Custom Lentiviral Vector Production Service

Overview of Lentiviral Vectors

The lentiviral vector system is a vector technology that is extremely effective in efficiently integrating foreign genes into mammalian cells. This approach is currently one of the most popular ways to introduce foreign genes into mammalian cells, along with traditional plasmid transfection. Due to its versatility in selecting target genes and promoters as well as its wide range of transfected cell types, the lentiviral vector system has emerged as a well-liked method of foreign gene expression.

Due to their broad host range, low immunogenicity, high gene capacity, and long-term expression, lentiviral vectors have emerged as one of the most widely utilized vectors in the field of gene therapy. Cultured tumor cells, liver cells, cardiomyocytes, neurons, endothelial cells, stem cells, and other cell types can all be successfully infected by it. In vitro, since lentivirus infection exhibits characteristics of integration, stable cell lines can be created for research. In vivo, stable tumor cell lines can be used to develop tumor animal models, test the effectiveness and pharmacokinetics of drugs, and perform imaging and other operations. Lentiviral vectors are also commonly used to modify T cells for CAR-T cell therapy.

Production

The third-generation lentivirus technology Creative Biolabs offers can be adjusted to different gene of interest (GOI)sizes. Lentivirus packaging employs a three- or four-substitute method. The four-substitute method includes two packaging plasmids, one envelope plasmid, and one transfer plasmid.

Given that recombinant lentiviruses originated from HIV-1, special care must be taken for gene transfer. The main worry is the potential for crossover events between components of the viral vector and the packaging cell line to produce replication-competent lentivirus. To increase safety, viral accessory genes are removed, and the packaging and envelope plasmids provide crucial genes in trans. The vesicular stomatitis virus's (VSV-G) glycoprotein is mostly used because it enables the transduction of a variety of target cells and VSV-G pseudo-typed.

LV production is mostly based on transient transfection protocols in 293T cells, a simian virus 40 (SV40) T-antigen transfected HEK293 cell line. The packaging cell line (often HEK293T cells) is co-transfected with the transfer plasmid carrying GOI, the envelope plasmid, and the packaging plasmid to create lentivirus particles. After the transfection, four days later, viral particles were recovered. The lentiviral was created in the manner described below.

Fig.1 Procedure of lentiviral vectors.

In the absence of any specific requests, we automatically offer third-generation lentiviruses (VSV-G). Additionally, we can offer second-generation lentiviruses and non-integrated lentiviruses.

Purification

LV vectors are frequently purified before further usage to get rid of contaminated DNA and foreign serum- and producer-cell-derived proteins. In addition to guaranteeing vector safety, purification vastly increases efficacy. For in vitro research, LV is purified using the PEG concentration virus method. Sucrose centrifugation is used to concentrate further and purify in vivo grade lentivirus. We may also provide other purification methods such as affinity chromatography, iodixanol gradient ultracentrifugation, and ion exchange chromatography. Please contact us if you require more thorough purification so we can offer you a tailored purification solution.

Quality Control

Titer determination, Bioburden test for viruses, and Mycoplasma test are performed by default. Transduction tests will be performed if the transfer vector encodes a fluorescent protein. In addition, for ultra-purified lentivirus, we can also detect endotoxin levels. Creative Biolabs has vast experience in lentivirus production. Please contact us in time to learn more about our highly customizable production of lentiviral vectors and a detailed quote.