It's interesting, but sometimes I feel as though the New York Times and I are strangely in sync with one another, especially in regards to their articles about science topics. Just yesterday I found these two articles, Genes Explain Race Disparity in Response to a Heart Drug and A Genetics Pioneer Sees a Bright Future, Cautiously
that made me pause for a moment. It was pharmacogenomics in mainstream news. Hooray! See? What I'm working on isn't so obscure after all. For current "real-world" applications of the kind of stuff I'm working on, please read these articles.
Anyway, back to my work. So I talked about how we aim to decrease the levels of a particular drug-clearing enzyme to make the cells we treat more sensitive to our chemo agent. The next thing I should explain is how we actually go about manipulating levels of said enzyme.
First, a brief lesson in molecular biology. DNA, or deoxyribonucleic acid, is considered to be the "code of life" as it prescribes nearly all the information required for organisms in all five kingdoms of life to fulfill the requirements of life, i.e. grow, reproduce, survive, etc. Living things, however, are not made of DNA alone (obviously). The information coded in DNA proceeds along the path to proteins (what we actually see and feel in living things, i.e. skin, hair, etc) through an intermediary molecule known as RNA, or ribonucleic acid. DNA and RNA are very closely related but have significantly different properties which suits them for their particular roles. RNA is much less stable (it's single-stranded instead of double-stranded) and is usually rapidly degraded after it's produced (compared to DNA which lasts until the cell dies). The type of RNA I'm interested in in particular is messenger RNA, or mRNA. The "messenger" bit comes from a famous molecular biologist who likened that molecule to a "messenger from God" for it's role in transducing DNA's message (so much for all scientists being determinedly irreligious!). What does the RNA send its divine message to? Structures called ribosomes which are synthesis stations in which mRNA is used as a template off of which proteins are built.
If you consider the percent composition of living things, we are possibly 99.9980% protein, 0.0015% RNA, and 0.0005% DNA. Do not take these percentages for fact; I literally made them up just for a sense of scale. But I digress.
If we want to knockdown protein levels, there are three conceivable ways we can go about doing that. One is to alter the gene (DNA) to make a substandard protein that doesn't do its job so well. Another would be to break down the final protein or inhibit its activity somehow by making it stick to another protein or small molecular (this is actually how many new fancy drugs work). A final way, and the way we're using, is to go after the mRNA and reduce its levels such that the protein isn't made in normal quantities in the first place.
Back in the early 1990s, two labs headed by molecular biologists Craig Mello and Andrew Fire discovered an incredibly potent viral defense mechanism in the roundworm C. elegans in which foreign RNA (many viruses have RNA instead of DNA as their primary "code of life"; bit of a misnomer here since viruses are technically not considered living things) was rapidly degraded by a unique type of RNA produced by the worm's cells. This defense mechanism had already been identified in plants, but what made Mello and Fire special (and eventually Nobel laureates) was their insight that could harness that natural defense mechanism to attack any RNA of their choice. This technique, called RNAi (short for RNA interference), won them the Nobel Prize in 2006 (just over a decade after their discovery) and is an extremely common and invaluable laboratory technique. (If you care to find an earlier post I made back in winter 2007, you can read about my experience of actually meeting Dr. Mello at the Field Museum here in Chicago).
Back to the lab. The siRNA experiment is actually quite simple:
1) I siphon off the volume of cell suspension I'll need from the flasks in which they grow (the cells we use, lymphoblastoid cells, do not grow flat on plates but suspended in media...think Guild Navigators for you Dune fans, except these cells fortunately can't bend space).
2) I then spin down the cells to form a tiny pellet that I then wash with a simple pH neutral solution to remove any remaining media.
3) Spin again to make a new, cleaner pellet.
4) Now I add in a "nucleofection solution" which basically makes lots of tiny holes in the cells such that stuff can get in, but they still stay alive.
5) I carefully plate these hole-ridden cells onto a dish full of tiny wells.
6) Quickly, else the cells will die, I add in my siRNA solution that is full of those attacking RNA molecules. Since I wish to knockdown the CYP1B1 protein, the siRNA I use is specific for the mRNA for that protein.
7) Once the cells have been dosed with the siRNA, I place them in a machine where they are "zapped" with an electric pulse to make the cells take up the siRNA. This takes seconds.
8) Once the cells are zapped, I give them some warm media and stick them in the incubator for some minutes to let them recover from the shock (a pun, haha).
9) Once they've relaxed a bit, I plate them out onto a new dish and add some more media so that they will be happy.
10) At a predetermined time point, in my case 24 and 48 hours after plating, I'll scoop up those cells and go on to the next experimental step: RNA isolation.
