Do you know for sure that all of the plasmids you are using look okay and are not degraded? I would run the psPAX2 and pMD2G on a gel to make sure.

Yes, it is possible to use psPAX2 and pMD2G plasmids in combination with a lentiviral vector such as pLVX-M-Puro to produce lentiviral particles for gene delivery.

When these plasmids are co-transfected into packaging cells (e.g., HEK293T cells) along with the pLVX-M-Puro lentiviral vector, they facilitate the production of lentiviral particles that contain the transgene encoded by pLVX-M-Puro. The lentiviral particles can then be harvested from the cell culture supernatant, purified, and used to transduce target cells for gene delivery.

It's important to note that appropriate biosafety measures should be followed when working with lentiviral vectors and viral particles. Additionally, the use of lentiviral vectors for gene delivery may be subject to regulatory requirements depending on the specific application and jurisdiction.

Yes, you can use psPAX2 and pMD2.G plasmids to produce viral particles to encapsulate a pLVX-M-Puro vector. Here's an overview of how this works:

1. **Plasmids Overview:**

- **psPAX2:** This is a packaging plasmid that provides the necessary structural proteins (gag, pol, and rev) for viral particle assembly.

- **pMD2.G:** This is an envelope plasmid that provides the VSV-G protein, which allows the virus to infect a wide range of cell types.

- **pLVX-M-Puro:** This is your transfer plasmid, containing the gene of interest and a puromycin resistance gene for selection.

2. **Co-Transfection:**

- You will co-transfect HEK293T cells with these three plasmids. HEK293T cells are commonly used for this purpose because they are easy to transfect and produce high titers of viral particles.

- Typically, a 3-plasmid system is used where psPAX2 and pMD2.G are helper plasmids that supply the necessary proteins in trans for packaging the pLVX-M-Puro vector into viral particles.

3. **Procedure:**

- **Cell Seeding:** Seed HEK293T cells in a suitable culture dish or flask and allow them to reach around 70-80% confluence.

- **Transfection Mix:** Prepare a transfection mix using a transfection reagent like Lipofectamine 2000 or PEI. Mix psPAX2, pMD2.G, and pLVX-M-Puro plasmids in a suitable ratio (e.g., 1:1:1 or 4:3:1, depending on the protocol you are following).

- **Transfection:** Add the transfection mix to the HEK293T cells and incubate for 6-8 hours, then replace the medium with fresh DMEM containing 10% FBS.

- **Viral Particle Collection:** After 48-72 hours, collect the supernatant containing the viral particles, filter it through a 0.45 µm filter to remove cell debris, and concentrate the virus if necessary using ultracentrifugation or a commercial lentivirus concentration kit.

4. **Transduction:**

- Use the collected viral supernatant to transduce your target cells. Add polybrene (a cationic polymer) to enhance infection efficiency.

- After transduction, select the successfully transduced cells using puromycin.

This process will allow you to produce lentiviral particles encapsulating your pLVX-M-Puro vector, which can then be used to infect target cells for gene expression studies or other applications.

Hello Mr Alejandro

I'm sorry, that is the topic of interaction between microbiology and immunology. I don't have any knowledge of the subject. But if one day you have a question about immunology and neuroinflammation, I will be happy to submit my ideas.

In the lab where I'm doing my PhD, don't use psPAX2 and pMD2G plasmids to produce viral particles to encapsulate a pLVX-M-Puro vector

However, you can check on the website of CRCHU the Pr in relation to the subject that is the link : Maladies infectieuses et immunitaires – Centre de recherche (ulaval.ca)

You have my encouragement.

Best regards

Yes, you can use psPAX2 and pMD2G plasmids to produce viral particles to encapsulate a pLVX-M-Puro vector. Here's why this combination works:

• psPAX2: This plasmid is a lentiviral packaging plasmid. It contains the essential genes required for packaging the lentiviral RNA genome into viral particles but lacks the genes for the viral envelope protein.
• pMD2G: This plasmid is an envelope plasmid. It encodes the vesicular stomatitis virus glycoprotein (VSV-G), which is a commonly used envelope protein for pseudotyping lentiviral particles. Pseudotyping allows the viral particles to infect a wider range of cells.
• pLVX-M-Puro: This is your transfer vector. It contains the gene of interest and a puromycin resistance gene for selecting cells that have been successfully transduced with the lentivirus.

When co-transfected into packaging cells (usually HEK293T cells), psPAX2 and pMD2G work together:

• psPAX2 provides the packaging machinery: This includes genes for structural proteins (gag and pol) and a regulatory protein (rev) necessary for viral particle assembly and RNA packaging.
• pMD2G supplies the envelope protein: VSV-G allows the viral particles to enter a broader range of cells compared to the native HIV envelope.

Overall, this three-plasmid system (psPAX2, pMD2G, and your transfer vector) is a widely used and efficient method for producing lentiviral particles for gene delivery experiments