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Cell Transfection Stanica Transfection

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. Ova stranica je Transfection Vaš portal za sve stvari vezane uz transfection of plasmid DNA stanica sa ili vektor, da li RNA siRNA ili RNAi, ili čak i rekombinantnim protein. 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! Pružamo Vam sve popratne informacije, protokole za ćelija transfection, optimizaciju i analizu korake, i stanica transfection informacije za rješavanje problema kako bi postao ultimativni stručnjak!

Transfection Table of Contents Transfection Table of Contents

• What is transfection? Što je transfection?
• Background Pozadina
• Applications Aplikacije
• Materials Materijali
• Transfection Methods Transfection Metode
• Buffers Buffers
  
• Preparation Priprema
• Protocol Protokol
• Other T ransfection Protocols Ostali T ransfection Protocols
• Discussion Topics for Transfection Teme za raspravu Transfection
• Get Help on your Transfection Problems Zatraži pomoć na vašem Transfection Problemi
• Tips Savjeti
• Storing Čuvanje
• Troubleshooting In Transfection UPOZORENJA U Transfection
• References Reference

What is Transfection? Što je Transfection?

Transfection describes the introduction of foreign material into eukaryotic cells using a virus vector or other means of transfer. Transfection opisuje uvođenje stranih materijala u eukaryotic stanice koristeći virus vektor ili drugim sredstvima prijenosa.

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. Pojam transfection za ne-virusnih metode se najčešće koristi u odnosu na stanice sisavaca, dok je pojam transformacije poželjna je opisati ne-virusnog prijenosa DNA u bakterijama i ne-životinjska eukaryotic stanica kao što su gljive, alge i biljke.

Transfection of animal cells typically involves opening transient pores or 'holes' in the cell plasma membrane, to allow the uptake of material. Transfection životinjskih stanica obično uključuje otvaranje prelazni pore ili 'rupe' u plasma cell membrane, kako bi omogućila unos materijala. Genetic material (such as supercoiled plasmid DNA or siRNA constructs), or even proteins such as antibodies, may be transfected. Genetski materijal (kao što su supercoiled plasmid DNA ili konstrukti siRNA), ili čak i proteina, kao što su protutijela, svibanj biti 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. Pored electroporation, transfection može biti obavljeno od strane jedne mixing cationic lipida s materijalom za proizvodnju liposomi, koji osigurač s plazma membrana stanica i njihovih tereta unutar deposit.

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. Izvorna značenja transfection je 'infekcije by transformacije', tj. uvođenje DNA (ili RNA) iz Eukarioti virus ili bakteriofag u stanice, što je rezultiralo u jednom zaraze. 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. Zato što je još jedan pojam transformacije u smislu životinjskih stanica biologija (a genetski izmijeniti omogućava dugoročno propagacija u kulturi, odnosno stjecanje svojstva tipična za opozvan stanica), pojam transfection stečena, za animalne stanice, njegovo sadašnje značenje promjene u stanici svojstva uzrokovana uvođenjem DNA.

Transfection is a method by which experimental DNA may be put into a cultured mammalian cell. Transfection je način na koji eksperimentalni DNA svibanj se stavi u uzgojenom stanica sisavaca. 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. Takvi eksperimenti su najčešće izvodi pomoću klon sadrži DNA sekvence kodiranja i kontrolu regije (promotora, itd.) kako bi se DNA test da li će se izrazio. 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. Budući da je klon DNA svibanj opsežno su modificirani (na primjer, protein binding sučelja na promicatelj svibanj su promijenili ili uklonili), transfection često se koristi za testiranje da li određeni modifikacija utječe na funkciju jednog gena.

Transfection Diagram: Transfection Diagram:

Transfection Applications Transfection Aplikacije

The ability to synthesize RNA in the laboratory is critical to many techniques. Mogućnost RNA sintetizirati u laboratoriju je najvažnije za mnoge tehnike. 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. Radiolabeled i non-radiolabeled RNA probe su obavezna za mnoge protokole, i oni mogu biti umjetni jednostavno u male scale in vitro transkripcije reakcije omogućava njihovo korištenje u izbrisat hybridizations nukleaze zaštitu i eseji.

Requirements for Transfection? Zahtjevi za Transfection?