All in all the nucleofection takes approximately 2 hours from start to finish. Not too bad, considering I performed an RNA isolation today which took approximately 4.5 hours, so I'm not exactly in the mood to describe that right now...but I will do so tomorrow! Stay tuned!
that made me pause for a moment. It was pharmacogenomics in mainstream news. Hooray! See? What I'm working on isn't so obscure after all. For current "real-world" applications of the kind of stuff I'm working on, please read these articles.
Anyway, back to my work. So I talked about how we aim to decrease the levels of a particular drug-clearing enzyme to make the cells we treat more sensitive to our chemo agent. The next thing I should explain is how we actually go about manipulating levels of said enzyme.
First, a brief lesson in molecular biology. DNA, or deoxyribonucleic acid, is considered to be the "code of life" as it prescribes nearly all the information required for organisms in all five kingdoms of life to fulfill the requirements of life, i.e. grow, reproduce, survive, etc. Living things, however, are not made of DNA alone (obviously). The information coded in DNA proceeds along the path to proteins (what we actually see and feel in living things, i.e. skin, hair, etc) through an intermediary molecule known as RNA, or ribonucleic acid. DNA and RNA are very closely related but have significantly different properties which suits them for their particular roles. RNA is much less stable (it's single-stranded instead of double-stranded) and is usually rapidly degraded after it's produced (compared to DNA which lasts until the cell dies). The type of RNA I'm interested in in particular is messenger RNA, or mRNA. The "messenger" bit comes from a famous molecular biologist who likened that molecule to a "messenger from God" for it's role in transducing DNA's message (so much for all scientists being determinedly irreligious!). What does the RNA send its divine message to? Structures called ribosomes which are synthesis stations in which mRNA is used as a template off of which proteins are built.
If you consider the percent composition of living things, we are possibly 99.9980% protein, 0.0015% RNA, and 0.0005% DNA. Do not take these percentages for fact; I literally made them up just for a sense of scale. But I digress.
If we want to knockdown protein levels, there are three conceivable ways we can go about doing that. One is to alter the gene (DNA) to make a substandard protein that doesn't do its job so well. Another would be to break down the final protein or inhibit its activity somehow by making it stick to another protein or small molecular (this is actually how many new fancy drugs work). A final way, and the way we're using, is to go after the mRNA and reduce its levels such that the protein isn't made in normal quantities in the first place.
Back in the early 1990s, two labs headed by molecular biologists Craig Mello and Andrew Fire discovered an incredibly potent viral defense mechanism in the roundworm C. elegans in which foreign RNA (many viruses have RNA instead of DNA as their primary "code of life"; bit of a misnomer here since viruses are technically not considered living things) was rapidly degraded by a unique type of RNA produced by the worm's cells. This defense mechanism had already been identified in plants, but what made Mello and Fire special (and eventually Nobel laureates) was their insight that could harness that natural defense mechanism to attack any RNA of their choice. This technique, called RNAi (short for RNA interference), won them the Nobel Prize in 2006 (just over a decade after their discovery) and is an extremely common and invaluable laboratory technique. (If you care to find an earlier post I made back in winter 2007, you can read about my experience of actually meeting Dr. Mello at the Field Museum here in Chicago).
Back to the lab. The siRNA experiment is actually quite simple:
1) I siphon off the volume of cell suspension I'll need from the flasks in which they grow (the cells we use, lymphoblastoid cells, do not grow flat on plates but suspended in media...think Guild Navigators for you Dune fans, except these cells fortunately can't bend space).
2) I then spin down the cells to form a tiny pellet that I then wash with a simple pH neutral solution to remove any remaining media.
3) Spin again to make a new, cleaner pellet.
4) Now I add in a "nucleofection solution" which basically makes lots of tiny holes in the cells such that stuff can get in, but they still stay alive.
5) I carefully plate these hole-ridden cells onto a dish full of tiny wells.
6) Quickly, else the cells will die, I add in my siRNA solution that is full of those attacking RNA molecules. Since I wish to knockdown the CYP1B1 protein, the siRNA I use is specific for the mRNA for that protein.
7) Once the cells have been dosed with the siRNA, I place them in a machine where they are "zapped" with an electric pulse to make the cells take up the siRNA. This takes seconds.
8) Once the cells are zapped, I give them some warm media and stick them in the incubator for some minutes to let them recover from the shock (a pun, haha).
9) Once they've relaxed a bit, I plate them out onto a new dish and add some more media so that they will be happy.
10) At a predetermined time point, in my case 24 and 48 hours after plating, I'll scoop up those cells and go on to the next experimental step: RNA isolation.
All in all the nucleofection takes approximately 2 hours from start to finish. Not too bad, considering I performed an RNA isolation today which took approximately 4.5 hours, so I'm not exactly in the mood to describe that right now...but I will do so tomorrow! Stay tuned!
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1 comment:
nice!
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