MICROINYECCIÓN DE LA PROTEÍNA CRE RECOMBINASA PARA EL TRAZADO DE LINAJES CELULARES DURANTE EL DESARROLLO EMBRIONARIO
Juan De Dios Hourcade Bueno , Miquel Sendra Ortola , Óscar H. Ocaña Terraza , Jorge Nicolás Domínguez Macías , Miguel Torres Sánchez 
- 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
The CNIC's Transgenesis Unit has as its main competence the development and generation of genetically modified animal models, mainly murines.
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 represented a new goal to be achieved. But let's start at the beginning...
What is transgenesis?
Originally transgenesis is the set of procedures and techniques that allow the insertion of exogenous genetic material into a cell. The purpose of this modification is to try to study that piece of DNA in the general context of an organism.
Today, we no longer just study how a piece 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 also in time (LoxP insertions through CrispR-Cas9)
All is possible?
The answer is, we do Science, but not magic. In the Transgenesis Unit we carry out many techniques and have good results, however, not always biologically good results can be achieved.
Considering that we work with experimental animals, we have to comply with strict requirements in animal welfare such as reduction, replacement and refinement of processes. In this way, our goal will be to make the process as efficient as possible.
In our hands, the different techniques that 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 offspring of the total born in GMOs Blastocyst / E8C
Fig. 3 Comparison of efficiencies between microinjection systems in zygotes and Blastocyst / E8C GMOs
And then...what can we do besides investigate with our model?
In the CNIC's Transgenesis Unit we have a wide range of techniques that allow us to preserve the models generated with the security of any unforeseen or inconvenience.
For this we have the following techniques:
We define germplasm as the set of germ cells that carry genetic information and are capable of giving rise to a new generation. The gametes, male and female of each species, that is, sperm and oocytes. The freezing of this cell type involves a series of precautions due to the fragility of these cells.
Given the wide catalog of protocols that we have in order to conserve our germplasm, at the UT we have opted for these two protocols.
In the case of sperm freezing, we used the protocol established by the European Mutant Mouse Archive (EMMA). In this way, any user will be able to exchange frozen material with other researchers at the CNIC, since it has recognized standards.
In our case, we have 396 lines preserved in the CNIC by freezing mouse sperm. Our recovery rates through In Vitro Fertilization are acceptable:
Another possibility is the freezing of mouse embryos. The advantage it presents is having a complete genome and there are no intermediate steps for the recovery of the line. The homozygous genome can be conserved if the line and the user require it. On the contrary, it will depend on the reproductive capacities of the line in question, the time to obtain a sufficient number of embryos could be delayed.
At the CNIC we have conserved 18 lines in the form of frozen embryos. From the UT we want to give a boost to this system for this we are already addressing new strategies to achieve it, such as reducing costs and increasing the efficiency of the technique.