1. MEA Preparazione <ol><li> MEA per l&#39;uso negli esperimenti di elettroporazione possono essere fabbricati usando la tecnica standard di fotolitografia come descritto in precedenza 18 o acquistati direttamente da fornitori di produttori MEA come i sistemi multicanale <a href="http://www.multichannelsystems.com" target="_blank">(http:/www.multichannelsystems.com/)</a> e Alpha MED Scientific, Inc. <a href="http://www.med64.com/" target="_blank">( http://www.MED64.com)</a> . Le MEA utilizzati nei nostri esperimenti sono stati realizzati in-house presso l&#39;impianto camera pulita in Arizona State University, che è gestito dal Centro per la Ricerca Elettronica dello Stato Solido (CSSER). I MEA utilizzati nei nostri esperimenti avuto 32 elettrodi indio ossido di stagno in un 8 del 4 array con dimensioni elettrodi diametro 50-200 micron all&#39;interno di un vetro 1 cm di diametro interno bene. Bene il vetro è stato incollato sul usando MEA polidimetilsilossano (PDMS). </li><li> Sterilizzare il MEA in autoclave prima cella seeding. Altri metodi di sterilizzazione come un ammollo in 70% etanolo, trattamento UV e trattamento con acqua calda come descritto altrove può anche essere utilizzato fintanto che non compromettere l&#39;integrità del MEA. </li><li> Trasferire il autoclavato MEA di una cappa a flusso laminare e metterla in un piatto polistirene standard sterile Petri. Prima della semina di cellule sulla MEA, sterilizzare la cappa a flusso laminare per accendere la luce UV per 20 min. Se la MEA è sensibile alla luce UV coprire la piastra Petri contenente la MEA con foglio di alluminio sterile mentre la cappa a flusso è sotto illuminazione UV. </li></ol> 2. Cellule semina sul MEA <ol><li> Cellule raccolto da una linea cellulare di esecuzione aderenti cellule di mammifero usando Tripsina / EDTA e alzarle una concentrazione di 100-200 cellule / microlitro in media ml 1 cella per placcatura. Nei nostri esperimenti abbiamo avuto successo nella coltura delle cellule NIH 3T3 fibroblasti linea, linea di cellule HeLa e primari neuroni corticali e ippocampali sulla MEA. </li><li&gt; Mettere a 20-30 ml di goccia media con celle del MEA. Trasferire la piastra di Petri con la MEA per l&#39;incubatore. È importante garantire che la dimensione della goccia cella è sufficiente a coprire tutti gli elettrodi sulla MEA. </li><li> 2 ore dopo la semina delle cellule trasferire la capsula di Petri con la MEA dall&#39;incubatore a cappa a flusso laminare e aggiungere delicatamente 400-500 microlitri di pre supporti riscaldati (37 ° C) al MEA bene. Controllare al microscopio per assicurare che le cellule siano ancora attaccato e distribuito uniformemente sulla superficie MEA. Trasferire il MEA per l&#39;incubatrice. Il periodo di attesa 2 ore prima dell&#39;aggiunta di supporto assicura tempo sufficiente per le cellule di aderire alla superficie MEA. Aggiunta del supporto delle cellule troppo presto dopo la semina le cellule possono lavare via le cellule. Se le cellule non aderiscono sulla MEA, la sua superficie può essere trattata con fattori di adesione cellulare quali aspolylysine, fibronectina e laminina per migliorare l&#39;adesione delle cellule. Nei nostri esperimenti sulle cellule HeLa che di solito trattare la superficie con MEA fibronectin (10 pg / ml) per 1-2 ore. </li><li> 8-12 ore dopo la semina delle cellule, eseguire 1-2 lavaggi con PBS per rimuovere le cellule morte. Medie cellulare può sostituire ogni 24-48 ore, se necessario. In un tipico esperimento, le cellule sono pronte per la trasfezione di solito 24-48 ore dopo la semina. Per mantenere la coerenza in termini di efficienza elettroporazione si consiglia di attendere fino a quando la cultura è almeno l&#39;80% confluenti prima di elettroporazione. </li></ol> 3. Specifici per un sito trasfezione di siRNA <ol><li> Preparare una soluzione di acido nucleico. Per la trasfezione siRNA, preparare una soluzione di 1-2 siRNA pM elettroporazione in tampone ghiacciato elettroporazione per ottenere un volume finale di 200-400 microlitri. In questo articolo il video che dimostrano la trasfezione di cellule HeLa con Alexa 488 coniugato strapazzate sequenza di siRNA. </li><li> Trasferire il MEA con le cellule dal termostato a cappa sterile a flusso laminare. Rimuovere il supporto delle cellule del MEA ed eseguire un lavaggio con PBS. Successivamente, aggiungere 200-400 microlitri della ice freddo 1 pM elettroporazione soluzione siRNA al pozzo. </li><li> Applicare anodici impulsi quadri elettrici agli elettrodi mirati (Fare riferimento al punto 4 per la determinazione dei parametri ottimali di impulso elettrico). Nei nostri esperimenti abbiamo utilizzato un Instruments pragmatiche 2414A generatore di forme d&#39;onda per generare impulsi elettrici e un sistema dual-inline-package (DIP) 16 clip passante di entrare in contatto con i rilievi dei titoli di MEA. Il generatore di forme d&#39;onda può essere connesso alla clip DIP demultiplexor tramite una scheda a circuito stampato per fornire selettività dei singoli elettrodi. Per l&#39;elettrodo di riferimento, posizionare un catodo di acciaio nei media cella. Riducendo l&#39;elettrodo di riferimento acciaio nei media è estremamente importante assicurare che non fa contatto con la superficie MEA. Al contatto, può causare danni alle cellule e il MEA. Si raccomanda di ridurre l&#39;elettrodo di riferimento a una posizione di 1 mm sopra le celle. Un distanziatore con un&#39;altezza di 1 mm può essere posizionato nel pozzo per assistere nel posizionamento del refrimento elettrodo. Un approccio alternativo utilizzando un elettrodo di riferimento esterno è utilizzare elettrodi adiacenti come catodo-anodo coppie. </li><li> Subito dopo l&#39;applicazione degli impulsi elettrici agli elettrodi mirati sulla MEA, trasferire il ritorno alla MEA l&#39;incubatrice. Dopo un periodo di incubazione di 5 minuti, delicatamente sostituire il 75% del tampone di elettroporazione con pre-riscaldato (37 ° C) supporti