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Transgenic mice express mRNA but no protein

Transgenic mice express mRNA but no protein - Knockout Mouse Forum

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Old 03-04-2010, 05:23 PM
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Default Transgenic mice express mRNA but no protein



Hy, we spent two years to set up a transgenic unit; at the end I got several founders transgenic mice nicely expressing my transgene under the control of CMV promoter, but none express the related protein! The sequence is OK, the polyA tail is added. I am very frustrated, no case in literature, have anibody had similar problems or can give me an explanation? Thanks, ciao roberto
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Old 03-05-2010, 12:11 AM
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Default Re: Transgenic mice express mRNA but no protein

What is the gene? Is it a mouse gene? Could it have anything to do with codon bias/availability? I have never done this personally but there was a question on our qualifier exams about this stuff.
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Old 03-05-2010, 08:56 AM
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Default Re: Transgenic mice express mRNA but no protein

Thanks for your prompt answer. The transgene is the human homologue of the mouse gene, EMILIN1. The use of a homologue is a quite common strategy in gene function studies, moreover we tested the expression of the human protein in mouse derived cells in vitro, and they nicely express our gene.
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Old 10-18-2011, 05:21 AM
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Default Re: Transgenic mice express mRNA but no protein

Look at your sequence of the gene again and make sure that it doesnt have any false stop codon in the middle
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Old 11-23-2011, 07:22 PM
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Default Re: Transgenic mice express mRNA but no protein

How are you detecting mRNA expression??? If you're using RT-PCR you may be detecting very low expression levels, whereas your antibody is almost certainly a much less sensitive assay.

CMV is not a good promoter to use for making transgenic animals. It seems the viral sequences are prone to getting silenced in vivo and if you do get expression it's likely to be very patchy and may be extremely unstable over generations. A much better choice if you want strong ubiquitous expression is the Chicken-Actin promoter:::rabbit-Globin polyA construct (pCAGGS generated by Hitoshi Niwa). Alternatively you could do knock-ins into the ROSA26 locus or use a promoter specific for the tissue that you're interested in (if one has been characterized).

I suspect the problem is not your coding sequence, but rather it's a problem that the CMV promoter is being silenced.
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