Transfections

Question 1

What is transfection?

Answer 1

The introduction of foreign DNA into eukaryotic cells

Question 2

What is a transfectant?

Answer 2

Cells which have incorporated foreign DNA

Question 3

What is a stable transfectant?

Answer 3

One which ahs integrated the foreign DNA into the genome

Question 4

What is a transient transfectant?

Answer 4

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

Question 5

At which confluency should cells be transfected?

Answer 5

40-80%

Question 6

How long are genes expressed for with transient transfectants?

Answer 6

24-96 hours

Question 7

Why should cells be transfected at this confluency?

Answer 7

Underconfluent - cultures grow poorly with no cell-cell contact

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

Question 8

Give examples of transfection methods

Answer 8

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

Question 9

How does electroporation work?

Answer 9

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

Question 10

What are the advantages of electroporation?

Answer 10

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

Question 11

What is a haemocytometer?

Answer 11

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

Question 12

Why use a haemocytometer?

Answer 12

The determine the concentration of viable cells in a smaple

Question 13

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

Answer 13

Tryptan blue

Question 14

Why is tryptan blue added to cells?

Answer 14

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

Question 15

What should cells be split before transfection?

Answer 15

1:3

Question 16

Why are cells split beforehand?

Answer 16

To keep them in the growth phase

Question 17

How does electroporation work?

Answer 17

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

Question 18

How does transfection with DEAE-dextran work?

Answer 18

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

Question 19

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

Answer 19

A few days

Question 20

What is DEAE-dextran not suitable for?

Answer 20

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

(Polybrene, dendrimers, polyethyleneimine)

Question 21

What is DEAE-dextran?

Answer 21

Cationic polymer

Question 22

When was calcium phosphate co-precipitation introduced?

Answer 22

Early 70s

Question 23

What is calcium phosphate precipitation favoured?

Answer 23

Easily available
Inexpensive
Simple
Effective with many types of cells

Question 24

Explain the calcium precipitate protocol

Answer 24

Mixing DNA with CaCl

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

Question 25

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

Answer 25

F | Can be used for stable and transient

Question 26

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

Answer 26

Endocytosis or phagocytosis

Question 27

What is calcium precipitant not useful for?

Answer 27

In vivo gene transfer to whole animal

Question 28

What are the main two drawbacks of calcium phosphate precipitant?

Answer 28

Variable | Small pH changes can compromise efficacy

Question 29

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

Answer 29

Much more efficient But much more expensive

Question 30

What is lipfection?

Answer 30

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

Question 31

Outline the basis of lipid mediated lipofection

Answer 31

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

Question 32

What are the clinical applications of lipid mediated lipofection?

Answer 32

Drug discovery and development Oncology research Gene silencing

Question 33

How are physical transfection methods hard on cells? | electroporation, injection

Answer 33

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

Question 34

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

Answer 34

T

Question 35

What is electroporation dependent on?

Answer 35

DNA concentration and the type of cells used

Question 36

What is microinjection commonly used for?

Answer 36

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

Question 37

Outline the process of microinjection?

Answer 37

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

Question 38

What are the pros and cons of microinjection?

Answer 38

Time consuming | Expensive

Question 39

Which viruses may be used to introduce DNA to cells?

Answer 39

Retrovirus, lentivirus, adenovirus or adeno-associated virus

Question 40

What is viral transfection termed?

Answer 40

Viral transduction | The cells are then said to be transducted

Question 41

Which viral vectors are common used for human virus?

Answer 41

Adenoviral

Question 42

What are the benefits of using lentiviral vectors?

Answer 42

Can transduce cells not currently undergoing mitosis