...6105. https://doi.org/10.1093/nar/gku241 Cong, L., Ran, F. A., Cox, D., Lin, S., Barretto, R., Habib, N..... Type I systems are also employed as CRISPR transposases, by leaving out Cas3. Type I is further subdivided...complex (blue subunits) with a guide RNA (green and orange) bound to the target DNA with Cas3 (pink). Complex...promoter, allowing for easy multiplexing. The double-stranded breaks caused by Cas12 create short 3’ overhangs...paradoxically called V-K, is a CRISPR-associated transposase (CAST) that can insert large fragments of DNA...use a Cas14 enzyme, and uniquely target single-stranded DNA non-specifically. Type VI Type VI is defined...interference, as it can target and degrade select RNA transcripts, or be engineered to conduct direct RNA editing...

...10.1146/annurev-biophys-062215-010822 Nishimasu, H., Ran, F. A., Hsu, P. D., Konermann, S., Shehata, S. I.... NUC HNH Cut DNA target strand RuvC Cut DNA non-target strand CTD (or PI) Recognize PAM...information on these regions.   Cas9’s transition from weak, random binding to precision gene editing requires...left) and transparent (right). The RNA guide sequence is extended as a single unpaired strand in the center...10 of the guide RNA, prearranged for base pairing with a complementary DNA strand. These 10 nucleotides...the non-target strand for the PAM sequence and doesn’t mind if the PAM complementary strand is mismatched...of the PAM in the non-target DNA strand (dark gray). Target DNA strand, light gray.   Once it has found...

... your membrane, run a double-membrane transfer with a psi membrane stacked behind the p membrane. If your... transfer buffer Small proteins: Over-transferring Transfer with two membranes* Psi membrane...alcohol in transfer buffer Mixed proteins: Transferring issues Transfer with two membranes* Electroblot...completely through the membrane or larger proteins not transferring completely onto the membrane. If you’re not...using two membranes during your transfer instead of one. If you see signal on both membranes, you know...large protein, you may be under-transferring.  Membrane pore size Membranes come in different pore sizes ...through the p membrane into the psi membrane, that’s a good indication that the p membrane pores are too...

...titrating the transposase to transposon ratios. High ratios of transposase to transposon result in a greater...]. Like other transposons, piggyBac™ has two components, a transposon and a transposase. The piggyBac™...piggyBac™ transposase facilitates the integration of the transposon specifically at ‘TTAA’ sites randomly dispersed... piggyBac™ transposon. These plasmids, when combined with a source of piggyBac™ transposase (available...distributor) allow you to quickly transfer a DNA sequence from the transposon vector to one of many TTAA sequences...folks at Transposagen. This post was contributed by Amar Singh and Mathew Goodwin from Transposagen What ...What is the piggyBac™ transposon? The piggyBac™ transposon was originally identified in moths in 1983, but...

...Cre-dependent transgenes can also be paired with Cre-expressing transgenic mouse lines to restrict transgene expression...with broad CNS or PNS transduction, cell type specificity, or retrograde transport in neurons. (b) Genetic...capsids or transgenes. Sensory neurons and liver appear especially susceptible to off-target transgene expression-induced...It has a similar transduction rate to AAV-PHP.eB in the brain, and a lower transduction rate in the spinal...2022). AAV-PHP.N specifically transduces NeuN+ neurons, with transduction efficiency varying from 10-70%... effectively transduces BALB/cJ, CBA/J, and FVB/NJ mice, with minimal liver transduction observed in both...effectively transduces neurons in the peripheral nervous system (PNS). It has been shown to transduce 80% of...

..., and enzymes for transmitter synthesis. Retrograde transport is used for transport of endocytosed material... facilitate transport: retrograde transport relies on dynein, while anterograde transport relies on kinesin...take advantage of existing transport pathways in neurons. Anterograde transport is typically used for the...mechanisms of trans-synaptic trafficking are not fully understood and may be a form of transcytosis where the...after intracellular transport without actually causing infection or leading to transgene expression within...endothelial transcytosis (Foust et al., 2009; Merkel et al., 2017), and these two modes of transport may share...optogenetically controlled transgenes, in combination with conditional transgenic mice, to reveal not only...

