Question

You would like to study Listeria monocytogenes (L.Mo) pore forming toxin listeriolysin O (LLO) first in murine bone marrow-derived macrophages and then in Mice animal model. Please answer the following in details:

A- In order to test the function of LLO in macrophages, you first have to create an LLO deletion in L.Mo. How would you create this deletion in vivo?

B- How would you verify that the deletion has been created? How does it work?

C- Once you have created the mutant, what is the first test you would perform to test the function of LLO in wild type macrophages?

D- Your results from “c” show that the transcription factor IRF3 is not activated in macrophages in the absence of LLO. Which test would you perform to confirm these results?

E- Describe how to create an mouse model with IRF3 deletion.

Answer 1

A - To create the mutant you can use Genome editing technique like transfection. since we want to first see in macrophages. this is stem cell culture. So first create an E. coli with the required deletion ( in this case introduce a faulty gene ) and add it in the culture where macrophage cells are growing.

B- you can add a marker floroscent gene along with the introduced sequence in e. Coli. Do cell culturing and then the second batch check for flouroscence if it is picked up then the gene is transferred to the next generation or next cell type.

C- wild type will not show fluorescence mutant will. Also second text can be you can grow it in a culture with an antibiotic or something where the poison can be detected if the cell has picked the mutation or not.

D- I will simply extract the DNA and run it on gel. If there is no sequence that is responsible for making the IF3 then it will not have a band on the gel.

E- Clustered regularly interspaced short palindromic repeats(CRISPR technology) can be used to create a mutant in the mouse. First prepare guide RNA from E Coli using a tramsfection method. Once you get the gRNA prepared run it on gel to see if it has the required sequence. Then inject the mouse with the guide RNA and Cas9 nuclease.