Would anyone be so kind as to give me a few examples of the equipment involved when modifying a virus? Otherwise my novel may end up reading
Eva entered the lab and modified a virus.
Not much back story there!
closed as too broad by WYSIWYG♦, The Last Word, Bez, daniel, rg255 Jul 2 '14 at 9:49
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I'm no expert at this and I would welcome any changes to this post. First you need to decide, what your target organism which you want to infect is (e.g. mammalian cells, bacterial cells) and based on that you choose your viral vector as listed in this article. Generally for mammalian cell stable transfection (usually you want the genetic materials you introduced to persist between cell divisions hence stable transfection) and transformation (just a general statement about changing cells as you have introduced, usually foreign genetic material but transformation is not necessarily the same as making something immortalised, which means the cells won't stop dividing i.e. becoming cell lines or in lay term cancerous) scientists tend to use lentiviruses, which are retroviruses, similar to HIV and they can even infect non dividing cells (so this should allow your imagination to run wild!). Once you have decided what the best viral vehicle for targeting your cells of interest is then you essentially transfect the plasmid DNA (packaging vector), which produces the virus protein package, into cells, whose job becomes to produce the virus as those cells have the required cellular machinery for this purpose. These cells are often 293T packaging cells. Along with the plasmid (circular) DNA, which produces the virus package, you co-transfect the packaging (e.g. 293T) cells with another plasmid, called lenti-vector, which does whatever you want it do, such as express a particular protein or microRNA or whatever but the idea is that stuff produces by the lenti-vector should not be toxic to the target cell (i.e. the cells you intend to target), unless thats what you want it to do.
Now as you can see, you can play around with two things here, first the packaging vector, to change the behaviour of the virus if you wish. And second the lenti-virus vector, to produce whatever you want it to! Once the packaging cells made your viruses, containing your lenti-vector, the virus get secreted into the media, which the packaging cells are in so you have to extract the media, congaing the virus, and concentrate the virus using centrifugation. Once you got the virus then you can use it to transform the (target) cells you wish.
For the lab based procedure of how you make the virus and what equipments you use and how to concentrate the virus then please see this link, although its a little technical. In this case, the packaging vector is fuGENE 6 and the lenti-vector or just plasmids of interests are listed (please not that I do not endorse any products mentioned here and I'm just using it as an example).
Now for making the packaging plasmid (making the virus), you often use the genetic materials from a virus and change it through a procedure called polymerase chain reaction (PCR). Same goes for the lenti-vector/plasmid as they are derived from somewhere and then change through time using PCR and restriction digests, which is an equivalent of genetic material cut and paste, to make the vectors and products of interest. Once all is done the the virus can infect the target cells and effect it based on its designed function (defined by the two vectors I described).
If the above response does not answer you question, since it is currently very broad, please make further clarification to the question so that I can modify my response accordingly.
Consider this as a summary of the previous answers. Also, I am elaborating more on how to make self-sustaining mutated viruses
Points to consider before you think about working with viruses:-
Having identified and obtained a suitable host, one can proceed to genetic manipulation. The lentiviral vectors that Bez described are basically partial viruses. One part codes for the capsid and cannot make active viruses by itself whereas the other holds the genes for replicative function. So in a way the lentiviral vectors are disarmed viruses that are used as molecular biology tools. But what you are looking for is different.
To modify a virus you need to make mutations in its genome. Once you have identified what mutations are to be made then as previously mentioned, PCR can be used to incorporate them. This technique is slightly different from a usual PCR (not in principle, though) and is called site-directed mutagenesis. For ease it is always better to clone (fix) the viral genome in a plasmid (circular DNA that can be propagated in a usual lab bacteria- E.Coli). After mutating the viral genome, you can excise it from the plasmid. Note that PCR is done with DNA. For RNA viruses you need to convert its genome into a DNA sequence before you go for site-directed mutagenesis. Conventional technique would be to reverse-transcribe the viral RNA. But nowadays chemical DNA synthesis has become quite easy and you can synthesize entire viral genome in a "test-tube".
Having done this, we proceed to the trickiest part: How to get this genome to form an active virus?
Some viruses, as mentioned before use RNA as their genome (poliovirus, retroviruses, common-cold virus etc) while some use DNA (chickenpox, smallpox, herpes etc). The mutagenesis, however, is performed on the DNA. For DNA viruses, introduction of the viral DNA into the host cell, using the conventional transfection methods, might work. For certain RNA viruses like poliovirus this DNA has to be transcribed to RNA. This can be done using a technique called in-vitro transcription (IVT) and the RNA product can be introduced inside the cell. IVT wont work well for long RNAs. In such cases you can use the cell itself to make the RNA instead of doing it in-vitro. Use a plasmid that can transcribe a DNA sequence inside a host cell- this is fairly common and almost all molecular bio labs will have such plasmids. Transfect this plasmid inside the host cell and it will make viral RNA, which in-turn will make active viruses.
Retroviruses work slightly different from other RNA viruses. They have an RNA genome that they convert to a DNA. This DNA gets integrated in the host genome and keeps producing viruses. To make a retroviral DNA integrate you need an enzyme encoded in the virus- integrase. You can clone integrase separately and co-transfect it with the viral genomic DNA.
A couple of final points to consider:
I would not start from so bottom as Bez in making viruses. This is very very long project. It is much less cumbersome task to consider modifying virus, since it already occurs passively in real life.
I would choose equipment based on the characteristics of the virus. I would start to develop equipment needed for the specific project, since there is no ready solution for this kind of task. The equipment can be targeted to
I would use iPS stem cells to as an target but to get this far is very very long process still. I would focus on antigen presentation - again very many fine regulation factors in Interferons etc should be considered in the special viral case - note that the interferon research is now in an active phase but early. Interferons are specific so specific parts of the equipment should exists to detect them - for instance, IFN-gamma.
Viruses are generally extremely difficult to control - shortly existing in one form. You can imagine how difficult they become after editing them real life. So the plan would be first to understand some easy virus and its early stages in the replication.
You can actually choose a virus that undergoes continuously point mutations (drift mutation) like influenza virus. However, I would pick something that is not so changing (not RNA virus). There is no good equipment to do regular analysis of the changes in the influenza virus. It always requires much work to recognize which genome part has changed and how much internationally. The vaccines are made based on reports from many countries about their influenza virus statuses and their genomes.
Some simple DNA virus with very slow but fixed point mutations in time so you have time to research them and you can estimate when the next change is going to be. It would be also useful if you know which part of the virus is going to change (particular plasmid). This makes the research much more manageable. Sufficient time-series analysis and group of Mathematicians are also needed to understand different data from different stages. Actually, your task is extremely broad.