1
$\begingroup$

I'm currently immersed in research involving the Rosa26-tdtomato (Ai9) mouse line. We performed scRNA seq and analysis focusing on tdtomato-expressing cells but faced a challenge with a lower-than-expected number of positive cells.

Our alignment process, using the tdtomato sequence (Genbank accession: AY678268), seems insufficient. Fellow researchers suggest success in similar cases by incorporating the full mRNA sequence, including the 3´UTR, along with tdtomato-WPRE mRNA.

I searched everywhere for the full mRNA sequence but couldn't find it. Can anyone share insights or provide the complete mRNA sequence for tdtomato-WPRE? This would greatly enhance our alignment process and improve the accuracy of tdtomato positive cell detection.

Appreciate your contributions to advancing our research!

$\endgroup$

2 Answers 2

2
$\begingroup$

I'd suggest looking at the mouse itself, which appears to be here on the JAX website.

This link includes RNA-seq experiments, so presumably you should be able to compare your pipeline and data to previous work to see whether there's anything weird there.

Looking at the original paper, you can see the structure of the transgene which might be enough info to work from, there is also information in the supplement. You should in principle be able to figure out the sequence of each of those components and include them.

If you are really having issues, you could do a transcriptome assembly to see whether you can recover the locus.

You can always write to the senior author asking about this line, or try to figure out why your transcript may truly not be expressed.

ai9 transgene structure

$\endgroup$
0
$\begingroup$

The Genbank sequence you've specified appears to be for RFP, not tdTomato.

Have you tried one of the other tdTomato sequences, for example a lentiviral sequence, or a sequence that is called tdTomato?

https://www.ncbi.nlm.nih.gov/nuccore/KJ081243.1

$\endgroup$

Your Answer

By clicking “Post Your Answer”, you agree to our terms of service and acknowledge you have read our privacy policy.

Not the answer you're looking for? Browse other questions tagged or ask your own question.