We will walk you through the expression and definition of your needs and objectives and take your scientific, budgetary, time or resources constraints into account.
We will suggest one or several technical solutions, resulting of our experience and creativity. We aim at designing the best solution fulfilling your needs and expectations.
We will validate with you the choice of the best solution and provide you with the designed tool after all required quality checks.
The specificity of transgene delivery by lentiviral vector is the result of a combination of several elements, such as:
We play with these parameters to obtain the perfect combination displaying the most specific tropism for your particular application.
The basic lentiviral vector is integrative, which is perfect if you are looking for long-term expression in dividing or non-dividing cells. However, if your project requires transient expression of the transgene, you might want to turn to other possibilities: integrase-deficient LV or mRNA vectors. According to your very particular needs, one or the other might be more suitable.
Once you have validated the proposed solution, the technical work begins.
This step also depends on your needs and constraints. We can either build the plasmids from scratch, or do so from your pre-existent plasmids.
In all cases, the constructions are submitted to several quality controls.
The GEG-tech production platform is equipped with advanced equipment, from L1 to L3 confinements laboratories. Production protocols are the result of years of experience and thousands of stocks have been produced. Multiple checkpoints during the production allow high quality and efficiency.
Depending on the future use of the vectors, different levels of purification and concentration can be chosen.
The titering method of lentiviral vector is a very tricky point, getting more and more complex as the technology evolves.
Some methods are based on determining the number of physical particles using the amount of capsid protein (p24 concentration). While this method gives you information of interest for in vivo use (will my vector induce toxicity or high immune reaction due to a high p24 concentration?), this is poorly reproducible and barely informative regarding the transduction capacity of the vector.
Titering methods based on transduction efficiency are thus increasingly developed, relying on transgene expression measurement or on the detection of the vector genome in the transduced cells. However, this transduction efficiency is highly dependent on a combination of numerous parameters such as the cell type, envelope, promoter and transgene that are used. In addition, transduction enhancers, such as polybren, DEAE dextran or sodium butyrate, are often used. Consequently, the titer determined by the provider will not necessarily reflect what you can obtain in your lab with your experimental conditions. In addition, you will hardly compare vectors with different promoters or different envelopes.
To prevent these inconveniences, we have developed a particular titering method that does not require transduction while not being subjected to p24 titration variabilities. Based on an RT-qPCR assay, the RNA titer enables us to quantify the amount of active particles in the concentrated stock. This assay is highly reproducible and can be related to transduction efficiency. Plus, it does not depend on any of the specificities of the vectors, which allows comparison between vectors and facilitates normalization of vectors quantities.