Cloning repetitive sequences in mammalian cells - (Apr/07/2013 )
Dear Forum members,
I need to clone a repetitive sequence (several kb in length) in a mammalian cell line. Since this sequence will be prone to recombination, I would need to work with some kind of REC minus cell line. My question is two-fold: 1) is there any way to integrate DNA into chromosomes without using the recombinant machinery of the cell? 2) is there any mammalian cell line available that is a mutant for essential recombination genes? Thanks a lot in advance for your kind replies.
Rec- only really applies to bacteria as far as I know. Typically you would clone the sequence into a plasmid and then transform that plasmid into bacteria so as to be able to grow up the bugs and get lots of the plasmid back.
There are a number of systems that can be used to integrate DNA into mammalian chromosomes, but most of these rely on either native recombination events or the use of a cloned bacterial recombinase and specific sites in the cell that have been generated earlier (e.g. Invitrogen's FLP-In T-REx system). If you have the right cell line containing an EBNA sequence, then plasmids can be retained and will be replicated by the cell, but they won't be integrated.
I don't know of any cell line that is recombinase deficient.
bob1 on Sun Apr 7 20:37:37 2013 said:
Rec- only really applies to bacteria as far as I know. Typically you would clone the sequence into a plasmid and then transform that plasmid into bacteria so as to be able to grow up the bugs and get lots of the plasmid back.
There are a number of systems that can be used to integrate DNA into mammalian chromosomes, but most of these rely on either native recombination events or the use of a cloned bacterial recombinase and specific sites in the cell that have been generated earlier (e.g. Invitrogen's FLP-In T-REx system). If you have the right cell line containing an EBNA sequence, then plasmids can be retained and will be replicated by the cell, but they won't be integrated.
I don't know of any cell line that is recombinase deficient.
Dear Bob1, thank you indeed for your reply. As a matter of fact, I really don´t need plasmid integration if the cells could keep the plasmid copy number. This is because I will use the transformed sequence as a quantitative control. So my following question would be if you know whether there is any plasmid that assures the copy number per cell constant (it would be sufficient with a single copy).
Thank you once more.
I don't think there is any way of ensuring that the copy number per cell is constant other than to make a single integrant cell line. This is quite time consuming (I would plan on more than 6 months) and relatively expensive.