NEURORIGINS started with the generation of a library of genetic enhancers that, based on previous evidence, are active in the cerebral cortex during embryonic stages. Previous work in transgenic lines encoding a reporter under the regulation of different enhancers suggested these enhancers might be expressed only in subsets of neural progenitors, which makes them very interesting candidates to mark different progenitor subpopulations. The library of genetic enhancers was tested by in utero electroporation (IUE) of the brain in E13 and E14 mouse embryos. In these experiments, the activity of the enhancers was much broader that expected, with most of enhancers being expressed in most neural progenitors in the cerebral cortex.
The lack of specific patterns of activity for at least some of these enhancers suggested that IUE was a poor system to screen for enhancers with specific activity in subsets of neural progenitors. The fact that with the IUE, multiple plasmids enter the cell and remain as episomes, increases the number of copies of the same enhancer in each cell. This is probably the reason behind the broader patterns observed by IUE: while in transgenic lines there is only one copy inserted into the genomic DNA, the multiple copies of the same enhancer probably lowers the threshold for a cell to exhibit enhacer activity. In this way, those cells not exhibiting enhancer activity with one copy, may exhibit activity when containing multiple copies. For this reason, I decided to use the LiON system, in which the expression of a reporter only occurs when integrated into the genomic DNA via a Piggybac transposon. This eliminates the expression coming from episomes. To test the library of enhancers with this new system, I activated a contingency plan to generate again the same library of enhancers encoded in LiON plasmids.
After the generation of the library in LiON plasmids, I repeated the screening for activity of the enhancers in specific neural progenitors. This time I found that for all the enhancers tested, their activity was variable across different experiments. Because the insertion site of the Piggybac transposon is random, the same enhancer may exhibit a different activity depending on the locus of insertion. This led me to activate a second contingency plan, seeking to generate transgenic lines to test the specific expression of each enhancer in the locus R26. For that I implemented iGonad, a novel approach for transgenesis based on the electroporation of the oviduct of a pregnant female mouse. I used this method to generate embryos in which a first enhancer was controlling the expression of the reporter gene tdTomato, with the construct inserted in a specific location in R26 via CRISPR.
