Question

A new gene is discovered. Its sequence has a significant similarity to the sequences of known Toll Like Receptor (TLR). You then decide to investigate its properties. Which of the following experiments would allow you to test if the gene indeed functions as a TLR and is important in pathogen detection? Give your reasoning for selecting a particular strategy.

a) A NF-kB knockout mouse. Antibody staining to evaluate the production of cytokines and antimicrobial peptides, compared to the wild type mouse

b) Transfection of this gene into cultured macrophage cells. Quantitative RT-PCR to measure the levels of transcription of NF-kB after stimulation with various pathogens, comparing it to the un-transfected cells

c) A knock in of the new gene in a mouse. Quantitative RT-PCR for to measure the levels of transcription of several cytokines, comparing it to the wild type mouse

Answer 1

When a pathogen invade the our body first line of defence in innate immune system. Toll like receptors ( TLR) can primarily detect wide range of microbial componanents and can initiate innate immune response. TLR signalling usually works through activation of transcription factor nuclear factor-kappaB (NF-kappaB) gene which inturn control several inflamatory cytokines to combat the microbial attack.

Now, to verify whether the new gene is indeed is functionaing is Toll like receptor, I would choose the experimental method b.

Transfection of the macrophages witht the newly identified gene. If it shows functional similarity to Toll like receptor, if the macrophages are stimulated with several pathogen , then Toll like receptor ( TLR) will be activated. Which in turn will activate NF-kappaB gene and this can be verified by RT-PCR method with the presence of increasing amount of NF-kappaB in comparison to non-transfected cell.

I would not choose strategy a as, NF-kappaB knock out mouse as we want to taste the funtion of a gene which act like Toll like like receptor. SO this strategy will not verify whether activation of NF-kappaB is happening because of TLR receptor or not.

Strategey C for knock-in the new gene in mouse is time consuming process.