cellulari. Successivamente, l&#39;imaging fluorescente può essere fatto per confermare la consegna di siRNA fluorescenti etichettato alle cellule. </li></ol> 4. Elettroporazione dei parametri di ottimizzazione <ol><li> Per determinare i parametri ottimali di impulsi elettrici per esperimenti di design elettroporazione, elettroporazione per un intervallo di valori di ampiezza dell&#39;impulso, durata dell&#39;impulso, e il numero di impulsi. Nella nostra esperienza con cellule HeLa, abbiamo osservato che un singolo impulso di tensione di ampiezza da 3 V a 9 V, e la durata di 1 msec a 10 msec per una dimensione di 100 pm elettrodo portato a elettroporazione successo con mila morte delle cellule Nimal. È nostra raccomandazione che un parametro variabile a un tempo e altri sono mantenute costanti. Per quantificare l&#39;efficienza di elettroporazione per ciascuno dei parametri di impulso, usiamo ioduro di propidio (PI), un colorante impermeant cella macchie materiale nucleico, e la metodologia basata assay vivo, come descritto nelle fasi 4,2-4,5. </li><li> Stabilire una coltura cellulare sulla MEA seguendo le indicazioni riportate ai punti 1 e 2. </li><li> Prima di preparare una elettroporazione 300-500 microlitri di 30 ug / ml soluzione PI in tampone ghiacciato elettroporazione e 400 pl di 4 um calcien AM soluzione in PBS. </li><li> Trasferire la MEA dall&#39;incubatore alla cappa a flusso laminare. Rimuovere il supporto delle cellule del MEA ed eseguire un lavaggio con PBS. Successivamente, aggiungere 300-500 microlitri del tampone freddo elettroporazione ghiaccio con PI. </li><li> Applicare impulsi elettrici con ampiezza di pulsazione desiderata e la durata agli elettrodi mirati (Le attrezzature utilizzate nei nostri esperimenti per la generazione e l&#39;applicazione del eletimpulso tric al MEA è descritto nel passaggio 3.3). Per l&#39;elettrodo di riferimento o abbassare un elettrodo di acciaio nel buffer elettroporazione nel MEA pozzo o utilizzare l&#39;elettrodo vicino come riferimento. Selettivamente applicando impulsi di ampiezza variabile o durate di elettrodi diversi esperimenti multipli può essere eseguita sul dispositivo stesso. Per esempio, su un elettrodo MEA 32, 8 esperimenti possono essere eseguiti suddividendo i 32 elettrodi in 8 gruppi, ognuno composto da 4 elettrodi. Ciascuno degli 8 gruppi possono essere stimolato da un singolo impulso rettangolare di durata tensione 5 msec con ampiezze diverse in 3 V-6 gamma V in incrementi di 0,5 V e un gruppo può essere utilizzato come controllo. </li><li> Immediatamente dopo l&#39;applicazione di impulsi di tensione trasferire il MEA per l&#39;incubatore per 5 min. Successivamente, rimuovere delicatamente il 75% del tampone di elettroporazione dal MEA pozzo e sostituirlo con supporti cellulari caldi. Trasferire il MEA per l&#39;incubatrice. </li><li> Dopo un periodo di incubazione di 2-4 hrsostituzione del supporto di cella nella MEA bene con 300-500 ml di 4 mM calceina AM soluzione in PBS. Trasferire il MEA di incubatrice per altri 30 min. </li><li> Dopo l&#39;incubazione delle cellule con calceina AM, sostituire la calceina AM soluzione nel MEA bene con PBS dopo due lavaggi con PBS. Ottenere immagini fluorescenti di cellule utilizzando con un microscopio ottico con filtri per la PI (eccitazione / emissione - 535 nm/617 nm) e calceina AM (eccitazione / emissione - 490 nm/520 nm). L&#39;assorbimento di PI, a causa di elettroporazione, induce le cellule a fluorescenza rossa e la AM Calcein induce le cellule che sono vivi a fluorescenza verde. Tre possibili scenari che si possono osservare sono: 1) cellule elettroporate e vivo (si fluorescenza sia rosso e verde), 2) cellule vive, ma non elettroporate (sarà solo fluorescenza verde) e 3) le cellule morte a causa di elettroporazione (sarà solo fluorescenza rossa) . La vitalità cellulare è stata calcolata come percentuale del numero totale di cellule vive sull&#39;elettrodo e l&#39;eff elettroporazioneiciency è stata calcolata come percentuale del numero totale di cellule caricate con PI / siRNA. </li></ol> 5. Risultati rappresentativi La figura 1 mostra il sito-specifica caricamento di cellule HeLa con PI. Si può osservare che le cellule solo sull&#39;elettrodo dimostrano un assorbimento di PI (Figura 1B). Un test dal vivo eseguita elettroporazione messaggio è stato utilizzato per valutare la vitalità delle cellule (Figura 1A). L&#39;efficienza elettroporazione e la vitalità cellulare per l&#39;esempio mostrato nella figura 1 sono 81,8% e 96,1% rispettivamente. Vitalità cellulare ed efficienza elettroporazione può essere ottenuto ottimizzando i parametri di impulso di elettroporazione. Sito-specifica trasfezione di cellule HeLa con siRNA fluorescente etichettato per un impulso di elettroporazione ottimizzato (8 V, 1 msec) è mostrato in Figura 2. L&#39;efficienza elettroporazione per il siRNA a 8 V, 1 msec è risultato essere 74,4 ± 16,0% evitalità cellulare è risultato essere 87,9 ± 8,4% (tutti i dati rappresentati come media ± deviazione standard, N = 5). <img alt="Figura 1" src="/files/ftp_upload/4415/4415fig1.jpg" /> Figura 1. Sito-specifica consegna di PI in cellule HeLa su un elettrodo 200 micron (6 V, 5 msec). A) Calcien saggio basato vivo eseguita due ore dopo l&#39;elettroporazione. Cellule vive sono indicati con fluorescenza verde. B) Red fluorescenza dimostra l&#39;assorbimento da parte delle cellule del PI sull&#39;elettrodo. C) Una immagine sovrapposta di A e B. Le frecce bianche indicano cellule che sono elettroporata e vivo. Le frecce nere indicano cellule che sono morti. Il cerchio bianco in A, B e C indica la sagoma dell&#39;elettrodo. <img alt="Figura 2" src="/files/ftp_upload/4415/4415fig2.jpg" /> Figura 2. Trasfezione di cellule HeLa con scrambl fluorescente etichettatoLa sequenza di siRNA. A) Immagine di cellule HeLa su un elettrodo di 100 um trasfettate con Alexa 488 siRNA tag (8 V, 1 msec). B) Immagine di nuclei di cellule colorate con DAPI. Il cerchio bianco segna il bordo dell&#39;elettrodo.

