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dgaughran
04-08-2011, 03:03 PM
Hi

I have just written a 5200k SF short and I need to check if I got some details right about GM food, genetic engineering, and transfection in particular.

Any experts out there?

Dave

DrZoidberg
04-08-2011, 03:39 PM
I suggest you write the details of how transfection works in your short and ask if this is how it really works. I suspect you're more likely to get an answer then.

/Tom, also stuck in Sweden

dgaughran
04-08-2011, 03:53 PM
Just to give a little background - this science fiction short is about a researcher, Dr. Peters, who is a university biologist. In the story, there is a food safety panic after a team of medical researchers find a link between GM food and cancer. I have tried just to cut out the relevant snippets, as suggest by Dr. Z above.

Here:

There were many stages in the complex process of genetic modification, but Dr. Peters knew that if something was going wrong, the root cause would be the foreign DNA that was being inserted into the host. He suspected he would find the answer if he monitored the transfection process.

And here:

As his assistant went into the office, separated from the lab by glass panes, Dr. Peters put on his gloves and pulled two petri dishes from the freezer, one containing the host cell and the other containing the desired gene, so tiny as to be invisible to the human eye. Under his specialised optical microscope, he injected the gene into the host cell membrane with a glass pipette, and watched the reaction through the micromanipulator.

Thanks,

Dave

OneWriter
04-08-2011, 05:59 PM
GM food is obtained throughout generation after generation of human-driven selection. Not in the lab. What you are hinting at is some gene-therapy experiment with live cultures. Are these plants or animals we're talking about? I don't know much about plants. If you're doing it with animals, you need to modify their germinal cells because if you do the normal cells the next generation will bear no change. It's like cutting a nose: the next generation will still have noses!

All of the above is done for therapeutic reasons. Too expensive to do on foods! Much easier to select, breed, repeat. What is indeed plausible, and people have been advocating against GM for this exact reason, is that GM foods may cause severe allergic reactions to the extreme of causing death. That's your most realistic scenario.

But then again, this is fiction, so theoretically you can do whatever you want.

LBlankenship
04-08-2011, 06:21 PM
Just to give a little background - this science fiction short is about a researcher, Dr. Peters, who is a university biologist. In the story, there is a food safety panic after a team of medical researchers find a link between GM food and cancer. I have tried just to cut out the relevant snippets, as suggest by Dr. Z above.

Here:

There were many stages in the complex process of genetic modification, but Dr. Peters knew that if something was going wrong, the root cause would be the foreign DNA that was being inserted into the host. He suspected he would find the answer if he monitored the transfection process.

And here:

As his assistant went into the office, separated from the lab by glass panes, Dr. Peters put on his gloves and pulled two petri dishes from the freezer, one containing the host cell and the other containing the desired gene, so tiny as to be invisible to the human eye. Under his specialised optical microscope, he injected the gene into the host cell membrane with a glass pipette, and watched the reaction through the micromanipulator.

Thanks,

Dave

Usually scientists get a virus to do the dirty work of injecting new DNA into cells, since injecting them one at a time? Even a small culture can contain thousands or millions of cells. Also, handling just one snippet of DNA directly with a pipette? Not with current technology, AFAIK.

When you see a video of one cell being injected, it's usually an egg cell. They're huge and you only need one.

There could be a connection between GM foods and cancer for any number of reasons, though. It could be some molecule the GM foods are producing because of the new DNA. Some bit of DNA that got in along with the desired DNA. That would be an error in isolating the desired DNA, though. Unless there's a rogue virus running around injecting itself into your host cells... :)

OneWriter
04-08-2011, 06:45 PM
The link between GM foods and cancer would be something that would take years to discover and it would take an epidemiological study at first. It would be done by taking the DNA profile of all cancer patients, taking all their anagraphic data, etc, and then compare it with healthy individual. Once you have that, you go back to the lab and try to understand how the GM food could have caused cancer at the molecular level. Think about smoke: it causes cancer because on the long run it irritates lungs, throat, etc. The inflammation lasts years, and eventually triggers cancer. You could have something similar happening with your GM foods. Nothing transpires on the surface, but the foods in the intestine or the in the stomach trigger some kind of long lasting inflammation which eventually leads to cancer. (though there would be some kind of symptoms on the outside too)

Gee, that's freaky.

dgaughran
04-08-2011, 07:00 PM
Are these plants or animals we're talking about? I don't know much about plants.

