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Cell Transfection Cell transfektering

This Transfection site is your portal for all things related to the transfection of cells with DNA plasmid or vector, RNA whether siRNA or RNAi, or even recombinant protein. Denne transfektering netsted er din portal for alle ting i relation til transfektering af celler med DNA plasmid eller vektor, RNA, om siRNA eller RNAi eller endda rekombinante proteiner. We provide you with all the background information, protocols for cell transfection, optimization and analysis steps, and cell transfection troubleshooting information to become the ultimate expert! Vi giver dig alle de baggrundsoplysninger, protokoller for celle transfektering, optimering og analyse trin, og cellen transfektering oplysninger om fejlfinding at blive den ultimative ekspert!

Transfection Table of Contents Transfektering Indholdsfortegnelse

• What is transfection? Hvad er transfektering?
• Background Baggrund
• Applications Ansøgninger
• Materials Materialer
• Transfection Methods Transfektering Metoder
• Buffers Buffers
  
• Preparation Forberedelse
• Protocol Protokol
• Other T ransfection Protocols Andre T ransfection protokoller
• Discussion Topics for Transfection Diskussionsemner på transfektering
• Get Help on your Transfection Problems Hjælp og support på din transfektering Problemer
• Tips Tips
• Storing Opbevaring
• Troubleshooting In Transfection Fejlfinding I transfektering
• References Referencer

What is Transfection? Hvad er transfektering?

Transfection describes the introduction of foreign material into eukaryotic cells using a virus vector or other means of transfer. Transfektering beskriver indførelsen af fremmed materiale i eukaryotiske celler ved hjælp af en virus vektor eller andre former for overdragelse.

The term transfection for non-viral methods is most often used in reference to mammalian cells, while the term transformation is preferred to describe non-viral DNA transfer in bacteria and non-animal eukaryotic cells such as fungi, algae and plants. Udtrykket transfektering for ikke-virale metoder er oftest bruges i reference til pattedyrceller, mens begrebet transformation er foretrukket at beskrive ikke-viral DNA-overførsel i bakterier og ikke-animalsk eukaryotiske celler såsom svampe, alger og planter.

Transfection of animal cells typically involves opening transient pores or 'holes' in the cell plasma membrane, to allow the uptake of material. Transfektering af animalske celler typisk indebærer åbning af forbigående porer eller 'huller' i cellen plasma membranen, til at tillade udnyttelsen af materialet. Genetic material (such as supercoiled plasmid DNA or siRNA constructs), or even proteins such as antibodies, may be transfected. Genetiske materiale (såsom supercoiled plasmid DNA eller siRNA konstruktioner), eller endog proteiner som antistoffer, kan transfected. In addition to electroporation, transfection can be carried out by mixing a cationic lipid with the material to produce liposomes, which fuse with the cell plasma membrane and deposit their cargo inside. Ud over elektroinjektion, transfektering kan udføres ved at blande en kationisk lipid med materiale til fremstilling af liposomer, som sikring med cellens plasma membranen og deponere deres last indeni.

The original meaning of transfection was 'infection by transformation', ie introduction of DNA (or RNA) from a eukaryote virus or bacteriophage into cells, resulting in an infection. Den oprindelige betydning af transfektering var "infektion ved omdannelse«, dvs indførelsen af DNA (eller RNA) fra en Eukaryot virus eller bakteriofag ind i cellerne, hvilket resulterer i en infektion. Because the term transformation had another sense in animal cell biology (a genetic change allowing long-term propagation in culture, or acquisition of properties typical of cancer cells), the term transfection acquired, for animal cells, its present meaning of a change in cell properties caused by introduction of DNA. Fordi begrebet transformation havde en anden mening i animalske celle-biologi (en genetisk ændring giver mulighed for langsigtet formering i kultur, eller erhvervelse af ejendomme typisk for kræft celler), er udtrykket transfektering erhvervet, for animalske celler, i sin nuværende betydning af en ændring i celle ejendomme som følge af indførelsen af DNA.

