1) Cella preparazione <ol><li> Quando si utilizzano cellule aderenti, è necessario trypsinize e raccogliere le cellule prima di elettroporazione. </li><li> Per confrontare trasfezione tra il mouse quattro fibroblasti embrionali (MEF) le culture, che rappresentano tre diversi numeri passaggio, effettuare quanto segue su ogni pallone. </li><li> Aspirare off terreni di coltura delle cellule. </li><li> Aggiungi PBS per lavare le cellule. </li><li> Rimuovere PBS, aggiungere tripsina sufficiente a coprire le cellule, e attendere qualche minuto per permettere tripsina per staccare le cellule. </li><li> Controllare fiaschi con un microscopio per verificare la condizione delle cellule, smack pallone per staccare le cellule, e quindi controllare fiasco di nuovo per rendere le cellule che sono tutti staccati. </li><li> Aspettare ulteriore tempo e ripetere se necessario. </li><li> Una volta che tutte le cellule si staccano, aggiungere siero supporti contenenti per neutralizzare tripsina. </li><li> Trasferire le cellule in una provetta da centrifuga, e le celle a pellet per centrifugazione (RCF = 300 xg). </li><li> Rimuovere il surnatante e risospendere le cellule in un volume noto di PBS. </li><li> Contare le celle. </li><li> Trasferimento in una nuova provetta il giusto volume di sospensione cellulare di fornire il numero di cellule per gli esperimenti (avrete bisogno di 150 ml di cellule per pozzetto ad una densità di 1 x 10 6 cellule / ml). </li><li> Centrifuga cellule. </li><li> Rimuovere il surnatante e risospendere le cellule nel volume adeguato di tampone di Gene elettroporazione Pulser per ottenere una densità cellulare di 1 x 10 6 cellule / ml. </li><li> Aggiungere 20 mg di plasmide per mL di sospensione cellulare e mescolare delicatamente. </li></ol> 2) navi di setup elettroporazione ed elettroporazione Piastra di configurazione ed elettroporazione <ol><li> Plug camera piastra nel modulo di potenza del sistema di elettroporazione Gene Pulser MXcell. </li><li> Aggiungere 150 microlitri miscela di cellule o tampone nei pozzetti di una piastra da 96 pozzetti elettroporazione. </li><li> Mettere piastra in camera di piatto e di impulsi. </li><li> Rimuovere la piastra dalla camera. </li><li> Mescolare bene il contenuto pipettando su e giù in ciascun pozzetto. </li><li> Trasferire le cellule da ogni pozzetto di pre-riscaldato a tampone 12-pozzetti. </li><li> Toccare piatto per distribuire le cellule e metterlo in incubatrice. </li><li> Lasciate che le cellule recuperare per 24 ore. </li></ol> Cuvette di configurazione ed elettroporazione <ol start="9"><li> Scollegare camera piatto dal modulo di potenza del sistema di elettroporazione Gene Pulser MXcell e collegare il ShockPod ™ cuvetta da camera. </li><li> Pipettare 600 l di sospensione cellulare in una cuvetta 0,4 elettroporazione divario cm. </li><li> Mettere cuvetta nella camera ShockPod e fornire impulso elettrico. </li><li> Togliere la cuvetta da camera. </li><li> Mescolare il contenuto cuvetta pipettando su e giù in cuvetta. </li><li> Trasferire le cellule da ogni pozzetto di pre-riscaldato a tampone 12-pozzetti. </li><li> Toccare piatto per distribuire le cellule e metterlo in incubatrice. </li><li> Lasciate che le cellule recuperare per 24 ore. </li></ol> 3) Rappresentante Risultati Dopo la trasfezione delle cellule e permettere loro di recuperare, analizzare l&#39;efficienza di trasfezione qualitativamente, usando la microscopia epifluorescente, e quantitativamente, in citometria a flusso. <img src="/files/ftp_upload/1662/1662fig1.jpg" alt="Figura 1" /> Cellule Figura 1. Che sono state con successo elettroporate e ora esprime il gene GFP appaiono al microscopio epifluorescente. <img src="/files/ftp_upload/1662/1662fig2.jpg" alt="Figura 2" /> Figura 2. Visualizzazione delle cellule in contrasto di fase permette la visualizzazione di