generating an array by gene amplification - (May/06/2008 )
I am not sure if this topic would be more relevant to cell biology but it`s also about cloning.
I am trying to genarate a tandem array of DNA-binding sites for a nuclear receptor and then integrate this DNA into mammalian cell line by stable transfection. The cloning part has been quite demanding. After a certain number of cloning steps where the array sequence doubles with each step, the array DNA becomes very unstable. I tried various bacteria including SURE cells however, I got short insert/array due to recombination/restriction (there must be some topics in the bioforum regarding this problems). I also tried to clone in a low copy plasmid backbone. It improved the cloning but this did not help in further cloning steps, either.
I`ve read about another way of generating such an array by gene amplification. One can avoid those many cloning steps and getting an unstable construct. One inserts the sequence in a plasmid (pSFV dhfr) that contains both a mammalian replication initiation origin and a matrix attachment region from the Chinese hamster dhfr gene: when integrated into the genome, it initiates events similar to gene amplification in cancer cells, and it generates tandem repeats of up to 10 000 copies (Bosisio et al, The EMBO Journal (Vol 25, No 4, 2006)).
Does anybody have experience with that? How would one keep the gene amplification stable after getting the transfectants?
Any suggestion would be appreciated.
You could create long (uncontrolled length) repetitive DNA by circularizing your sequence, followed by in-vitro phi29 amplification using random (or non-random) primers. I don't know if this would help you or not.
Thank you phage434!
I will read more about it.