Transfection is a method by which experimental DNA may be put into a cultured mammalian cell. Transfektering er en metode, hvormed eksperimentel DNA kan sættes i en dyrkede pattedyrceller. Such experiments are usually performed using cloned DNA containing coding sequences and control regions (promoters, etc) in order to test whether the DNA will be expressed. Sådanne eksperimenter er normalt udført med klonede DNA indeholder kodende sekvenser og kontrol regioner (projektledere, osv.) med henblik på at teste, om DNA bliver udtrykt. Since the cloned DNA may have been extensively modified (for example, protein binding sites on the promoter may have been altered or removed), transfection is often used to test whether a particular modification affects the function of a gene. Da det klonede DNA-mai er blevet grundigt ændret (for eksempel, proteinbinding websteder på initiativtageren kan have været ændres eller fjernes), transfektering ofte bruges til at teste, om en bestemt ændring påvirker funktionen af et gen.

Transfection Diagram: Transfektering Diagram:

Transfection Applications Transfektering Applications

The ability to synthesize RNA in the laboratory is critical to many techniques. Muligheden for at sammenfatte RNA i laboratoriet er kritisk for mange teknikker. Radiolabeled and non-radiolabeled RNA probes are required for many protocols, and they can be synthesized easily in small scale in vitro transcription reactions allowing their use in blot hybridizations and nuclease protection assays. Radioaktivt og ikke-radioaktivt RNA-sonder er behov for mange protokoller, og de kan syntese let i lille skala in vitro transkription reaktioner gør det muligt at anvende dem i skamplet hybridizations og nuclease beskyttelse assays.

Requirements for Transfection? Krav til transfektering?

• Vector or DNA Vector eller DNA
• Cells Celler
• Transfection Reagent Transfektering Reagent

Optimizing Transfection Optimering transfektering

In vitro transcription requires a purified linear DNA template containing a promoter, ribonucleotide triphosphates, a buffer system that includes DTT and magnesium In vitro transkription kræver en renset lineære DNA-skabelon indeholder en initiativtager, ribonucleotid triphosphater, en buffer, der omfatter DTT og magnesium

Which Transfection Reagent to Use? Hvilke transfektering Reagent bruges?

In vitro transcription requires a purified linear DNA template containing a promoter, ribonucleotide triphosphates, a buffer system that includes DTT and magnesium In vitro transkription kræver en renset lineære DNA-skabelon indeholder en initiativtager, ribonucleotid triphosphater, en buffer system, der omfatter DTT og magnesium

Transfection Methods Transfektering Metoder

There are various methods of introducing foreign DNA into a eukaryotic cell. Der er forskellige metoder til at indføre fremmed DNA i en eukaryot celle. Many materials have been used as carriers for transfection, which can be divided into three kinds: (cationic) polymers, liposomes and nanoparticles. Mange materialer har været anvendt som bærestoffer for transfektering, der kan inddeles i tre typer: (kationiske) polymerer, liposomer og nanopartikler.

One of the cheapest (and least reliable) methods is transfection by calcium phosphate, originally discovered by FL Graham and AJ van der Eb in 1973[citation needed] (see also [1]). En af de billigste (og mindst troværdige) metoder er transfektering af calcium fosfat, som oprindeligt blev opdaget af FL Graham and AJ van der Eb i 1973 [citation behov] (se også [1]). HEPES-buffered saline solution (HeBS) containing phosphate ions is combined with a calcium chloride solution containing the DNA to be transfected. HEPES-buffer saltoploesning (HeBS) indeholdende fosfat-ioner er kombineret med en calciumchlorid opløsning indeholder det DNA, der skal transfected. When the two are combined, a fine precipitate of the positively charged calcium and the negatively charged phosphate will form, binding the DNA to be transfected on its surface. Når de to ting kombineres, en bøde bundfald af de positivt ladede calcium og den negativt ladet fosfat vil form, der binder DNA, der skal transfected på dens overflade. The suspension of the precipitate is then added to the cells to be transfected (usually a cell culture grown in a monolayer). Suspensionen af Bundfaldet tilsættes derefter til de celler, der skal transfected (normalt en cellekultur dyrkes i et monolayer). By a process not entirely understood, the cells take up some of the precipitate, and with it, the DNA. Ved en proces ikke helt forstået, at cellerne tage op nogle af de udfældes, og med det, DNA.