cellule sia transfettate e untransfected. Queste sono le cellule che sono stati esposti a l&#39;impulso di tensione più bassa elettroporazione a 200V. Le cellule sono in gran parte confluenti a causa della elevata densità cellulare. <img src="/files/ftp_upload/1662/1662fig3.jpg" alt="Figura 3" /> Figura 3. Lo stesso campo visivo in epifluorescenza mostra un numero di cellule esprimono il marcatore GFP, ma questi sono solo una piccola percentuale di cellule visibile nell&#39;immagine precedente. <img src="/files/ftp_upload/1662/1662fig4.jpg" alt="Figura 4" /> Figura 4. A 250V, il numero totale delle cellule vive visto in contrasto di fase diminuisce leggermente. <img src="/files/ftp_upload/1662/1662fig5.jpg" alt="Figura 5" /> Figura 5. Sotto epifluorescenza, si può vedere che il numero di cellule che esprimono GFP è aumentato. <img src="/files/ftp_upload/1662/1662fig6.jpg" alt="Figura 6" /> Figura 6. Al massimo della tensione applicata, 375V, ci sono meno cellule vive visibili. <img src="/files/ftp_upload/1662/1662fig7.jpg" alt="Figura 7" /> Figura 7. Tuttavia, una grande percentuale di cellule rimanenti esprimono GFP. Quale condizione è ottimale dipende dal disegno sperimentale. In alcuni esperimenti il ​​maggior numero di cellule transfettate potrebbe essere ottimale, in altri esperimenti la transfezione più alta percentuale potrebbe essere migliore. Siamo interessati a la percentuale di cellule che sono GFP positivo sotto ogni condizione e di come le percentuali variano con l&#39;età delle cellule. Citometria a flusso in grado di fornire informazioni quantitative sui risultati trasfezione per ciascuna delle diverse condizioni di elettroporazione. <img src="/files/ftp_upload/1662/1662fig8.jpg" alt="Figura 8" /> Figura 8. Qui la percentuale di cellule GFP positive che sono nel passaggio sono mostrati 5 celle in ciascuna delle 12 condizioni di elettroporazione. La percentuale massima di trasfezione è stata di circa l&#39;80% sotto l&#39;impulso di alta tensione di decadimento esponenziale, condizione 6, e 70% sotto il forte impulso di onda quadra testato, condizione 12. <img src="/files/ftp_upload/1662/1662fig9.jpg" alt="Figura 9" /> Figura 9. Con le cellule superato 9 volte prima della elettroporazione, lo schema generale di transfezione percentuale è quasi identica, ma con un calo molto lieve nelle percentuali di trasfezione. <img src="/files/ftp_upload/1662/1662fig10.jpg" alt="Figura 10" /> Figura 10. Visualizzati sono le percentuali di cellule GFP nel passaggio 13 celle che mostrano una marcata riduzione in percentuale rispetto alla trasfezione delle cellule più giovani. Le più alte percentuali di trasfezione sono stati circa la metà di quello che è stato ottenuto con le cellule più giovani che dimostrano l&#39;importanza di utilizzare le cellule sane non appena possibile dopo l&#39;isolamento. <table border="1"><tbody><tr><td colspan="3"> Condizioni Elettroporazione usati per le cellule MEF trasfezione con il gene Pulser MXcell </td></tr><tr><td> Condizione (1-6) Impulsi di decadimento esponenziale, tutti con 350 uF, 1000ohm </td><td> Tensione (V) </td><td></td></tr><tr><td> 1 </td><td> 200 </td><td></td></tr><tr><td> 2 </td><td> 250 </td><td></td></tr><tr><td> 3 </td><td> 300 </td><td></td></tr><tr><td> 4 </td><td> 326 </td><td></td></tr><tr><td> 5 </td><td> 350 </td><td></td></tr><tr><td> 6 </td><td> 376 </td><td></td></tr><tr><td> Condizione (7-12) Onda quadra legumi, tutti con 2000 uF, 1000 ohm, e 1 impulso </td><td> Tensione (V) </td><td> Durata impulso (ms) </td></tr><tr><td> 7 </td><td> 200 </td><td> 10 </td></tr><tr><td> 8 </td><td> 250 </td><td> 10 </td></tr><tr><td> 9 </td><td> 300 </td><td> 10 </td></tr><tr><td> 10 </td><td> 200 </td><td> 20 </td></tr><tr><td> 11 </td><td> 250 </td><td> 20 </td></tr><tr><td> 12 </td><td> 300 </td><td> 20 </td></tr></tbody></table>

