Minigene splicing assay is undoubtedly useful and popular method to dissect splicing regulation, its aberrations, etc. When used for detecting the effect of mutations on splicing, most of its reported results are generally consistent with the results from patient's blood. However, sometimes the method just can not be used. As has been reported in Lastella et al., 2006, some of the cloned exons might be totally excluded from the final transcript (even in the wild type form), preventing any investigation of mutations effect. Possible explanation is that the cloned exon needs a larger genomic context for its proper recognition, as in Baralle et al., 2006. But, we have encountered a case that can not be explained such easily.
In our case, we have cloned exon 10 of the was gene into the pET01 vector (MoBiTec). In 2011, we saw partial exon inclusion in HeLa cells (circa 80 % exon inclusion, 20 % exon skipping by estimation). However, in 2013, we repeatedly detected only exon skipping – both in HeLa and U-937 cell lines. There was no change of the cell lines between these experiments, only ordinary cultivation. As we really sought to work with this exon, we have cloned it into the pET01 in a larger genomic context: exons 8–11 with parts of surrounding introns. Still, we could detect only exon 10 skipping in HeLa cells, as well as in U-937, HepG2 and EBV-infected lines. Later, we have purchased a new stock of the HeLa cell line. No surprise came: once again, we have detected only exon 10 skipping (tested only in the larger context). Of note: the first HeLa cell line was originally ordered from ATCC and the second one from ECACC, both credible sources.
Do you have any idea, what might have happened? Or do you have any similar experience, some minigene-related troubles? Maybe we could make a leap forward to find a solution together. I really look forward to discussing these issues.
http://www.ncbi.nlm.nih.gov/pubmed/16995940
http://www.ncbi.nlm.nih.gov/pubmed/16870183
Hi Dr. Grodecká,
I was particularly interested on this discussion because I have faced a similar problem few months back. I was analyzing the COLQ exon 16 alternative splicing in human KD3 myoblast cells. From a sample extracted a year ago (an early passage of KD3 cells), I've got ~70% inclusion; whereas, from a recent sample it was almost 80% skipping!
The probable explanation for this is:
1. the number of passage have changed the properties of the cells
2. the cells are transformed
However, your scenario seems to be different from mine. I will follow this discussion in hope that someone can give a better insight about this, as you mentioned, 'pitfall'!
Dear Dr. Nazim,
in agreement with your proposals, the change in the intracellular environment (e.g. the levels of splicing factors) was the most probable explanation that came to our minds. However, our case shows not just the change during the cultivation (even if we regularly freeze the low-number passages), but also the difference between the different stocks of the same cell lines. This is something we should be always aware of when we do the minigene assays and when working with cell lines in general.
This brings another answer - is there any solution, how to avoid such differences or rather how to accomodate the experiments to count on with such differences?
Dear Lucie,
If I understand well, you detected exon10 inclusion only once in 2011. Could you imagine that cells were in different conditions at this time? A critical parameter might be cell density, since this may affect alternative splicing of genes for cytoskeletal proteins. Another parameter might be the plasticware, which may have different coating properties, hence different outcomes on AS.
Besides, it seems that the nature of alternative transcripts of the WAS gene is not well annotated in databases. Annotation of the Ensembl ENSG00000015285 gene shows only two and probably incomplete transcripts. In NCBI (http://www.ncbi.nlm.nih.gov/gene/7454), two mRNAs differ from what is shown as Exon10, which contains two alternative acceptor sites. Is it what you're looking at?
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