Other methods use highly branched organic compounds, so-called dendrimers, to bind the DNA and get it into the cell. Andre metoder bruger meget forgrenet organiske forbindelser, de såkaldte dendrimers, til at binde DNA og få det ind i cellen. A very efficient method is the inclusion of the DNA to be transfected in liposomes, ie small, membrane-bounded bodies that are in some ways similar to the structure of a cell and can actually fuse with the cell membrane, releasing the DNA into the cell. En meget effektiv metode er optagelse af det DNA, der skal transfected i liposomer, dvs små, membran-afgrænset organer, der er på nogle måder ligner strukturen i en celle, og kan rent faktisk sikring med celle membranen, frigivelse af DNA i cellen . For eukaryotic cells, lipid-cation based transfection is more typically used, because the cells are more sensitive. For eukaryotiske celler, lipid-stemmelser baseret transfektering er mere typisk anvendes, fordi de celler er mere følsomme.

Another method is the use of cationic polymers such as DEAE-dextran or polyethylenimine. En anden metode er anvendelse af kationiske polymerer såsom DEAE-dextran eller polyethylenimine. The negatively charged DNA binds to the polycation and the complex is taken up by the cell via endocytosis. De negativt ladet DNA binder sig til polycation og de komplekse er optaget af cellen via endocytose.

A direct approach to transfection is the gene gun, where the DNA is coupled to a nanoparticle of an inert solid (commonly gold) which is then "shot" directly into the target cell's nucleus. En direkte tilgang til transfektering er det gen pistoler, hvor DNA er koblet til et nanopartikler af en inert fast (almindeligt guld), som derefter "skud" direkte i mål celles kerne. DNA can also be introduced into cells using viruses as a carrier. DNA kan også indføres i celler ved hjælp af vira som et luftfartsselskab. In such cases, the technique is called viral transduction, and, the cells are said to be transduced. I sådanne tilfælde kan den teknik kaldes viral transduktion, og cellerne siges at være transduced.

Other methods of transfection include nucleofection, electroporation, heat shock, magnetofection and proprietary transfection reagents such as Lipofectamine, Dojindo Hilymax, Fugene, jetPEI, Effectene or DreamFect. Andre metoder til transfektering omfatter nucleofection, elektroinjektion, varme stød, magnetofection og proprietære transfektering reagenser såsom Lipofectamine, Dojindo Hilymax, Fugene, jetPEI, Effectene eller DreamFect.

Stable vs Transient Transfection Methods Stabil vs Transient transfektering Metoder

For most applications of transfection, it is sufficient if the transfected gene is only transiently expressed. For de fleste anvendelser af transfektering, er det tilstrækkeligt, hvis transfected genet er kun forbigående udtryk. Since the DNA introduced in the transfection process is usually not inserted into the nuclear genome, the foreign DNA is lost at the later stage when the cells undergo mitosis. Siden DNA indført i transfektering proces er normalt ikke medtages i den nukleare genom, den fremmede DNA er tabt på det senere tidspunkt, når cellerne undergår mitose. If it is desired that the transfected gene actually remains in the genome of the cell and its daughter cells, a stable transfection must occur. Hvis det er ønsket at transfected genet faktisk forbliver i genom af cellen og dens datterceller, en stabil transfektering må forekomme.

To accomplish this, another gene is co-transfected, which gives the cell some selection advantage, such as resistance towards a certain toxin. For at opnå dette, et andet gen er co-transfected, hvilket giver cellen nogle udvælgelse fordel, såsom modstand mod et bestemt toksin. Some (very few) of the transfected cells will, by chance, have inserted the foreign genetic material into their genome. Nogle (meget få) af de transfected celler vil, ved en tilfældighed, har indsat fremmed genetisk materiale i deres genom. If the toxin, towards which the co-transfected gene offers resistance, is then added to the cell culture, only those few cells with the foreign genes inserted into their genome will be able to proliferate, while other cells will die. Hvis toksin, mod hvilke co-transfected genet giver resistens, er derefter lagt til cellekultur, er det kun de få celler med den fremmede gener indsættes i deres genom vil være i stand til at formere sig, mens andre celler vil dø. After applying this selection pressure for some time, only the cells with a stable transfection remain and can be cultivated further. Efter at anvende denne markering pres for et stykke tid, er det kun celler med en stabil transfektering forblive og kan dyrkes yderligere.