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In questo articolo il video che dimostrano l&#39;uso di MEA per il sito-specifica trasfezione di cellule HeLa con strapazzate sequenza di siRNA. Uno dei vantaggi di questa tecnologia è la sua applicabilità a diverse linee cellulari incluse linee cellulari primarie. Il nostro laboratorio ha già dimostrato l&#39;uso di questa tecnologia per il sito-specifica trasfezione di colture primarie neuronali dell&#39;ippocampo di ratto E18 dal giorno di vita e NIH-3T3 strapazzate con sequenze siRNA e GFP plasmide 18, 19.</...

<table border="1"><tr><td> Nome del reagente </td><td> Azienda </td><td> Numero di catalogo </td><td> Commenti (opzionale) </td></tr><tr><td> Cellulari media: Avanzata MEM L-Glutammina 200 mM Penicillina / streptomicina Siero fetale bovino </td><td> Gibco / Invitrogen Himedia Laboratori / VWR Lonza Group Ltd. Gibco / Invitrogen </td><td> 12492-013 95057-448 09-757F 16000-044 </td><td> Composizione cella media: 2% FBS, Pennstrep 2% L-glutammina e il 2% in Advance MEM </td></tr><tr><td> Tripsina EDTA </td><td> Mediatech, Inc. </td><td> 25-053-CL </td><td></td></tr><tr><td> PBS </td><td> Mediatech, Inc. </td><td> 21-040-CV </td><td></td></tr><tr><td> Alexa 488 e rodamina tagged strapazzate sequrienza siRNA </td><td> Qiagen, Inc. </td><td> 1027292 </td><td></td></tr><tr><td> Elettroporazione tampone </td><td> Biorad Laboratories </td><td> 165-2677 </td><td></td></tr><tr><td> Generatore di forme d&#39;onda </td><td> Pragmatico </td><td> 2414A </td><td> Qualsiasi forma d&#39;onda / generatore di impulsi che può fornire gli impulsi desiderati possono essere utilizzati. </td></tr></table>

high efficiency, site-specific transfection of adherent cells with sirna using microelectrode arrays (mea)

