This FAQ at Addgene nearly has it all!. Learned a lot there myself. I think they answer your question and more.

http://www.addgene.org/lentiviral/faqs/

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

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.

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).

Emmanuel

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!

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.

BW

Al

Hi,

See the following papers to fully understand:

- http://www.actabp.pl/pdf/4_2009/531.pdf

- http://www.ncbi.nlm.nih.gov/pubmed/22507128)

In summary, concerning packagin vectors:

- 1st generation packaging: you will cotransfect

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.).

There is a Lentivirus Webinar coming up that may able to help address your concern and any additional questions.

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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?

Thanks!

Hi Eric

From what I have understood so far addgene 70182 is a third generation transfer plasmid as it has a truncated 5'LTR.

The primary differences between second and third generation transfer plasmids or vectors, especially in the context of lentiviral vector systems, lie in their safety profiles and the elements they contain for gene expression regulation. These differences are designed to enhance safety and minimize the risk of generating replication-competent lentivirus (RCL) during vector production and use. 1. **Safety Enhancements**: - **Second Generation**: These vectors were developed as an improvement over first-generation vectors, primarily in terms of safety. They typically lack viral genes like vif, vpr, vpu, and nef, which are not necessary for the vector's basic function. This reduces the chance of generating RCL. - **Third Generation**: These vectors take safety a step further by further reducing the number of viral genes present. They typically lack all viral genes except for those necessary for packaging (gag and pol) and envelope (env) proteins. This further reduces the risk of recombination events that could lead to the generation of RCL. 2. **Regulatory Elements**: - **Second Generation**: They generally contain a stronger promoter in the transfer vector (like CMV) to drive the expression of the transgene. This can lead to higher levels of gene expression. - **Third Generation**: These vectors often use internal promoters that are weaker than those used in second-generation vectors. This might result in lower but safer and more controlled levels of gene expression. 3. **Components of the System**: - **Second Generation**: A typical second-generation system includes three plasmids: a transfer vector (carrying the gene of interest), a packaging plasmid (providing gag and pol genes), and an envelope plasmid (usually providing the VSV-G protein for pseudotyping). - **Third Generation**: This system usually involves four plasmids: the transfer vector, a packaging plasmid (gag and pol), an envelope plasmid (like second-generation), and a separate plasmid for the Rev protein, which enhances the export of RNA from the nucleus. 4. **Applications**: - **Second Generation**: Due to their relatively simpler design and higher expression levels, these vectors are often used in basic research and some clinical applications where high transgene expression is required. - **Third Generation**: These are more commonly used in clinical settings due to their enhanced safety profile. They are suitable for applications where controlled expression is more important than high expression. 5. **Complexity of Production**: - The third-generation system, due to its additional components, can be more complex and challenging to work with compared to the second generation. In summary, the main differences between the second and third generation transfer vectors in lentiviral systems are based on the safety features, the number of viral genes present, the strength of promoters used for transgene expression, and the overall complexity of the system. The choice between these generations depends on the specific requirements of the experiment or therapy, balancing between expression levels and safety considerations.

l With this protocol list, we might find more ways to solve this problem.