CUSTOM LENTIVIRAL VECTOR PRODUCTION: QUALITY CONTROLS
For every lentiviral vector produced by our team, we perform mandatory quality controls before validating the vector and shipping it to our customer. These controls include quality checkpoints along the production process, as well as the final titration of the vector. This titration is provided as an RNA genome titer, as well as a TU equivalent titer.
In addition to these controls, we also offer Extra Quality Controls or titers upon your request.
The measurement of Transducing Units is very informative on the efficiency of a viral stock. For each of our lentiviral vectors, we provide a TU equivalent titer. This titer is an estimation of what the TU would be if the vector is used in optimal conditions (i.e. appropriate promoter and envelope for the cell type). However, if you wish to know the actual amount of TU in your vector, we can measure it in the cell line of your choosing. GEG Tech already disposes of several cell lines of various origins (human, mouse, pig…) and multiple types (epithelial, glial, neuronal…). We can also measure TU on a cell line specific of your project. If you want to know more about this, please contact us.
The P24 titer is commonly used for the quantification of lentiviral vectors. Though not the most relevant, it can be useful to assess the potential toxicity of the vector. Indeed, this titer measures the total amount of P24 capsid protein, including ones that are not part of an effective particle. Consequently, a high P24 titer does not necessarily mean that the vector will be highly efficient. However, it most likely will induce toxicity and/or inflammatory reactions when used in vivo.
Furthermore, this titer might be a requisite from the health and safety services of some companies. If you need this titer for your vector, please contact us.
Endotoxins: titration and/or removal
Endotoxins (or Lipopolysaccharides) are pyrogenic molecules from the membrane of certain bacteria. The most common source of contamination is the use of bacteria-produced plasmids and/or unpurified water. They also strongly adhere on lab equipments (plasticware, glassware…). Endotoxins can have heavy negative effects on experiments in vivo by causing strong immune response, and induce various cell culture issues in vitro.
Taking great precautions at every step of the production, we make sure that our vectors show low rates of endotoxin in the final product. As an extra QC, we can measure the endotoxin level of your vector. Moreover, for highly sensitive experiments, we can also perform an additional removal of endotoxins from your vector. If you need more details about these options, please contact us.
Lentiviral vectors are built to be non-replicative, which means that after they transduce the target cell, no new vector particle is formed. However, there is a theorical risk of recombination between the transfer plasmid and the packaging plasmid. It may induce the formation of replicative particles. The RCL (Replication Competent Lentivirus) testing is made to make sure that in a given vector sample, no RCL are present. To do so, the sample is used on a permissive cell line, allowing the replication of RCL particles if there are any. After weeks of subculture, the culture medium is tested using a p24 ELISA assay detecting the capsid protein.
This QC is often required in order to use lentiviral vectors in Biosafety Level 1 laboratories, or by the health and safety services of some companies. For detailed informations about this, contact us.
Number of integrated copies
For most experiments, it is useful to know how many copies of the vector genome are integrated in your target cell. Indeed, the transgene expression level is crucial for the outcome of an experiment, and it greatly depends on the vector genome integration. Moreover, for applications related to genome editing (like CRISPR editing for example), control over the number of integrated copies may be critical for a successful editing.
With that in mind, we offer the possibility to determine the number of integrated copies of your vector using a specific qPCR assay. It can be done two ways:
- We can transduce one of our cell lines with your vector, and determine the copy number by µl of vector. That way, you will be able to predict how many copies will integrate in your target cell. This method is appropriate if your cell line matches one of our own, or if you only require an estimation of the integrated copy number.
- You cand send us gDNA of your transduced cells, and we will quantify the copy number on it. This way, you will know exactly how many vector genomes are integrated in your cells.
If you want to know more about this, please contact us.
This method is particularly useful to assess the actual expression of the protein of interest. When the transgene expression cannot be quantify by direct fluorescence, Western Blot is the method of choice.
Thanks to specific antibodies, it allows the detection and quantification of the protein of interest, thus giving precious informations on the transgene expression. Moreover, it gives valuable informations about the protein, allowing to determine if the expressed protein has the expected size. Western Blot is a very sensitive assay, which means that even small quantities of protein can be detected and measured.
If you are interested in having your lentiviral vectors tested using this QC, please contact us.
If your project require an Extra QC that is not part of the list above, don’t hesitate to contact us. We will do our best to accomodate your needs.