a ssDNA whose entire complementary strand was degraded. Beta: Beta protects the ssDNA created by...higher recombination frequency when the lagging strand of DNA is targeted, it’s best to determine the ...design an oligo that is complementary to the lagging strand. In practice, it’s easier to not determine the ...replication and instead design oligos that target both strands. One of the two oligos will recombine with a higher...you’ll find out empirically which is the lagging strand. ssDNA substrate is more efficient than dsDNA with...

...host range and tissue tropism (Borkenhagen et al., 2019). AdVs are non-enveloped double-stranded DNA viruses... protein translation. Its genome consists of “early” (E) and “late” genes, and the transition between ...sequence (transgene) instead.    Although the late genes are usually present in the transfer plasmid for... (and therefore between early and late gene transcription) is demarcated by viral genome replication (...vectorization strategies include creating an AdV transfer plasmid with the E1 and E3 (Early 1 and Early ...cells that are infected by the vector, only the transgene will be expressed, since the early genes are absent...possible to design a vector that achieves persistent transgene expression in certain cell types (Tatsis et al...

...(single vs. double stranded) all depends on the size of the knock-in. Single strand oligonucleotide donors...knock-out. The goal of a knock-out is to disrupt DNA translation by generating a frameshift mutation. The majority...pathway cells already have for repairing DNA double strand breaks – (HR). HR is the dominant homology-directed... Fig. 1: Early steps in repair of a DNA double strand break by homologous recombination.    Designing...have larger homology arms in the 500 bp to 1 kb range, however, shorter homology tracts have recently ...recognition sequence in the donor DNA, you can guarantee there won’t be re-targeting after HDR. If your...cycle regulatory proteins responsible for the transition out of S phase, so cells stay in an HDR-promoted...

...Forster Resonance Energy Transfer (FRET). Through the non-radiative transfer for energy between neighboring...Whether BiFC constructs are expressed transiently by transfection or stably impacts the success of BiFC..., kinase signaling, integrin signaling, and transcription factor interactions (Kerppola, 2006). One of...interactions between basic leucine zipper and Rel transcription factors (Hu et al., 2002). This would be more...overexpression of fusion proteins it requires can lead to aberrant subcellular localization of the studied PIPs. ...frame (ORF) library to screen PPIs among 450 transcription factors in live fruit flies. Although BiFC open-reading... high background fluorescence levels due to the random association between FP fragments independent of...

...nickase. Nickases create a single-strand rather than a double-strand break, and when used with two adjacent...the target strand. Conversely, the H840A mutation in the HNH domain creates a non-target strand-cleaving...strand-cleaving nickase. Instead of cutting both strands bluntly with WT Cas9 and one gRNA, you can create a staggered...to each other? The gRNAs must target different strands to create a DSB, but this can be accomplished with... benefit of using nickases for HDR is targeting range: using an individual gRNA with WT Cas9, repair levels...experiment to insert an EcoRI site using a single-stranded oligonucleotide (ssODN) donor. Using a D10A nickase...across the 51 nt region using either a top or bottom strand ssODN donor with 40 bp homology arms. The HDR efficiency...

...increase as the DNA amplicon transitions from single-stranded to double-stranded, and finally, drop once more... by abundant transcripts, leading to falsely lowered levels for less abundant transcripts. However, adding...can even be used to detect a single copy of a transcript. But this sensitivity can sometimes cause as ...signals are produced by the presence of double-stranded DNA, when temperatures are high, signal intensity...reflecting the presence of primarily denatured (single-stranded) DNA. As the temperature drops, single intensity...assumption that there is a smooth, single-step transition between ssDNA and dsDNA. This is not true for...effects, cation concentrations and a temperature range) to predict PCR melting curves and dynamic melting...

...References: Amir RE, Van den Veyver IB, Wan M, Tran CQ, Francke U, Zoghbi HY (1999) Rett syndrome is caused...collection of molecular biology tools so that both veteran and new Rett researchers can find the reagents ...maturation (Tillotson & Bird, 2019) and is a global transcriptional regulator of thousands of genes. Studies have...have suggested roles in transcriptional repression, activity dependent de-repression, chromatin remodeling...groove of AT-rich DNA sequences to regulate transcription (Baker et al., 2013). Mutations that affect ...gene editing, MECP2 reactivation, RNA editing, RNA trans-splicing, and protein replacement to reverse the...in animal models, Rett syndrome is a strong translational candidate for gene replacement therapy. In 2014...

...the Netherlands. Transformation of HEK cells with adenovirus 5 DNA was postdoc Frank Graham’s 293rd experiment...endothelial, epithelial, or fibroblast. However, Frank Graham showed HEKs display many immature neuron-...with kidney cells (Shaw et al.). Since then, transcriptome studies of the cell line have determined it ...replication. To generate a virus, you need only transfect the ‘helper’ plasmids (viral packaging machinery...cell or organism. This pipeline is utilized to transduce cell lines for basic science research all the ...Protein production  HEKs are extremely easy to transfect in bulk and produce recombinant proteins at high...cell line model for cancer-specific research. Membrane receptors and ion channels are often exogenously...

...three different membrane-targeting flavors – PDGFR, GPI, and SGZ, to improve neuronal membrane trafficking...properties such as kinetics, ∆F/F0, SNR, dynamic range, photostability, quantum yield, and expression. ...segment (PDGFR TMD) that anchors the sensor to the membrane and displays it on the extracellular side. In .... PDGFR is a C-terminal fusion to the PDGFR transmembrane domain in the mammalian expression pMinDisplay...glycosylphostidylinositol anchor. SGZ contains a PDGFR transmembrane domain, followed by a modified form of the cytosolic...fluorescent probe for visualizing glutamate neurotransmission.” Nature Methods (2013) PubMed PMID: 23314171...kinetics and localiation for imaging synaptic transmission” bioRxiv (2022)...

... engineered to detect a wide array of neurotransmitters ranging from dopamine to glutamate, all the way... or animal system. This ‘thing’ could be a neurotransmitter, such as norepinephrine, or an electrolyte...last component of the biosensor is the signal transducer which will quantitatively give a readout for ...bio-recognition event is (fluorescence, pH, etc.). The transducer will generate a numerical value which represents...activated GFP expression (receptor), then the transducer could be a microscope, plate reader, or any other...—keep reading to find out how!   Neurotransmitters Many canonical fluorescent as well as FRET-based...fluorescence-based and have been optimized for a wide-range of uses to monitor the fundamental cellular building...