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The Sci-Files is hosted by Mari Dowling and Dimitri Joseph. Together they highlight the importance of science, especially student research at Michigan State University.
Welcome to the Sci Files, an impact 89 FM series that explores student research here at Michigan State University. We're your cohosts, Mari Dowling
Dimitri Joseph:and Dimitri Joseph. Hi. Today, we have Veona Cutinho. Veona, could you introduce yourself and tell us what your major is?
Veona Cutinho:Hi. So I'm Veona, and I'm a junior in the department of microbiology and molecular genetics.
Veona Cutinho:That's my major as well. I'm majoring in genomics and molecular genetics. And this is my 4th semester here, and I love it.
Mari Dowling:And what kind of research do you do, and what lab are you in?
Veona Cutinho:I work with doctor Jose Sebeli in the cellular reprogramming laboratory. And simply put, we are just looking at improving the efficiency of cloning. And my research is looking at one specific protein and how that impacts the overall success rate of cloning.
Dimitri Joseph:Cloning. That used to be a hot topic. I I remember back in the early 2000 where cloning was the thing that we can replicate. I think it was a goat.
Veona Cutinho:Sheep.
Dimitri Joseph:A sheep. Yes. Alright. Cool. Could you just give us a foundational, a basic of what cloning
Veona Cutinho:is? Yeah. It's very interesting that you mentioned that, especially cloning was really hot topic back in nineties and 2000. And our biggest thing is, why is it not that impactful anymore? And that is why, because the efficiency of cloning is becoming very less.
Veona Cutinho:It's not entirely feasible to apply that science everywhere. And you asked me about the foundation of cloning, and it's theoretically, it's really simple. All you have to do is get the genetic material from a body cell, a somatic cell, get that, transfer it into an enucleated oocyte, the environment of an egg without a nucleus. You transfer it and then it grows, and that's your clone. Theoretically, really simple.
Veona Cutinho:Right? But it's not working out as much as we'd expect it to, and that is what we're looking at right now.
Dimitri Joseph:Your lab seems to focus on the genomics and cellular reprogramming. What is your approach to connecting these 2?
Veona Cutinho:So what we do and what exactly is the difference between a clone and a naturally developed embryo, for example, is the manipulated genetic material that we introduce. And that is what cellular reprogramming is, where we are manipulating the genetic information. And this doesn't necessarily imply that we're mutating genes, introducing new genes, functional. Although we can do that, what we are more interested in is the histone proteins. Now histone, they become a very important component of wrapping DNA.
Veona Cutinho:Right? They are responsible for the condensed structure of DNA. They hold it together. That is what histones do. They are an important structural and functional part of the DNA.
Veona Cutinho:The nucleosome is what we call it. Now when a cell is developing, and this is what makes a cell at its starting stage, the first single celled organism from a multicellular organism where each cell has a different function, What makes that difference is what we call cell differentiation, where you're acquiring histone modifications. And how this happens, we have epigenetic factors, we have methylations, acetylations, all that physically change the structure of the histone protein. This is very interesting because your experiences can actually change who you are, and this is how we can physically manifest it in our histones and the structure of our genes. So in our lab, we have noticed that one particular histone protein, we call it h three k nine m e three, 3.
Veona Cutinho:Histone 3 lies in 9 trimethylated. This protein is associated with heterochromatin. And how that works is the trimethylation helps the DNA bind to it more strongly. And as a result, because the DNA is so tightly coiled, it is very difficult for it to be replicated. And we don't want this to happen especially when a cell is developing.
Veona Cutinho:We want DNA replication to happen really quickly and in large amounts.
Mari Dowling:So you said that cloning was a hot topic at some point, but it's died down a little bit, and that's because there's some challenges in cloning. So what are some of the challenges that have come up that make cloning not as desirable to do?
Veona Cutinho:Now I explained the very theoretical concept of cloning. You have the genetic material of a somatic cell, and you transfer that into an enucleated oocyte. That is it. Theoretically simple. But at each stage, it's very complex.
Veona Cutinho:So with the somatic cell, it has histone modifications, epigenetic factors that make it difficult for DNA to replicate. At the same time, when you are removing the genetic material of that egg, you are hurting it as well because it's not natural. So there could be something very subtle, the changes that you introduce, that it could impact the way the WHO site may be able to function. It could also be where you're growing it, the culture media. That could also have an impact of how it's because you're making this all outside the natural process.
Veona Cutinho:We are working with zebrafish. I should have mentioned that. Zebrafish, so they breed outside. They breed externally. But there's something about what we do is also unnatural in a way because we're removing it out of its natural environment.
Veona Cutinho:We're growing it in the lab. There could be very little differences that could amount a lot in developing this clone that we would like. So those are all the factors that could impact cloning.
Mari Dowling:Okay. So you mentioned this h three k nine m e three histone that's associated with heterochromatin as being something that causes problems for cloning and DNA replication because of the tight coiling.
Veona Cutinho:Right.
Mari Dowling:So how exactly are you investigating that and the challenges that it presents for cloning?
