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Team:Freiburg/Content/HumanPracticeAndSafety/Safety/SafetySheet
From 2014.igem.org
Safety-Sheet Regarding The Work With Viral Vectors
To expand the usual safety- and check-in-forms we developed an additional safety-sheet which not only increases safety for scientists working directly with viral vectors but also aims to provide an easy to understand tool for people not involved in laboratory work. This is also part of our goals we want to achieve with Link-It, to increase the acceptance of the public by giving them greater insights.
We are providing the pMIG (viral vector) as a tool for easy gene delivery and generating cell lines under BSL-1 conditions to the iGEM community. This spreadsheet, as a checklist for safety standards, supplies future teams with the possibility to fast and on the point inspection of their projects safety .
General Safety Of The Viral Vector
| Criteria | Explanation | Our Project | Check |
|---|---|---|---|
| Human pathogenesis | Is it possible that the viral vector causes any illness or irritation in humans | We tested our viral vector for potential infection of human cells. Therefore we tried to infect HEK293T (human embryonic kidney) and A549 (lung cancer) cells. Non of our results indicated any infection of those cells. Specificity and Safety of MuLV | ✔ |
| Viral vectors differ from natural viruses. A virus needs at least three different genes to replicate. These are the gag, pol and env genes. In order to ensure higher safety a lot of viral vectors lack these genes. The viral vectors themselves can not reproduces themselves. The vector can integrate itself into the hosts genome, a process coined transduction, but cannot create new viral particles. Its more or less a cul-de-sac for the viral vector. | We sequence our viral vector to ensure the lack of the three viral genes (gag, pol, env). In order to generate the viral vector we used the Phoenix eco cell line which harbors the three genes under different non-viral promoters to minimize the risk of recombination. View the sequence in the registry | ✔ | |
| Transmissibility | How can the viral vector be transferred between cells/organisms. There are different means by which pathogens can be transmitted: by air, (body) fluids or by contact. | We performed a extensive literature research on our viral vector prior to our lab work. We found that the viral vector we are using is solely transmittable via fluids. Nevertheless we performed all steps involving the viral vector with maximum carefulness.General lab safety | ✔ |
| Hostrange | Cells from which animals can be infected by the viral vector. | The MuLV used in our project is highly specific for the mCAT-1 receptor. This receptor variation of the CAT-1 receptor is only found in rodent cells (mouse and rat). Human cells also carry a CAT-1 receptor but with a different glycosylation motive and are therefore not recognized by the viral vector. Receptor specificity of viral vectors | ✔ |
| Survival outside of host | How long is the time range of survival of the viral vector until it becomes non-infectious. | Besides literature research we wanted to test our viral vector for its half-life time. We started at a given time point and measured the exact percentage of infected murine cells. We repeated this for several time points using the same viral vector which was stored at 37°C. Our findings of a half-life of ~6 h matched those found in literature. Half life of our viral vectors | ✔ |
| Presence of transmitter | Is it possible for natural transmitter to come in contact with the viral vector. | The only natural transmitter of our viral vector are rodents. Besides not having any rodents in our lab, the vector itself is replication-defective, which ensures the safety standard. Learn more about the non-replicability of our viral vectors | ✔ |
| Usage | Is the vector only used under lab conditions for research or is it going to be used in humans for clinical applications. | The MuLV is, in our project only used for research purposes. The viral vector can cause leukemia. In clinical applications using viral vectors the amount of used vector is considerably higher. Furthermore the specificity viral vectors must be altered to enable the infection of human cells. With our significant lower virus titer and lower used amounts of viral vector suspension transduction is more than unlikely. | ✔ |
| Additional safety measurements | Every lab that works with genetically modified organisms (GMO) must ensure a certain standard of safety (Germany: Anhang I.2 GenTSV). | After careful research and consultation of our Biosafety officers we know that lab work with our viral vector is categorized as BSL-1. To ensure safety at BSL-1 we received a safety training. Check out our safety form and our safety sheet | ✔ |
References
- Cornetta K, Moen RC, Culver K, Morgan RA, McLachlin JR, Sturm S, Selegue J, London W, Blaese M and Anderson WF (1990) Amphotropic murine leukemia retrovirus is not an acute pathogen for primates. Hum. Gene Ther. 1 (1): 15-30
- Rother RP, Squinto SP, Mason JM and Rollins SA (1995) Protection of retroviral vector particles in human blood through complement inhibition. Hum Gene Ther 6: 429-235
- Battini JL, Rodrigues P, Müller R, Danos O, Heard JM (1996) Receptor-binding properties of a purified fragment of the 4070A amphotropic Murine Leukemia Virus envelope glycoprotein. JVirol. 70 (7): 4387-4393