Gli autori sono utilizzati da Bio-Rad Laboratories che produce reagenti e strumenti utilizzati in questo articolo

In questo articolo video viene illustrato come utilizzare il sistema di elettroporazione MXcell di identificare facilmente le condizioni ottimali per l&#39;elettroporazione MEF o altre linee di cellule primarie. Il formato a 96 pozzetti piastra permette di replicare molte delle condizioni sperimentali o di ottimizzazione da eseguire simultaneamente, il che può eliminare la necessità per molti esperimenti separati. Mentre si esegue questa procedura, si deve ricordare di utilizzare le cellule sane non appena possibile dopo l&#39;isolamento e ad utilizzare l&#39;elettroporazione condizioni che sono abbinati al buffer di elettroporazione.

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<td>Gene Pulser&reg; Electroporation Buffer</td>
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<td>Gene Pulser MXcell&trade; Electroporation System</td>
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using the gene pulser mxcell electroporation system to transfect primary cells with high efficiency

E &#39;sempre più evidente che elettroporazione è il modo più efficace per introdurre DNA plasmidico o siRNA in cellule primarie. Il gene Pulser sistema di elettroporazione MXcell e Gene buffer di elettroporazione Pulser (Bio-Rad) sono stati specificamente sviluppati per trasfettare facilmente acidi nucleici nelle cellule dei mammiferi e di difficile trasfezione delle cellule, come le cellule primarie e staminali. Dimostreremo come eseguire un semplice esperimento per identificare rapidamente le migliori condizioni di elettroporazione. Dimostreremo come eseguire diversi campioni attraverso una serie di condizioni elettroporazione in modo che un esperimento può essere condotto al tempo stesso l&#39;ottimizzazione viene eseguita. Mostreremo anche come condizioni ottimali identificati utilizzando piastre a 96 pozzetti elettroporazione può essere utilizzato con cuvette elettroporazione standard, facilitando il passaggio da piastre di elettroporazione ad cuvette elettroporazione, pur mantenendo la stessa efficienza elettroporazione. Nel video, si discuterà anche alcuni dei fattori chiave che possono portare al successo o al fallimento degli esperimenti di elettroporazione.

Watch this Scientific Journal Video about Using the Gene Pulser MXcell Electroporation System to Transfect Primary Cells with High Efficiency at JoVE.com

Using the Gene Pulser MXcell Electroporation System to Transfect Primary Cells with High Efficiency

Questa procedura mostra come utilizzare il sistema di elettroporazione Gene Pulser MXcell di identificare rapidamente e facilmente le migliori condizioni di elettroporazione di fibroblasti embrionali di topo (MEF) o altre cellule primarie. Considerazioni per la risoluzione dei problemi vengono discussi anche nel video associato.

Utilizzando il sistema di elettroporazione Gene Pulser MXcell per trasfettare cellule primarie con alta efficienza

It is becoming increasingly apparent that electroporation is the most effective way to introduce plasmid DNA or siRNA into primary cells. The Gene Pulser ...

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Nanyang Technological University

Research

JoVE Journal

Biology

24.1K Views.  Bio-Rad Laboratories, Inc.. Questa procedura mostra come utilizzare il sistema di elettroporazione Gene Pulser MXcell di identificare rapidamente e facilmente le migliori condizioni di elettroporazione di fibroblasti embrionali di topo (MEF) o altre cellule primarie. Considerazioni per la risoluzione dei problemi vengono discussi anche nel video associato.

