Search for tag: "genetically"

Interviewer: A group of concerned scientists have raised the question: Should we be able to genetically engineer people? We'll talk about that next on The Scope.

Announcer: Examining the latest research and telling you about the latest breakthroughs. The Science and Research Show is on The Scope.

Interviewer: Scientists have called for a moratorium on genetically engineering human embryos until the safety and ethics have been worked out. I'm talking with Dr. Dana Carroll, a distinguished professor of biochemistry at the University of Utah. He and his colleagues have made their voices heard in a commentary that was just published in the journal Science. Dr. Carroll, why is this topic coming under scrutiny now?

Dr. Carroll: The CRISPR technology is very, very simple in its design and application and it's turned out to be extremely effective. More and more laboratories are now using this technology for a variety of purposes for research, even for agriculture. Because it's so easy and so many people are using it, the issues, which were sort of in the background for a number of years, have now moved to the front and amongst them is this issue of potentially engineering the human germ line.

Interviewer: One of the points under discussion right now is germ line modification. Can you explain what the germ line is and what that means?
Dr. Carroll: The thing that makes the germ line changes more serious than things you might do to somatic cells is that the changes are permanent and heritable. So if mom and dad are carrying mutations that could cause disease in their offspring, the idea is that the genome engineering would allow the correction back to what we would call the normal DNA sequence of that potential disease gene.

Interviewer: Yes, well exactly, and that's why this technology is so exciting is that we may be able to eliminate inherited diseases within a family. Correct?

Dr. Carroll: That's right.

Interviewer: So you told us about the promise of this technology, but what are people worried about? Why is this under discussion right now?

Dr. Carroll: There are a few things to be concerned about. One, from a scientific perspective is that, although I said we can make very specific targeted changes in the genome, the reagents that we are using, these nucleases, are not 100.00% specific. There may be other changes that occur, not just at the gene we're trying to target, but elsewhere in the genome. So we need to perfect the technology to a point where we can be quite confident that we aren't making things worse at the same time we're making things better.
There is some concern that because the technology is easy, that it will begin to be used for what you might call trivial purposes, like a couple trying to endow their children with what they consider favorable characteristics: blonde hair, blue eyes, athletic prowess.

Interviewer: How close are scientists to being able to do these types of experiments?

Dr. Carroll: In humans . . .

Interviewer: In humans, yes.

Dr. Carroll: This sort of manipulation has been done in a lot of different organisms, including mice and rats and pigs and cows and even monkeys. So the technology is ready to be applied to humans.

Interviewer: I think one thing that is interesting is that there is not even consensus among informed scientists. I think you're in the camp that can see a future where this technology is permissible, under the right control, but there are others who think that we shouldn't go forward with this technology at all.

Dr. Carroll: The commentary that was published in Nature basically said, the authors stated that they could never envision a situation where germ line genome modifications should be allowed or would be necessary. I think that's a little bit short-sighted. Like I said, I don't know what the best candidate genetic diseases are for this kind of genome engineering, but I'd love to hear from physicians and other biologists who are sort of closer to the human disease arena to see what they have to say about it.
The other thing is that we don't want to get into a situation where the public in general, individuals around the country and around the world, think that scientists are just trying to make monsters. We want to engage the public to see what the public thinks about it. If it turns out that there's, at this stage, close to zero public acceptance of going forward, then I think scientists should be governed by that.

Announcer: Interesting, informative, and all in the name of better health. This is The Scope Health Sciences radio.

Interviewer: A new practical method that will boost the productivity of genetic engineering labs, coming up next on The Scope.

Woman: Examining the latest research and telling you about the latest breakthroughs, The Science and Research Show is on The Scope.

Interviewer: Using principles borrowed from population biology, Dr. Luca Brunelli, an assistant professor in pediatrics at the University of Utah, has developed a rapid, inexpensive, one step method for isolating rare DNA recombinants. Dr. Brunelli, what motivated you to come up with this method?

Dr. Luca Brunelli: Our interest in these techniques really stems from our research focusing both on human genetics and mouse genetics and our interest in trying to use mice to elucidate key mechanisms of human disease.

Interviewer: This addresses an intermediate step in genetic engineering.

