Conferences
ELECTROPORATION OF MOUSE ZYGOTES WITH THE CRISPR/CAS9 SYSTEM FOR THE GENERATION OF GENETICALLY MODIFIED MICE
José María Fernández Toro [1], María Clemente Maltes [1], Laura Cernadas González [1], Juan De Dios Hourcade Bueno [1]
- Centro Nacional de Investigaciones Cardiovasculares, Unidad de Transgénesis, Madrid, España
XVII Congreso de la Sociedad para el estudio del animal de laboratorio (SECAL) Santiago de Compostela 2023
MICROINJECTION OF CRE RECOMBINASE PROTEIN FOR TRACING CELL LINEAGES DURING EMBRYONIC DEVELOPMENT
Juan De Dios Hourcade Bueno [1], Miquel Sendra Ortola [2], Óscar H. Ocaña Terraza [2], Jorge Nicolás Domínguez Macías [3], Miguel Torres Sánchez [2]
- Centro Nacional de Investigaciones Cardiovasculares, Unidad de Transgénesis, Madrid, España
- Centro Nacional de Investigaciones Cardiovasculares, Laboratorio Control genético del Desarrollo de Órganos y Regeneración, Madrid, España
- Universidad de Jaén, Dpto Biología, Jaén, España
XVI Congreso de la Sociedad para el estudio del animal de laboratorio (SECAL) Lleida 2021
Know more
The main competence of the CNIC Transgenesis Unit is the development and generation of genetically modified animal models, mainly murine.
The concept of transgenesis has been displaced by that of genetic modification, since thanks to the development of new tools in molecular biology and micromanipulation, the “modeling” of genetic material in vivo has been a new goal to achieve. But let's start at the beginning...
What is transgenesis?
Originally transgenesis is the set of procedures and techniques that allow exogenous genetic material to be inserted into a cell. The purpose of this modification is to try to study that DNA fragment in the general context of an organism.
Today, we no longer only study how a fragment of genetic material behaves; We have gone further thanks to the new tools that have been developed. We have the ability to directly modify the genetic material of an individual, selectively eliminating sequences (CrispR-Cas9) not only in space but in time (LoxP insertions through CrispR-Cas9).
Can we have whatever we want?
The answer is, we do Science, but not magic. In the Transgenesis Unit we carry out many techniques and we have good results, however, good results cannot always be achieved biologically.
Considering that we work with experimental animals, we have to comply with strict animal welfare requirements such as reduction, replacement and refinement of processes. In this way, our objective will be to make the process as efficient as possible.
In our hands, the different techniques we carry out have acceptable results.
Fig. 1 Percentage of positive offspring of the total born in microinjections on zygote
Fig. 2 Percentage of positive pups of the total born in GMOs Blastocyst/E8C
Fig. 3 Comparison of efficiencies between microinjection systems in zygotes and GMOs Blastocyst/E8C
And then... what can we do besides research with our model?
At the CNIC Transgenesis Unit we have a wide range of techniques that allow us to preserve the generated models with the security of any unforeseen event or inconvenience.
For this we have the following techniques:
Germplasm cryopreservation:
We define germplasm as the set of germ cells that carry genetic information and are capable of giving rise to a new generation. The male and female gametes of each species, that is, sperm and oocytes. Freezing this type of cell involves a set of precautions due to the fragility of these cells.
Given the extensive catalog of protocols we have to conserve our germplasm, at UT we have opted for these two protocols.
In the case of sperm freezing, we resort to the protocol established by the European Mutant Mouse Archive (EMMA). In this way, any user will be able to exchange material frozen at the CNIC with other researchers, since it has recognized standards.
In our case, we have 396 lines conserved at the CNIC by freezing mouse sperm. Our recovery rates through In Vitro Fertilization are acceptable:
Embryo cryopreservation:
Another possibility is freezing mouse embryos. The advantage it presents is that it has a complete genome and there are no intermediate steps for the recovery of the line. The genome can be kept conserved in homozygosity if the line and the user so require. On the contrary, it will depend on the reproductive capabilities of the line in question; the time to obtain a sufficient number of embryos could be delayed.
At the CNIC we have 18 lines preserved in the form of frozen embryos. From the UT we want to give a boost to this system, so we are already addressing new strategies to achieve it, such as reducing costs and increasing the efficiency of the technique.
Electroporation of mouse zygotes:
An alternative for the generation of genetically modified organism models is the electroporation of mouse cigarettes.
In this sense, during the 2021-2022 period, the CNIC transgenesis unit has launched the electroporation protocol to obtain animals genetically modified by this technique.
The efficiency of the technique ranges between 30-50% efficiency for the generation of KO animals.
