Retrovirus Packaging Protocol for Retrovirus Transfection

BioInnovatise Viral Vector Team

Updated November 11, 2024

What Is a Retrovirus Packaging Protocol and Which Is Right for My Research?

A retrovirus packaging protocol outlines the steps to produce retroviral particles that deliver a genetic payload to a target cell for research. Retroviral packaging involves inserting a gene of interest into a retroviral vector and producing infectious retroviral particles that can transfer this gene into target cells. But depending on your research application, you may need to pick a specific retrovirus packaging protocol that matches your application best.

Retroviral transfection protocols can vary for several reasons, depending on the specific experimental requirements, the characteristics of the retroviral plasmid construct being used, and the target cells for transduction. Important factors affecting the method of retrovirus transfection may include:

  • Type of retroviral system:
  • Packaging system and helper plasmids
    • Single-plasmid packaging
    • Multiple-plasmid packaging
  • Transfection method
  • Purity requirements

Our retroviral packaging team will always use the specific transfection protocol requested by the customer. If you are unsure which retrovirus transfection protocol is right for you, contact our team. The below transfection protocol is a general protocol for educational purposes and should not be used outside of BioInnovatise productions.

Learn about our quick turnaround retrovirus packaging services

Retrovirus Packaging Transfection Process Overview, Retrovirus Packaging Protocol

The above diagram illustrates the retrovirus packaging process at BioInnovatise.

Materials Required

  1. Cell Line: HEK293T is the preferred cell line for retrovirus and lentivirus packaging
  2. Media
  3. Plasmids:
    1. Retroviral vector plasmid (contains gene of interest)
    2. Packaging plasmids (pGag/Pol and pEnv for packaging retroviral structural proteins)
  4. Transfection Reagents
  5. Polybrene
  6. 0.45 µm Syringe Filter

Procedure

  1. Prepare cells
    1. Plate HEK293T cells in a T75 flask or 6-well plate with DMEM + 10% FBS without antibiotics. Cell density should be around 60-70% confluence by the time of transfection.
    2. Allow cells to adhere and grow overnight at 37°C with 5% CO₂.
  2. Transfection
    1. Prepare Transfection Mix:
      1. Use the following plasmid mix per T75 flask: Retroviral vector plasmid (1 µg) + Packaging plasmid(s) (1 µg pGag/Pol + 1 µg pEnv if separate, or 2 µg total if combined)
      2. Dilute Plasmids: Add plasmids to 250 µL Opti-MEM (serum-free medium) in one tube.
      3. Dilute Transfection Reagent: Add 10 µL Lipofectamine 3000 (or 20 µL PEI) to 250 µL Opti-MEM in a separate tube.
      4. Combine and Incubate: Mix the plasmid and transfection reagent solutions and incubate for 10-20 minutes at room temperature.
    2. Add Transfection Mix to Cells:
      1. Add the transfection mix dropwise to the HEK293T cells in fresh DMEM + 10% FBS.
      2. Swirl the plate/flask gently to ensure even distribution.
      3. Incubate: Return cells to the incubator at 37°C with 5% CO₂.
  3. Change Medium
    1. Replace the medium with fresh DMEM + 10% FBS after 6-8 hours to remove toxic byproducts of transfection.
  4. Harvest Viral Supernatant
    1. Collect Supernatant:
      1. 48 hours post-transfection, collect the viral supernatant by carefully removing the medium.
      2. Filter Supernatant: Filter the collected supernatant through a 0.45 µm syringe filter to remove cell debris.
    2. Ultracentrifuge: Concentrate virus by ultracentrifugation at 20,000 x g for 2 hours at 4°C.
  5. Titration
    1. Prepare Target Cells: Plate target cells in a 12- or 24-well plate at 30-40% confluence.
    2. Serial Dilutions: Perform serial dilutions of the virus to determine viral titer.
    3. Infect Cells and Determine Titer: Add viral dilutions to the target cells with 8 µg/mL polybrene to enhance infection efficiency. Incubate for 48 hours, then assay the titer by counting GFP-positive cells (if the virus has a GFP reporter), performing qPCR, or using another suitable assay. Learn why GFP lentivirus is so prevalent in molecular biology for its inherent features.

Notes:

  • Optimization: The transfection conditions may need optimization for different cell types and retroviral vectors. This includes the MOI, incubation time, and the presence of transfection enhancers.
  • Safety: Follow appropriate safety guidelines for working with retroviral vectors, including the use of biosafety cabinets and personal protective equipment.
  • Validation: Validate transduction efficiency and expression levels for your specific experimental goals.

Notes from the BioInnovatise laboratory:

  • Our team uses HEK293T cells for retrovirus packaging. Learn more about other cell lines available for retrovirus packaging.
  • We use RT-qPCR (Real-time polymerase chain reaction) to measure titer levels
  • Our preferred QC methods includes fluorescence microscopy, flow cytometry, and western blot.

Want to learn more about the latest in retroviral based research? Our colleagues at ScienceDirect and Genetic Engineering & Biotechnology News are always collecting and publishing the latest information on retrovirus based research.

Insertion mutagenesis, Deletion mutagenesis, Point mutagenesis, Random mutagenesis, Site Directed Mutagenesis, Site Directed Random Mutagenesis

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