Video: Using the Gene Pulser MXcell Electroporation System to Transfect Primary Cells with High Efficiency

Electroporation of Mycobacteria

High efficiency transformation is a major limitation in the study of mycobacteria. The genus Mycobacterium can be difficult to transform; this is mainly caused by the thick and waxy cell wall, but is compounded by the fact that most molecular techniques have been developed for distantly-related species such as Escherichia coli and Bacillus subtilis. In spite of these obstacles, mycobacterial plasmids have been identified and DNA transformation of many mycobacterial species have now been described. The most successful method for introducing DNA into mycobacteria is electroporation. Many parameters contribute to successful transformation; these include the species/strain, the nature of the transforming DNA, the selectable marker used, the growth medium, and the  conditions for the electroporation pulse. Optimized methods for the transformation of both slow- and fast-grower are detailed here. Transformation efficiencies for different mycobacterial species and with various selectable markers are reported.

Mycobacterial pathogenic strategies remain poorly understood. The slow growth rate of most species, the impenetrable nature of the cell-wall, and the hazards of working with pathogens make mycobacteria difficult to study and are largely responsible for our poor understanding of these organisms. In this video we will demonstrate the technique of electroporation, which involves subjecting cells to a brief high electrical impulse to allow the entry of DNA. It is the most widely used method for introducing DNA into mycobacterial cells.

Elettroporazione di micobatteri

High efficiency transformation is a major limitation in the study of mycobacteria. The genus Mycobacterium can be difficult to transform; this is mainly ...

Ricerca

Biologia

The Preparation of Primary Hematopoietic Cell Cultures From Murine Bone Marrow for Electroporation

It is becoming increasingly apparent that electroporation is the most effective way to introduce plasmid DNA or siRNA into primary cells. The Gene Pulser MXcell  electroporation system and Gene Pulser  electroporation buffer were specifically developed to transfect nucleic acids into mammalian cells and difficult-to-transfect cells, such as primary and stem cells.This video demonstrates how to establish primary hematopoietic cell cultures from murine bone marrow, and then prepare them for electroporation in the MXcell system. We begin by isolating femur and tibia. Bone marrow from both femur and tibia are then harvested and cultures are established. Cultured bone marrow cells are then transfected and analyzed.

This procedure describes how to establish primary hematopoietic cell cultures from murine bone marrow and is followed by transfection using the Gene Pulser MXCell electroporation system.

La preparazione di colture primarie di cellule emopoietiche dal midollo osseo murino per elettroporazione

In utero and ex vivo Electroporation for Gene Expression in Mouse Retinal Ganglion Cells

The retina and its sole output neuron, the retinal ganglion cell (RGC), comprise an excellent model in which to examine biological questions such as cell differentiation, axon guidance, retinotopic organization and synapse formation[1]. One drawback is the inability to efficiently and reliably manipulate gene expression in RGCs in vivo, especially in the otherwise accessible murine visual pathways. Transgenic mice can be used to manipulate gene expression, but this approach is often expensive, time consuming, and can produce unwanted side effects. In chick, in ovo electroporation is used to manipulate gene expression in RGCs for examining retina and RGC development. Although similar electroporation techniques have been developed in neonatal mouse pups[2], adult rats[3], and embryonic murine retinae in vitro[4], none of these strategies allow full characterization of RGC development and axon projections in vivo. To this end, we have developed two applications of electroporation, one in utero and the other ex vivo, to specifically target embryonic murine RGCs[5, 6]. 
With in utero retinal electroporation, we can misexpress or downregulate specific genes in RGCs and follow their axon projections through the visual pathways in vivo, allowing examination of guidance decisions at intermediate targets, such as the optic chiasm, or at target regions, such as the lateral geniculate nucleus. Perturbing gene expression in a subset of RGCs in an otherwise wild-type background facilitates an understanding of gene function throughout the retinal pathway. Additionally, we have developed a companion technique for analyzing RGC axon growth in vitro. We electroporate embryonic heads ex vivo, collect and incubate the whole retina, then prepare explants from these retinae several days later. Retinal explants can be used in a variety of in vitro assays in order to examine the response of electroporated RGC axons to guidance cues or other factors. In sum, this set of techniques enhances our ability to misexpress or downregulate genes in RGCs and should greatly aid studies examining RGC development and axon projections.