La scoperta della via di RNAi in eucarioti e il conseguente sviluppo di agenti RNAi, come siRNA e shRNA, hanno raggiunto un metodo efficace per mettere a tacere i geni specifici 1-8 per la genomica funzionale e terapie. Una sfida importante coinvolto in studi basati su RNAi è la consegna di agenti RNAi alle cellule bersaglio. Tradizionali non virali tecniche di parto, come l&#39;elettroporazione di massa e metodi di trasfezione chimici spesso non hanno il controllo spaziale necessaria su consegna e permettere l&#39;efficienza di trasfezione poveri 9-12. I recenti progressi nei metodi di trasfezione chimici come lipidi cationici, polimeri cationici e nanoparticelle hanno portato ad efficienze di trasfezione altamente avanzate 13. Tuttavia, queste tecniche ancora non riescono a offrire un controllo preciso dello spazio sulla consegna che possono beneficiare immensamente miniaturizzata tecnologie high-throughput, studi cella singola e le indagini di interazioni cellula-cellula. nt &quot;&gt; I recenti progressi tecnologici nella consegna del gene hanno permesso alta produttività trasfezione di cellule aderenti 14-23, la maggioranza dei quali utilizzano elettroporazione microscala. elettroporazione Microscale offre spazio-temporale preciso controllo di consegna (fino a singole cellule) ed è stato dimostrato per ottenere alte efficienze 19, 24-26. Inoltre, approcci basati elettroporazione non richiedono un lungo periodo di incubazione (tipicamente 4 ore) con complessi siRNA e DNA come necessario in metodi di trasfezione base chimica e causare l&#39;immissione diretta di siRNA nudo e DNA molecole nel citoplasma cellulare. Di conseguenza espressione genica può essere ottenuto fino a sei ore dopo la trasfezione 27. nostro laboratorio ha precedentemente dimostrato l&#39;utilizzo di matrici di microelettrodi (MEA) per sito-specifica trasfezione aderenti in colture cellulari di mammifero 17-19. Nell&#39;approccio basato MEA, la consegna del carico utile genetica avviene tramite localizzato micro-scala electroporatisu di cellule. Una applicazione di impulsi elettrici agli elettrodi selezionati genera locale campo elettrico che conduce alla elettroporazione di cellule presenti nella regione degli elettrodi stimolati. Il controllo indipendente delle micro-elettrodi fornisce controllo spaziale e temporale sopra trasfezione e permette anche esperimenti di trasfezione multipli basati da eseguire sulla stessa cultura aumentando la produttività e riducendo sperimentale cultura a cultura variabilità. Qui si descrive l&#39;apparato sperimentale e il protocollo per la trasfezione mirata di aderenti cellule HeLa con una fluorescente tag criptato siRNA sequenza usando elettroporazione. Lo stesso protocollo può essere utilizzato anche per la trasfezione di vettori plasmidici. Inoltre, il protocollo qui descritto può essere facilmente estesa ad una varietà di linee cellulari di mammifero con modifiche secondarie. Disponibilità commerciale degli accordi ambientali multilaterali con i modelli di elettrodi sia predefiniti e personalizzati rendono questa t accessibile tecnicala maggior parte dei laboratori di ricerca o con attrezzature di base di coltura cellulare.

Watch this Scientific Journal Video about High efficiency, Site-specific Transfection of Adherent Cells with siRNA Using Microelectrode Arrays MEA at JoVE.com

High efficiency, Site-specific Transfection of Adherent Cells with siRNA Using Microelectrode Arrays MEA

L&#39;articolo descrive il protocollo per sito-specifica di transfezione criptato sequenza di siRNA in una coltura cellulare di mammifero aderente utilizzando una matrice di microelettrodi (MEA).

Alta efficienza, sito-specifica trasfezione di cellule aderenti con siRNA mediante matrici microelettrodi (MEA)

The discovery of RNAi pathway in eukaryotes and the subsequent development of RNAi agents, such as siRNA and shRNA, have achieved a potent method for ...

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Research

JoVE Journal

Bioengineering

High efficiency, Site-specific Transfection of Adherent Cells with siRNA Using Microelectrode Arrays (MEA)

13.4K Views.  Arizona State University. L'articolo descrive il protocollo per sito-specifica di transfezione criptato sequenza di siRNA in una coltura cellulare di mammifero aderente utilizzando una matrice di microelettrodi (MEA).

