Molecular biology service: producing tailor-made DNA constructs for scientific research
Since 2013, the Molecular Biology and Virus Service of the IGBMC has been providing scientists with custom DNA constructs, essential elements for many of the projects conducted within the institute. Led by Paola Rossolillo, this service is an essential tool when specific genes or sequences need to be expressed within a cell or organism for in vitro or in vivo studies. This department is involved in a large number of studies and produces more than 250 plasmids (circular DNA fragments) and 90 viral vectors on average per year.
Producing easy-to-trace biological elements
The aim of the Molecular Biology and Virus Service is to provide the biological elements that research projects need. The department's staff will construct plasmids, fragments of DNA capable of self-replicating, tailored to the needs of the studies. These constructs will contain all the elements needed to express genes or sequences in different organisms (bacteria, mammals, yeast etc.).
The service is useful when one wishes to produce particular proteins, localise specific elements within the cell, inactivate or modify genes or construct viral vectors.
For example, to observe where a specific protein acts within a particular cell type, the service adds a DNA sequence to make the protein being studied fluorescent. The principle is as follows:
- The engineer selects the DNA sequence responsible for producing a fluorescent protein and the part that recruits the factors that initiate transcription in the cells used (promoter).
- The engineer fuses this DNA sequence with the one that produces the protein of interest.
- When the DNA is read and translated into protein, the protein produced will have both its usual characteristics and fluorescence.
Reconstructing harmless viruses to transport genetic material into cells or organisms
Another aspect of the service led by Paola Rossolillo is to produce viral vectors. Viruses are extremely efficient at entering different types of cells and transporting their genetic material (RNA or DNA). Viral vectors are constructed by modifying the virus genome in such a way as to :
- Keep the elements that allow it to enter cells.
- Substitute the elements that allow it to replicate with the DNA under study.
To produce a viral vector, the Molecular Biology and Virus Service will construct different plasmids, each containing part of the information. In total, the unit will construct 3 plasmids:
- A plasmid containing the information on the surface proteins that allow the virus to enter a cell.
- A plasmid to produce the structural proteins and enzymes that are used to make the virus genome.
- A plasmid that contains the genes that make up the RNA or DNA strands of the virus.
These three plasmids are then integrated into a so-called "producer" cell. The information contained in these three plasmids is translated to produce the different elements and put them together in a single structure: the viral vector.
The only difference with the virus found in nature is that the genome sequence coding for the element that allows it to reproduce in an infected cell has been removed. In this way, the virus can still enter a new cell and introduce the DNA that we want to study and express, but it will not be able to replicate itself to infect another cell.
A facility capable of working with very small viruses and having contributed to coronavirus research
Like other organisations in Strasbourg, the IGBMC's Molecular Biology and Virus Department works on lentiviruses, viruses derived from HIV, and 'commonly used in laboratories', explains Paola Rossolillo, head of the service.
What "sets our unit apart is that we are the only ones in Strasbourg that are also able to work on AAV viruses, or adeno-associated viruses". These non-pathogenic viruses, found in nature associated with adenoviruses (responsible for cold sores etc.), are 'smaller than HIV and have a DNA genome rather than RNA. The purification process for rAAV vectors is quite long and laborious. Moreover, 'they are injected into organisms in order to carry out in vivo studies on animal models and to study the interaction of the viruses with the organism, which requires a very high level of purity', explains Paola Rossolillo.
Thanks to this particular expertise, the unit headed by the engineer is currently 'very much in demand by several academic laboratories in Strasbourg because rAAVs are very effective tools'.
Recently, the Molecular Biology and Virus Service has been involved in research on the coronavirus. The latter has produced viruses with the Spike protein (and all its variants during the pandemic) as the surface protein that allows the virus to penetrate a cell. The service integrated a fluorescent protein into the RNA of the virus so that it could detect, and count, the number of viruses infecting the cells. This allowed us to observe whether the antibodies produced as a result of an infection or following the 3rd dose of vaccine enabled immunocompromised patients to fight against the invasion of their cells by the virus or not.
For more information, you can consult the corresponding articles:
- Charmetant X, Espi M, Benotmane I, Barateau V, Heibel F, Buron F, Gautier-Vargas G, Delafosse M, Perrin P, Koenig A, Cognard N, Levi C, Gallais F, Manière L, Rossolillo P, Soulier E, Pierre F, Ovize A, Morelon E, Defrance T, Fafi-Kremer S, Caillard S, Thaunat O. Infection or a third dose of mRNA vaccine elicits neutralizing antibody responses against SARS-CoV-2 in kidney transplant recipients. Sci Transl Med. 2022 Mar 16;14(636):eabl6141. doi: 10.1126/scitranslmed.abl6141. Epub 2022 Mar 16. PMID: 35103481; PMCID: PMC8939774.
- Gallais F, Gantner P, Planas D, Solis M, Bruel T, Pierre F, Soulier E, Rossolillo P, Fourati S, Sibilia J, Schwartz O, Fafi-Kremer S. Case Report: Evolution of Humoral and Cellular Immunity in Two COVID-19 Breakthrough Infections After BNT162b2 Vaccine. Front Immunol. 2022 Feb 23;13:790212. doi: 10.3389/fimmu.2022.790212. PMID: 35281046; PMCID: PMC8905643.
Paola Rossolillo, Inserm research engineer, head of the molecular biology and viruses service
Paola Rossolillo obtained her PhD in genetics and molecular biology in 2000. She did her thesis on the molecular biology of the Gram-positive bacterium Bacillus subtilis at the University of Pavia in Italy. She then completed a post-doctorate at the University of Verona where she studied the HIV envelope protein.
The scientist arrived in Strasbourg in 2008 where she joined Matteo Negroni's team at the IBMC to work on a system for the evolution of cellular genes through lentiviral vectors. She joined the IGBMC in 2013 as head of the molecular biology and viruses service.