Induced stable expression in MDCK cells
CONGRATULATIONS! 
You have chosen to focus on characterization of the receptor's mode of operation and the development of new drugs, and therefore selected the induced stable expression in MDCK cells.

The tetracyclin-regulated expression system allows tight control of expression and is commonly used. In this tutorial the tet-off system will be used. A detailed description is given here.




























































First you have to choose what vector to use for introducing BHR1 into the cells.
You have an antibody for the identification of BHR1, and you don't intend to purify the protein. On the other hand, you want the protein to be folded correctly, so you can rely on the agonist/antagonist screens.
 
Do you need to attach a His-tag to your gene?






































































Well, after all this elaborate brain-work (illustrated to the right) you finally start to do the actual work. 
The initial step is to create stable cells with the trans-activator cloned on a vector containing the gene encoding resistance to  G418 antibiotics. 
Three students, who work with you on a part time basis, transfected the MDCK cells with the appropriate plasmid and started a seven days selection period with the  G-418. 
Their observations are presented below.
Name
Day 1
Day 2
Day 3
Day 4
Day 5
Day 6 
Day 7
Lior
Confluent
Confluent
Confluent
Confluent
Confluent
Confluent
Cell death starts
Ronnie
Confluent
70%
50%
10%
0%
 
 
Noam
Confluent
70%
50%
5%
1%
Colonies formation
Colonies formation

What happened to the different cultures? (Choose between wrong and possible.)

a. Lior probably used an old stock of antibiotics that was not efficient. The cell death observed on the 7th day is due to the aging of the cells and over-confluency.  Some of the cells might have contained the transfected DNA incorporated in their genome, however due to the inefficient stock of antibiotics they could not be isolated.
b. Lior's transfection was inefficient. None of the cells incorporated the DNA and so they all remained alive.
c. Apparently, Ronnie's cultures were exposed to antibiotics in excessive amounts (x5 times the required concentration) and so no cells survived.
d. In Ronnie's cultures the transfection was inefficient. None of the cells incorporated the DNA and so they all died.
e. In Noam's cultures a small fraction of the cells incorporated the transfected DNA into their genome. They should be further characterised.
f. Noam's cultures were grown in low concentrations of antibiotics. Thus, most cells died and only a small fraction of the cells survived despite the fact that they did not necessarily contain the transfected DNA.