Difference between a 2nd and 3rd generation transfer plasmid/vector?
It's very clear with detailed explanations describing the differences of 2nd and 3rd generation lentiviral packaging systems. But I can hardly find any information on how to classify a transfer plasmid as 2nd or 3rd generation.
The information I have on hand is 2nd generation transfer plasmids have wt 5'LTR while 3rd generation have modified 5'LTR conjunction with a promoter.
Is this the only difference and how does the modified 5'LTR make it Tat independent & safer at the same time?
The 2nd generation packaging systems contain Tat, which is necessary when the wild type HIV 5'LTR U3 region does control the viral transcription. When the U3 enhancer/promoter is replaced by another promoter (e.g. RSV, CMV), you can use the so-called third generation packaging plasmids (that do not contain Tat).
2nd generation vector: Modified by 1st generation plasmid, deleting all HIV affiliate genes which will not affect transfection efficiency and viral titer and safer than 1st one as well.
3rd generation vector: (1)Lentiviral vector was inactivated due to lacking of enhancer element and promoter sequence via deleting 3’LTR in U3 region, so that viral RNA could be got although viral proteins are there.(2) Tat gene was replaced by heterology promoter gene sequence, that means there are only 3 original viral genes (including gag, pol and rev) were left in total 9 of lentiviral vector. So comparing 2nd generation plasmid, the 3rd one is safer!
The sequence differences are outlined above (ie wt LTR > CMV/RSV/etc). As far as I'm aware there are no other sequence differences in the transfer vector that exclusively differentiate a 2nd from a 3rd gen transfer vector.
As for the added 'safety', the arguement, as I understand it, is that having a foreign promoter embedded in an LTR 'may' make it less likely to recombine with wt HIV that may be latently infecting your target cell, thus reducing the chances of a replication competent virus (RCV) arising. Further, it means you can exclude one more lenti gene (tat) from your virus production cocktail - which purportedly bolsters the safety profile of 3rd gen with respect to RCVs.
However, when assessing the relative 'safety' of lentiviral systems (especially 2nd vs 3rd gen) it is important to keep in mind that (as far as I'm aware) there has never been a bonafide case of RCV reported for 2nd gen (according to Dull and Trono - pioneers of lenti safety) and thus it's perhaps a bit meaningless to say that one system is 'safer' than the other (despite what the bureaucrats tell us!). In fact, one of the biggest problems in designing assays for RCV is that it is so difficult to 'engineer' a RCV outside of a bioreactor.
I think the issue is simply this: if you have a plasmid map and sequence, how can you tell whether the 5'UTR is wt (2nd generation) or chimeric (3rd generation)? Is there a labeling convention on the plasmid map?
For this question, just use Free Snapgene Viewer (http://www.snapgene.com/products/snapgene_viewer/) software and insert you sequence (select "detect common features). The sequence will be annotated as HIV LTR for 2nd generation or RSV promoter and 5' LTR (truncated) for 3rd generation. Regarding only the gene transfert vector. Indeed, this gene transfert plasmid (3rd generation) can be use with packaging system of the second generation.
As I described before, you should also have a separate plasmid for Rev expression to have a "real" third generation system :
1. gene transfert vector (with hybrid 5' Promoter, no need to have TAT expression). Can also be SIN or not.
2. packagin system: >= 3 plasmids: pREV, pGAG-POL (w/o Rev and TAT), pVSV-G (or other glycop. env.).
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There are so many answers here. But somehow the most practical question is still unclear. Let's take this transfer plasmid: https://www.addgene.org/70182/ . How can I tell if it is second generation or third generation? There is the CMV promoter in front of the LTR. So does that make it third generation?
Can someone show me an addgene plasmid that is a second generation transfer plasmid?
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