Veona Cutinho:So our basic hypothesis is that because it is associated with heterochromatin and the DNA is tightly coiled, we don't want the cells with a lot of histone h three k nine m e three to be used as donor for our cells. So that is our basic hypothesis. We expect that the cells with lower expression might be ideal donors, and these cells will have a better chance of developing into a functioning clone.
Dimitri Joseph:That's very interesting. So from from what I'm understanding is that h three k nine m e three coils the DNA so tightly that genes aren't being expressed. And it seems that what you're saying is you're taking an approach to kind of reduce the amount of h three m e k nine, and therefore, the genes in this oocyte that can grow into any cell will be allowed to express its genes. So just to connect that to something that you mentioned earlier, you said that sometimes the environment can impact h three k nine m e three expression. But I'm curious to know, how are you manipulating this expression?
Dimitri Joseph:Are you doing something to their environment, or are you changing their genetics somehow?
Veona Cutinho:Right. So we, right now, are at a very preliminary stage of our investigation. Right now, we just want to know what cells have a higher expression of the histone that we are focusing on. And while we do that is we're using very basic immunocytochemistry. We are labeling these histones with fluorescent markers.
Veona Cutinho:And that is antibody specific binding. So we have specific antibodies that bind to this histone, and we label them with fluorescent markers, and we introduce that. And this helps us to actually visualize what cells have a higher expression of histone because they will glow brightly. And as a result, we can visualize in real life under a microscope. We can physically separate those cells, those genetic material from the ones we need, the lower expression, and that is what we're using.
Mari Dowling:And how does this tie into you using zebrafish in your research?
Veona Cutinho:Zebrafish as a model for studying human diseases, it's increasing right now, and there are a lot of reasons why. Now with the zebrafish, you might think we are so different, genetically different from a zebrafish. Right? We don't look alike. We are not the same size.
Veona Cutinho:We are not the same species. But why do we use this as a model? You'll be interested to find out that 70% of our genes are similar to a zebrafish. And in addition to that, zebrafish just like us, they have 2 eyes. They've got a mouth.
Veona Cutinho:They've got blood. They've got ears. They have got pancreas, intestines. They have all of these things that we already have. So if we want to study any diseases pertaining to these organs, we can theoretically study them in a zebrafish.
Veona Cutinho:So they make a really good model. In addition to that, a zebrafish can be bred very consistently every 10 days. And each time they breed, they can give us up to 50 to 300 x at a time. So this just makes it more simpler for you to study something. You make a mistake, well, it's not of a consequence.
Veona Cutinho:It does not have much repercussions because you have plenty more x to spare. Another very important and interesting fact about a zebrafish, it's its embryo. It's very transparent in a way. And now if I mentioned about fluorescent marking. Right?
Veona Cutinho:If I label specific tissues with the fluorescent marker into a zebrafish embryo, we can see it develop. We can see where it goes, all because it's transparent. Imagine this in a mouse model. You're not able to do that. You're not able to study each molecular mechanism under the microscope, but to the zebrafish, you can.
Veona Cutinho:And that's what makes it really interesting.
Mari Dowling:Very cool. Thank you for that explanation. So do these zebrafish also have these histone proteins as well?
Veona Cutinho:Yes. They do. And that is what I'm looking at. I'm looking at both zebrafish, and I'm also looking at bovine cells, bovine fibroblasts. And, again, DNA is not very different.
Veona Cutinho:We have so many genes that are similar. Our structures are also similar. Histones, DNA, it's just eukaryotic genetics that comes into play here. So a zebrafish does have a lot of h3knime3. And I am looking at isolating cells with higher expression so we can use the ones with lower expression as ideal donors.
Dimitri Joseph:And how is this connected to the cloning, or have you not reached that far in your experiments to to see if you can develop a clone.
Veona Cutinho:Right. So it takes us really long time to clone. We clone, let's say, 3 times each week, every week, and we get maybe a successful clone in 3 months. That is how low the efficiency is right now. And my project, we are looking at low expression of h three ks, and we're trying to isolate it still.
Veona Cutinho:We have not yet been able to remove those cells with low expression and form a successful clone as of this. But our hypothesis is that because it has low expression of the histone, the DNA is more accessible to be replicated. And because of that, the clone has a higher chance of developing and surviving.
Dimitri Joseph:Just to clarify the process of your experiments, from what I'm understanding is that the zebrafish are able to, release their eggs, and you're able to then label their transparent or their translucent eggs with fluorescence marking to identify whether these cells have the histone. And you're separating those with high histone expression versus those with low histone expression. And then what exactly are the steps you're taking from there?
Veona Cutinho:So starting from the beginning, we need the somatic cells. That is our entire thing about cloning. It's cloning in the scientific terms is called somatic cell nuclear transfer. So we will be using the body cells of zebrafish. What we like to use is the tail, zebrafish tails, and this is because you know how we have cells but not all of our cells are able to replicate.
Veona Cutinho:Only some have that capacity, something that we call potency, multipotency, pluripotency. The tails of the zebrafish still have that. So that is the somatic cell that we are interested in. That will be our donors. As for the zebrafish eggs, they are going to be our recipient.
