Asked 10th Sep, 2013

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?

Most recent answer

9th Apr, 2019
Birunthi Niranjan
Monash University (Australia)
Hi Eric
From what I have understood so far addgene 70182 is a third generation transfer plasmid as it has a truncated 5'LTR.

All Answers (16)

10th Sep, 2013
Steven Applequist
Karolinska Institutet
This FAQ at Addgene nearly has it all!. Learned a lot there myself. I think they answer your question and more.
1 Recommendation
10th Sep, 2013
Nick Li
University of South Australia
Thanks Steven for the reply! I have looked in Addgene FAQ sections for a few times and it does contains a lot of information there.
But again, it stated a lot about PACKAGING SYSTEM and the only information they have about TRANSFER PLASMID classification I found is in their packaging plasmid page
"Q: How can you tell if your transfer vector is 2nd generation?
A: In general, lentiviral vectors with a wildtype 5’ LTR need the 2nd generation packaging system because these vectors require TAT for activation."
Please tell me if I have missed anything from the FAQ page which actually explain the difference of 2nd and 3rd gen TRANSFER PLASMID
10th Sep, 2013
Steven Applequist
Karolinska Institutet
Sorry. :-( I hereby retract my poor answer. I'll also watch to see if you get one. I would also like to find the answer.
1 Recommendation
10th Sep, 2013
Emmanuel Payen
French Institute of Health and Medical Research
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).
2 Recommendations
10th Sep, 2013
Yongxin Hao
University of Lethbridge
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!
1 Recommendation
12th Sep, 2013
Alasdair Russell
University of Cambridge
Hi Nick
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 hope this is of some use.
3 Recommendations
7th Mar, 2014
Emmanuel Di Valentin
University of Liège
See the following papers to fully understand:
In summary, concerning packagin vectors:
- 1st generation packaging: you will cotransfect <= 3 plasmids containing the viral accessory proteins genes.
- second generation: 3 plasmids without viral accessory proteins genes.
- third generation: more than 3 plasmids (no viral accessory proteins genes)
The gene transfert vector can also be modified. One of the most important modification for biosafety is the delta U3.
For biosafety consideration:
2 Recommendations
24th Mar, 2016
Alison Mungenast
Eisai AiM Institute, Eisai Inc.
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?
4 Recommendations
25th Mar, 2016
Emmanuel Di Valentin
University of Liège
For this question, just use Free 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.).
4 Recommendations
11th May, 2018
Joanne Worobec
There is a Lentivirus Webinar coming up that may able to help address your concern and any additional questions.
Title: Knockdown, Knockout, Validate: Lentivirus delivers payload in vitro and in vivo Date: Wednesday, May 16, 2018 Duration: 60 minutes Times: 10:00 AM (CST) Speaker: @Christy Hoffmann
What you will learn: Lentivirus biosafety features and best handling practices Flexible customization options to fit your needs Techniques to incorporate lentivirus into your gene studies Insight from a leading Lenti manufacturing expert
1 Recommendation
4th Apr, 2019
Eric Danner
Max-Delbrück-Centrum für Molekulare Medizin
There are so many answers here. But somehow the most practical question is still unclear. Let's take this transfer plasmid: . 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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