Lentivirus Packaging

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. We use 3rd generation lentivirus systems for the highest biosafety standards. Several additional viral sequences must be acquired from the packaging plasmids via recombination for successful DNA transfection to possible.

Description of Lentiviral Vector Design

  • Transfer plasmid: The Lentiviral vector encoding your insert fragment of interest. The transgene sequence is flanked by long terminal repeat (LTR) sequences, which facilitates the integration of the transfer plasmid sequences into the host genome. Typically, it is the sequences between and including the LTRs that are integrated into the host genome upon viral transduction. For safety reasons, transfer plasmids are all replication-incompetent and may contain an additional deletion in the 3'LTR, rendering the virus “self-inactivating” (SIN) after integration.
  • Packaging plasmids: One encoding Rev and one encoding Gag and Pol.
  • Envelope plasmid: Encodes components of the viral capsid and envelope proteins, such as the VSV-G envelope glycoprotein.
Self-inactivating VSV-G pseudotyped lentivirus

Lentivirus Production

Transfect 293T packaging cells with transfer plasmid, envelope plasmid, and packaging plasmids. Remove media, and replace it with fresh media. After 48~72 hours, harvest the virus. The harvested virus can be used in vitro study. After further purification, the resulting ultra-purified lentivirus can be used in vivo study.

Quality Control for Virus

Lentiviral titer test will be conducted to make sure only qualified high titer high purity lentivirus vectors enter your laboratory. For the luciferase reporter gene labeled lentivirus, we will perform a viral transduction assay and further confirm the titer by qPCR. The qPCR technique allows quantification of the transducing units (TU) according to the viral copy number integrated into the cell genome.

Service Details

Type

Cell number

Titer

Turnaround

Lentivirus (LV)

 

CAR Lentivirus

≥×108 TU

≥1×108 TU/mL

   5-8 weeks

Conventional overexpression lentivirus

≥×108 TU

≥1×108 TU/mL

Conventional  interference lentivirus

≥×108 TU

≥1×108 TU/mL

Conventional knockout lentivirus

≥×108 TU

≥1×108 TU/mL

Purified Lentivirus

≥×109 TU

≥1×109 TU/mL

Advantages of Cyagen’s Lentivirus packaging service:

      Design of CAR molecules and vectors for various targets to meet individual needs.
      A variety of vector catalogs shorten the experimental turnaround.
      Mature lentivirus packaging technology, with many articles published by customers.
      Strict virus quality control standards, and high infection efficiency.
      The third-generation lentivirus packaging system further improves biological safety.

Case studies

Case 1. Lentiviral Titer via Flow Cytometry - CD19 Antigen

The lentiviral purification solution expressing the CD19 antigen was diluted in four gradients (0.01, 0.1, 1, 10 μL) and added to the same number of 293T cells separately. After 72 hours, the positive rate of 293T cells was detected by flow cytometry. The percentage of virus-infected 293T cells to the total number of cells), and then calculate the transduction titer of the lentivirus according to the following formula:

A: The percentage of positive cells in the 0.1 μl group. B: The percentage of positive cells in the 0.01 μl group. N: Number of cells when the virus is added V: Volume of virus added in group A

 
The number of CD19 antigen-positive cells gradually increases with the increase of the transfection virus gradient, indicating that the lentivirus has infectious activity. The calculated virus titer is 1.4×108 TU/mL.

Case 2. Lentiviral Titer via Flow Cytometry - FMC63 CAR

The FMC63 CAR lentivirus titer was detected by the same method as in Case 1. After calculation, the virus titer is about 4.33×108 TU/mL.


Figure 2. The results of FMC63 CAR lentivirus titer test

Case 3. Images of Lentivirus-Infected Cells

HepG2
A549
HT1080
THP-1
Ploy
HepG2

Figure 3. The effects of lentivirus infection on various cells

Inquiries and Quote Requests

Request a quote now. Alternatively, you can always email cell-service@cyagen.com to inquire about our services or obtain a quote for your project.