...attractive due to the ease of transduction in difficult-to-transfect cell types ranging from primary to embryonic... traditional transfection and random genomic integration of a plasmid. Viral transduction is also generally...integrated into the genome of the target cell or transiently expressed. It’s important to note that for all...vector can express your construct in target cells transiently or constitutively depending on the virus used...generally less toxic to cells than transfections, preserving cell viability. Library delivery Screening... entire genome, overexpression of all human transcription factors, barcoded libraries to trace cell lineage... with viral vectors instead of a traditional transfection method. This is because viral systems have a...

...Efficient gene transfer in C. elegans: extrachromosomal maintenance and integration of transforming sequences...easily manipulated with tools like CRISPR to make transgenic lines. The fruit fly (Drosophila melanogaster... in only 6 weeks compared to months to make a transgenic mouse line. Over the years the fly has become...DNA or RNA to edit their genomes or to create transgenic animals. Zebrafish are an ideal model organisms...development studies as their embryos are completely transparent. Thus scientists can easily observe early developmental...can be difficult in other vertebrate species. Transparency also allows scientists to easily observe fluorescently... control the temporal expression of different transgenes, the Stainer group determined that there are ...

...The development of the transient transfection platform for assaying transcriptional activity, for example...such as p300 and CBP histone acetyltransferases or CARM1 methyltransferase. This attendant signaling feature...dietary origin) that are diffused or actively transported from cells, traverse the body through the bloodstream...ligands NRs represent the largest family of transcription factors found in metazoans. The superfamily ... govern expression of genes involved in a broad range of reproductive, developmental, metabolic, and immune...multiple cell signaling systems. Activation of membrane receptors and signaling cascades conceivably allows...profiling datasets in to our data mining tool, Transcriptomine. We are currently nearing the completion of...

..., expressing Ad E1A and E1B, can be transfected with your transfer plasmid, carrying your gene of interest... performed 12–18 hours post-transfection to help remove toxic transfection reagents and keep the cells...helper plasmids, commonly called “triple plasmid transfection” (Figure 1).    Figure 1: Overview of key steps...cells happy. Harvesting virus Once you have transfected your cells, wait 2–5 days and then collect the supernatant...the concentration of viral particles that can transduce cells, which can vary based on target cells and... capsids, thus leading to an overestimate of transducible units in the virus preparation.  rAAVs  Now...Whitney, O., Walther, N., & Maurer, A. C. (2023). Transgene Expression in Cultured Cells Using Unpurified ...

...respondents (transfection method, amount of each component per transfection etc.). Take Pooran's NgAgo Survey...Doudna Lab Many thanks to our Guest Blogger Pooran Dewari! Pooran Dewari is a postdoc at the MRC Centre for...TOOL This post was contributed by guest blogger Pooran Dewari. Any views in this post are those of the...an ssDNA guide. Therefore, researchers must co-transfect cells with 5’-P-ssDNA guides and an NgAgo expression...delivery methods. Researchers can conveniently transfect/inject cells with in vitro preassembled Cas9/gRNA...). View the Complete and Up-to-Date Results of Pooran's NgAgo Survey The take-home message While most ...researchers will have to carry out repeated transfections with the ssDNA guide, although many may find...

...target and nontarget DNA strands upstream of the PAM site found in the nontarget strand. Protein Cas: ...sequences and keeping the array organized. Target strand: The strand of foreign DNA that Cas proteins recognize...recognize using an RNA guide. Non-target strand: The paired strand of foreign DNA cut by Cas9, whose sequence... and variable spacer sequences. The array is transcribed and processed into crRNAs, each including one...protospacer-adjacent motif” present in the non-target strand of DNA to be cut, which varies between CRISPR systems...from Addgene!   RNA crRNA: “CRISPR RNAs” are transcribed from the CRISPR array and include a spacer and...track down its suspect—the target DNA. tracrRNA: A “trans-activating CRISPR RNA” base pairs to the repeat ...

...between the edited strand and wild-type strand of the host DNA. When these strands hybridize, the nuclease...nuclease can detect strands with mismatches and cleave them. The results are then visualized using gel electrophoresis...is also key to perform these validations as translational researchers begin to use CRISPR as a therapy... search criteria are not exhaustive. Mismatch tolerance settings are often limited to off-target sites...nucleotides) that may still lead to off-target double-stranded breaks (Tsai et al. 2015). Additionally, current... genome-wide, unbiased identification of double-strand breaks (DSBs) evaluated by sequencing (GUIDE-seq...et al. 2013). GUIDE-seq and BLESS detect double-stranded breaks and do not require high sequencing read...

...different DNA strand comes along, it can attack this covalent bond thus joining the two DNA strands and releasing...planning to transform right away OR you can store the reaction at -20C overnight. 4. Transform TOPO Cloning...DNA sequence 5´-(C/T)CCTT-3' and digests double stranded DNA at this sequence. The energy from this breakage...stored as a covalent bond between the cleaved 3′ DNA strand and a tyrosyl residue of topoisomerase I (1). If...researcher to perform blue-white screening after transformation - self joining of the vector ends results in... minutes will not significantly improve the transformation efficiency).  5. Select and Analyze 10 White...add will depend on whether you are planning on transforming your reaction into chemically or electro-competent...

...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...

...lentiviral vector to be transported retrograde and also has higher neuronal transduction efficiency and a better...virus to either restrict or broaden the host cell range (tropism). Pseudotyping is only done on enveloped...Lentiviral production uses three plasmids: (1) The transfer plasmid, (2) the envelope plasmid, and (3) the...the packaging plasmid. These are transfected into target cells which produce lentiviral particles.   ... titer is one of the reasons VSV-G is used to transduce stem cells and neurons. However, VSV-G’s broad...for off-target infectivity as it can bind and transduce many cell types before reaching the target cells...find tables and information on different viral membranes, tropism, and advantages and disadvantages: Table...

...scientists to quickly and easily generate zebrafish transgenic lines to study neurobiology and neuorodgenerative...pluripotent stem cells) through the expression of transcription factors OCT3/4, SOX2, KLF4, and c-MYC is the... method for generating iPSCs to express the transcription factors from a polycistronic cassette. However...comparable experimental results. Using PiggyBac transposons, the Woltjen Lab compared different variants ...reprogramming discrepancies. The polycistronic piggyBac transposon vectors tested in this work are available at ...approach Genetically modified organisms like transgenic mice are important tools for understanding the...a donor in FRET (fluorescence resonance energy transfer) and FLIM (Fluorescence Lifetime Imaging Microscopy...

...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...

...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...

... 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...

... 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...

...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...

... 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...

...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...

...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...