Sorry I should have made that clear. It is plants I am talking about.


Usually scientists get a virus to do the dirty work of injecting new DNA into cells, since injecting them one at a time? Even a small culture can contain thousands or millions of cells. Also, handling just one snippet of DNA directly with a pipette? Not with current technology, AFAIK.

I found something called a glass micropipette (http://en.wikipedia.org/wiki/Microinjection) which seems to be used in the transfection process and could be small enough.

Ok, so I don't know a lot about this, so please feel free to correct me any of the following (and a lot of what I based this on is from Wikipedia so I could be off). This is my understanding and is probably wrong.

Genetic modification is the direct human manipulation of an organism's genetic material (in a way that does not occur under natural conditions).

One of the ways that this modification can take place is through the introduction of a virus (known as viral transformation or transduction)

Transfection seems to be a word that is used in different ways by different people to describe different (but related) processes, and it appears to have been originally used to describe working with animal cells, but the terms seems to have spread (and I like it!) to cover viral transformation which uses plant viruses.

My basic thesis (which could be weak), is that the scientist discovers that there is a short burst of radiation emitting from the host cells at the moment of transfection, and that his is what is probably leading to the increased cancer-risk. I know it's a stretch, but too much?

dgaughran
04-08-2011, 07:04 PM
The link between GM foods and cancer would be something that would take years to discover and it would take an epidemiological study at first. It would be done by taking the DNA profile of all cancer patients, taking all their anagraphic data, etc, and then compare it with healthy individual. Once you have that, you go back to the lab and try to understand how the GM food could have caused cancer at the molecular level. Think about smoke: it causes cancer because on the long run it irritates lungs, throat, etc. The inflammation lasts years, and eventually triggers cancer. You could have something similar happening with your GM foods. Nothing transpires on the surface, but the foods in the intestine or the in the stomach trigger some kind of long lasting inflammation which eventually leads to cancer. (though there would be some kind of symptoms on the outside too)

Gee, that's freaky.

I suspected that, I think, because in the story, the medical researchers who discover the cancer-link are working with animals fed exclusively on GM foods. The link to humans remains unproven, but is sufficient to cause a collapse in the share price of the GM food companies.

Does that work?

OneWriter
04-08-2011, 07:12 PM
I wouldn't buy the radiation thing. It just doesn't happen. Again, I seriously doubt GM plants are obtained through viruses. Plants are bred, selected, and then inbred for several generations. It's fast and cheap, and that's how they are modified.

Now, like I said, it's fiction. What I would find more plausible, if you really wanna go the virus route, is that these viruses are not completely cleared by the plant. They somehow remain dormant inside the plant (that can happen) and have no harmful effect. BUT they do in the animals, hence the cancer. This is all plausible, as there are viruses like that. Now of course in your case the viruses would be engineered as to transfer genetic material, they wouldn't be natural viruses.

Hope this helps. Sorry, the radiation just doesn't fly. You need energy to generate radiation. You can have radioactive cells being injected, but you don't "create" radiation out of the blue.

dgaughran
04-08-2011, 07:22 PM
Ok this is where I might really start to annoy you.

I accept what you are saying, but I need the radiation for various reasons. This scientist goes mad trying to decipher the radiation bursts, eventually becoming convinced that they are some kind of language or code, but he is ignored, becomes more obsessed, turns to alcohol, breaks up with his wife, gets fired, and eventually becomes homeless.

A year or so later, his research assistant is packing away some stuff into storage and comes across his old research, and gives it another look, and begins to think there was something to it after all, and publishes a research paper. This kicks off a debate on whether these radiation bursts can be considered a "language", what level of consciousness plants have, about how "alive" plants are and whether it is ethical to eat them.

Because that plants were considered "alive", the vegetarians of the world (which are now a significant portion of the population after various food scares) decided only to eat GM food (as the seeds from which it is grown are already "dead"), despite the health risks, and the GM food companies' profits soar.

The previously homeless scientist is catapulted into international celebrity, but one day as he is looking at his old data, he notices something is off, that the data had been changed, and his investigation leads to the door of the GM companies.

Maybe it's a stretch!

dgaughran
04-08-2011, 07:29 PM
I wouldn't buy the radiation thing. It just doesn't happen. Again, I seriously doubt GM plants are obtained through viruses. Plants are bred, selected, and then inbred for several generations. It's fast and cheap, and that's how they are modified.