In utero and ex vivo Electroporation for Gene Expression in Mouse Retinal Ganglion Cells

Here we present two techniques for manipulating gene expression in murine retinal ganglion cells (RGCs) by in utero and ex vivo electroporation. These techniques enable one to examine how alterations in gene expression affect RGC development, axon guidance, and functional properties.

In elettroporazione in vivo ed ex utero per l&#39;espressione genica in cellule gangliari della retina del mouse

The retina and its sole output neuron, the retinal ganglion cell (RGC), comprise an excellent model in which to examine biological questions such as cell ...

Primary Culture and Plasmid Electroporation of the Murine Organ of Corti. 

In all mammals, the sensory epithelium for audition is located along the spiraling organ of Corti that resides within the conch shaped cochlea of the inner ear (fig 1). Hair cells in the developing cochlea, which are the mechanosensory cells of the auditory system, are aligned in one row of inner hair cells and three (in the base and mid-turns) to four (in the apical turn) rows of outer hair cells that span the length of the organ of Corti. Hair cells transduce sound-induced mechanical vibrations of the basilar membrane into neural impulses that the brain can interpret. Most cases of sensorineural hearing loss are caused by death or dysfunction of cochlear hair cells.
An increasingly essential tool in auditory research is the isolation and in vitro culture of the organ explant 1,2,9. Once isolated, the explants may be utilized in several ways to provide information regarding normative, anomalous, or therapeutic physiology. Gene expression, stereocilia motility, cell and molecular biology, as well as biological approaches for hair cell regeneration are examples of experimental applications of organ of Corti explants.
This protocol describes a method for the isolation and culture of the organ of Corti from neonatal mice. The accompanying video includes stepwise directions for the isolation of the temporal bone from mouse pups, and subsequent isolation of the cochlea, spiral ligament, and organ of Corti. Once isolated, the sensory epithelium can be plated and cultured in vitro in its entirety, or as a further dissected micro-isolate that lacks the spiral limbus and spiral ganglion neurons. Using this method, primary explants can be maintained for 7-10 days. As an example of the utility of this procedure, organ of Corti explants will be electroporated with an exogenous DsRed reporter gene. This method provides an improvement over other published methods because it provides reproducible, unambiguous, and stepwise directions for the isolation, microdissection, and primary culture of the organ of Corti.

Primary Culture and Plasmid Electroporation of the Murine Organ of Corti.

This procedure describes a method for the isolation and culture of the murine organ of Corti with or without the spiral limbus and spiral ganglion neurons. We also demonstrate a method for the expression of an exogenous reporter gene in the organ of Corti explant by electroporation.

Cultura primaria e elettroporazione plasmidi dell&#39;Organo murini di Corti.

In all mammals, the sensory epithelium for audition is located along the spiraling organ of Corti that resides within the conch shaped cochlea of the ...

Using an Automated Cell Counter to Simplify Gene Expression Studies: siRNA Knockdown of IL-4 Dependent Gene Expression in Namalwa Cells

The use of siRNA mediated gene knockdown is continuing to be an important tool in studies of gene expression.  siRNA studies are being conducted not only to study the effects of downregulating single genes, but also to interrogate signaling pathways and other complex interaction networks.  These pathway analyses require both the use of relevant cellular models and methods that cause less perturbation to the cellular physiology.  Electroporation is increasingly being used as an effective way to introduce siRNA and other nucleic acids into difficult to transfect cell lines and primary cells without altering the signaling pathway under investigation. There are multiple critical steps to a successful siRNA experiment, and there are ways to simplify the work while improving the data quality at several experimental stages.  To help you get started with your siRNA mediated gene knockdown project, we will demonstrate how to perform a pathway study complete from collecting and counting the cells prior to electroporation through post transfection real-time PCR gene expression analysis.  The following study investigates the role of the transcriptional activator STAT6 in IL-4 dependent gene expression of CCL17 in a Burkitt lymphoma cell line (Namalwa).  The techniques demonstrated are useful for a wide range of siRNA-based experiments on both adherent and suspension cells.  We will also show how to streamline cell counting with the TC10 automated cell counter, how to electroporate multiple samples simultaneously using the MXcell electroporation system, and how to simultaneously assess RNA quality and quantity with the Experion automated electrophoresis system.

