Will the viral packaging work with a combination of a retroviral target plasmid and lentiviral packaging plasmids (pMD2.G and pPAX2)? Although I saw Syncytia formation in the HEK293FT cells post transfection, does this also indicate virus production?
Hi Prasad,
On Addgene site you can find the Lentiviral Guide (https://www.addgene.org/viral-vectors/lentivirus/lenti-guide/). It's very useful and easy to follow. This guide is good from the basics to the advanced level.
Here you can find a a short citattion from the above page:
"Frequently Asked Questions (FAQ) about Lentiviral Plasmids
What is the difference between a lentivirus and a retrovirus?
Lentiviruses are a subtype of retrovirus. From an experimental standpoint the main difference between lentiviruses and standard retroviruses (γ-retroviruses) is that lentiviruses are capable of infecting non-dividing and actively dividing cell types whereas standard retroviruses can only infect mitotically active cell types. This means that lentiviruses can infect a greater variety of cell types than retroviruses.
Both lentiviruses and standard retroviruses use the gag, pol, and env genes for packaging; however, they are different viruses and thus use slightly different isoforms of these packaging components. Therefore, lentivirus may not be efficiently packaged by retroviral packaging systems, and vice versa."
Here you can read an another very good paper about the lentiviral systemArticle Nucleic Acid Delivery: Lentiviral and Retroviral Vectors
You have said that you use 3 plasmids, therefore this is a 2nd generation system. The 3rd genetartion system is more safer. All of the required plasmids are available from Addgene site (https://www.addgene.org/) also.
I think you can't exactly estimate the virus propagation only from target cell morphology. I have been used Reverse Transcriptase Assay to check virus titer. But you can use p24 Antigen Assay which is very common. Here you can read a paper about titration method comparison.Article Comparison of lentiviral vector titration methods.
I have some experience in lentivirus (3rd generation) propagation and transduction. If you need any help just feel free to write! I have got detailed protocols for the entire process (from transduction to clone screening)
Bests,
Tamas
Hi Prasad,
On Addgene site you can find the Lentiviral Guide (https://www.addgene.org/viral-vectors/lentivirus/lenti-guide/). It's very useful and easy to follow. This guide is good from the basics to the advanced level.
Here you can find a a short citattion from the above page:
"Frequently Asked Questions (FAQ) about Lentiviral Plasmids
What is the difference between a lentivirus and a retrovirus?
Lentiviruses are a subtype of retrovirus. From an experimental standpoint the main difference between lentiviruses and standard retroviruses (γ-retroviruses) is that lentiviruses are capable of infecting non-dividing and actively dividing cell types whereas standard retroviruses can only infect mitotically active cell types. This means that lentiviruses can infect a greater variety of cell types than retroviruses.
Both lentiviruses and standard retroviruses use the gag, pol, and env genes for packaging; however, they are different viruses and thus use slightly different isoforms of these packaging components. Therefore, lentivirus may not be efficiently packaged by retroviral packaging systems, and vice versa."
Here you can read an another very good paper about the lentiviral systemArticle Nucleic Acid Delivery: Lentiviral and Retroviral Vectors
You have said that you use 3 plasmids, therefore this is a 2nd generation system. The 3rd genetartion system is more safer. All of the required plasmids are available from Addgene site (https://www.addgene.org/) also.
I think you can't exactly estimate the virus propagation only from target cell morphology. I have been used Reverse Transcriptase Assay to check virus titer. But you can use p24 Antigen Assay which is very common. Here you can read a paper about titration method comparison.Article Comparison of lentiviral vector titration methods.
I have some experience in lentivirus (3rd generation) propagation and transduction. If you need any help just feel free to write! I have got detailed protocols for the entire process (from transduction to clone screening)
Bests,
Tamas
Tamas Kerekes Thank you so much for the very useful information. Looks like there is an incompatibility issue between the retro and lentiviral packaging systems.
For retroviral packaging you will need gag, pol and env genes from the same virus type of which you are using the retroviral vector (for transgene expression). The vectors psPAX (encodes gag, pol and other accessory proteins (Tat, rev, vpu) derived from subclass of retroviruses called lentivirus e.g. HIV) and pMD2.g (codes for VSVG env gene) . Thus, retrovirus packaging should work with the above said packaging vector (psPAX and pMD2.G) but efficiency of virus preparation can be low due compatibility issue because ideally the retroviral vector and packaging vector should of same origin or derived from same virus.
One can check this with GFP encoding retroviral vector and above said packaging vector, it might work but efficiency of transduction can be low.
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