Hi Nireekshit,

I suspect, in advance, that your mouse DHC will be able to compensate the loss of the human DHC, because they are 99% identical in protein sequence (do a blastp of the mouse protein sequence you have expressed in your cell line and the endogenous human one). Now, there's probably a high chance that the proteins are different enough at the mRNA level that you can design (or buy from a company) a/a set of siRNA/s that can only target the human DYNC1H1/2 without knocking down the overexpressed mouse protein (be aware that there are two isoforms, and to see something, you might want to KD both to avoid redundancy). We have done it in the past and it works, if the siRNA is specific enough and/or the species are far from each other in evolution. So check the sequences of the siRNAs, try to double-check that they align against the human mRNA but not the mouse mRNA that you are also overexpressing. The 'problem' or difficult step will be to simply verify/confirm/show good evidence that the siRNA is working properly and KD just the human protein since you will have the overexpressed protein doing its work in that cell (unless the phenotypes are strikingly different). WB probably will show specificity for both proteins (again, they are highly similar at the protein level) and it's not a very quantitive approach to convince that the levels of one of the two proteins in that cell has changed after a siRNA treatment. But probably you have a tag in your overexpressed protein that could help you to distinguish the human from the mouse...but I don't know...If you have a chance, maybe you can measure by RTPCR the levels of both mRNAs, the human and the mouse ones, using different primers, in WT, overexpressed, and KD of hDYNC1H in the overexpressed. If the KD is short, you could also test the efficiency of the KD in the WT human cell line with those siRNAs, by RT-PCR again and also see dispersion of most of the organelles that depend on dynein for retrograde movement (eg lysosomes). But I expect the cell to be not happy for a long time if it lacks DHC for long periods of time.

You could go further if you want: design a CRISPR experiment in that stable cell line for kocking out the human DHC. You could confirm the KO by sequencing the genomic DNA, but you can show that you still have DHC by WB, because of the overexpressed one.

Hope some of these suggestions work for you! Please, let me know if it's helpful. I may have additional advice if you want to test the role of dynein in a certain controlled experiment, so you could provide a little more detail in a future answer.

Cheers, 

Charly

Hello Charly,

Thanks for your response. I understood what needs to be done and what could go wrong. I am slightly afraid I do not have the resources at my disposal to do a CRISPR.

For a start, this is what I did. On siDirect, I found all the possible siRNA sequences for mouse dynein. I do have some siRNA sequences against human dynein from literature. I cross checked if any of the human dynein siRNAs match with the mouse dynein ones from siDirect. Thankfully none did. I am hoping this means that the siRNA sequence I have right now is specific to human dynein only.

It would be great if you can give your inputs on this.

Cheers,

ATN.

Hi there!

There's always a probability of having off-target effects with siRNAs (and also with CRISPR). Being said that, I'm sure that you are in the right way to solve your problem. Remember to check by WB if the transfected mouse DHC is still there after you did the KD of the human endogenous one.

CRISPR KO is becoming cheaper and cheaper, so try to find out if it's a possibility. If not, I make my KOs "from scratch" in the lab. In two months, you can have a KO. Boom! The only thing that you need is the plasmid with the Cas9 system (check addgene!) and that's almost all!

Good luck!

Charly