I want to clone a target gene into a lentiviral vector to produce a mouse cell line that overexpresses the target gene.
I want to know the difference between inserting the full-length cDNA and ORF sequence of the target gene when producing these cell lines.
Also, if the restriction site contained in the cDNA sold by origene does not match the site of the expression vector, please let me know how it would be better to insert.
Thanks
Hi Jihyun,
The ORF or open reading frame is just the DNA sequence that codes for the protein of interest.
Most likely the cDNA clone is very similar to the mRNA, as in it includes the 3'UTR, ORF and 5'UTR.
For your cloning question you could try to do blunt end cloning. Open your expression vector blunt the ends and then directly clone in the cDNA you bought.
The other thing you could try is to PCR amplify the cDNA clone with an overhang on one or both primers including the restriction site you want to use. Then cut the back bone and cDNA PCR product and clone from there.
Hope that helps.
Hi Jihyun,
If you are sure that there are no common restriction sites in the MCS of the two vectors (since sub-cloning is easier), you could amplify the target gene from the cDNA vector by incorporating the desired restriction sites into the primers like Christopher mentioned. Good luck with the cloning!
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