This procedure describes a quick and easy workflow to introduce siRNA into difficult to transfect cell lines and follow gene expression by real-time PCR. Use of an automated cell counter, multi-well electroporation plate, and automated electrophoresis station provide quick and reliable results without the need for expensive robotic handling.

L&#39;utilizzo di un contatore automatico cella per semplificare gli studi sull&#39;espressione genica: Knockdown siRNA di IL-4 genica dipendente nelle celle Namalwa

The use of siRNA mediated gene knockdown is continuing to be an important tool in studies of gene expression.  siRNA studies are being conducted not only ...

Quantification of dsDNA using the Hitachi F-7000 Fluorescence Spectrophotometer and PicoGreen Dye

Quantification of DNA, especially in small concentrations, is an important task with a wide range of biological applications including standard molecular biology assays such as synthesis and purification of DNA, diagnostic applications such as quantification of DNA amplification products, and detection of DNA molecules in drug preparations. During this video we will demonstrate the capability of the Hitachi F-7000 Fluorescence Spectrophotometer equipped with a Micro Plate Reader accessory to perform dsDNA quantification using Molecular Probes Quant-it PicoGreen dye reagent kit.
The F-7000 Fluorescence Spectrophotometer offers high sensitivity and high speed measurements. It is a highly flexible system capable of measuring fluorescence, luminescence, and phosphorescence. Several measuring modes are available, including wavelength scan, time scan, photometry and 3-D scan measurement. The spectrophotometer has sensitivity in the range of 50 picomoles of fluorescein when using a 300 &#956;L sample volume in the microplate, and is capable of measuring scan speeds of 60,000 nm/minute. It also has a wide dynamic range of up to 5 orders of magnitude which allows for the use of calibration curves over a wide range of concentrations. The optical system uses all reflective optics for maximum energy and sensitivity. The standard wavelength range is 200 to 750 nm, and can be extended to 900 nm when using one of the optional near infrared photomultipliers. The system allows optional temperature control for the plate reader from 5 to 60 degrees Celsius using an optional external temperature controlled liquid circulator. The microplate reader allows for the use of 96 well microplates, and the measuring speed for 96 wells is less than 60 seconds when using the kinetics mode.
Software controls for the F-7000 and Microplate Reader are also highly flexible. Samples may be set in either column or row formats, and any combination of wells may be chosen for sample measurements. This allows for optimal utilization of the microplate. Additionally, the software allows importing micro plate sample configurations created in Excel and saved in comma separated values, or "csv" format. Microplate measuring configurations can be saved and recalled by the software for convenience and increased productivity. Data results can be output to a standard report, to Excel, or to an optional Report Generator Program.

Demonstration of quantification of dsDNA using Molecular Probes PicoGreen dye and Hitachi F-7000 Fluorescence Spectrophotometer equipped with a microplate reader accessory.

Quantificazione di dsDNA con il F-7000 Hitachi Fluorescenza spettrofotometro e PicoGreen Dye

Quantification of DNA, especially in small concentrations, is an important task with a wide range of biological applications including standard molecular ...

