Workshop 3

Question 1

What are the four principal mechanisms by which DNA can be introduced into animal cells ?

Answer 1

1. Physical transfection : Microinjection, particle bombardment, electroporation
2. Chemical-mediated transfection : Lipofection (e.g. cationic lipids), polyamines, Ca2+-phosphate DNA co-precipitate.
3. Transduction: DNA packaged inside a virus.
4. Bactofection : DNA packaged inside a bacterium.

Question 2

What happens to DNA introduced into mammalian cells that is not integrated in nuclear genome ?

Answer 2

DNA introduced in to mammalian cells DNA will be degraded/diluted over time if not integrated into nuclear genome.

Question 3

What is the CMV promoter ?

What are its characteristics ?

Answer 3

CMV promoter = strong mammilan promoter from human cytomegalovirus
May contain enhancer region. Can be silenced in some cells.

Question 4

What is the SV40 promoter ?

What are its characteristics ?

Answer 4

SV40 promoter = mammalian expression promoter from simian virus 40
May include enhancer.

Question 5

What is the CAG promoter ?

What are its characteristics ?

Answer 5

CAG promoter = strong hybrid mammalian promoter

Contains CMV enhancer, chicken beta actin promoter, and rabbit beta-globin splice acceptor.

Question 6

What is the CamKIIa promoter ?

What are its characteristics ?

Answer 6

CamKIIa promoter = Ca2+/calmodulin-dependent protein kinase II promoter
Used for neuronal/CNS expression

Question 7

Why do scientists use viral based ORIs ?

Answer 7

Because there are no “natural” mammalian origin of replications, so scientists use viral based ORIs - trans expression of Epstein-Barr virus nuclear antigen 1 (EBNA1) or SV40 large-T antigen in cells with vector carrying EBV or SV40, respectively, results in episomal amplification.

Question 8

What are the 5 main steps for designing a high-level eukaryotic expression construct ?

Answer 8

1. Strong and constitutive promoter
2. Inclusion of intron
3. Polyadenylation signal
4. Removal of unnecessary untranslated sequences
5. Optimization of the transgene for translational efficiency (Kozak sequence CCRCCAUGG and codon usage)

Question 9

Why is recombinant technology in yeast useful ?

Answer 9

• Saccharomyces cerevisiae and other fungi are easy to
  handle eukaryotic system
• Overproduction of proteins (research & commercial value)
• Ability to clone very large pieces of DNA

Question 10

Whata re the 2 primary carbon sources used by E. Coli ?

Why does the cel prefer to use glucose ?

Answer 10

E. coli can use either glucose (monosaccharide) or lactose (disaccharide).
E. Coli preferentially use glucose because lactose needs to be hydrolyzed first by beta-galactosidase into D-galactose and D-glucose.