My understanding, again from Wikipedia mostly and some hazy memories of college, is that what you are describing is how plants are generally modified. But with GM-food, when they want to produce something that is resistant to pesticides or viruses, or has a longer shelf-life (like that tomato they have been trying to engineer for ages), a different process is involved - usually the addition of genes from either a virus, bacteria, an enzyme or another plant.

There are various methods of doing this, but one, according to Wikipedia is this: "To do this artificially may require transferring genes as part of an attenuated virus genome or physically inserting the extra DNA into the nucleus of the intended host using a microsyringe, or as a coating on gold nanoparticles fired from a gene gun."

LBlankenship
04-08-2011, 07:38 PM
Ok this is where I might really start to annoy you.

I accept what you are saying, but I need the radiation for various reasons. This scientist goes mad trying to decipher the radiation bursts, eventually becoming convinced that they are some kind of language or code, but he is ignored, becomes more obsessed, turns to alcohol, breaks up with his wife, gets fired, and eventually becomes homeless.

A year or so later, his research assistant is packing away some stuff into storage and comes across his old research, and gives it another look, and begins to think there was something to it after all, and publishes a research paper. This kicks off a debate on whether these radiation bursts can be considered a "language", what level of consciousness plants have, about how "alive" plants are and whether it is ethical to eat them.

Because that plants were considered "alive", the vegetarians of the world (which are now a significant portion of the population after various food scares) decided only to eat GM food (as the seeds from which it is grown are already "dead"), despite the health risks, and the GM food companies' profits soar.

The previously homeless scientist is catapulted into international celebrity, but one day as he is looking at his old data, he notices something is off, that the data had been changed, and his investigation leads to the door of the GM companies.

Maybe it's a stretch!

How about a tiny burst of bioluminescence? It's still a stretch, but not as big as spontaneous radiation.

I think the argument is how "conscious" plants are -- their being alive isn't arguable. Actually, there are enough papers out there on the generation of stress proteins in plants that one could argue they do feel pain. ;)

LBlankenship
04-08-2011, 07:39 PM
My understanding, again from Wikipedia mostly and some hazy memories of college, is that what you are describing is how plants are generally modified. But with GM-food, when they want to produce something that is resistant to pesticides or viruses, or has a longer shelf-life (like that tomato they have been trying to engineer for ages), a different process is involved - usually the addition of genes from either a virus, bacteria, an enzyme or another plant.

There are various methods of doing this, but one, according to Wikipedia is this: "To do this artificially may require transferring genes as part of an attenuated virus genome or physically inserting the extra DNA into the nucleus of the intended host using a microsyringe, or as a coating on gold nanoparticles fired from a gene gun."

Yes, but none of those are going to cause spontaneous bursts of radiation.

Ultimately, how accurate you want to be is up to you, though...

Saul Tanpepper
04-08-2011, 07:50 PM
GM food is obtained throughout generation after generation of human-driven selection. Not in the lab. What you are hinting at is some gene-therapy experiment with live cultures.

NO! THIS IS WRONG, WRONG, WRONG!

I have a PhD in molecular genetics and I write genetic thrillers. Hybrid foods may be the product of breeding and cross breeding, but GMO refers to the process of artifically inserting exogenous (foreign) DNA into a host cell's genome (whether plant or animal). Transfection is the process by which animal cells are genetically altered, usually by some mechanical, chemical, enzymatic, or viral method. There are a host of ways to do it. Plant cells are often altered by biolistic methods (using DNA-coated gold beads) or viral methods, though so-called transposable elements (non-viral but pathogenic DNA) can also be used.

And, yes, it's done in the lab.


If you're doing it with animals, you need to modify their germinal cells because if you do the normal cells the next generation will bear no change. It's like cutting a nose: the next generation will still have noses!

This is accurate.


All of the above is done for therapeutic reasons. Too expensive to do on foods! Much easier to select, breed, repeat. What is indeed plausible, and people have been advocating against GM for this exact reason, is that GM foods may cause severe allergic reactions to the extreme of causing death. That's your most realistic scenario.

Oh dear, so much misinformation out there.

No, it's not too expensive. And as far as the allergen theory, this is more hyperbole. Just like vaccinations cause autism. Yes, introduction of new genetic material into plants will cause the plant to express new proteins (in most cases, though not necessarily restricted to this), but most proteins are enzymatically neutralized in the stomach. GMO plants pose no more risk to the general population than do mixed breed (hybrids) or newly introduced exotic breeds.

