6 replies to this topic

[searcher]

It says that some genes need introns for expression hence I have to add it in my cDNA. How do introns look like and where should I add it?

thanks for any inputs.

[paulina]

Hi,

You don't need intron in your sequence. What you need is just the coding seqence (CD).

[searcher]

paulina, on Mar 22 2005, 06:44 PM, said:

Hi,

You don't need intron in your sequence. What you need is just the coding seqence (CD).

<{POST_SNAPBACK}>

Thanks pauline, but my article says this; " There are several examples of genes that have strong needs for the presence of an intron, so that it is better to include introns in cDNA expression vectors for mammalian cells. Examples are when transcription is driven by an immunoglobulin U promoter-enhancer..."  

Hope for ideas.

Thanks.

[paulina]

Yes, a few genes use promoters or enhancer that are located within introns.

[searcher]

paulina, on Mar 22 2005, 07:03 PM, said:

Yes, a few genes use promoters or enhancer that are located within introns.

<{POST_SNAPBACK}>

So does it mean that i don't need to add the intron into my insert, since it is already on the vector? Since the vector has the introns should it also have splicing sites to cut these introns off from the transcript?

Thanks.

[fred_33]

hi
normally if vector contains an intron there's no need to add it on your cdna.

[george@CASE]

to add to this, most expression vectors today are driven by strong viral promoters. All you need is a complete cDNA.

It is very difficult to start with a cDNA with no introns and then re-engineer the introns back into it. You'll have more luck by just taking out the genomic version of the gene itself, but they are almost always too large for mol bio purposes.