Lentiviral Packaging Plasmids

BioInnovatise Viral Vector Team

Updated December 18, 2024

The role of lentiviral packaging plasmids within the lentivirus packaging process

Packaging plasmids are important in the production of functional lentiviral particles in lentivirus packaging. They ensure the proper assembly and packaging of the lentiviral particles. These proteins work together to facilitate the encapsulation of the viral RNA genome into the particles (see diagram below of the lentivirus packaging process).

The lentivirus packaging process involves the co-transfection of three types of plasmids into a packaging cell line:

  1. Transfer Vector (or Expression Vector): This plasmid contains the gene of interest (GOI) or the transgene to be delivered into the target cells. It typically includes regulatory elements, such as promoters and terminators, to control gene expression.

  2. Packaging Plasmids (or Helper Plasmids): These plasmids provide the necessary viral proteins in trans that are essential for the packaging of the lentiviral RNA genome into viral particles. The packaging plasmids usually include:

    • Gag/Pol: Codes for the structural proteins (Gag) and enzymes (Pol) necessary for the formation of viral particles.

    • Rev: Codes for the Rev protein, which is crucial for the export of unspliced or partially spliced viral RNA from the nucleus to the cytoplasm.

    • Tat: Codes for the Tat protein, which enhances the efficiency of transcription from the viral promoter.

  3. Envelope Plasmid: This plasmid provides the gene encoding the viral envelope glycoprotein, determines the host cell specificity and is responsible for mediating the entry of the lentiviral particles into target cells. The commonly used envelope protein is derived from the vesicular stomatitis virus (VSV-G) due to its broad tropism. Our viral vector production team can package lentivirus in VSV-G cells and other types. Read more on our lentivirus packaging services

Lentiviral Packaging Plasmids
Lentivirus Packaging Transfection Process Overview Lentivirus Packaging Protocol

The use of multiple plasmids allows for better control over the packaging process and helps avoid recombination events that could result in replication-competent lentiviruses. The above diagram illustrates the lentivirus packaging process at BioInnovatise where our team uses HEK293T cells for lentivirus packaging.

Do packaging plasmids effect titer levels?

Packaging plasmids can significantly impact both the titer levels and transfection efficiency in lentivirus production. The proper balance and functionality of the various elements encoded by packaging plasmids are necessary for the successful assembly and packaging of lentiviral particles. 

Having the right lentivirus packaging protocol for your research application is important for optimizing both transfection efficiency and titer levels. Learn more about lentivirus packaging protocols. If you are unsure which protocol is right for your research, contact our team to discuss your project.

Our team has made this list to help highlight important packaging plasmid considerations when packaging lentivirus:

  1. Plasmid Ratio: Adjust the ratios of the three plasmids (transfer vector, packaging plasmids, and envelope plasmid) to find the optimal combination for high titer production. The relative amounts of each plasmid can impact the efficiency of lentivirus packaging.

  2. Promoter Strength: Use strong and well-regulated promoters for the expression of essential viral proteins (Gag, Pol, Rev, Tat) to ensure robust and efficient lentivirus production.

  3. Cell Line: Choose an appropriate packaging cell line with characteristics favorable for lentivirus production. Our lab uses HEK-293T cell line when packaging lentivirus. However there are many cell line options, read more about lentivirus packaging cell lines.

  4. Plasmid DNA: The purity and integrity of plasmid DNA used for transfection is incredibly important. If your plasmid DNA requires molecular cloning services to increase integrity, we can help.

Notes from the laboratory at BioInnoatise:

  • Our team uses HEK293T cells for lentivirus packaging
  • Our team offers pseudotype coronavirus spike protein lentivirus, bald lentivirus lacking viral envelope protein, and integrase-deficient lentivirus cell options
  • We use RT-qPCR (Real-time polymerase chain reaction) to measure titer levels
  • We currently offer 2nd generation lentivirus and 3rd generation lentivirus packaging options. Read more about lentivirus packaging generations
 
Want to learn more about the latest in lentivirus based research? Our colleagues at Science Direct and Genetic Engineering and Biotechnology News are always collecting and publishing the latest information on lentivirus based research.
ELISA plate to measure OD with microplate reader.