A common agent for stable transfection is Geneticin, also known as G418, which is a toxin that can be neutralized by the product of the neomycin resistant gene. En fælles agent for stabile transfektering er Geneticin, også kendt som G418, som er et toksin, der kan neutraliseret med produktet af neomycin resistente gen.

Which Transfection Reagent or Method to Use? Hvilke transfektering Reagent eller metode bruges?

In vitro transcription requires a purified linear DNA template containing a promoter, ribonucleotide triphosphates, a buffer system that includes DTT and magnesium In vitro transkription kræver en renset lineære DNA-skabelon indeholder en initiativtager, ribonucleotid triphosphater, en buffer, der omfatter DTT og magnesium

Tips for Transfection Tips til transfektering

• Use RNAse inhibitors always! Brug RNAse hæmmere altid!
• Saving Money with T7 RNA Phage Polymerase: Clones with an N-terminal His-6 tag are available. Spare penge med T7 RNA-fag polymerase: Clones med en N-terminal Hans-6-tag er til rådighed. If you obtain this clone, you can purify large amounts of T7 polymerase with high activity (ref: He B, Rong M, Lyakhov D, Gartenstein H, Diaz G, Castagna R, McAllister WT, Durbin RK. Protein Expr Purif. 1997, 9, 142–151). Hvis du hente denne klon, kan du rense store mængder T7 polymerase med høj aktivitet (ref: Han B, Rong M, Lyakhov D, Gartenstein H, Diaz G, Castagna R, McAllister WT, Durbin RK. Protein expr Purif. 1997, 9, 142-151).
• To remove the DNA template, use an RNAse-free DNAse. At fjerne DNA-skabelon, skal du bruge en RNAse-fri DNAse. We have used RQ1 DNase (Promega) added for 15 minutes and it works well. Vi har brugt RQ1 DNase (Promega) tilsættes i 15 minutter og det virker godt.

Storing Trasfection Reagent Opbevaring Trasfection Reagent

• RNA is best stored at a neutral pH with EDTA. RNA er bedst opbevaret ved en neutral pH med EDTA.
• TE buffer is recommended (10 mM Tris-HCl, pH 7.5, 1 mM EDTA), however this should be prepared with RNAse free purified (preferablly Millipore water). TE buffer anbefales (10 mM Tris-HCl, pH 7,5, 1 mM EDTA), men dette skulle være parat med RNAse gratis renset (preferablly Millipore vand).

Troubleshooting Transfection Problems Fejlfinding transfektering Problemer

Failed Transfections Det lykkedes ikke Transfections

• Old transfection reagent Old transfektering reagens
• Improperly stored transfection agent Forkert lagret transfektering agent
• Poor quality DNA or vector Dårlig kvalitet DNA eller vektor
• Insufficient DNA amount Utilstrækkelig DNA beløb
• Insufficient transfection reagent Utilstrækkelig transfektering reagens
• Too much DNA For meget DNA
• Too much transfection reagent For meget transfektering reagens

References on Transfection Referencer om transfektering

References on Transfection Referencer om transfektering

1. Bacchetti S, Graham F (1977). Bacchetti S, Graham F (1977). "Transfer of the gene for thymidine kinase to thymidine kinase-deficient human cells by purified herpes simplex viral DNA". "Overførsel af genet for thymidin kinase til thymidin kinase-mangelfuld menneskelige celler ved renset herpes simplex viral DNA". Proc Natl Acad Sci USA 74 (4): 1590-4. Proc Natl Acad Sci USA 74 (4): 1590-4. PMID 193108. PMID 193108.