Video: High efficiency, Site-specific Transfection of Adherent Cells with siRNA Using Microelectrode Arrays MEA

Therapeutic Gene Delivery and Transfection in Human Pancreatic Cancer Cells using Epidermal Growth Factor Receptor-targeted Gelatin Nanoparticles

More than 32,000 patients are diagnosed with pancreatic cancer in the United States per year and the disease is associated with very high mortality 1. Urgent need exists to develop novel clinically-translatable therapeutic strategies that can improve on the dismal survival statistics of pancreatic cancer patients. Although gene therapy in cancer has shown a tremendous promise, the major challenge is in the development of safe and effective delivery system, which can lead to sustained transgene expression. 
Gelatin is one of the most versatile natural biopolymer, widely used in food and pharmaceutical products. Previous studies from our laboratory have shown that type B gelatin could physical encapsulate DNA, which preserved the supercoiled structure of the plasmid and improved transfection efficiency upon intracellular delivery. By thiolation of gelatin, the sulfhydryl groups could be introduced into the polymer and would form disulfide bond within nanoparticles, which stabilizes the whole complex and once disulfide bond is broken due to the presence of glutathione in cytosol, payload would be released 2-5. Poly(ethylene glycol) (PEG)-modified GENS, when administered into the systemic circulation, provides long-circulation times and preferentially targets to the tumor mass due to the hyper-permeability of the neovasculature by the enhanced permeability and retention effect 6. Studies have shown over-expression of the epidermal growth factor receptor (EGFR) on Panc-1 human pancreatic adenocarcinoma cells 7. In order to actively target pancreatic cancer cell line, EGFR specific peptide was conjugated on the particle surface through a PEG spacer.8
Most anti-tumor gene therapies are focused on administration of the tumor suppressor genes, such as wild-type p53 (wt-p53), to restore the pro-apoptotic function in the cells 9. The p53 mechanism functions as a critical signaling pathway in cell growth, which regulates apoptosis, cell cycle arrest, metabolism and other processes 10. In pancreatic cancer, most cells have mutations in p53 protein, causing the loss of apoptotic activity. With the introduction of wt-p53, the apoptosis could be repaired and further triggers cell death in cancer cells 11. 
Based on the above rationale, we have designed EGFR targeting peptide-modified thiolated gelatin nanoparticles for wt-p53 gene delivery and evaluated delivery efficiency and transfection in Panc-1 cells.

Type B gelatin-based engineered nanovectors system (GENS) was developed for systemic gene delivery and transfection in the treatment of pancreatic cancer. By modification with epidermal growth factor receptor (EGFR) specific peptide on the surface of nanparticles, they could target on EGFR receptor and release plasmid under reducing environment, such as high intracellular glutathione concentrations.

Consegna dei geni e trasfezione in cellule umane di cancro al pancreas con Epidermal Growth Factor Receptor mirati Nanovectors Progettato a base di gelatina

More than 32,000 patients are diagnosed with pancreatic cancer in the United States per year and the disease is associated with very high mortality 1. ...

Ricerca

Bioingegneria

Site-specific Bacterial Chromosome Engineering: &#934;C31 Integrase Mediated Cassette Exchange (IMCE)

The bacterial chromosome may be used to stably maintain foreign DNA in the mega-base range1. Integration into the chromosome circumvents issues such as plasmid replication, plasmid stability, plasmid incompatibility, and plasmid copy number variance. This method uses the site-specific integrase from the Streptomyces phage (&Phi;) C312,3. The &Phi;C31 integrase catalyzes a direct recombination between two specific DNA sites: attB and attP (34 and 39 bp, respectively)4. This recombination is stable and does not revert5. A "landing pad" (LP) sequence consisting of a spectinomycin- resistance gene, aadA (SpR), and the E. coli &szlig;-glucuronidase gene (uidA) flanked by attP sites has been integrated into the chromosomes of Sinorhizobium meliloti, Ochrobactrum anthropi, and Agrobacterium tumefaciens in an intergenic region, the ampC locus, and the tetA locus, respectively. S. meliloti is used in this protocol. Mobilizable donor vectors containing attB sites flanking a stuffer red fluorescent protein (rfp) gene and an antibiotic resistance gene have also been constructed. In this example the gentamicin resistant plasmid pJH110 is used. The rfp gene6 may be replaced with a desired construct using SphI and PstI. Alternatively a synthetic construct flanked by attB sites may be sub-cloned into a mobilizable vector such as pK19mob7. The expression of the &Phi;C31 integrase gene (cloned from pHS628) is driven by the lac promoter, on a mobilizable broad host range plasmid pRK78139.
A tetraparental mating protocol is used to transfer the donor cassette into the LP strain thereby replacing the markers in the LP sequence with the donor cassette. These cells are trans-integrants. Trans-integrants are formed with a typical efficiency of 0.5%. Trans-integrants are typically found within the first 500-1,000 colonies screened by antibiotic sensitivity or blue-white screening using 5-bromo-4-chloro-3-indolyl-beta-D-glucuronic acid (X-gluc). This protocol contains the mating and selection procedures for creating and isolating trans-integrants.

