Initially, I have thought to use siRNA to study the role of specific genes in mo-DCs infected with T.cruzi, but I found this work these days:Article Induction of the interferon response by siRNA is cell type- ...
I need to evaluate what the pathogen modulate in the host cell and this would be a problem in my experiments.
What do you reccomended to achive my purpose?
This is technically challenging but:
1-The easiest way is to get the knockout or inducible-knockout mouse but I am guessing this is not available.
2-You can use CRISPR editing. Not sure how hard this will be in DCs but is worth a try. Not sure either whether the gRNA will activate IFN
https://www.idtdna.com/pages/education/decoded/article/successful-crispr-genome-editing-in-hard-to-transfect-cells
https://www.cell.com/molecular-therapy-family/nucleic-acids/fulltext/S2162-2531(18)30013-1
https://www.researchgate.net/post/Does_anyone_have_experience_using_the_CRISPR-Cas9_in_primary_human_cells
3-Finally, if the gene encodes a protein that is surface expressed, maybe antibody that blocks the protein can help uncover mechanism?
4-Can you try the knockdown in a primary cell that is easier to manipulate first? There are multiple monocyte cell lines that can give you some insight into the function of the gene
You can have similar effects with antisense oligonucleotides (see https://www.oligotherapeutics.org/immune-activation-therapeutic-oligonucleotide/), though morpholino is supposed to not activate the immune system.
Whichever you choose, you should start out with >1 siRNA/ASO and at a minimum a scrambled control oligo (see https://www.oligotherapeutics.org/guidelines-for-experiments-using-antisense-oligonucleotides-and-double-stranded-rnas/).
For Morpholinos, two non-overlapping translation-blocking oligos and a Standard Control oligo (negative control) are a commonly used set in cell cultures. A recent paper describes cell stress responses, including upregulation of interferon genes, from knockdown of some targets. However, these responses are target-specific and appear related to the knockdown outcome rather than the oligo itself. https://www.biorxiv.org/content/early/2018/11/27/479188
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