Genome-wide Gene Deletions in Streptococcus sanguinis by High Throughput PCR

Transposon mutagenesis and single-gene deletion are two methods applied in genome-wide gene knockout in bacteria 1,2. Although transposon mutagenesis is less time consuming, less costly, and does not require completed genome information, there are two weaknesses in this method: (1) the possibility of a disparate mutants in the mixed mutant library that counter-selects mutants with decreased competition; and (2) the possibility of partial gene inactivation whereby genes do not entirely lose their function following the insertion of a transposon. Single-gene deletion analysis may compensate for the drawbacks associated with transposon mutagenesis. To improve the efficiency of genome-wide single gene deletion, we attempt to establish a high-throughput technique for genome-wide single gene deletion using Streptococcus sanguinis as a model organism. Each gene deletion construct in S. sanguinis genome is designed to comprise 1-kb upstream of the targeted gene, the aphA-3 gene, encoding kanamycin resistance protein, and 1-kb downstream of the targeted gene. Three sets of primers F1/R1, F2/R2, and F3/R3, respectively, are designed and synthesized in a 96-well plate format for PCR-amplifications of those three components of each deletion construct. Primers R1 and F3 contain 25-bp sequences that are complementary to regions of the aphA-3 gene at their 5' end. A large scale PCR amplification of the aphA-3 gene is performed once for creating all single-gene deletion constructs. The promoter of aphA-3 gene is initially excluded to minimize the potential polar effect of kanamycin cassette. To create the gene deletion constructs, high-throughput PCR amplification and purification are performed in a 96-well plate format. A linear recombinant PCR amplicon for each gene deletion will be made up through four PCR reactions using high-fidelity DNA polymerase. The initial exponential growth phase of S. sanguinis cultured in Todd Hewitt broth supplemented with 2.5% inactivated horse serum is used to increase competence for the transformation of PCR-recombinant constructs. Under this condition, up to 20% of S. sanguinis cells can be transformed using ~50 ng of DNA. Based on this approach, 2,048 mutants with single-gene deletion were ultimately obtained from the 2,270 genes in S. sanguinis excluding four gene ORFs contained entirely within other ORFs in S. sanguinis SK36 and 218 potential essential genes. The technique on creating gene deletion constructs is high throughput and could be easy to use in genome-wide single gene deletions for any transformable bacteria.

Genome-wide Gene Deletions in Streptococcus sanguinis by High Throughput PCR

An efficient genome-wide single gene mutation method has been established using Streptococcus sanguinis as a model organism. This method has achieved via high throughput recombinant PCRs and transformations.

Genome-wide gene delezioni in<em&gt; Streptococcus sanguinis</em&gt; Da Throughput PCR ad alta

Transposon  mutagenesis and single-gene deletion are two methods applied in genome-wide  gene knockout in bacteria 1,2. Although transposon mutagenesis is ...

siRNA Screening to Identify Ubiquitin and Ubiquitin-like System Regulators of Biological Pathways in Cultured Mammalian Cells

Post-translational modification of proteins with ubiquitin and ubiquitin-like molecules (UBLs) is emerging as a dynamic cellular signaling network that regulates diverse biological pathways including the hypoxia response, proteostasis, the DNA damage response and transcription.&#160; To better understand how UBLs regulate pathways relevant to human disease, we have compiled a human siRNA &#8220;ubiquitome&#8221; library consisting of 1,186 siRNA duplex pools targeting all known and predicted components of UBL system pathways. This library can be screened against a range of cell lines expressing reporters of diverse biological pathways to determine which UBL components act as positive or negative regulators of the pathway in question.&#160; Here, we describe a protocol utilizing this library to identify ubiquitome-regulators of the HIF1A-mediated cellular response to hypoxia using a transcription-based luciferase reporter.&#160; An initial assay development stage is performed to establish suitable screening parameters of the cell line before performing the screen in three stages: primary, secondary and tertiary/deconvolution screening. &#160;The use of targeted over whole genome siRNA libraries is becoming increasingly popular as it offers the advantage of reporting only on members of the pathway with which the investigators are most interested.&#160; Despite inherent limitations of siRNA screening, in particular false-positives caused by siRNA off-target effects, the identification of genuine novel regulators of the pathways in question outweigh these shortcomings, which can be overcome by performing a series of carefully undertaken control experiments.

Here, we describe a methodology to perform a targeted siRNA &#8220;ubiquitome&#8221; screen to identify novel ubiquitin and ubiquitin-like regulators of the HIF1A-mediated cellular response to hypoxia.&#160; This can be adapted to any biological pathway where a robust read out of reporter activity is available.

siRNA screening per identificare ubiquitina e ubiquitina-come regolatori del sistema di percorsi biologici in cellule di mammifero coltivate

Post-translational modification of proteins with ubiquitin and ubiquitin-like molecules (UBLs) is emerging as a dynamic cellular signaling network that ...

