This is great question, and the comments are helpful.

1) Adding a frameshift in an early exon should result in downstream exons being converted to non-coding exons. Nonsense-mediated mRNA decay mechanisms should then be expected to reduce expression, which is one reason the OP is listing this as the expected result.

2) The other known mechanism we think a lot about in these cases is gene compensation. The same gene - or other genes - getting upregulated when one or both alleles is/are defective.

1. Why do you expect a knock-down on the RNA level when the frame-shift only matters on the protein level? 

2. If it is true that you have a significant increase on RNA level of the heterozygous mutant compared to WT, it might be a hint that the protein might induce a negative feedback (directly or indirectly) on the expression of itself.

As you have a heterozygous mutant you have effectively less protein expressed in the beginning altough you have the same RNA concentration (sum of WT RNA and mutant RNA); but only half of the translation products form the active WT protein meaning if the protein act as a hypothetical (in)direct repressor on its expression itself, their will be less effective repression in the beginning (compared to WT) and the expression of your protein increases to (near) physiological level.

I guess if you quantify your protein you will have nearly the same level like in the WT mouse but an increased RNA level?

I agree with Jeffery, The inroduced deletiojn in the exon shouldn't exactly knockdown the expression. The stop codon would just render the protein non -functional. The increase may be because of the regulatory mechanism in the cell type which would increase the expression at the transcriptional level since there isn't a functional protein produced which could tune down its own expression by performing the designated function. 

This is great question, and the comments are helpful.

1) Adding a frameshift in an early exon should result in downstream exons being converted to non-coding exons. Nonsense-mediated mRNA decay mechanisms should then be expected to reduce expression, which is one reason the OP is listing this as the expected result.

2) The other known mechanism we think a lot about in these cases is gene compensation. The same gene - or other genes - getting upregulated when one or both alleles is/are defective.

The mutant RNA may be degraded faster but the turnover of the TFs and RNA poly may be faster thus increasing the transcription rate. If you move the detection region (no idea how many exons/introns in your gene) closer to the 1st premarture stop codon you may see different expression level. Like stated above other compensatory feedback loops are possible.

Thank you all for your comments. And Stephen - good to hear from you. This is very helpful. I do believe it is most likely a case of compensation. Unfortunately, there is no specific antibody for this gene  as it has a separate homolog that is 96% homologous. The antibody that we are using has an epitope that is shared with both genes. We see absolutely zero differences in expression at the protein level with this antibody (striatum) which produces very nice single band staining. For now, we are just trying to demonstrate some sort of functional molecular consequence of the mutation that is directly tied to the gene and will be sufficient for publication. We already have a behavioral phenotype that we replicated in two separate lines (two founders, same TALENs).

A frameshift mutation will create a premature stop codon in most cases with a shorter protein produced, but in some cases a longer protein is obtained because the stop codon is downstream of the wt stop codon.

Nonsense mediated decay (NMD) will  cause breakdown of the mRNA, but only if the stop codon is in the 5' part of the transcript, the further downstream the less NMD, when present in the last or the second last exon or within 50 bp of the donor splicesite of the second last exon, NMD does not occur.

The deletion will create a shorter PCR fragment compared to wt, the PCR efficiency will increase with the shorter PCR fragment, which result in more PCR product.

Hi Camron,

Did you ever get this resolved or publish your findings? I have had a similar issue with some Crispr mutations...

Hi Sarah,

we published it. https://doi.org/10.1371/journal.pgen.1005713

• we demonstrated 50% reduction of the wild-type transcript level (as assessed via designing primers that were specific for the wild-type transcript (that contained the otherwise deleted region). total transcript levels (WT + mutant) are upregulated though. at the protein level, we have not been able to demonstrate 50% reduction b/c there is no specific antibody (H1 and H2 are 96% a.a. sequence similarity as i mentioned above) so there could be an upregulation of H2. we are using mass spec to hopefully sort this out.