Now, as to your story, dgaughran,

that if something was going wrong, the root cause would be the foreign DNA that was being inserted into the host. He suspected he would find the answer if he monitored the transfection process.

It's a bit more complex than this. What do you mean by "monitor"? That's key. Any number of things can happen yielding failure. In fact, chromosomal alteration is a rare process and is usually the result of millions of trials and errors (often conducted in the same test tube), which is why scientists have developed methods for screening for the altered cells that eventuall become progenitor cells of the new organism/strain/etc. So, he would find the answer by screening for cells (usually for linkage to antibiotic resistance, colorometric or luminescent biomarkers, etc).
And here:

As his assistant went into the office, separated from the lab by glass panes, Dr. Peters put on his gloves and pulled two petri dishes from the freezer, one containing the host cell and the other containing the desired gene, so tiny as to be invisible to the human eye. Under his specialised optical microscope, he injected the gene into the host cell membrane with a glass pipette, and watched the reaction through the micromanipulator.

The petri dishes wouldn't be in the freezer but an incubator, and they're called tissue culture flasks. The petri dish would be filled with thousands to tens of millions of cells and the DNA would be in a tube called an eppendorf tube. The actual insertion of the DNA would not be done under a microscope (though one might be used), but using sophisticated instrumentation, usually a biolistic gun. The DNA would enter the cell, traverse the cytoplasm and enter the nucleus, which is where the chromosomes are located. The process is not monitored visually, nor done by a micromanipulator. It would be done "blindly" and monitored for detection of the appropriate biomarkers (color, antibiotic resistance etc) for weeks and weeks, the cells carefully cultured until the untransfected cells die off and the transfected ones survive, divide into colonies and eventually form whole tissues or organisms.

JayMan
04-08-2011, 08:04 PM
I got my BS degree in molecular and cell biology and I worked in a lab that worked with plants for two years, so I think I'm semi-qualified to comment on this.

OneWriter, I think you're confusing regular plant breeding to select for certain traits with actual genetically modified food.

It is very possible to transform plant genomes with foreign DNA using viral or bacterial pathogens, and this is one of the methods used widely today. In fact, my honors thesis work was just that. I had to transfer a cell wall related gene into a bacterium called Agrobacterium tumefaciens (http://en.wikipedia.org/wiki/A._tumefaciens). Agrobacterium is a plant pathogen, ie, it infects plants, and when it does, it transfers a certain section of DNA into the plant's genome. So what we did was insert the gene of interest into that section which gets transferred. This way, when the bacteria infect the plant (in my case, the plant was Arabidopsis thaliana... the thale cress, the most commonly used plant model in plant research) and transfer their DNA into the plant, the gene of interest gets transferred with it.

OP (and others who are interested), I highly suggest you check out GM corn, specifically Bt corn (corn that was modified by inserting a gene for Bt toxin, a toxin that affects insects but not humans) and Starlink corn (which used a Bt protein that was found to be allergic in some people and caused a pretty big controversy). Check out the Wikipedia link:
http://en.wikipedia.org/wiki/GM_corn


I don't think any kind of "radiation burst" or "bioluminescence" would make sense as a side effect for a gene inserted into a GM plant, but there are plenty of mechanisms that could cause more plausible side effects, or cancer, or something, for a fictional story. Again, check out Starlink corn.


Regarding transfection vs transformation: my professor told me (after I'd written the word "transfection" in the rough draft of my thesis) that transfection is generally only used in regard to animal cells. For plant purposes, it's just "transformation."


edit: ninja'd by the above post! Saul is probably a lot more qualified than I am, but I'll be happy to answer any other questions within my scope of knowledge.

dgaughran
04-08-2011, 08:20 PM
Is there a doctor in the house!

Thanks guys for those lengthy, informative, educational posts. I will take some time to read through them all, then post back if I have any questions - and I'm sure I will.