• Vector or DNA Vektor ili DNA
• Cells Stanica
• Transfection Reagent Transfection reagens

Optimizing Transfection Optimiziranje Transfection

In vitro transcription requires a purified linear DNA template containing a promoter, ribonucleotide triphosphates, a buffer system that includes DTT and magnesium In vitro transkripcije zahtijeva očistili linearna DNA sadrži predložak promicatelj, ribonucleotide triphosphates, tampon sustav koji uključuje DTT i magnezija

Which Transfection Reagent to Use? Koji reagens Transfection koristiti?

In vitro transcription requires a purified linear DNA template containing a promoter, ribonucleotide triphosphates, a buffer system that includes DTT and magnesium In vitro transkripcije zahtijeva očistili linearna DNA sadrži predložak promicatelj, ribonucleotide triphosphates, tampon sustav koji uključuje DTT i magnezija

Transfection Methods Transfection Metode

There are various methods of introducing foreign DNA into a eukaryotic cell. Postoje razne metode za uvođenje stranih DNA u eukaryotic cell. Many materials have been used as carriers for transfection, which can be divided into three kinds: (cationic) polymers, liposomes and nanoparticles. Mnogi su materijali koji se koriste kao nosače za transfection, koja se može podijeliti na tri vrste: (cationic) polimeri, liposomi, nanočestice.

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]). Jedan od najjeftinije (i najnepouzdanija) metoda je transfection by kalcijev fosfat, izvorno otkrio FL Graham i AJ van der je rekao in 1973 [citat potreban] (vidi također [1]). HEPES-buffered saline solution (HeBS) containing phosphate ions is combined with a calcium chloride solution containing the DNA to be transfected. HEPES-bufferom slan rješenje (HeBS) sadrže fosfatnih iona je u kombinaciji s kalcij klorid otopina koja sadrži DNA da se 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. Kada se dvije su u kombinaciji, novčanom kaznom od precipitate pozitivno se tereti i kalcijev fosfat će negativno naplaćeno form, binding DNA da se transfected na svoju površinu. The suspension of the precipitate is then added to the cells to be transfected (usually a cell culture grown in a monolayer). Obustave na precipitate je zatim dodao do stanice da se transfected (obično stanične kulture uzgajaju u monolayer). By a process not entirely understood, the cells take up some of the precipitate, and with it, the DNA. By proces nije u potpunosti shvatio, ćelije zauzimaju neke od precipitate, i s njim, DNA.

Other methods use highly branched organic compounds, so-called dendrimers, to bind the DNA and get it into the cell. Ostale metode koristiti vrlo račvast organski spojevi, tzv dendrimers, obvezivanja na DNA i dobiti ga u ćeliju. 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. A vrlo efikasna metoda je uključivanje u DNA da se transfected u liposomi, tj. mali, membranskih ograničen tijela koja su u nekim načina slična strukturi ćeliju, a čak može sjediniti s stanična membrana, u objavljivanju u DNA stanica . For eukaryotic cells, lipid-cation based transfection is more typically used, because the cells are more sensitive. Za eukaryotic stanice, lipida-cation temelji transfection je više obično koristi, jer stanice su više osjetljivi.

Another method is the use of cationic polymers such as DEAE-dextran or polyethylenimine. Drugi način je korištenje cationic polimeri kao što su DEAE-dextran ili polyethylenimine. The negatively charged DNA binds to the polycation and the complex is taken up by the cell via endocytosis. The negativno naplaćeno DNA veže na polycation i kompleksa je preuzet od strane stanica putem endocitoza.

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. A direktni pristup transfection je gene pištolj, gdje se DNA je spregnuti u nanoparticle od inertnih čvrstih (obično zlato) koji je tada "pucao" izravno u Target Cell's nucleus. DNA can also be introduced into cells using viruses as a carrier. DNA također mogu biti uvedene u stanice pomoću virusa kao nosilac. In such cases, the technique is called viral transduction, and, the cells are said to be transduced. U takvim slučajevima, tehnika se zove virusni pretvaranje, i, ćelije su, rekao je da se 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. Ostale metode transfection uključuju nucleofection, electroporation, toplinski stres, magnetofection i vlasnički transfection Reagensi kao što su Lipofectamine, Dojindo Hilymax, Fugene, jetPEI, Effectene ili DreamFect.