Veona Cutinho:So we take the eggs and the eggs are just haploid. They have half the chromosomal makeup. And that's all we need. We are going to remove all of it actually, keeping the eggs intact. We're removing the genetic material and we call it enucleation.
Veona Cutinho:And once we have the environment and here we have our somatic cells, we transfer the genetic material into that. Now before we choose what kind of somatic cells are we using even within our population of tails, we want the cells with the lower expression of histone protein. So what I'm doing is when I get my population of zebrafish tail cells, I am characterizing the h three k nine expression by labeling them with the markers. And the ones that glow less tell me that they have lower expression, and I'm separating those. And that's what I give to the people that are actually involved in cloning and transferring the genetic material.
Dimitri Joseph:Okay. Cool. Cool. So it seems that you're not cloning an entire animal. You're cloning specific cells or, particularly, it seems that you're cloning the genetic information from the tail.
Veona Cutinho:Right. And that is what we call cellular reprogramming. When I put this genetic information into an oocyte, it doesn't necessarily just give me all tail cells. It has the potential to develop into a fully functioning organism. The thing that you're also mentioning is something what stem cells do, for example.
Veona Cutinho:Stem cells and organoids, they grow cells, particular tissues, hearts, lungs, whatever you're interested in. And that's what makes a difference, the amount of differentiation that the donor cell has reached, and that is with the histone markers, the gene regulated gene expression, all of that are different in every single cell of our body. So whatever you want to focus and how much you want to revert the time cycle back. How much you want to induce pluripotency, for example, tells you the potential of that organism to grow into what you want. That is gene manipulation, cellular reprogramming.
Dimitri Joseph:It seems that these techniques can take a lot of different directions, and there can be a lot of fine tuning at several points. Has there been any findings that you've seen? I I know you mentioned that it's been challenging to successfully generate clones, but have you noticed any other changes in the cells that that have developed from your experiments?
Veona Cutinho:From research from other labs, published articles that I have been reading, there are many papers that do completely agree with the expression of histone relating to success of cloning. That is the reason we want to try that because it backed up by very, very strong scientific evidence. One more thing is that the cells that have reached a higher level of differentiation, cells that are more specific within their function. That is how, you know, how differentiated, how much they've progressed in differentiation. So the cells that are more specific in their function, they gain more methylation patterns.
Veona Cutinho:And the entire idea of methylation is very interesting because cancer, for example, it's also related to the number of methylations that you gain in your course of life. That can tell you about the possibility that you have for the development of cancer. So it tells a lot about the differentiation of cells is my point. And I hope that answers your question in terms of what cells we use based on the histone expression.
Dimitri Joseph:Okay. So just a quick clarification. What I'm understanding here is that hypomethylation or few methylated histone sites are more closer to a blank state. And as cells begin to become more specific, like a specific neuronal cell or a specific tissue in the pancreas, they gain these methylation sites, and that kind of sets them on a path to become that specific cell. So the focus of your project is to just extract more cells that have this blank state or are closer to the blank state, and you guide them.
Dimitri Joseph:Do do you guide them towards developing in any particular manner?
Veona Cutinho:That that is a very neat way of putting what I'm doing, and thank you for that because you've explained it really well. As far as guiding them into developing, that is a really cool concept, but it's not something we have considered yet simply because it requires more manipulations, more things that we do not know. Right now, our focus is, do these even work? Can we even improve cloning with having less histone expression? Does that work better?
Veona Cutinho:That is our primary focus right now. But you simply saying that a cell in its progression of differentiation acquires these histone patterns. That is a very interesting way of putting it because that is exactly right. It is shifting the way we approach medicine, for example, with understanding, let's say, a pharmaceutics, understanding the effectiveness of a drug. You don't have to necessarily have human subjects, animal subjects even anymore.
Veona Cutinho:You can test them out on these clones that you're making because it's going to remove some of the ethical restraints that you have, and you can make so many of them in your lab. You can study them in greater replications, greater trials. It's changing the way we approach medicine. We can transform lives with just this basic simple science of cloning and ancillary programming.
Mari Dowling:You've given us a really cool understanding of your research and an explanation of some of the challenges to cloning and how you're trying to overcome those. But what are some of the directions that you're hoping to take this in the future?
Veona Cutinho:With improving cloning, with improving the efficiency of our clones actually surviving and developing into a fully functioning organism. I think the direction that we are headed at is how can these clones be modeled for human diseases, for example. If we understand gene expression, gene mutations in certain diseases, we can observe the development of these diseases and the clones that we make because we have more control because we made those clones. We can create knockouts and knock ins and decide how basically track the development. Not even just disease, anything else, life processes.
Veona Cutinho:We have so much to learn. And all of these things we can understand if we can really track from the pre embryonic stage, basically, the conception of life to where it ends. That is the direction we're headed.
Dimitri Joseph:Thank you, Vianna. That that was a lovely explanation. And then yeah. Thank you.
Mari Dowling:Very interesting. Thank you.
Veona Cutinho:It's really exciting. And thank you for having me.