Thanks a lot,

Dave

JayMan
04-08-2011, 08:21 PM
The petri dishes wouldn't be in the freezer but an incubator, and they're called tissue culture flasks. The petri dish would be filled with thousands to tens of millions of cells and the DNA would be in a tube called an eppendorf tube. The actual insertion of the DNA would not be done under a microscope (though one might be used), but using sophisticated instrumentation, usually a biolistic gun. The DNA would enter the cell, traverse the cytoplasm and enter the nucleus, which is where the chromosomes are located. The process is not monitored visually, nor done by a micromanipulator. It would be done "blindly" and monitored for detection of the appropriate biomarkers (color, antibiotic resistance etc) for weeks and weeks, the cells carefully cultured until the untransfected cells die off and the transfected ones survive, divide into colonies and eventually form whole tissues or organisms.
Another method, in addition to the biolistic method Saul Tanpepper described, is electroporation: mixing a suspension of cells with the DNA, then briefly running a current through it to permeabilize the cell membranes and allow the DNA to enter. I'm not sure if or how frequently this is used in industry GM, though.

OneWriter
04-08-2011, 09:02 PM
Saul, thanks for the clarification and correction. I didn't say it couldn't be done, I just said I didn't think it would be done in mass produced foods. The original question had led me into thinking that there was some common food that was causing cancer. That's why I found the scenario unlikely. I've worked in cancer epidemiology and it takes years to prove associations. If, on the other hand, you do have some special food that's been lab produced and fed to pigs, that's a different story. Sorry I misunderstood, Dave.

Oh, and I still stand by the allergic reactions being far more common than possible carcinogenic effects. Of course it's rare, just like with vaccines. But it happens. In fact, what you're saying about the proteins being broken up by the digestive system holds for the cancer scenario as well, if you assume it's the proteins causing cancer. The carcinogens (or allergens, for that matter) are molecules which can be absorbed through the skin, inhaled, etc.

I also stand by the virus (the one engineered as to deliver the new genetic material) being dormant in the food and eventually causing cancer in the organism that consumes the food. That is plausible UNLESS the food is cooked. Of course, being this a fictional and engineered virus, you could make it survive high cooking temperatures, too.

And finally, it IS done for therapeutic reasons. That's when it's called gene therapy.

veinglory
04-08-2011, 09:18 PM
Any cell you get out of a freezer will be dead. I think your description needs a lot of work and the process will almost certainly be done by a team, not one person.

OneWriter
04-08-2011, 09:20 PM
Any cell you get out of a freezer will be dead.

No, that's not true. Why do people freeze embryos? Of course it's not a household freezer they store them in!

veinglory
04-08-2011, 10:02 PM
Any cell in a petri dish in a regular freezer will be dead. They need to be in a matrix to prevent ice crystals from forming.

OneWriter
04-08-2011, 10:09 PM
Ah, yes... :)

JayMan
04-08-2011, 10:09 PM
Yeah, like stored at -80C in 15% glycerol for most bacterial cells.

Mmm... glycerol.

MAP
04-09-2011, 12:49 AM
I agree with Saul and Jayman. They have given good advice.



Ok this is where I might really start to annoy you.

I accept what you are saying, but I need the radiation for various reasons. This scientist goes mad trying to decipher the radiation bursts, eventually becoming convinced that they are some kind of language or code, but he is ignored, becomes more obsessed, turns to alcohol, breaks up with his wife, gets fired, and eventually becomes homeless.

A year or so later, his research assistant is packing away some stuff into storage and comes across his old research, and gives it another look, and begins to think there was something to it after all, and publishes a research paper. This kicks off a debate on whether these radiation bursts can be considered a "language", what level of consciousness plants have, about how "alive" plants are and whether it is ethical to eat them.

Because that plants were considered "alive", the vegetarians of the world (which are now a significant portion of the population after various food scares) decided only to eat GM food (as the seeds from which it is grown are already "dead"), despite the health risks, and the GM food companies' profits soar.

The previously homeless scientist is catapulted into international celebrity, but one day as he is looking at his old data, he notices something is off, that the data had been changed, and his investigation leads to the door of the GM companies.

Maybe it's a stretch!

Radiation comes from radioactive isotope (atoms usually with an unstable number of neutrons like 14C, 3H, etc.) not genes. So where are these plants picking up the radioactive isotopes? Granted that all living things have trace amounts of radioactive isotopes, but these plants would need more than normal amounts for the radiation levels to register above background.

From what I understand of your story, you need plants to be able to communicate in some way. You are proposing that they do so by controlled emission of radiation. I think this could work if there was a reason these plants are grown in a medium of radioactive isotopes. What is the professor researching? Could he be looking at a way to make plants that are more resistant to the effects of radiation? Then he might have a good reason to expose the plants to radioactive isotopes.

Someone else suggested bioluminescence which would work better IMO, but the scientists have to have a reason to introduce bioluminecence genes into the plants. Those genes aren't going to get there by themselves.