Dr. Luca Brunelli: Yes. We have described this technique that we call F.P.E., founder principle-driven enrichment, in E. coli in a technique that is commonly called recombineering or recombination mediated genetic engineering, which is an intermediate step that allows, for example, among many other things to produce the targeting vectors that are required to produce genetically modified mice.

Interviewer: What were some of the problems that you were trying to address?

Dr. Luca Brunelli: If one thinks of genetic combination, the general idea is that these events are rare. The key problem is the general conception that it's really impossible to isolate that one event out of a hundred thousand unless you link a marker to that event because of how rare it is.
In reality, we have found that by applying principles from population genetics, specifically the founder principle, you are able to very effectively, rapidly, cheaply, and simply isolate these rare variants without using markers. What population genetics and the founder principle tell us is that when you establish founder populations, meaning very small populations, from large populations some of these alleles, some of the genetic background that existed in the original population, either is wiped out or is enriched, is fixed in this new population.
As long as you have a screening technique, which in our case was P.C.R., to be able to detect whether in liquid cultures your event is present or not, what happens when you decrease the size of that culture to, let's say, 5000 cells is that in some of those liquid cultures you will still have one recombinant, but you've actually enriched for your recombinant for your rare event. So, going through two or three enrichment cycles such as this that I've just described you can actually isolate your variant, bring it to a frequency that is 1 in 20, 1 in 50, such that you can easily plate your liquid cultures on an agar plate and isolate the clone easily.

Interviewer: And just pick individual ones.

Dr. Luca Brunelli: Yeah, just picking the ones.

Interviewer: Basically, you're taking your original pool that has your recombinant in it, then you're diluting it out.

Dr. Luca Brunelli: How many days it's going to take to isolate your rare variant and how many P.C.R.s you're going to want to do really depends on your preference. In our manuscript we've actually included a mathematical model with a web available calculator that allows a researcher to sort of import their desired level of effort, if you will, and days that they want to do the isolation in, and be able to reselect the preferred conditions. The calculator will advise them on what kind of enrichment, what kind of dilution, to use under those circumstances.

Interviewer: Right. How much quicker is it?

Dr. Luca Brunelli: To generate something seamless you need to introduce both selection markers and counter selection markers, so at a minimum ten days. With our approach that takes us down to two to five days at the most.

Interviewer: Oh wow. And I imagine it's cheaper.

Dr. Luca Brunelli: Cheaper also, yes, because you save reagents, antibiotics, a number of...

Interviewer: I'm struck by this as really a very straightforward technique.

Dr. Luca Brunelli: It is, and what's striking in a way is that it had not been described before.

Interviewer: Exactly.

Dr. Luca Brunelli: I have to say that once we were developing this method and we thought to include the full mathematical model of this, we went to a mathematician to help us out with that. The first comment from the mathematician was well, this is so easy, why has nobody else thought about this before.

Interviewer: Yeah.

Dr. Luca Brunelli: It was extremely easy for the mathematician to understand that if you decrease the number of cells below a number that breaks the symmetry of how rare and more common variants are present it's pretty clear that you can enrich for those. Interviewer: What's the advantage over other selection techniques like zinc fingers or crispers?

Dr. Luca Brunelli: With zinc fingers and crispers people have used a different approach which is to create double stranded breaks in the DNA so the DNA would effectively make these changes at a much higher frequency. But the double stranded break, it's easy to understand, also creates, if you will, stress on the DNA and so has a chance to produce what people call off target effects . . .

Interviewer: I see.

Dr. Luca Brunelli: . . . making changes and modifications also in regions of the DNA where you wouldn't want to have those changes.

Interviewer: What are the applications for this method?

Dr. Luca Brunelli: One of the direct implications would be to ask the question whether what we have done in E. coli can also be replicated in mouse embryonic stem cells.
Other possible applications that we see that are really exciting are in recombineering pipelines with the idea of studying gene function, large scale modifications also, for example, to produce biofuels, so bacteria acquire certain characteristics that will enable them to perform better at certain functions like to do production of a biofuel or cleaning water or whatever you might think of.
Although it's outside of the medical field, we're certainly interested in some of those applications, and we are considering possibly collaborating with some investigators who work in those fields.

Woman: Interesting, informative, and all in the name of better health. This is The Scope Health Sciences Radio.