FQA

What is called “Ready to Use”?

It means that all you need to do is to add our product to your cell cultures. That’s it! The viral particles are ready to infect cells, no extra work needed.

What are lentiviral vectors?

Lentiviral vectors are a type of retrovirus that can infect both dividing and nondividing cells. They can be used for nondividing or terminally differentiated cells such as neurons, macrophages, hematopoietic stem cells, retinal photoreceptors, muscle and liver cells and those cells which previous gene transfer methods could not achieve.

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How do lentiviral vectors work?

To obtain a lentiviral gene expression vector, a reporter gene or functional gene is cloned into a vector sequence that is flanked by LTRs and the Psi-sequence of HIV. The LTRs are necessary to integrate the gene into the genome of the target cell, just as the LTRs in HIV integrate the dsDNA copy of the virus into its host chromosome. The Psi-sequence acts as a signal sequence and is necessary for packaging RNA with the reporter or functional gene in virions. Viral proteins which make virus shells are provided in the packaging cell line, but are not in context of the LTRs and Psi-sequences and so are not packaged into virions. Thus, virus particles are produced that are replication deficient, so are designed to be unable to continue to infect their host after they deliver their content.

How are your lentiviral vectors made?

Lentiviral vectors are usually created in a transient transfection system in which a cell line is transfected with 3-5 separate plasmid expression systems. These include the transfer vector plasmid, the packaging plasmid, and a plasmid with the heterologous envelop gene (ENV) of a different virus (e.g. VSV-G). Viral particles in the supernatant of packaging cell culture are collected and concentrated to high titer values (usually > 10^7 Transfection Units per mL) with our proprietary technology.

Is it safe to use lentivirus vector products?

The CDC suggests that Lentiviral stocks be treated as Biosafety Level 2 organisms. For further information on BL-2 guidelines and lentivirus handling, please refer to “Biosafety in Microbiological and Biomedical Laboratories”, 4th Ed. Centers for Disease Control and their website: www.cdc.gov. We strongly recommend users strictly follow all CDC published guidelines for BL- 2.

What about safety concern on your lentiviral products?

There are some key safety features for our lentiviral products.

1. HIV lentiviral vectors are being produced whose packaging plasmid does not contain the necessary HIV genes. This does not interfere with efficient vector production and is a great increase in safety.
2. All our lentiviral products are the third generation “self-inactivating” vectors. This is another significant safety feature because a deletion in the LTR region at the 3′ end of the virus genome theoretically abolishes any chance of virus self replication.
3. Other safety methods include using specific internal promoters that regulate gene expression either temporally or with tissue or cell specificity so as to prohibit gene expression potentially cause replication of HIV in the target cells.

Why should I use Polybrene for virus transduction?

Polybrene is a small, positively charged molecule that binds to cell surfaces and neutralizes surface charge and greatly enhances transduction by lentiviruses. Polybrene can be added to the viral dilution before adding the virus to the target cells. Our viral supernatants usually contain 16ug/ml polybrene. When use 1:1 dilution for cell transduction, polybrene will be diluted out to 8ug/ml in the culture medium. Alternately, users can add the same concentration of polybrene in the virus-cell mixture right before starting transduction. Please note that polybrene can be toxic to terminally differentiated neurons and dendritic cells. Lower concentration of polybrene should be used for these types of cells (e.g., 1,2,4,6 ug/ml). If toxicity is a problem, then cells can be transduced in the absence of polybrene, but the MOI (multiplicity of infection) will have to be increased.

For more information, Cantact Us.