We recently had the opportunity to interview Prof. Dr. Joaquín Gadea and Ms. Celia Piñeiro-Silva about their paper “Production of Genetically Modified Porcine Embryos via Lipofection of Zona-Pellucida-Intact Oocytes Using the CRISPR/Cas9 System”, which was published in Animals (ISSN: 2076-2615) in 2023 and has received a significant amount of positive feedback from readers.

The following is a brief interview with Prof. Dr. Joaquín Gadea and Ms. Celia Piñeiro-Silva:

1. Can you tell us a little bit about yourself and your current research?
Prof. Dr. Joaquín Gadea: I studied veterinary medicine and graduated with a Ph.D. in animal reproduction. I worked for some time as a veterinary inspector and ultimately returned to the university, which was 30 years ago. I have been working in the Department of Physiology and my teaching activities are related to veterinary physiology and reproductive biology. I am teaching master's and doctorate students. My research activity is mainly focused on animal reproduction, but I also research human reproduction. The main topics I have been working on are sperm functionality and the cryopreservation of sperm, and I also conduct research on genetically modified pigs, which has been my main research topic for the last few years. I am part of the research group “Physiology of Reproduction” which consists of 8 permanent professors and 35 people right now. We are working on different topics related to reproductive biology and our main research species is pig, but we also have some other experimental models such as bull, cow, human, mice, and so on.
Ms. Celia Piñeiro-Silva: I have a masters in the reproduction of mammals and now I’m doing my Ph.D. I started my research on the genetic modification of porcine embryos to generate mutant pigs with my master’s thesis and now I’m continuing this work with the Ph.D. This article is part of my thesis work.

2. What inspired you to conduct this research?
We have been working on this topic of gene editing in pigs since 2015, more or less. The first gene-edited pigs were studied one year beforehand, and later the same year we started working in collaboration with Dr. John Parrington in Oxford. Because we had previously been working together in sperm-mediated gene transfer methodology, we had some experience generating transgenic animals, and when the CRISPR/Cas technology worked with pigs, we started our research very quickly. In the beginning, it was very difficult, because all of it was new to us and because some of the work was done in Oxford, and some of it happened in Murcia. It was very difficult, but we learned a lot. In 2019, we obtained the first gene-edited TPC2KO pigs by microinjection. Later, we started to optimize alternatives to the microinjection. One alternative is electroporation and another is lipofection. This article in Animals was our first related to lipofection and the objective was to optimize a new system with lower cost in terms of equipment and complexity. Previously, a Japanese group led by Prof. Tanihara had achieved lipofection, but they had to remove the Zona Pellucida, and this is a problem, because when you remove the Zona Pellucida, on the one hand, you facilitate the interrelationship between the lipofectamine and plasmatic membrane, and on the other hand you encounter some problems with embryo development and management later on. Our article is the first publication, to the best of our knowledge, to show lipofection with the Zona Pellucida intact. In the article, the rate of mutation was low, and we have since improved this technology with different systems and published a new paper last year related to this. It's a very, very interesting technology to develop, but it's in the beginning stages of development.

3. What do you think we can learn from your article?
I think we can learn that it is possible to generate genetically modified embryos more efficiently instead of through microinjection because microinjection is a very complex technique and you need all the equipment for it. Lipofection is very easy and any researcher can do it without learning or practicing a lot.

4. What do you think made the academic community respond so well to your research?
First of all, gene editing in pigs is a bit new for the academic community. A small number of groups in the world are working with this technology. Not only lipofection but also electroporation, for instance, is also very new. Usually, research groups work with the first technology, microinjection, and for that, you need very expensive laboratory equipment. Personally, I think that, at the moment, pigs as a model are not very well-studied. The second reason for this is that people who use pigs as models have no access to large and expensive equipment to carry out microinjection. We are probably pioneers in this research, and in several years this study will be more and more recognized than it is now. Also, the pig industries need to know that we can produce animals resistant to various diseases. When it was accepted, companies were even more interested in the technology. For instance, in commercial companies or different laboratories, researchers know a lot about lab animals, and mice, but they do not have enough knowledge of pigs. Pigs can be a very nice lab model for human diseases or xenotransplantation.

5. What challenges did you encounter while writing this commentary, and how did you overcome them?
We had a lot of them. The most difficult things are to get money and to keep staff. We have some grants for only a short period of time, but you have to keep staff, especially talented people, for a long period of research. We started this research several years ago and now we are starting to get good results, so it takes a lot of time. On the other hand, this is a very multidisciplinary activity because we have to manage people who are very good in different fields of research—in surgery, embryo culture, molecular biology, managing pregnancies, phenotyping, etc. You have to participate in a very large network to be able to improve, and this is not easy. Additionally, it's difficult when you are the first person in the field to discover something, it is very hard because you have no reference at all. You have to optimize everything.

6. What do you think are the future directions for your research?
We are doing many things at once. Lipofection is one of the techniques we use, and we have to optimize the electroporation. We are now writing a grant in which we try to optimize every important phase in this technique. One factor is to obtain the best quality embryo. We have to develop better techniques to obtain oocytes of the best quality. At the same time, we have optimized in vitro fertilization and in vitro embryo culture. Celia is working right now on treatments for achieving better epigenetic status of the oocyte. We are working on four different techniques: microinjection, electroporation, and lipofection, but also on cloning (somatic cell nuclear transfer). So, all these techniques have to be improved and adapted to the pig model, which is different from the mouse model. We have a lot to improve. Regarding the CRISPR/Cas9 system, we were using the 1st generation. Now, we are using the 2nd generation, and base editors, which also require a lot of work for optimization. We have to improve the embryo transfer and the number of pigs obtained from the embryos, so there is a large space to improve. And finally, the application of the animals. At the moment, we have only two life models, and with every life model, you have to phenotype for a long period to know exactly what the characteristics of this animal are. It's a large area for research. We have work for the next two or three generations.

7. Why did you choose Animals as the best platform for your work?
We have no issues with Animals. I have published several papers there and I didn't encounter problems with the Animals journal. From a reviewer and an editor's perspective, the Animals journal has developed and is getting better over the years. Also, I am always looking for the right audience for our articles, and in this case, Animals was the best choice.

8. What do you feel is your goal as a researcher?
Now, the objective is to be able to provide good quality research so that this research can solve problems. We spend a lot of time, a lot of effort, and a lot of resources to carry out the best science, but it's not only for publishing. It's important, but it's only one part of the science. The other very important side is being able to transfer science to offer solutions to society, to full application. In this field, this is very interesting, because it will be applied very quickly. The other one is to do my best as a teacher. To participate in the growth of new researchers, this my main objective. I am in the maturity phase of my professional life and I think the best I can do is to participate in the formation of the best researchers.

9. What advice would you give to young scholars seeking to get into academia or publish their work?
Our advice is to learn how to write, how to give answers to the reviewers and publish step by step, according to the circumstances and situation of your lab. Being clever, learning everything you can and from every person you meet. Networking is very important. Also, traveling, and trying different laboratories; it's crucial and beneficial to going out of your comfort zone to explore new things.