Of course this all depends on how realistic you want the story to be. Good luck with this.

JayMan
04-09-2011, 02:23 AM
MAP brings up good points as well.


Also, I just thought about how the cause could be something completely incidental or accidental.

For example, maybe there's a lab next door doing research with radioactive isotopes, and unbeknownst to them, one of their machines is defective and leaking radioactivity. Maybe when the researchers in the plant lab come to work everyday, they're tracking something in on their shoes or clothes, etc.

This is just a random example and there'd have to be more details to make it plausible (not to mention that I don't know the plot of your story), but I'm just trying to illustrate that it could be because of something unrelated to the plant lab altogether.

DrZoidberg
04-09-2011, 02:03 PM
I think the argument is how "conscious" plants are -- their being alive isn't arguable.

Yes, it is. What we define as life is pretty arbitrary and there are many different ways to define it. Does any inorganic matter that assume self replicating stable count as being alive? Are self replicating pyramidines alive? It's the exact same philosophical conundrum when we're talking about the definition of consciousness.

http://en.wikipedia.org/wiki/Life

But this is philosophy, not biology. The only reason this is as at all a topic of contention is because of religion. Religious definitions of life are very shallow and shouldn't really be applied to anything. But that doesn't prevent it from informing our laws and legal frameworks. Hence the issue, abiogenesis and all that. ...again... this is philosophy... not biology/science.

This is also maths. Do complex systems, (like entire nations) operate as a super organism? Should all of humanity count as one shared consciousness? Is the Internet a emergent consciousness in it's own right?

http://en.wikipedia.org/wiki/Complex_system

dgaughran, it's a great idea for a story and I'm also working on a similar manuscript. I'm in computer science by the way. I'm a systems theorist. I was lured away from a PH D by big bags of money, so my title isn't as impressive as the rest here. But I still know a thing or two about systems.

dgaughran
04-09-2011, 02:12 PM
I majored in Philosophy and did my Masters in Cognitive Science (specialising in cognitive neurobiology), so I love dealing with classifications of life and questions of consciousness.

I really enjoyed writing this story, and I would like to make it as scientifically accurate as possible, but I may have written myself into a corner here. One thing I have to be careful with is not writing all the "story" out in a quest for accuracy.

still working on it...

Saul Tanpepper
04-09-2011, 08:28 PM
I majored in Philosophy and did my Masters in Cognitive Science (specialising in cognitive neurobiology), so I love dealing with classifications of life and questions of consciousness.

I really enjoyed writing this story, and I would like to make it as scientifically accurate as possible, but I may have written myself into a corner here. One thing I have to be careful with is not writing all the "story" out in a quest for accuracy.

still working on it...

YES! While it's best to couch your fiction solidly in reality, at the end of the day, it's still a piece of fiction. As long as you make it believable (by making it reasonable based on current knowledge of the real world), you'll be fine.

dgaughran
04-12-2011, 10:20 PM
All this information has been great. And I am learning a bunch of stuff. I will probably never have to use this information again, but it's fascinating! Such is the life of a writer (and makes a change from trying to figure out what kind of handkerchief they used in Chile in 1806).

So I am still rewriting the bits where the genetic manipulation takes place (that bit is tough to get right and even tougher to make it exciting).

But I thought of a sidestep, that could allow me to keep radiation in the story (and it really is quite crucial).

Could this work (given that it's a science fiction short)?


It was confirmed: a short burst of radiation just at the moment the transfection process began. But this didn’t make any sense. Radiation comes from radioactive isotopes. And while all living things have trace amounts, these numbers were way off.

“I don’t know,” said Jim. “There’s something about not right about this. Maybe they’re doing some research on isotopes downstairs. Maybe some of their machines are defective and leaking radiation.”

“But then why did the Geiger counter only pick it up when we started the transfection?”

Jim shook his head. Neither of them had an answer, as there was no plausible scientific explanation for what they had witnessed. Dr. Peters made Jim swear to keep this quiet, then went for a short walk to clear his head. He kept murmuring the same thing over and over. “This is huge.”

dgaughran
04-13-2011, 02:40 PM
I have been looking at the biolistic gun, and that seems to be the way forward (even if it messes with my story a bit). Can I ask something though (which may save some hassle)? Is there no circumstances where inserting foreign DNA into plant cells would be called transfection (it's a good title for the story)? Also, is there no circumstances where a manual insertion of the DNA into one test cell would occur (even for the purposes of experimentation)?