Stable vs Transient Transfection Methods Stabilne vs Transient Transfection Metode

For most applications of transfection, it is sufficient if the transfected gene is only transiently expressed. Za većinu aplikacija, transfection, to je dovoljno ako transfected gena je samo transiently izrazio. 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. Od DNA transfection uvedeni u proces se obično ne umetnuta u nuklearni genocid, stranih DNA je izgubio u kasnijoj fazi kada je stanica podvrći Mitoza. 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. Ako je željeni da transfected gena zapravo ostaje u genom od stanica i njezine kćeri stanice, stabilne transfection moraju pojaviti.

To accomplish this, another gene is co-transfected, which gives the cell some selection advantage, such as resistance towards a certain toxin. Da biste ostvarili ovo, drugi je gene co-transfected, što daje neki selekciji stanica prednost, kao što je otpornost prema određenom toksin. Some (very few) of the transfected cells will, by chance, have inserted the foreign genetic material into their genome. Neki (vrlo malo) od transfected ćelije će se, slučajno, su ubačeni u stranim genetski materijal u njihovoj 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. Ako je toksin, prema kojem je co-transfected gena pruza otpor, a zatim je dodao da su stanične kulture, te samo nekoliko stanica sa stranim geni ubačeni u njihov genom će moći proliferate, dok druge stanice će umrijeti. After applying this selection pressure for some time, only the cells with a stable transfection remain and can be cultivated further. Nakon primjene ovog selekcija pritisak za neko vrijeme, samo ćelije s transfection ostati stabilan i može se dodatno obrađuje.

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. A common agent za stabilne transfection je Geneticin, također poznat kao G418, koja je otrov koji se može neutralizirala je proizvod otporan na neomycin gena.

Which Transfection Reagent or Method to Use? Koji Transfection reagens ili metodu koristiti?

In vitro transcription requires a purified linear DNA template containing a promoter, ribonucleotide triphosphates, a buffer system that includes DTT and magnesium In vitro transkripcije zahtijeva očistili linearna DNA sadrži predložak promicatelj, ribonucleotide triphosphates, tampon sustav koji uključuje DTT i magnezija

Tips for Transfection Savjeti za Transfection

• Use RNAse inhibitors always! Koristite RNAse inhibitori uvijek!
• Saving Money with T7 RNA Phage Polymerase: Clones with an N-terminal His-6 tag are available. Spremanje Money s T7 RNA fag Polymerase: Knin s N-terminal-6 tag mu stoje na raspolaganju. 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). Ako ste nabavili ovaj klon, možete očistiti velike količine T7 polymerase s visokim aktivnosti (ref: B On je, Rong M, Lyakhov D, Gartenstein H, G Diaz, Castagna R, McAllister WT, Durbin RK. Protein expr Purif. 1997, 9, 142-151).
• To remove the DNA template, use an RNAse-free DNAse. Da biste uklonili DNA predložak, koristiti jedan RNAse-free DNAse. We have used RQ1 DNase (Promega) added for 15 minutes and it works well. Mi smo koristili RQ1 DNase (Promega) dodao za 15 minuta i to dobro radi.

Storing Trasfection Reagent Spremanje Trasfection reagens

• RNA is best stored at a neutral pH with EDTA. RNA je najbolje pohranjena na neutralnom pH s 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 je preporučeno (10 mm-Tris HCL, pH 7,5, 1 mm EDTA), no ovo bi trebao biti pripremljen s RNAse besplatno očistili (preferablly Millipore vode).

Troubleshooting Transfection Problems UPOZORENJA Transfection Problemi

Failed Transfections Neuspješno Transfections

• Old transfection reagent Stara transfection reagens
• Improperly stored transfection agent Nepropisno pohranjena transfection agent
• Poor quality DNA or vector Loša kvaliteta DNA ili vektor
• Insufficient DNA amount Nedovoljna količina DNA
• Insufficient transfection reagent Nedovoljna transfection reagens
• Too much DNA Previše DNA
• Too much transfection reagent Previše transfection reagens

References on Transfection Reference na Transfection

References on Transfection Reference na Transfection

1. Bacchetti S, Graham F (1977). Bacchetti S, F Graham (1977). "Transfer of the gene for thymidine kinase to thymidine kinase-deficient human cells by purified herpes simplex viral DNA". "Transfer od gena za thymidine kinaza na thymidine kinaza-deficijentni ljudskih stanica po očistili herpes simplex virusnim DNA". Proc Natl Acad Sci USA 74 (4): 1590-4. Proc Natl Acad Sci USA 74 (4): 1590-4. PMID 193108. PMID 193108.