We have generated a novel animal model, the Kiss1Cre::Cas9eGFP mouse, for implementation of CRISPR/Cas9 protocols for genome editing of selected genes only in KNDy neurons in vivo. In addition, we have designed CRISPR reagents for targeting genes expressed in adult KNDy neurons, namely those encoding kisspeptins, neurokinin B (NKB) and dynorphin, which were initially validated by in vitro assays.
Viral particles carrying the coding sequences for CRISPR reagents were produced by external resources. Stereotaxic injections of viral vectors were performed in male and female Kiss1Cre::Cas9eGFP mice to target genes expressed in KNDy neurons, as a way to interrogate the KNDy paradigm. Cellular validation of double-strand breaks by guided Cas9 nuclease was achieved by detection of on-target indel mutations in our loci of interest by fluorescence-activated cell sorting (FACS), allowing isolation of infected Kiss1 neurons, coupled to SANGER sequencing of complementary DNA from virus-infected cells in the Arc, as chromatogram sequence analysis evidenced the presence of reading frame shifts in the coding DNA sequence of targeted genes.
By applying the above tools, we have assessed the physiological relevance of NKB from KNDy neurons in the neural control of the reproductive axis. To this end, we have monitored estrous cyclicity from AAV-injected females, as proxy marker of the function of the hypothalamus-pituitary-gonadal axis. In addition, LH pulsatility was assessed in AAV-injected mice, under basal conditions and after gonadal hormone removal by gonadectomy (GNX). Also, GNX, AAV-injected females were subjected to tail skin temperature recordings, as a means to further assess putative NKB actions in thermal (de)-regulation in response to ovarian hormone deprivation.
We have also evaluated the physiological role of Dyn output from KNDy neurons in the neural regulation of the reproductive axis. To this end, we monitored estrous cyclicity and assessed LH pulsatility in AAV-injected females with gene edition of the Pdyn locus. In addition, we explored also the role of Dyn signal from KNDy neurons in the modulation of the preovulatory LH surge.
We have initiated protocols and experimental studies for gene editing of the Kiss1 locus in Arc KNDy neurons in vivo. Validation of the capacity to disrupt Kiss1 expression has been obtained by immunohistochemistry (as well as direct sequencing in FACS cells). Functional studies are currently in progress to define, in a physiological setting, the impact of Kiss1 invalidation in KNDy neurons on the effects of NKB and Dyn in the dynamic control of pulsatile LH secretion.
Finally, regarding dissemination activities, and despite the impact of COVID-19 pandemic, these have been carried out, as described in the original proposal. The results of the Action have been shared with the scientific community through multidisciplinary seminars, laboratory reports in monthly meetings in Tena-Sempere lab at IMIBIC-UCO, and in international scientific conferences (e.g. “Modern Approaches to Research in Physiology” organized by the American Society of Physiology), as well as in specialized workshops (e.g. Current Trends in Biomedicine Workshop – CRISPR/Cas: from Microbiology to Biomedicine at the International University of Andalusia), where the fellow presented the work “Challenging the KNDy hypothesis: in vivo evaluation of the role of Neurokinin B and Dynorphin in Kiss1 neurons using CRISPR-Cas9 gene editing”. Additionally, the fellow has actively participated in different outreach activities, including one seminar at IMIBIC-UCO, and two European Researcher's Night events at UCO (2020 & 2021). Anyhow, since the core of the experimental work of this Action is under final validation and confirmation with ongoing experiments, no papers directly emerging from this Action have been published as yet. We plan to complete data generation and analysis during the coming months, with a perspective of getting high quality publications within the next 1.5 years.
Note that no specific website has been developed for this project.