Saul Tanpepper
04-13-2011, 08:12 PM
Got your PM, dgaughran, and I'll try and lend a hand here, though I may be off base with where I think you're going with all this.

In answer to your question above, yes, "transfection" is often used to describe the process of introducing DNA into plant cells (the scientific literature supports this), though transformation is the more "correct" term. Either term would be acceptable in a fictional work. When referring to viral-mediated methods, transduction and and transformation are the correct terms. Transfection would apply for biolostic, electroporative (using a buffered, salt-free bath and a strong electrical "jolt" to force the nucear material through the de-walled plant cell membrane), chemical (such as calcium phosphate/calcium chloride or DMSO fusion, PEG, etc), or other "mechanical" methods. You could even invent one and I think you'd be fine. Basically, the obstacle is that you're trying to pass a negatively charged water-loving polymer (DNA) through a lipophillic (fatty, or water-hating) barrier (the cell membrane). Not easy. So, scientists use chemicals, electrical current, cell-fusion or viral-mediated methods to do the job. Since plant cells tend to be larger than animal cells (and much, much larger than bacterial or yeast cells), it's feasible to use a glass micropipette, though this is very tedious and results in a single cell being modified. Most laboratory methods do these sorts of things on a scale of thousands to millions of cells and then screen for what they're looking for by eliminating what they don't want. And, at least with chemical and viral methods, the process is not instantaneous. With electroporation and biolostics, it is.

As far as the radiation burst, first, there'd have to be a reason the scientist is monitoring for such. Geiger counters are standard laboratory fare, but because of background radiation, they're not left on unless in specific use. DAn can be made radioactive by making it in vitro (in the test tube) or growing the cells from which it will be harvested in the presence of phosphorus-32 (P32). The scientists might do this to detect cells that have incorporated the DNA into their cells, making them radioactive, which would then provide a basis for the cells giving off counts. Because radiodecay occurs randomly, the counts are measured over time and averaged (often with a scintillation counter, a large machine that measures radiactive decay by coupling the process with luminescence). You could have your cells behave strangely by giving off non-random bursts of counts rather than a steady, though random, stream.

Given that this is a short and the reader essentially takes what little is presented as "factual" or based in true science or at least valid, I think you have a lot of leeway. You won't/shouldn't have to explain a lot of technical details. Just say the donor DNA is radioactive for purposes of following the process and leave it at that.

Hope this helps.

Saul

Saul Tanpepper
04-13-2011, 08:17 PM
“I don’t know,” said Jim. “There’s something about not right about this. Maybe they’re doing some research on isotopes downstairs. Maybe some of their machines are defective and leaking radiation.”


This is a highly unlikely scenario. Leaking radiation is serious business and no scientist would take such a possibility lightly. Also, most laboratories using isotopes use P32 or S35 or radioactive Iodine, which are relatively weak emitters (the radiation wouldn't pass through ceilings and walls and even several feet of air).

dgaughran
04-13-2011, 08:31 PM
Ok!

This is great stuff, exactly what I needed.

I think I know exactly how to approach it now, without having to rewrite the whole story, and as far as the Geiger counter goes, I already have that covered.

And with regard to the "highly unlikely scenario", your response just gave me the perfect way to rewrite it.

Thank you for taking the time to explain all of this to a science dunce, you have really helped me figure this out.

It's appreciated,

Dave

JayMan
04-13-2011, 08:48 PM
I have been looking at the biolistic gun, and that seems to be the way forward (even if it messes with my story a bit). Can I ask something though (which may save some hassle)? Is there no circumstances where inserting foreign DNA into plant cells would be called transfection (it's a good title for the story)? Also, is there no circumstances where a manual insertion of the DNA into one test cell would occur (even for the purposes of experimentation)?
I decided to do a bit of Googling, and it turns out that DNA microinjection has been used in plants. Everybody generally agrees that microinjection of DNA into plant cells is usually avoided because it's more difficult than other methods, like this site (http://www.aces.uiuc.edu/vista/html_pubs/irspsm91/transfor.html):
"DNA can also be microinjected into target plant cells using very thin glass needles in a method similar to that used with animals. Microinjection, however, has produced only a few transgenic plants. The technique is laborious, technically difficult, and limited to the number of cells actually injected. "

