Gene technology 2: Expression vectors

Question 1

What’s a shuttle vector?

Answer 1

It’s a vector which is maintained (stable) in e.coli and eukaryotes.

Question 2

There are two types of plasmid vectors, episomal- and integration vectors. Describe them briefly.

Answer 2

Episomal vectors are extrachromosomal. If transformed into e.coli, chi-sequence and bac-sequence are necessary. When the cells undergo mitosis, the plasmids aren’t necessarily divided properly. The plasmid requires an origin of replication (ORI).

Integration vectors don’t require an ORI since the plasmid contents are integrated into a chromosome. When the genetic material has been introcued, there’s no need for selection methods after the initial one.

Question 3

What five domains are required in a eukaryotic plasmid vector?

Answer 3

1. Multiple cloning site (MCS)
2. Promoter in ocnunction with MCS (viral promoters are strong)
3. Translation termination signal
4. Poly-adenylation signal
5. Resistance genes.

Question 4

Therea re 7 ways to transfect eukaryotic cells. Which?

Answer 4

1. Microinjection of DNA
2. Calcium phosphate co-precipitation with DNA on cell surfaces. Requires high [DNA] for high efficiency.
3. Liposomal transfer using an artificial phospholipid vesicle with bound DNA. Liposomes fuse with the plasma membrane and/or get endocytosed.
4. Cell electroporation. Electric shock o cells causes a temporary membrane depolarisation, which creates transient pores through which the DNA can pass through.
5. Gene gun: Metal particles are covered with DNA vectors that can pierce the cells.
6. Viral transfer, is the most efficient method.
7. Cell-penetrating peptides, conjugated with DNA or other cargo molecules. Low toxicity, can target specific intracellular functions.