I still don't figure it out yet how exactly a virus particle is formed by adding three plasmids (transfer plasmid with transgene, packaging plasmid and envelope plasmid) in a cell by transfection?
How the plasmids connect to each other in the host cell so that the transfected cell produces virus particles?
In addition to that, how does this method improves the security? Why the virus is not able to reproduce again after the infection of the host cell? I thought it is necessary, so that more cells can be infected with the viral genome to integrate the transfer gene/ gene of interest (so that the cell is able to express it).
And is it even right that I achieve my gene of interest/ the expression of the transgen through lentiviruses? Through the stable integration in the host cell genome?
I would be very glad, if someone could describe to me the process with lentiviruses in a detailed but easy way. I would like to be able to fully understand the method.
With lentiviruses you have the wild type genome which contains an origin of replication, packaging signal, proteins for DNA replication and genome packaging into a protein coat (envelope). The original lentiviral systems where your gene of interest was inserted into the genome were under selective pressure to lose the transgene as genomes without it would replicate faster and be packaged more efficiently. The second generation lentiviral systems used a helper plasmid where a viral genome with a heavily mutated origin of replication was inserted into a plasmid and the origin of replication and packaging signal were cloned onto a second plasmid with the gene of interest. This means all genes needed for replication of the ori containing plasmid and packaging it into virus like particles would be present when both plasmids were transfected while the helper plasmid would have greatly impaired replication and packaging. There was the problem though that the origins could undergo recombination resulting in the formation of wildtype like viral genomes lacking the gene of interest. By moving the envelope protein from the helper plasmid to a third plasmid lacking a viral origin the probabilities for the formation of a DNA sequence with all elements of the wild type genome is greatly reduced as the envelope containing plasmid is not transferred to new host cells.
The plasmids do not "connect" to each other in the host cell, they're just co-expressed in the cell which means all the different components needed to make the lentiviral particle are expressed in the cell.
This improves safety because the genome of the lentiviral particle that is produced in this way will only contain the transgene from your transfer plasmid*, since that's the only plasmid containing LTRs which are required for the RNA genome to get packaged into the lentiviral particle. The lentiviral particle won't contain the envelope genes or packaging genes (which are on the other plasmids), and those genes are necessary for making the lentiviral particle. So the only time the lentiviral particle can be made is when you co-transfect all three of those plasmids. Once you isolate the lentivirus and then use it to infect other cells, it won't be able to replicate because it doesn't express the necessary genes.
And for your last question, it is completely normal to test overexpression of a gene by integrating copies into the genome with lentivirus. This is very common. But it's important to keep in mind that your gene of interest may behave differently because it will be expressed at different levels than endogenously.
*not completely true but I'm simplifying things.
Ciaran Daly Thank you for your answer! I've written a text with the information I've understood. Would you mind taking the time to read through it and correct me if I've misunderstood anything?
So, as I understand it now, the three transfected plasmids remain in the cytoplasm and are transcribed by RNA polymerase (there is no integration of genes into the cell's genome). The transgene then gets packaged as an RNA molecule into the synthesized envelope protein, along with the necessary expressed structural proteins, forming a lentivirus particle. This particle is secreted into the supernatant and remains there. If the viral supernatant gets harvest, it could be used to infect another cell line with the lentivirus particles. The lentivirus particle would then enter the host cell, and with the help of reverse transcriptase, the RNA carrying the transgene information would indeed be converted into a DNA molecule and integrated into the host cell genome, right? However, a new lentiviral particle could not be formed in the host cell because the necessary genetic elements for replication are missing, right?
Lea-Sophie Ziegler yes that paragraph is correct. The only small mistake is "the three transfected plasmids remain in the cytoplasm and are transcribed by RNA polymerase (there is no integration of genes into the cell's genome)."
The plasmids will make it into the nucleus (there is never any transcription in the cytoplasm), and the transgene will get integrated into the genome because the reverse transcriptase and integrase genes on the transfected plasmids will be expressed.
So the cells that you use to make the virus will get the transgene integrated into the genome, and also when you take the virus in the supernatant and infect other cells then the transgene will also get integrated there. But in no cells will the packaging/envelope/etc genes necessary for replication get integrated. They will only ever be expressed from the transfected plasmid.
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