Animal Biotechnology

Question 1

Differentiate between transfection, transformation and transduction.

Answer 1

Transfection: To introduce genetic materials such as DNA, RNA or nucleic acids into eukaryotic cells
Transformation: To introduce recombinant plasmid DNA into competent bacterial or plant cells
Transduction: To introduce DNA by using virus.

Question 2

What is the difference between stable and transient transfection?

Answer 2

Stable Transfection: Sustain transgene expression even after host cells replicate, Integrated into host genome, genetic materials that usually have a marker gene for selection

Transient Transfection: Genes are expressed for a limited period of time, Not integrated into host genome, Can be lost by environmental factors and cell division.

Question 3

Describe the three categories of transfection methods.

Answer 3

• Biological: Using virus to transduce foreign gene into the host cells.
• Chemical: Using cationic materials such as cationic polymer, calcium phosphate and cationic lipid to form complex with negatively charged nucleic acids
• Physical: Using physical tools to deliver nucleic acids. Such as microinjection, electroporation, laser-based transfection, biolistic particle delivery.

Question 4

Describe the steps in ES cell mediated gene transfer

Answer 4

1. Prepare DNA
2. Transfect the embryonic stem cells in culture.
3. Select for successfully transfected cells.
4. Inject the successfully transfected cells into inner cell mass of mouse blastocysts.
5. Transfer embryos into the uterus of a pseudopregnant mouse.
6. Examine the DNA of the offspring for the desired gene.

Question 5

Describe the pronuclear microinjection method.

Answer 5

1. Foreign DNA is injected into one of the pronuclei of fertilized mouse egg.
2. The injected eggs are transferred into a foster mother.
3. 10-30% of offspring will contain the injected foreign DNA, those who express foreign DNA are bred.

Question 6

Describe laser-based transfection.

Answer 6

• Laser-based transfection uses a pulse laser to irradiate a cell membrane to form a transient pore.
• Nucleic acids in the medium can be transferred into the cell due to the osmotic difference between the medium and cytosol
• Advantage: Pores can be made at any location on the cells
• Disadvantage: Expensive

Question 7

What are the advantages of transfecting RNA instead of DNA?

Answer 7

(!!!) mRNA does not need to be located in a nucleus to be expressed.

• No risk of integration into host genome
• Transfection efficiency is independent from cell cycle
• No need for immune inducible vectors
• Rapid expression
• Adjustable: Expression levels can be adjusted by changing the amount of mRNA transfected and the frequency of transfection.

Question 8

How does one detect if a transgene or a mRNA has been inserted into a cell?

Answer 8

• Transgene integration is confirmed by southern hybridization.
• mRNA expression is detected by northern hybridization.