In the previous post of this series I explained the significance of using cell lines in cancer research. Today I would like to continue by sharing some details about my research, and to show you how interesting and challenging at the same time science actually is!
Missing an adequate model system for your research? Make one!
The idea of our project was to investigate transcriptional and translational changes in Multiple Myeloma cell line induced by over-expression of particular oncogene.
However, to be able to properly assess changes going on in your cells, you need to have cell line that has high levels of gene, and the SAME cell line with low levels of that gene, so you could compare them and eventually attribute all observed changes to high expression of your gene of interest, because everything else must stay the same.
But, where to find cell line that has both low and high expression of the same gene? It doesn't exist, of course!
This means that you have to make your own model system, a cell line that will have high or low levels of whichever gene you need.
So I did it!!! Want to know how? ;)
Making my own transgenic cell line by lentiviral transduction
For our experiments we decided to use a cancer cell line which has by default low expression of our gene on interest, and to create a transgenic cell line, which will be the same as parental line, except for having high expression of our gene of interest.
To do that, you need to insert your gene somehow into parental cell line, and to force that cell line to produce a protein product of that gene, in other words - you need to have a stable expression of your gene of interest to be able to do experiments with such cell line.
This is achieved by using lentiviral vectors in a process called lentiviral transduction.
Lentiviruses are subtype of retroviruses (viruses with RNA genomes) and are specific by the fact that they can infect both non-dividing and actively dividing cells, unlike retroviruses, which can infect only mitotically active cells. HIV virus is an example of lentivirus.
Lentiviral vectors are basically lentiviruses that are modified in such way that your gene of interest is inserted into their genome, and they are capable of infecting your cell line, deliver your gene which becomes stably integrated into the genome of your cell line, BUT they do not have the capability of reproducing themselves, in other words, they cannot form new viral particles and infect other cells.
Once lentiviral vector integrates your gene into the genome of your cell line, that gene is being transcribed into mRNA and translated into protein product, in other words - you got yourself a transgenic cell line that produces your protein of interest like crazy!
Modified from Wikimedia Commons, public domain
One batch of cells was transfected with lentiviruses carrying control vectors - vectors without our gene of interest (GOI), and the other batch was transfected with lentiviruses that contained our GOI. Vectors also contained virus-specific genes necessary for integration of vector into genome of human cells, and very important gene for antibiotic (in this case puromycin) resistance.
Why do we need gene for antibiotic resistance? Because it represents selection marker, based on which we are able to tell which cells were successfully transfected, and which did not integrate the vectors.
Selection is made simply by growing cells in selective media, which contains antibiotic (puromycin). Cells that were successfully transfected will continue to grow in media containing puromycin, whilst cells that did not take up the plasmid will die, because they don't have puromycin resistance and the antibiotic will kill them.
After you have confirmed that you have uniform population of cells (cells that have taken up the plasmid), you have to perform qPCR and Western Blot experiments with your transgenic cell line to confirm that you have stable expression of your GOI .
When you see amplification in qPCR and get bands in Western Blot - congratulations, you have created your model system successfully!!!
You can finally proceed to your experiments using your cells... and this is where you realize that the party has just begun :)
In the next Lab Diaries post I will write about the experiments and methods I use in my research.
Until then, relax and keep steemSTEM! ;)
Image credits pixabay.com and myself, unless otherwise stated