Not sure what is the aim of the question... are you looking for such a mouse model or are you asking if something like this could happen, that you insert the Neo cassette in an intron and you reduce the endogenous gene expression?

This last thing happens regularly when you produce conditional mouse models, you insert a LoxP site and a Neo cassette in an intron and a second LoxP site in a subsequent downstream intron. This almost inevitably reduces the expression of the gene you are targeting, because Neo expression is driven by a strong promoter (Actin, PGK, etc) in order to select the cells and it both sequesters transcription factors from the usually much weaker promoter of the gene you are targeting and stops whatever upstream transcription of the targeted gene can occur because of DNA occupancy by the transcription machinery functioning on the Neo gene. For this reason the Neo cassette is regularly flanked by Frt sites so that you can remove it using a Flp recombinase, cleaning up the locus and leaving only the LoxP sites for future complete inactivation of the targeted gene with a Cre recombinase.

But it can also happen that the insertion interferes with a strong transcription enhancer present in the intron.

In any case your reduction in protein production is a consequence of impaired mRNA transcription.

Pietro,

Thanks for the information. We have a mouse model that the Neo insertion in the intron 1, results in a low expression of the protein, but the transcript level seems to be not much difference although KO mice also contain ~30% transcript, which makes the assay to be difficult. We have to look at the transcript closely since the hypothesis is that the insertion could disrupte gene transcription and /or RNA splicing, not much theory about affecting on translation of protein, am I right? I need do more works on gene transcription and RNA splicing rather than protein translation part, this is my key question. Please let me know if have more information concerning on this.

Ruiting,

Yes Ruiting, sorry for the late reply, but you are essentially right. One would not expect anything at the level of translation from an intron, unless you have a splicing problem that for some reason is not undergoing NMD (non-sense mediated decay). This means that due to the Neo cassette insertion you might disturb splicing but still produce the mRNA. When the mRNA arrives in the cytoplasm, nevertheless will never be able to translate the endogenous protein. Depending on how efficiently the splicing mistake happens, you'll have different degrees of protein loss. You can test this by RT-(q)PCR using primers on the Neo cassette, from your total RNA (but be sure to have a good negative control, RNA without the RT step).

If you have any other doubts don't hesitate to contact me again! Good luck.

Hi Pietro,

Thanks again. I have done some RT-PCR to examine the splicing problem, but I did not find the problem so far, qPCR data suggest that the transcript level is not much changed either with the negative controls within a single mouse sample. I think I need repeat the tests with more mouse samples to collect some more information.

Have a great weekend!

Ruiting,

Hi Elsa,

A great point. We were thinking about this approach, but just do not have lots of time to do it. Nevertheless, we know that the mRNA level is down too by qPCR besides low level of the protein was found in mouse line, that maybe ok for us right now.

Thanks for the suggestion.

Ruiting,