I have a strange problem. I am using this mouse model:
http://jaxmice.jax.org/strain/009604.html
I designed primers to amplify the mutation site (gDNA). This works fine, the primers are designed vs. human-LRRK2 and do not amplify in my WT-mice (mouse-LRRK2) at all.
I sent my product for sequencing to confirm the mutation but I am only sequencing the non-mutated sequence of human-LRRK2. The 4321C>G exchange is simply not there.
Sequencing was repeated once including an additional animal, same result.
BTW:
I did the same for the JAX G2019S mice (http://jaxmice.jax.org/strain/009609.html), for these guys everything was as expected, so my procedure itself should be fine, I guess.
Did you get any amplification in your negative control? Could it be possible that you are amplifying your own DNA?
There are quite a few differences between the human LRRK2 and mouse Lrrk2 at the gDNA level - are you able to confirm that you *are* sequencing the human version? Absence of a band would be expected if you have human-specific primers, but can be caused by so many other experimental variables (DNA quality, forgotten reagent, contamination, lab gnomes) that it's not the most reliable way to go about it. If you are just looking at the 4321C position, it is a conserved C in both human and mouse reference sequences.
You could also try contacting JAX technical services: [email protected]
Hi Stephen & Joanne,
thanks for your helpful comments.
The chromatogram of the sequencing results shows, without doubts, only one peak for all of my replicates. If there was contamination with my DNA, there should be two peaks.
The sequence is quite conserved but I am looking at a 200bp long sequence around the mutation site, there is not a single base exchanged!! And when I am aligning both to the annotated mouse-LRRK2 sequence there are many basepairs exchanged.
And as I already mentioned I am not amplifying the mouse LRRK2 in the non-carrier mice using my primers.
All my NTCs were also negative.
At the moment I am thinking that we either got the wrong mice or the wrong ones were ordered... Because as I saw Jax lab hast also mice available carrying the human wildtype LRRK2.
I need to discussion with my PI next week, afterwards we will decide what to do.
Hi Jonas,
Did you have any luck working out what was going on with this experiment?
I have heard people spending time working on a particular strain of mice (ie. a K/O), only to realise it was not the right one. That lab now spends quite a bit of time genotyping their mice before running the experiment.
I hope it was an easy fix - let us all know how you went.
CHeers,
Steve
Hi Stephen,
thanks for asking!
Since we're having the G2019S and R1441G mice, my last idea was that these guys were just mixed - otherwise it would be a big mystery because I sequenced 3 other R1441G-mice, showing the R1441G muation, in the meanwhile.
If they were mixed, I am sequencing of course wt-human LRRK2-sequence as it should be for the G2019S mice at the R1441G-mutation site.
I just sent everything for final sequencing, I let u know as soon as I get the results.
Best, Jonas
- Badges
- Science topic
- Similar topics
- Medicine
- Disease
- Disease Models
- Animal Models
- Rodent Models
- Mouse Models