Common questions

What is lentiviral packaging?

What is lentiviral packaging?

Lentiviral particles are packaged in producer cell lines such as HEK293T cells. Upon co-transfection of the plasmids, all required sequences are available to produce and package a viral particle containing the transgene of interest.

What is a packaging plasmid?

Some plasmids (typically called packaging and envelope plasmids) encode components of the viral capsid and envelope and are used in conjunction with the plasmid that encodes the viral genome (typically called the transfer plasmid).

How do you perform lentiviral transduction?

c. Transduction

  1. Thaw the lentivirus on ice. Mix 8 µl Polybrene (1 mg/ml aliquot) with 957 µl culture.
  2. The next day, exchange Lentivirus/Polybrene mixture by fresh culture medium. Incubate cells at standard cell culture conditions.
  3. concentrations range from 0.1-10 μg/ml. Replace the culture medium 48-72 hours.

How do you store lentiviral particles?

If storing for less than a day, lentivirus can be kept at 4°C. For long-term storage, viral preps should be divided into single-use aliquots, and stored at -80°C. Some reports suggest rapidly freezing virus in a dry ice/ethanol bath or liquid nitrogen prior to storing.

How does the lentiviral system work?

More specifically, lentiviruses attach to the CD4 glycoproteins on the surface of a host’s target cell. The viral material is then injected into the host cell’s cytoplasm. Within the cytoplasm, the viral reverse transcriptase enzyme performs reverse transcription of the viral RNA genome to create a viral DNA genome.

How do you pack lentivirus?

Procedure

  1. Seed 293T packaging cells at 3.8×106 cells per plate in DMEM complete in 10 cm tissue culture plates.
  2. Incubate the cells at 37 ℃, 5% CO2 for ~20 hours.
  3. Gently aspirate media, add 10 mL fresh DMEM complete containing 25 μM cloroquine diphosphate and incubate ~5 hours.

Can lentiviruses infect resting cells?

However, lentiviruses are critically limited in their ability to infect resting CD4 T cells in the peripheral blood and lymphoid tissues due to the expression of SAMHD1.

How long can lentivirus be stored?

For short term storage, keep the vector at 4degrees (fridge) for 3 or 4 days, rather than freezing and thawing, but for longer storage definitely store at -80.

Why are lentiviral vectors used?

Lentiviral vectors are gene transfer tools derived from viruses. They are used to deliver specific genes into target cells. All pathogenic and replicative properties of the viruses are removed, preserving only their gene transporting capabilities. Lentiviral vectors can integrate into dividing or non-dividing cells.

How do lentiviruses work?

Lentiviruses (a genus of retrovirus) express reverse transcriptase, which converts the viral RNA to double stranded DNA, and integrase, which inserts this viral DNA into the host DNA. Once the viral DNA is integrated into the host DNA, it divides along with host cell and none are the wiser.

What kind of packaging is used for lentiviral?

In addition to Lenti-X Packaging Single Shots, we also offer packaging systems for generating VSV-G pseudotyped lentivirus and ecotropic pseudotyped lentivirus in a traditional format. These kits include packaging mix and Xfect Transfection Reagent as separate liquid components.

How does the Lenti-PAC HIV expression packaging system work?

Lenti-Pac™ HIV expression packaging kit. Comes with an optimized lentiviral packaging plasmid mix, eGFP positive control plasmid, EndoFectin™ Lenti, a transfection reagent optimized for virus production, and TiterBoost™ reagent, which increases titers 5-10 fold.

What is the plasmid mix for lentiviral expression?

The Packaging Plasmid Mix is a mixture of the human immunodeficiency virus (HIV) lentiviral packaging plasmid psPAX2 and the pMD2.G plasmid containing VSV-G. These plasmids provide all the necessary structural, regulatory, and replication proteins required to produce pseudotyped packaged lentiviral expression constructs.

How many infectious units per mL of lentiviral?

This modal can be closed by pressing the Escape key or activating the close button. Our Lenti-X packaging systems generate lentiviral titers that far exceed most other commercially available packaging systems—you can expect titers of 10 7 –10 8 infectious units (IFU) per ml, 25 times what other popular systems generate.