Site-specific Bacterial Chromosome Engineering: &amp;#934;C31 Integrase Mediated Cassette Exchange (IMCE)

A quick and efficient method to integrate foreign DNA of interest into pre-made acceptor strains, termed landing pad strains, is described. The method allows site-specific integration of a DNA cassette into the engineered landing pad locus of a given strain, through conjugation and expression of the &#934;C31 integrase.

Site-specific Ingegneria batterica Cromosoma: ΦC31 integrasi Mediated Cassette Exchange (IMCE)

The bacterial chromosome may be used to stably maintain foreign DNA in the mega-base range1. Integration into the chromosome circumvents issues such as ...

Engineering Adherent Bacteria by Creating a Single Synthetic Curli Operon

The method described here consists in redesigning E. coli adherence properties by assembling the minimum number of curli genes under the control of a strong and metal-overinducible promoter, and in visualizing and quantifying the resulting gain of bacterial adherence. This method applies appropriate engineering principles of abstraction and standardization of synthetic biology, and results in the BBa_K540000 Biobrick (Best new Biobrick device, engineered, iGEM 2011).
The first step consists in the design of the synthetic operon devoted to curli overproduction in response to metal, and therefore in increasing the adherence abilities of the wild type strain. The original curli operon was modified in silico in order to optimize transcriptional and translational signals and escape the "natural" regulation of curli. This approach allowed to test with success our current understanding of curli production. Moreover, simplifying the curli regulation by switching the endogenous complex promoter (more than 10 transcriptional regulators identified) to a simple metal-regulated promoter makes adherence much easier to control.
The second step includes qualitative and quantitative assessment of adherence abilities by implementation of simple methods. These methods are applicable to a large range of adherent bacteria regardless of biological structures involved in biofilm formation. Adherence test in 24-well polystyrene plates provides a quick preliminary visualization of the bacterial biofilm after crystal violet staining. This qualitative test can be sharpened by the quantification of the percentage of adherence. Such a method is very simple but more accurate than only crystal violet staining as described previously 1 with both a good repeatability and reproducibility. Visualization of GFP-tagged bacteria on glass slides by fluorescence or laser confocal microscopy allows to strengthen the results obtained with the 24-well plate test by direct observation of the phenomenon.

The design of a synthetic operon encoding both the secretory apparatus and the structural monomers of curli fibers is described. Overproduction of these amyloids and adherent polymers allows a measurable gain of adherence of the E. coli chassis1. Easy ways to visualize and quantify adherence are explained.

Ingegneria batteri aderenti mediante la creazione di un unico sintetico curli Operon

The  method described here consists in redesigning E. coli adherence properties by assembling the minimum number of  curli genes under the control of a ...

Characteristics of Precipitation-formed Polyethylene Glycol Microgels Are Controlled by Molecular Weight of Reactants

This work describes the formation of poly(ethylene glycol) (PEG) microgels via a photopolymerized precipitation reaction. Precipitation reactions offer several advantages over traditional microsphere fabrication techniques. Contrary to emulsion, suspension, and dispersion techniques, microgels formed by precipitation are of uniform shape and size, i.e.&#160;low polydispersity index, without the use of organic solvents or stabilizers. The mild conditions of the precipitation reaction, customizable properties of the microgels, and low viscosity for injections make them applicable for in vivo purposes. Unlike other fabrication techniques, microgel characteristics can be modified by changing the starting polymer molecular weight. Increasing the starting PEG molecular weight increased microgel diameter and swelling ratio. Further modifications are suggested such as encapsulating molecules during microgel crosslinking. Simple adaptations to the PEG microgel building blocks are explored for future applications of microgels as drug delivery vehicles and tissue engineering scaffolds.

This work describes the formation of poly(ethylene glycol) (PEG) microgels via a photopolymerized precipitation reaction. Increasing the PEG molecular weight increased microgel diameter and swelling ratio. Simple adaptations to the PEG microgel precipitation reaction are explored for future applications of microgels as drug delivery vehicles and tissue engineering scaffolds.

This work describes the formation of poly(ethylene glycol) (PEG) microgels via a photopolymerized precipitation reaction. Precipitation reactions offer ...

Ordering Single Cells and Single Embryos in 3D Confinement: A New Device for High Content Screening

Biological cells are usually observed on flat (2D) surfaces. This condition is not physiological, and phenotypes and shapes are highly variable. Screening based on cells in such environments have therefore serious limitations: cell organelles show extreme phenotypes, cell morphologies and sizes are heterogeneous and/or specific cell organelles cannot be properly visualized. In addition, cells in vivo are located in a 3D environment; in this situation, cells show different phenotypes mainly because of their interaction with the surrounding extracellular matrix of the tissue. In order to standardize and generate order of single cells in a physiologically-relevant 3D environment for cell-based assays, we report here the microfabrication and applications of a device for in vitro 3D cell culture. This device consists of a 2D array of microcavities (typically 105 cavities/cm2), each filled with single cells or embryos. Cell position, shape, polarity and internal cell organization become then normalized showing a 3D architecture. We used replica molding to pattern an array of microcavities, &#8216;eggcups&#8217;, onto a thin polydimethylsiloxane (PDMS) layer adhered on a coverslip. Cavities were covered with fibronectin to facilitate adhesion. Cells were inserted by centrifugation. Filling percentage was optimized for each system allowing up to 80%. Cells and embryos viability was confirmed. We applied this methodology for the visualization of cellular organelles, such as nucleus and Golgi apparatus, and to study active processes, such as the closure of the cytokinetic ring during cell mitosis. This device allowed the identification of new features, such as periodic accumulations and inhomogeneities of myosin and actin during the cytokinetic ring closure and compacted phenotypes for Golgi and nucleus alignment. We characterized the method for mammalian cells, fission yeast, budding yeast, C. elegans with specific adaptation in each case. Finally, the characteristics of this device make it particularly interesting for drug screening assays and personalized medicine.

