I want to clone a gene of 413kb size into drosophila system. Can anyone suggest the success rate of transformation of such sizes of genes and also what is the gene size limit for transformation in a model system like Drosophila?
Hi Gargi,
413 kb is indeed a very large fragment, ruling out systems such as P-element transformation. In this case, I think it would be best to look into cloning your fragment via the PhiC31 system, as this system is well established in D. melanogaster and fragments of any size can theoretically be cloned with it (see Geisinger and Calos 2013 Top Curr Genet 23: 211-239). You would just need to get your fragment in a vector containing an attB site for integration into one of the many available attP insertion sites.
I agree with Jason. Go for the PhiC31 approach. You can get flies with the appropriate attP landing sites from the Bloomington stock center. Good luck.
the largest insertion I remember published was 146 kb, and the efficiency will be low. Can you break your gene into smaller parts and add them sequentially using phiC31? This may leave small scars, but at least it would be a start.
http://www.sciencemag.org/content/314/5806/1747.long
Hi,
Like others have said, ØC31 is your best bet as p element plasmids have a size limit. The biggest p element plasmid I successfully transformed is 17.5kb and the efficiency drops to almost nil after that. I have made 200kb BAC using the ØC31 system and even then the efficiency is low compared to 10-15 kb plasmids. But 400kb constructs - the chance of this working is anyone's guess. We injectionist usually inject 200 embryos for a construct, for 200kb BAC, I'd inject 1000 to have any chance of success.
There is unstable repeat sequence in the intron of this gene which I want to interrogate for having any physiological effects. I wish to make many clones having variable sizes of this unstable repeat. Should I clone the entire gene or just few flanking exons surrounding the repeat?
This might be a good opportunity for using the CRISPR technique, because you can use the flanking exons as the homology arms of your CRISPR guide strand, and then alter the number of intronic repeats.
This gene has an ortholog in fly.. Which is of not much help. I was wondering if only few exons surrounding the intron of interest (containing unstable repeats) can be cloned insteasd of the entire gene or not. And if so, to check splicing disregulation, what should be my best vector?
- Badges
- Science topic
- Similar topics
- Books