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BMSc - Labs > Transfections > Flashcards

Transfections Flashcards

1
Q

What is transfection?

A

The introduction of foreign DNA into eukaryotic cells

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2
Q

What is a transfectant?

A

Cells which have incorporated foreign DNA

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3
Q

What is a stable transfectant?

A

One which ahs integrated the foreign DNA into the genome

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4
Q

What is a transient transfectant?

A

One which has NOT integrated the foreign DNA into the genome and only expresses the genes for a limited time

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5
Q

At which confluency should cells be transfected?

A

40-80%

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6
Q

How long are genes expressed for with transient transfectants?

A

24-96 hours

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7
Q

Why should cells be transfected at this confluency?

A

Underconfluent - cultures grow poorly with no cell-cell contact

Overconfluent cells - Contact inhibition, cells resistant to the uptake of DNA

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8
Q

Give examples of transfection methods

A 
Electroporation (what we used)
Calcium phosphate precipitation
Lipid mediated lipofection
Retroviral infection
Microinjection
DEAE-dextran
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9
Q

How does electroporation work?

A

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

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10
Q

What are the advantages of electroporation?

A

Non-chemical, so doesn’t alter function or biological structure of the cell
Easy
Highly effective
Done on a wide range of cell types

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11
Q

What is a haemocytometer?

A

Thick microscope slide with grids of known size/volume used to count the number of viable cells in a sample

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12
Q

Why use a haemocytometer?

A

The determine the concentration of viable cells in a smaple

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13
Q

What is added to cells so that they can be counted?

A

Tryptan blue

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14
Q

Why is tryptan blue added to cells?

A

Penetrates dead cell membranes and stains them blue so that they are easy to differentiate from viable cells

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15
Q

What should cells be split before transfection?

A

1:3

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16
Q

Why are cells split beforehand?

A

To keep them in the growth phase

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17
Q

How does electroporation work?

A

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

18
Q

How does transfection with DEAE-dextran work?

A

Creates an excess of positive charge in the DNA:polymer complex to cause strong attraction
Transfection occurs through endocytosis

19
Q

How long is the foreign DNA expressed for with DEAE-dextran?

A

A few days

20
Q

What is DEAE-dextran not suitable for?

A

Stable or long-term transection studies which rely on the integration of transferred DNA into cells

(Polybrene, dendrimers, polyethyleneimine)

21
Q

What is DEAE-dextran?

A

Cationic polymer

22
Q

When was calcium phosphate co-precipitation introduced?

A

Early 70s

23
Q

What is calcium phosphate precipitation favoured?

A

Easily available
Inexpensive
Simple
Effective with many types of cells

24
Q

Explain the calcium precipitate protocol

A

Mixing DNA with CaCl

Adding this to a buffered saline/phosphate solution and incubating at room temp

25
Q

Calcium phosphate precipitant can only be used for transient transfection
T/F

A

F

Can be used for stable and transient

26
Q

How is the precipitate taken up into cells in calcium phosphate precipitant?

A

Endocytosis or phagocytosis

27
Q

What is calcium precipitant not useful for?

A

In vivo gene transfer to whole animal

28
Q

What are the main two drawbacks of calcium phosphate precipitant?

A

Variable

Small pH changes can compromise efficacy

29
Q

Why may lipid mediate lipofection be used over chemical transfection and why may it not?

A

Much more efficient

But much more expensive

30
Q

What is lipfection?

A

The use of liposomes to fuse with the cell membrane and release their contents

31
Q

Outline the basis of lipid mediated lipofection

A

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

32
Q

What are the clinical applications of lipid mediated lipofection?

A

Drug discovery and development
Oncology research
Gene silencing

33
Q

How are physical transfection methods hard on cells?

electroporation, injection

A

Disruption of the membrane occurs (which may lead to cell death)

34
Q

Physical methods are used for cells which are difficult to transfect
T/F

A

T

35
Q

What is electroporation dependent on?

A

DNA concentration and the type of cells used

36
Q

What is microinjection commonly used for?

A

Introduce DNA and RNA into single cells (like stem cells)

37
Q

Outline the process of microinjection?

A

DNA/RNA directly injected into the cytoplasm or the nucleus of a single cell
With the aid of a micromanipulator and microscope

38
Q

What are the pros and cons of microinjection?

A

Time consuming

Expensive

39
Q

Which viruses may be used to introduce DNA to cells?

A

Retrovirus, lentivirus, adenovirus or adeno-associated virus

40
Q

What is viral transfection termed?

A

Viral transduction

The cells are then said to be transducted

41
Q

Which viral vectors are common used for human virus?

A

Adenoviral

42
Q

What are the benefits of using lentiviral vectors?

A

Can transduce cells not currently undergoing mitosis

BMSc - Labs (6 decks)

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