We report a device and a new method to study cells and embryos. Single cells are precisely ordered in microcavity arrays. Their 3D confinement is a step towards 3D environments encountered in physiological conditions and allows organelle orientation. By controlling cell shape, this setup minimizes variability reported in standard assays.

Ordinazione di singole cellule e singola embrioni in 3D confinamento: Un nuovo dispositivo per l&#39;alta Screening contenuti

Biological cells are usually observed on flat (2D) surfaces. This condition is not physiological, and phenotypes and shapes are highly variable. Screening ...

Porous Silicon Microparticles for Delivery of siRNA Therapeutics

Small interfering RNA (siRNA) can be used to suppress gene expression, thereby providing a new avenue for the treatment of various diseases. However, the successful implementation of siRNA therapy requires the use of delivery platforms that can overcome the major challenges of siRNA delivery, such as enzymatic degradation, low intracellular uptake and lysosomal entrapment. Here, a protocol for the preparation and use of a biocompatible and effective siRNA delivery system is presented. This platform consists of polyethylenimine (PEI) and arginine (Arg)-grafted porous silicon microparticles, which can be loaded with siRNA by performing a simple mixing step. The silicon particles are gradually degraded over time, thereby triggering the formation of Arg-PEI/siRNA nanoparticles. This delivery vehicle provides a means for protecting and internalizing siRNA, without causing cytotoxicity. The major steps of polycation functionalization, particle characterization, and siRNA loading are outlined in detail. In addition, the procedures for determining particle uptake, cytotoxicity, and transfection efficacy are also described.

Delivery remains the main challenge for the therapeutic implementation of small interfering RNA (siRNA). This protocol involves the use of a multifunctional and biocompatible siRNA delivery platform, consisting of arginine and polyethylenimine grafted porous silicon microparticles.

Poroso Silicon microparticelle per consegna di siRNA Therapeutics

Small interfering RNA (siRNA) can be used to suppress gene expression, thereby providing a new avenue for the treatment of various diseases. However, the ...

Gene Transfection toward Spheroid Cells on Micropatterned Culture Plates for Genetically-modified Cell Transplantation

To improve the therapeutic effectiveness of cell transplantation, a transplantation system of genetically modified, injectable spheroids was developed. The cell spheroids are prepared in a culture system on micropatterned plates coated with a thermosensitive polymer. A number of spheroids are formed on the plates, corresponding to the cell adhesion areas of 100 &#181;m diameter that are regularly arrayed in a two-dimensional manner, surrounded by non-adhesive areas that are coated by a polyethylene glycol (PEG) matrix. The spheroids can be easily recovered as a liquid suspension by lowering the temperature of the plates, and their structure is well maintained by passing them through injection needles with a sufficiently large caliber (over 27 G). Genetic modification is achieved by gene transfection using the original non-viral gene carrier, polyplex nanomicelle, which is capable of introducing genes into cells without disrupting the spheroid structure. For primary hepatocyte spheroids transfected with a luciferase-expressing gene, the luciferase is sustainably obtained in transplanted animals, along with preserved hepatocyte function, as indicated by albumin expression. This system can be applied to a variety of cell types including mesenchymal stem cells.

This protocol describes a cell transplantation system using genetically modified, injectable spheroids. Cell spheroids are cultured on micropatterned culture plates and recovered after gene introduction using polyplex nanomicelles. This system facilitates prolonged transgene expression from the transplanted cells in host animals while maintaining the innate function of the cells.

Gene Transfection verso Celle sferoide su lastre micropatterned coltura per trapianto di cellule geneticamente modificati

To improve the therapeutic effectiveness of cell transplantation, a transplantation system of genetically modified, injectable spheroids was developed. ...

