functional studies - (Sep/02/2008 )

Dear mates,

I am working in a gene that has two alternatively spliced isoforms. My main project now is to the important sequences that could affect the alternative splicing towards one of the isoforms, i am performing minigenes study by constructing different deletion in the noncoding area around the two exons, and then transfect them into mammalian cells and study the expression levels for both isoforms.


My question is, if i want to start simultaneously with another strategy that studies how the differenet splicing or transcription factors could affect the exon skipping in my case, by probably , cotransfecting these factors with the gene plasmid into mammalian cells and study the affect on splicing. But the thing is how can i select the best splicing and transcription factor candidates for my gene of interest. Is there any software that can help finding that or???????????????????

Thanks

---

thegen,

Hi there. I did my PHD thesis on an alternative splicing factor (same name as my alias if you want to look it up). One of the things I came across in my research was the idea that small changes in the makeup of the general splicing machinery can lead to changes in alternatively spliced products (no big surprise there). But the big question is whether these changes were really relevant to how splicing of a particular gene actually occurs in vivo. In other words, if you were to overexpress a general splicing factor in your transfection experiments, is that really telling you anything about how splicing occurs for your gene? My guess is that by taking a hit and miss approach, you might see small changes in your alternatively spliced products even if your factor isn't all that important to splicing of your particular gene. You might waste a lot of time generating data that is essentially meaningless. Therefore, you need to know what factors are SPECIFIC for alternative splicing of your gene. By specific, I mean that the factor only affects splicing of a small subset of genes. If you have no current information on this then it might be very difficult to proceed with your idea. If you were using a genetic model species (such as Drosophila or C. elegans), then I would suggest a genetic screen to identify a specific factor, but since it doesn't sound as if your lab works on these organisms, then I think your task is going to be more difficult. One option might be to look for factors that bind specifically to your pre-mRNA in a sort of affinity pull-down assay. Then you could use your minigene experiment as evidence to back up your claim that this factor is specific to splicing of your particular gene. Another option might be a yeast three hybrid assay. There may be other types of targeted screens that I'm not aware of (I haven't looked through the literature for several years), but the idea is that you need some way of targeting specific factors rather than just looking through a computer database and selecting your best guess.

Hope this helps,
smu

---

Thank you so much smu, your answer helped me alot .


I want to ask you if you know any nice books, websites and very good litreture that can help me, for example about bigger overview about aterantive splicing or splicing factors or????????????????

and in my case do you think that 2d-gels and mass spectometry might help in selection the splicng factors binding to my gene.??? a

and thanks again

---

Hi again,

I am wondering about affinity pull_down assay and yeast three hybrid system. These techniques are for studying protein-protein interaction, and in my case i want to know what proteins are binding to my DNA sequence to study the splicing method and the splicing factors. or???????.

So do you think that i can use them in my case????
Or if you can tell me your idea about how to imply them in my experiment.

Thanks alot

---

11

---