Gene not expressed in wild-type and knockout yet differences in microarray - (Dec/08/2010 )

Hi Bioforumers,

I have a puzzling situation on my hands. I am working with embryonic fibroblasts derived from knockout (KO) mice for a particular gene. These cells showed clear differences in behavior compared to wild-type cells, e.g. they proliferate faster. In order to find out the basis of these differences, I performed microarray analyses to compare wild-type and KO transcriptional profiles. To my big surprise, the target gene for the knockout was not expressed in both wild-type and KO cells. However, I do see distinct differences in the transcriptional profiles between the two types of cells and this is reproducible in a knockout in a different exon created independently in a lab located in another country. If the target gene is not even expressed, what then could account for the differences in transcriptional profiles between wild-type and KO?

If any of you have encountered similar situations, hope you can share some wisdom.

Thanks.

Neuropath

Where did the wild-type cells come from (same line of mice, siblings of the KO mice, skin...)?

bob1 on Thu Dec 9 19:32:36 2010 said:

Hi bob1,

Yes, the wild-type cells came from the same line of mice as the KO. I did PCR genotyping and saw deletions in the KO cells are as they should be. Bear in mind that I am seeing the same phenomenon in 2 different lines of mice. The KO cells from the two lines share highly similar gene expression profiles. Any ideas?

Are the wild type cells MEFs as well?

Is the target gene cell cycle dependent? Low level expression (check with qRT-PCR)? How was the KO generated?

Believe the biology. Clearly, this is an anomaly of the microarray analyses -- how did you do them? How many technical and biological replicates did you do? What statistical analysis says the gene is not expressed? Not expressed compared to what?

bob1 on Sun Dec 12 22:10:08 2010 said:

Hi bob1,

Yes, the wild-type cells are MEFs as well. The wild-type and KO MEFs were prepared and cultured the same way.

So far, there is no evidence to suggest that this gene is cell cycle dependent and it is not known to be involved in any cell cycle regulated functions. However, it is something worth checking. The probe intensities for the gene is either at background value or even negative relative to background. Housekeeping genes levels are normal and all the QC analyses are ok so I do not doubt the quality of the microarray data. I have done semi-quantitative RT-PCR which but did not get the expected band size even at high cycle numbers. I am going to try doing RT-PCR on freshly isolated MEFs and see if the gene was shut down during in vitro culturing or passaging. However, it would still not answer the question of why there are differences between the transcriptional profiles of WT and KO.

KO for both lines (two differenct exons) were generated by cre-lox excision. The KO were not done in my lab but in 2 established labs, one in the US and the other in Japan.

HomeBrew on Mon Dec 13 01:45:17 2010 said:

Hi HomeBrew,

I did two biological replicates and two technical replicates for each on the Illumina Mouse WG-6 Beadchip. Probe signals with detection p-value <0.05 was normalized and background subtracted using GenomeStudio. It is basic but it is sufficient to give me information about the expression of the gene. The signal intensities for the 4 different probes for the gene are at either background or below background levels. All the QC analyses checked out fine. From my experience with other Illumina beadchip experiments in the past, I know that the data tend to be quite reliable.

I was hoping someone out there would have encountered a similar phenomenon - i.e. KO target gene not expressed in both WT and KO yet there are phenotypic/functional/transcriptional differences between the two. My search of the literature has yielded nothing so far. It is even difficult to come up with a good search string

neuropath on Mon Dec 13 16:00:54 2010 said:

This is a self-contradictory statement -- if a gene is not expressed in WT, how can knocking it out have any phenotypic effect? That is why I was asking about how you concluded it was not expressed in wild type...

HomeBrew on Mon Dec 13 17:31:02 2010 said:

That is the whole purpose of my post......to shed some light on this paradoxical phenomenon. Signal intensities from WT are either zero or negative values, just like KO. The data has been normalized.

There has been a new development. I have found some papers to support my observation that this gene is not expressed in MEFs. This gives us some assurance about the validity of the microarray data. So the question remains......why are there differences in transcriptional profiles between WT and KO when the target gene is not even expressed in WT cells?

Did the papers you found also rely on microarray expression profiles to conclude that this gene is not expressed in wild type?

I can't get past the fact that if a gene is not expressed in wild type, then it can have no influence on wild type phenotype, thus knocking it out would also have no effect.

Perhaps the Illumina probes for this gene are faulty?