Science Insider: Isolation of Rare Recombinants Made Cheap and EasyJul 12, 2014
Using principles borrowed from population biology, Dr. Luca Brunelli, an associate professor in pediatrics at the University of Utah, has developed a rapid and inexpensive one-step method for isolating rare recombinants without using selectable markers. The work was published online in Nature Methods on July 13. Brunelli describes the method, its advantages, and its wide applicability, from creating animal models to biofuels.
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.
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.
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.