Hi,
I would like to carry out an in vivo mice infection experiment with Leishmania parasites where I want to knock down my gene of interest in once the parasite is in the mice. Initially I would like to maintain the parasites in the WT form, so that the initial stages of the infection remain unchanged. I would like to see how the mice respond at later stages when I knock down the particular gene in the parasite. So, I would like to knock down the gene only after infection establishment in mice. I know that there are tet-off systems available, where we can add tetracyclin to a cell culture to knock down particular gene. But my understanding is people do this only in cell cultures (in vitro). Is it possible to carry out similar thing in vivo? Is there a way to feed the mice with tetracyclin, may be with water or by direct injection to knock down genes in the parasites inside them? Alternatively, is there any other approach that I can adopt to carry out this experiment?
I would really appreciate any comment or suggestions!
Thanks
Sumit
Is there a marker of the "infection" stage? As a proof of concept, you could engineer a plasmid that expresses CRISPR/Cas9 system or alternatively Cre after detecting the infection stage marker. (http://www.ncbi.nlm.nih.gov/pubmed/25939677)
@ Swapnil: My aim is not to knock out/ down a mouse gene. It is to knock out/ down gene from the parasite when it is inside the mouse. However, do you know if Cre-lox system works for the parasites?
I work with zebrafish and we use morpholinos for (transient) gene knockdown. While I have never worked with parasites, I know morpholinos are used in other model systems. Checking pubmed, it looks like someone has used MOs (morpholinos) in parasites. Here is the article, and good luck!
http://www.ncbi.nlm.nih.gov/pubmed/26351679
Looks as if the cre-lox system has been used to knock out genes in parasites.
http://www.ncbi.nlm.nih.gov/pubmed/23489321
I know nothing of parasites, however, an inducible cre recombinase construct would allow you to time the excision of your target sequence.
I don't think that you would be able to express cre recombinase in the mice to knockdown a loxP flanked gene in a parasite. A lot of the inducible cre-lox systems rely on chimeric fusion proteins, such as cre recombinase fused to a modified estrogen receptor in which binding of the ligand (tamoxifen in our case) initiates translocation of cre to the nucleus. Not sure how you could get cre into the nucleus of the parasites cells.
Matt, yes, I was suggesting a Cre-lox system engineered into the parasite. Unclear if this will affect growth or other parameters of interest that OP may want to measure un disturbed. CRISPR/Cas9 has also been used in the parasite that OP is interested in.
I've no experience with CRISPR/Cas9 but I've heard that its cheap and easy to perform. Because I doubt there will be a commercially available transgenic parasite (perhaps there is?), CRISPR might be preferable.
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