...contains the transposase gene (red) for expressing the transposase enzyme.2.) The transposase enzyme is ...which cuts the DNA.3.) The released transposon can now bind a strand of DNA with a TA dinucleotide (there...removal of the transposon; the plasmid is then degraded by the cell.4.) The transposase creates a double...double strand break in the DNA and allows the tranposon to integrate. Additionally, the TA sequence is duplicated....   The Sleeping Beauty transposon system The Sleeping Beauty (SB) transposon system is one such system...perform gene transfer in vertebrates. The system is comprised of 2 components: 1) an SB transposase, the enzyme...enzyme required for catalysis of transposition; and 2) a transposon containing the gene-expression cassette...

...piggyBac (PB) transposon: The PB transposon is a mobile genetic element that transposes via a “cut and...Generates transgene-free and vector-free cells Limited genomic integration Multiple transfections required...Delivery DNA-Free High High Transgene-free and vector-free Multiple transfections required Protein Delivery...vector, but transient expression of Cre recombinase allows for deletion of the inserted transgenes that are.... This system is transfected into cells as a donor plasmid containing the transposon, along with a helper...sequences, which the PB transposase recognizes. Following reprogramming, transgenes can be removed by re-...also includes measles and mumps. Sendai transduces a wide range of cell types and replicates in the cytoplasm...

...forming membrane pores) transfection. These techniques are still used and efficiently transfer plasmid...and stable expression of the transgene   Transfer plasmids The transfer plasmid is the vector you clone...comes to transferring DNA into primary cells or non-dividing cells. To target these un-transfectable cells...Dox), the Tet-controlled transactivator (tTA, also expressed by your transfer vector) binds to the TRE...production and transduction Once you have cloned your gene of interest into lentiviral transfer vector, the...will first need to transfect producing cells, usually 293T cells, with your transfer plasmid and your packaging...you don’t have any experience in transfection, CaCl2 transfection is a simple and cheap technique that...

...covers a size range appropriate for the target(s) and transfer method. Membranes The two membrane options ...) steps to the transfer: you’ll need to pre-wet most PVDF membranes before the transfer and allow them...Types of transfers Your transfer options are wet, semi-dry, or dry/electroblot. A wet transfer is the most... while semi-dry transfers require a semi-dry transfer cell. Dry/electroblot transfers only work in the...low-background PVDF membrane, it also has higher background noise than nitrocellulose membranes. Pro tip! You...visualize proteins on a PVDF membrane without staining; simply wet your membrane with 20% methanol and place..., meaning that it will transfer the most protein from your gel to your membrane. It requires anywhere ...

...variant of PSmOrange, a photoswitchable fluorescent protein which transitions from emitting orange (λ= 565nm...enhancement of transcriptional activation via binding of multiple copies of a transcription factor to a ...the stop codon. Thus a non-translated mRNA will appear yellow, but a translated mRNA will be red. This system... were untranslated in the nucleus, confirming the hypothesis that most mRNAs are not translated until ...P-bodies are translationally repressed whereas the nonsequestered pool goes on to initiate translation. These...tool can also be used to visualize translocations, genome rearrangements and cancer-associated chromosome... been used to study transport characteristics of various Golgi and plasma membrane reporters as well as...

...systems efficiently transfer a range of protein sizes in minutes without any transfer buffer. These systems...proteins are: (1) separated by size, (2) transferred to a membrane, and  (3) detected using antibodies. ...mixture of protein is separated in a gel, transferred to a membrane, stained with antibodies, and finally ...a broad size range to be separated on a single gel.  Immobilizing proteins on a membrane Once separated...migrate out of the gel and attach to the membrane.  In a wet transfer system the sandwich is completely submerged...filled with a transfer buffer formulated to conduct the electric current. The wet transfer process is very... very effective in transferring a broad range of protein sizes but takes hours to overnight.  In contrast...

...initiation step of gene transcription; today we'll provide an overview on how transcription stops, or termination...from the transcription machinery. Terminators are found downstream of the gene to be transcribed, and typically...generally serves to promote transcript longevity in eukaryotes and promote transcript degradation in prokaryotes...thought to destabilize the transcription complex, initiating cleavage of the transcript.   Figure 1: ...Hepatitis Virus Posttranscriptional Regulatory Element Enhances Expression of Transgenes  Delivered by ...terminators in the context of AAV transgene expression levels. Transcription Termination Rho-dependent termination...both prokaryotic and eukaryotic systems must be transcribed into RNA, which occurs in three phases: initiation...