Easy and Accurate Mechano-profiling on Micropost Arrays

Cell culture substrates with integrated flexible microposts enable a user to study the mechanical interactions between cells and their immediate surroundings. Particularly, cell-substrate interactions are the main interest. Today micropost arrays are a well-characterized and established method with a broad range of applications that have been published over the last decade. However, there seems to be a reservation among biologists to adapt the technique due to the lengthy and challenging process of micropost manufacture along with the lack of easily approachable software for analyzing images of cells interacting with microposts. 
The force read-out from microposts is surprisingly easy. A micropost acts like a spring with the cell ideally attached at its tip. Depending on size a cell applies force from its cytoskeleton through one or multiple focal adhesion points to the micropost, thus deflecting the micropost. The amount of deflection correlates directly to the applied force in direction and in magnitude. The number of microposts covered by a cell and the post deflection patterns are characteristic and allow determination of values like force per post and many biologically relevant parameters that allow &#8220;mechano-profiling&#8221; of cell phenotypes.
A convenient method for mechano-profiling is described here combining the first generation of ready-to-use commercially available microposts with an in-house developed software package that is now accessible to all researchers. As a demonstration of typical application, single images of bone cancer cells were taken in bright-field microscopy for mechano-profiling of cell line models of metastasis. This combination of commercial traction force sensors and open source software for analysis allows for the first time a rapid implementation of the micropost array technique into routine lab work done by non-expert users. Furthermore, a robust and streamlined analysis process enables a user to analyze a large number of micropost images in a highly time-efficient manner.

Described are protocols for quantifying mechanical interactions between adherent cells and microstructured substrates. These interactions are closely linked to essential cell behaviors including migration, proliferation, differentiation, and apoptosis. The protocols present an open-source image analysis software called MechProfiler, which enables determination of involved forces for each micropost.

Facile e preciso meccano-profiling su micropost Array

Cell culture substrates with integrated flexible microposts enable a user to study the mechanical interactions between cells and their immediate ...

Delivery of Therapeutic siRNA to the CNS Using Cationic and Anionic Liposomes

Prion diseases result from the misfolding of the normal, cellular prion protein (PrPC) to an abnormal protease resistant isomer called PrPRes. The emergence of prion diseases in wildlife populations and their increasing threat to human health has led to increased efforts to find a treatment for these diseases. Recent studies have found numerous anti-prion compounds that can either inhibit the infectious PrPRes isomer or down regulate the normal cellular prion protein. However, most of these compounds do not cross the blood brain barrier to effectively inhibit PrPRes formation in brain tissue, do not specifically target neuronal PrPC, and are often too toxic to use in animal or human subjects. 
We investigated whether siRNA delivered intravascularly and targeted towards neuronal PrPC is a safer and more effective anti-prion compound. This report outlines a protocol to produce two siRNA liposomal delivery vehicles, and to package and deliver PrP siRNA to neuronal cells. The two liposomal delivery vehicles are 1) complexed-siRNA liposome formulation using cationic liposomes (LSPCs), and 2) encapsulated-siRNA liposome formulation using cationic or anionic liposomes (PALETS). For the LSPCs, negatively charged siRNA is electrostatically bound to the cationic liposome. A positively charged peptide (RVG-9r [rabies virus glycoprotein]) is added to the complex, which specifically targets the liposome-siRNA-peptide complexes (LSPCs) across the blood brain barrier (BBB) to acetylcholine expressing neurons in the central nervous system (CNS). For the PALETS (peptide addressed liposome encapsulated therapeutic siRNA), the cationic and anionic lipids were rehydrated by the PrP siRNA. This procedure results in encapsulation of the siRNA within the cationic or anionic liposomes. Again, the RVG-9r neuropeptide was bound to the liposomes to target the siRNA/liposome complexes to the CNS. Using these formulations, we have successfully delivered PrP siRNA to AchR-expressing neurons, and decreased the PrPC expression of neurons in the CNS.

The goal of this protocol is to use cationic/anionic liposomes with a neuro-targeting peptide as a CNS delivery system to enable siRNA to cross the BBB. The optimization of a delivery system for treatments, like siRNA, would allow for more treatment options for prion and other neurodegenerative diseases.

La consegna dei terapeutica siRNA al SNC Utilizzando cationici e anionici liposomi

Prion diseases result from the misfolding of the normal, cellular prion protein (PrPC) to an abnormal protease resistant isomer called PrPRes.  The ...

Using Synthetic Biology to Engineer Living Cells That Interface with Programmable Materials

We have developed an abiotic-biotic interface that allows engineered cells to control the material properties of a functionalized surface. This system is made by creating two modules: a synthetically engineered strain of E. coli cells and a functionalized material interface. Within this paper, we detail a protocol for genetically engineering selected behaviors within a strain of E. coli using molecular cloning strategies. Once developed, this strain produces elevated levels of biotin when exposed to a chemical inducer. Additionally, we detail protocols for creating two different functionalized surfaces, each of which is able to respond to cell-synthesized biotin. Taken together, we present a methodology for creating a linked, abiotic-biotic system that allows engineered cells to control material composition and assembly on nonliving substrates.

This paper presents a series of protocols for developing engineered cells and functionalized surfaces that enable synthetically engineered E. coli to control and manipulate programmable material surfaces.

Utilizzando biologia sintetica per Ingegnere cellule viventi che si interfacciano con materiali programmabili

We have developed an abiotic-biotic interface that allows engineered cells to control the material properties of a functionalized surface. This system is ...