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Cloning repetitive sequences in mammalian cells


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#1 Chelo

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Posted 07 April 2013 - 06:53 AM

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.

#2 bob1

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Posted 07 April 2013 - 12:37 PM

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.

#3 Chelo

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Posted 08 April 2013 - 04:44 AM

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.

#4 bob1

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Posted 08 April 2013 - 01:54 PM

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.




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