...gene transcription (6). The EBOV genome contains seven viral genes, each flanked by a transcription start...replicated and transcribed in the cell when the appropriate viral proteins are provided in trans (see Other...genes and the minigenome are transcribed (and the viral mRNAs are translated) by cellular machinery. NP ...altering the amount transfected (see Technical Tips). Minigenome replication and transcription In order to replicate...it will terminate transcription and polyadenylate the mRNA. These mRNAs are translated by cellular host... lines that are highly transfectable.  The amounts of each plasmid transfected into cells are also extremely...interested in genome replication and transcription of negative strand RNA viruses.   References 1. Buchholz...

...plates. Step 2: Express random phage DNA fragments in E. coli Now that random DNA fragments have been ...nitrocellulose membranes and exposed to IPTG in order to induce protein expression. Cells on the membranes will ...orientation of each membrane on its plate before moving on. Then peel each membrane off the plate (saving...high salt.  To probe membranes with target bacterial cells, wash your membranes with PBST to remove salt... on the membrane to bind their target cells, pulling them out of solution. Wash your membranes well with...searches return no candidates. Our strategy: Express random fragments of the phage genome and probe with live...identified using this method. Step 1: Prepare a random fragment library of phage DNA This first step involves...

... non-edited strand, they can cause the cell to remake that strand using the edited strand as the template...the nicked target strand. The initial result is a heteroduplex, with overlapping strands of edited and unedited...Improving reverse transcriptase efficiency Building upon prior reverse transcriptase research, the Liu...the edit into one strand, there’s a mismatch between the original sequence on one strand and the edited ... the unedited strand away from the initial nick site (to avoid creating a double strand break), increasing...binds the edited strand, as in the PE3b system, guides nicking of the unedited strand only after editing...process and promote copying of the edited strand to the unedited strand (Chen et al., 2021). The team applied...

... does not guarantee 100% transfection efficiency. Stable transfection A stable transfection is used to...process called transfection. Transfection is somewhat comparable to bacterial transformation (the introduction...the transfected plasmid to be expressed transiently. Since the DNA introduced in the transfection process...the genome to form a stable transfection. Unlike bacterial transformation, scientsts do not "select" for...fluorophore in a later post, so stay tuned! Transient transfection and the elusive "mammalian ORI" For many...plasmid depleted, but still want to use transient transfection methods. Since there are no "natural" mammalian...must be compatible with the cell type they are tranfected into – a bacterial origin of replication (ORI...

...considered. Q2: Can I use lentiviral transfer vectors for transient transfections? A2: Technically, yes. Will ...produced by co-transfecting three essential ingredients: the lentiviral packaging vector, a transfer vector including...specifically for transient expression (find some useful backbones here) or from viral transduction. While not...3rd-generation transfer vectors apart? A3: Addgene defines 2nd- or 3rd-generation transfer vectors based... Are transfer plasmids replication competent? What is SIN? A6: Most (if not all) Addgene transfer plasmids...enhancers and the transgene; such interactions can alter expression of not only the transgene but also that...which is rarely more than 30% of the transfer plasmid. Many transfer vectors are SIN. Packaging plasmid...

... your gel and membrane transfer as normal. Immediately after transfer, dry the membrane to maximize protein...you from having to run a whole new gel and membrane transfer. Let’s get started! Before you begin In the...manufacturer’s instructions for the membrane you are using. Dry nitrocellulose membranes are brittle, so proceed ... as normal! Stripping your membrane Alright, so you’ve dried your membrane and returned it to buffer, ...’ll want to: Rinse the membrane in water or fresh buffer. Cover the membrane in stripping solution and...a fume hood. Rinse the membrane in water or fresh buffer. Cover the membrane in stripping solution and...you need to start a whole new blot? Never fear: membrane stripping is here! Rather than start over, you...

...γ-Retroviral transfer plasmid encoding a transgene, sgRNA, or shRNA of interest: The transgene sequence is...packaging and transduction are not encoded by the transfer plasmid, but instead are provided in trans by other...integrated into the host genome upon viral transduction. Most transfer plasmids available from Addgene were ...packaging, and transfer) are supplied by three types of plasmids, which are cotransfected into the 293T...cell line. γ-Retroviruses can transduce target cells, but the transduced target cell does not produce ...protein containing Reverse Transcriptase and Integrase components. Transfer cPPT Central polypurine...proviral DNA synthesis. Increases transduction efficiency and transgene expression. Psi (Ψ) RNA target...