Tutorial 2

Question 1

What is the selection system used to select for cells with a high expression of the vector?

Answer 1

DHFR/MTX

Question 2

Why is DHFR expression beneficial in cell culture mediums where GHT is absent?

Answer 2

DHFR can allow for the synthesis of GHT from folate precursor

Question 3

How does one synthesize two proteins from one mRNA strand?

Answer 3

1. Using p2a between the two genes which creates a self cleaving peptide
2. It causes ribosomal skipping during translation which results in a missing peptide bond and separates the two proteins

Question 4

How many amino acids does p2a have?

Answer 4

19

Question 5

What is IRES and how is it beneficial in genetic engineering?

Answer 5

1. Internal ribosomal entry site
2. Allows for the ribosome to initiate translation at locations within the mRNA molecule without the need of a 5’ cap
3. Allows the GOI and DHFR to be transcribed and translated together

Question 6

Why is it beneficial for DHFR and GOI to be translated together?

Answer 6

Their production can be controlled together

Question 7

What are two ways where the vector can get integrated into the CHO cell chromosome?

Answer 7

1. Random integration of linearized vector
2. Site specific integration

Question 8

How many times are the cell cultures exposed to MTX after transfection with the vector

Answer 8

Twice
1st is a low level of MTX to select for cells that have the vector
2nd is a high level of MTX to select for cells that express high levels of the vector

Question 9

What does the basal medium contain?

Answer 9

inorganic salts, amino acids, vitamins, pH buffer and indicator, glucose