pLenti-puro vector- effect of insert on plasmid stability?
I am trying to clone a 2.9 kb cDNA for a human long non-coding RNA (lncRNA) into Addgene vector pLenti-puro (7.1 kb). I am inserting directionally into NotI (sticky) and PmeI (blunt). The vector, which has lentiviral repeat sequences, has a tendency to be unstable. It tends to recombine after transformation even in recA-mutant host strains. I am using the recommended host strain Stbl3 which is supposed to reduce these recombinational events. The vector was dephosphoryated prior to ligation and the vector-only ligation transformation plate had no colonies. The colonies I got on the experimental (vector + insert) plate all produce what appear to be recombined plasmids. The desired construct would have been about 10 kb. These are varying in size from about 2.9 to 3.1 kb- just big enough to have the backbone ori and the AmpR gene, but not the lentiviral insert. I can cut them and get a single band with Asp700 (isoschizomer of XmnI; site located in the Amp gene, but there should be a second site in the lentiviral insert) but they do not cut with either NotI or PmeI. I also tried the same ligations in DH5alpha with the same results. I also tried incubating the transformation plates at 30°C, as recommended by some literature, to reduce recombination; this produced some late-appearing colonies, but again, they had spat out the lentiviral insert.
Yes, I know pLenti-puro has 2 NotI sites and I am using partial digests to get the vector cut only at the site located in the MCS. I have done partials before, and I have reasonable confidence in my prep of vector and insert, and my ligations. I am going to try testing the vector prep by cloning an innocuous 1.3 kb fragment of bacterial origin and see if I get anything from that ligation. But I know there are things you just can’t clone, or at least, they won’t go in in certain orientations, due either to the sequence being too “foreign” for E. coli, or too repetitive, or because it encodes something lethal to E. coli. And this lncRNA sequence has a lot of difficult regions: dinucleotide repeat runs, etc. I am beginning to suspect that the construct, if it does not recombine to spit out the insert, is lethal in E. coli. I suspect that the more I suppress recombination, the fewer clones I’m going to get.
The 2.9 kb lncRNA insert was originally cloned in pCR4-Blunt-TOPO (Thermo) by its originator, and I have been told that, despite the non-directionality of TOPO cloning, only one orientation of the insert was ever obtained, even though many clones were screened. The NotI and PmeI sites are provided by the pCR4 vector.
Meanwhile, if anyone has any experience with this vector, how large an insert have you successfully added? Has anyone else had trouble with inserts this size or larger in this vector? Or any other advice? It would be much appreciated.