On the other hand, there's also this paper (http://jxb.oxfordjournals.org/content/46/9/1157.full.pdf) that says the researchers found a relatively efficient method of using microinjection to transform plant cells:
"20% of the targeted cells developed into microcalli and roughly 50% of these calli were stably transformed. Transient expression of the firefly luciferase gene {Luc) was nondestructively analysed 24 h after injection of pAMLuc. Approximately 50% of the injected cells that were alive at this time point expressed the Luc gene transiently. Apparently, stable integration of the injected genes occurred in essentially all transiently expressing cells that developed into clones"

Basically, 20% of the cells continued to divide and grew into calluses (mass of undifferentiated cells... see Wikipedia (http://en.wikipedia.org/wiki/Callus_%28cell_biology%29)), and 50% of those successfully took up the gene of interest, ie 10% efficiency.

By comparison, this particular facility (http://www.agron.iastate.edu/ptf/service/biolisticmaize.aspx)'s success rate using the gene gun/biolistic method to transform corn calluses is 15%. Incidentally, the site also gives a timeline of how long each stage takes. Inserting the DNA, waiting for calluses that have taken up the gene to grow, confirming that the surviving calluses indeed took up the gene, putting the calluses into soil and generating a plant, etc.

Going back to the paper, though, the researchers talk about a few disadvantages and advantages of using DNA microinjection on plant cells.
Disadvantages:
"(1) The plant cell wall is hard to penetrate with injection capillaries. (2) Plant cells are normally under turgor pressure. (3) A lytic compartment, the vacuole, generally makes up a large proportion of the plant cell volume. (4) Single plant cells do not adhere firmly enough to the supporting matrix to anchor them for microinjection."

Advantages
"(1) Only very small amounts of DNA are required for successful transformation. (2) DNA transfer is possible through cell walls into virtually any type of target cell. (3) Any biologically active substance can be coinjected together with DNA and the number of transferred molecules can be crudely controlled. (4) Individual target cells can be monitored during and after the DNA transfer. (5) Extremely high transformation efficiencies (percentage of stably transformed clones per cell surviving DNA delivery) can be achieved."
(I bolded number 3 because perhaps that can be the mechanism by which some cancer-causing stuff gets into the cells in your story?)

So, to answer question 1: I think transformation is the preferred term for plants, however, if you do a search for "plant transfection" you'll get a bunch of hits, so it's not as if using transfection to describe plant transformation is taboo. You'd be fine using "transfection" for your story.

And to answer question 2: It turns out that you can in fact insert DNA into single plant cells and monitor them, and that this has been done.

By the way, with any kind of cell transformation, you won't see anything immediately, but you monitor them as they grow over the next few days/weeks/months into calluses.





Also, just for fun (but not what you're looking for if you want to microinject DNA into the cells), I found these in my search of biolistics:
Picture of a gene gun being used on a plant:
http://www.genomicon.com/wp-content/uploads/2010/01/gene-gun.jpg

Video demonstrating how to use a gene gun (in the video, it's used on a mass of neural cells, I think):
http://www.wired.com/wiredscience/2008/06/video-how-to-us/

dgaughran
04-13-2011, 09:10 PM
Thanks JayMan,

That's the final pieces of the puzzle, sorted.

I appreciate your (and everyone else's) help on this thread.

Great stuff.

Dave

P.S. That gene gun looks like something my granny used to put prices on stuff in her shop.

MAP
04-13-2011, 09:20 PM
I agree with Saul.

The scientists need a reason to use the geiger counter. Also it would be really serious if the lab downstairs was leaking radiation, not something that the scientists would take lightly. It would be a serious health risk to everyone in the area.

Radiation is closely monitored in lab settings that use it, and there are radiation safety officers who stop by randomly to make sure the labs using radiation are using it responsibly.

I'm not saying contamination can't happen only that the proper authorities would have to be alerted so that the situation can be contained. No one wants to wants to work in an environment in which they could be randomly exposed to unknown amounts of radiation.

I like Saul's idea of giving the plant cells P32 labeled DNA to monitor the transformation. Then there is a reason for the plant cells to be radioactive and a reason for the scientists to monitor the cells with a geiger counter.

Then if the plants emitt the radiation in a seemingly controlled patterm rather than the normal random decay, that would be shocking.

Of course this all depends on how accurate you want the science to be. Give me a compelling story with interesting characters, and I don't care if the science is perfect. The story is the most important part. :)