Transfections Flashcards
What is transfection?
The introduction of foreign DNA into eukaryotic cells
What is a transfectant?
Cells which have incorporated foreign DNA
What is a stable transfectant?
One which ahs integrated the foreign DNA into the genome
What is a transient transfectant?
One which has NOT integrated the foreign DNA into the genome and only expresses the genes for a limited time
At which confluency should cells be transfected?
40-80%
How long are genes expressed for with transient transfectants?
24-96 hours
Why should cells be transfected at this confluency?
Underconfluent - cultures grow poorly with no cell-cell contact
Overconfluent cells - Contact inhibition, cells resistant to the uptake of DNA
Give examples of transfection methods
Electroporation (what we used) Calcium phosphate precipitation Lipid mediated lipofection Retroviral infection Microinjection DEAE-dextran
How does electroporation work?
Exposes cell to a high intensity electric field which temporarily destablises the membrane
This allows exogenous molecules to get through and into the cell
When field switched off, molecules trapped into
What are the advantages of electroporation?
Non-chemical, so doesn’t alter function or biological structure of the cell
Easy
Highly effective
Done on a wide range of cell types
What is a haemocytometer?
Thick microscope slide with grids of known size/volume used to count the number of viable cells in a sample
Why use a haemocytometer?
The determine the concentration of viable cells in a smaple
What is added to cells so that they can be counted?
Tryptan blue
Why is tryptan blue added to cells?
Penetrates dead cell membranes and stains them blue so that they are easy to differentiate from viable cells
What should cells be split before transfection?
1:3
Why are cells split beforehand?
To keep them in the growth phase
How does electroporation work?
Creates an electric field which creates a positive charge causing DNA to move into the cell
Creates pores on the membrane so that DNA can move in
Pores go away when field does
How does transfection with DEAE-dextran work?
Creates an excess of positive charge in the DNA:polymer complex to cause strong attraction
Transfection occurs through endocytosis
How long is the foreign DNA expressed for with DEAE-dextran?
A few days
What is DEAE-dextran not suitable for?
Stable or long-term transection studies which rely on the integration of transferred DNA into cells
(Polybrene, dendrimers, polyethyleneimine)
What is DEAE-dextran?
Cationic polymer
When was calcium phosphate co-precipitation introduced?
Early 70s
What is calcium phosphate precipitation favoured?
Easily available
Inexpensive
Simple
Effective with many types of cells
Explain the calcium precipitate protocol
Mixing DNA with CaCl
Adding this to a buffered saline/phosphate solution and incubating at room temp
Calcium phosphate precipitant can only be used for transient transfection
T/F
F
Can be used for stable and transient
How is the precipitate taken up into cells in calcium phosphate precipitant?
Endocytosis or phagocytosis
What is calcium precipitant not useful for?
In vivo gene transfer to whole animal
What are the main two drawbacks of calcium phosphate precipitant?
Variable
Small pH changes can compromise efficacy
Why may lipid mediate lipofection be used over chemical transfection and why may it not?
Much more efficient
But much more expensive
What is lipfection?
The use of liposomes to fuse with the cell membrane and release their contents
Outline the basis of lipid mediated lipofection
Cationic polymers carry a positive charge and bind well to negatively charged nucleic acids
Cells as plated out, optimise the lipophectant and dilute in optimum
Combine 1:1 to open the cell membrane and let DNA into the cell
What are the clinical applications of lipid mediated lipofection?
Drug discovery and development
Oncology research
Gene silencing
How are physical transfection methods hard on cells?
electroporation, injection
Disruption of the membrane occurs (which may lead to cell death)
Physical methods are used for cells which are difficult to transfect
T/F
T
What is electroporation dependent on?
DNA concentration and the type of cells used
What is microinjection commonly used for?
Introduce DNA and RNA into single cells (like stem cells)
Outline the process of microinjection?
DNA/RNA directly injected into the cytoplasm or the nucleus of a single cell
With the aid of a micromanipulator and microscope
What are the pros and cons of microinjection?
Time consuming
Expensive
Which viruses may be used to introduce DNA to cells?
Retrovirus, lentivirus, adenovirus or adeno-associated virus
What is viral transfection termed?
Viral transduction
The cells are then said to be transducted
Which viral vectors are common used for human virus?
Adenoviral
What are the benefits of using lentiviral vectors?
Can transduce cells not currently undergoing mitosis
