Inducible gene expression

Question 1

What do the tet-on and tet off systems require?

Answer 1

-Requires 2 transgenes, one making a drug responsive repressor (or activator), the other has a response element with the gene of interest

Question 2

How does the Tet-on system work?

Answer 2

• A four amino acid change occurs to TetR which alters its binding characteristics
• This creates reverse TerR (rtTa), which binds the TRE in the presence of doxycycline and activates transcription

Question 3

How does the tet-off system work?

Answer 3

-The tet-controlled transcriptional activator (tTA) is a fusion of the wild-type Tet repressor (TetR) to the VP16 activation domain (AD) of herpes simplex virus. tTA binds the Tet-responsive element (TRE) and activates transcription in the absence of tetracycline or doxycycline.

Question 4

How does the Tet repressor work?

Answer 4

• Wild-type TerR is a repressor protein from bacteria
  
  • When present in the same cell, it will bind the TRE and turn OFF gene expression
  • When it binds Tet, it can’t bind the TRE and turns ON gene expression
    
    • A basis for antibiotic response in bacteria

Question 5

What can the oestrogen receptor pathway be adopted to generate?

Answer 5

The oestrogen receptor pathway can be adopted to generate other oestrogen responsive proteins

Question 6

Steps in oestrogen receptor pathway

Answer 6

• The oestrogen receptor is usually held in the cytoplasm and its held in a complex with a protein called HSP90 (Heat shock protein 90).
  
  • If this cell encounters oestrogen, it diffuses into the cell and bind to the oestrogen receptor
  • This binding causes the dissociation of HSP90 from the oestrogen receptor
    
    • This allows the oestrogen receptor to translocate from the cytoplasm into the nucleus where it dimerises with other oestrogen receptors and drive the transcription of downstream target genes

Question 7

How can the oestrogen receptor pathway be adopted?

Answer 7

• This system can be adopted by taking the ligand binding domain (oestrogen receptor ligand binding domain) and fuse it to a strong trans activator in order to make them translocate to the nucleus in an oestrogen-dependent manner
  
  • A reporter gene can be placed downstream of the ER-responsive element