We have Tet-ON mice transgenic for our human gene of interest that have been on doxy diet (food pellets at 2000mg/kg ) for at least 10 weeks. We have not been able to detect our protein of interest by ELISA or WB in liver, spleen, large and small intestine samples. We confirmed by qRT-PCR that messenger RNA is being made for our gene of interest using DNAse treated RNA samples, but we don't understand why we are not able to detect protein. What could be the reasons as to why I am not able to detect protein? We always include positive controls for both ELISA and WB experiments. The antibody we use can detect protein as low as 65ng, and the tissue lysates derived from these mice contain 100ug of total protein. Unfortunately the expression of gene of interest is not accompanied by GFP. Any direction or answer will be much appreciated.
Doxicycline is an inhibitor of protein synthesis.
see Research TETRACYCLINES AGAINST CANCER. A REVIEW
- DOI
- 10.13140/RG.2.1.4097.16
Hello Mirian.
I agree with Jiang's comments. Best to evaluate the transgenic vector-transfected cells in vitro first. In this experiment, you could know what is the problem.
The other point is that number of copies of the transgene inserted into mouse genome. Most of transgenic mice would have several to over ten copies (i.e., overexpression).
Considering positive PCR products of cDNA, but not the protein may suggest that the mice happen to have only a or a few transgene copies. Then, it would be hard to see any expression at protein level (i.e., no GFP under fluorescence microscopy even if the transgene contains GFP reporter) or Western blot etc.
Test in cells in vitro first.
Best wishes.
first check the sequence of the vector AND by sequencing the genomic integrated DNA...
then check where it is integrated - it could be that during integration the sequence was changed e.g. by recombination or it also could be that the stuff is silenced so also check the integration environment...
another thing could indeed be the RNA, unstable, 3D folding (which also could happen to the DNA), perhaps you also check the amino acid compostion of your protein? rare codon usage?
but I suspect the best is to get genomic DNA and sequence your integrated vector sequence !
As Tomas said, Doxycyclin is an inhibitor of protein synthesis. So it could be that the diet you're feeding your mice is actually inhibitory to your tet-on system.
Hello. Did you resolve the problem you had with the transgenic mice? I have the same issue.
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