Reverse Genetic Morpholino Approach Using Cardiac Ventricular Injection to Transfect Multiple Difficult-to-target Tissues in the Zebrafish Larva

The zebrafish is an important model to understand the cell and molecular biology of organ and appendage regeneration. However, molecular strategies to employ reverse genetics have not yet been adequately developed to assess gene function in regeneration or tissue homeostasis during larval stages after zebrafish embryogenesis, and several tissues within the zebrafish larva are difficult to target. Intraventricular injections of gene-specific morpholinos offer an alternative method for the current inability to genomically target zebrafish genes in a temporally controlled manner at these stages. This method allows for complete dispersion and subsequent incorporation of the morpholino into various tissues throughout the body, including structures that were formerly impossible to reach such as those in the larval caudal fin, a structure often used to noninvasively research tissue regeneration. Several genes activated during larval finfold regeneration are also present in regenerating adult vertebrate tissues, so the larva is a useful model to understand regeneration in adults. This morpholino dispersion method allows for the quick and easy identification of genes required for the regeneration of larval tissues as well as other physiological phenomena regulating tissue homeostasis after embryogenesis. Therefore, this delivery method provides a currently needed strategy for temporal control to the evaluation of gene function after embryogenesis.&#160;

An adaptable reverse genetic method for zebrafish to assess gene function during later stages of development and physiological homeostasis such as tissue regeneration using intraventricular injections of gene-specific morpholinos.

Reverse genetica Morpholino approccio Uso cardiaca ventricolare iniezione per transfettare diversi tessuti difficili da bersaglio in Zebrafish Larva

The zebrafish is an important model to understand the cell and molecular biology of organ and appendage regeneration. However, molecular strategies to ...

High Efficiency Differentiation of Human Pluripotent Stem Cells to Cardiomyocytes and Characterization by Flow Cytometry

There is an urgent need to develop approaches for repairing the damaged heart, discovering new therapeutic drugs that do not have toxic effects on the heart, and improving strategies to accurately model heart disease. The potential of exploiting human induced pluripotent stem cell (hiPSC) technology to generate cardiac muscle &#8220;in a dish&#8221; for these applications continues to generate high enthusiasm. In recent years, the ability to efficiently generate cardiomyogenic cells from human pluripotent stem cells (hPSCs) has greatly improved, offering us new opportunities to model very early stages of human cardiac development not otherwise accessible. In contrast to many previous methods, the cardiomyocyte differentiation protocol described here does not require cell aggregation or the addition of Activin A or BMP4 and robustly generates cultures of cells that are highly positive for cardiac troponin I and T (TNNI3, TNNT2), iroquois-class homeodomain protein IRX-4 (IRX4), myosin regulatory light chain 2, ventricular/cardiac muscle isoform (MLC2v) and myosin regulatory light chain 2, atrial isoform (MLC2a) by day 10 across all human embryonic stem cell (hESC) and hiPSC lines tested to date. Cells can be passaged and maintained for more than 90 days in culture. The strategy is technically simple to implement and cost-effective. Characterization of cardiomyocytes derived from pluripotent cells often includes the analysis of reference markers, both at the mRNA and protein level. For protein analysis, flow cytometry is a powerful analytical tool for assessing quality of cells in culture and determining subpopulation homogeneity. However, technical variation in sample preparation can significantly affect quality of flow cytometry data. Thus, standardization of staining protocols should facilitate comparisons among various differentiation strategies. Accordingly, optimized staining protocols for the analysis of IRX4, MLC2v, MLC2a, TNNI3, and TNNT2 by flow cytometry are described.

The article describes the detailed methodology to efficiently differentiate human pluripotent stem cells into cardiomyocytes by selectively modulating the Wnt pathway, followed by flow cytometry analysis of reference markers to assess homogeneity and identity of the population.

Alta efficienza differenziazione delle cellule staminali pluripotenti umane di cardiomiociti e Caratterizzazione mediante citometria di flusso

There is an urgent need to develop approaches for repairing the damaged heart, discovering new therapeutic drugs that do not have toxic effects on the ...