Dissecting Estrogen Positive Feedback Mechanisms Using CRISPR-Cas9
Orange, Luke
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Orange, L. (2018). Dissecting Estrogen Positive Feedback Mechanisms Using CRISPR-Cas9 (Thesis, Bachelor of Biomedical Sciences with Honours). University of Otago. Retrieved from http://hdl.handle.net/10523/8584
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http://hdl.handle.net/10523/8584
Abstract:
Estrogen feedback mechanisms are arguably the most important component of mammalian fertility regulation. Estrogen positive feedback is mediated by a population of estrogen receptor alpha (ESR1)-expressing neuronal afferents to gonadotropin-releasing hormone neurons located in the rostral periventricular region of the third ventricle (RP3V). Due to limitations in present neuroscience techniques, the precise role of γ-amino butyric acid (GABA)-ergic ESR1-expressing neuronal afferents is currently uncertain. There is a need to develop new methodologies to manipulate ESR1 expression in GABAergic neurons of the RP3V. A novel application of the genetic CRISPR-Cas9 system may enable elucidation of the role of RP3V-GABA-ESR1 neurons in estrogen positive feedback. Firstly, this study aims to assess CRISPR-Cas9 as a tool for ESR1 knockdown in vivo, by injecting guide RNA (gRNA) and Cas9 in two separate adeno-associated viral vectors (AAV) to the arcuate nucleus (ARN). Secondly, this study aims to develop and characterise transgenic mice expressing Cas9 exclusively in GABAergic neurons for future CRISPR-Cas9 mediated experiments. I hypothesised that CRISPR-Cas9 effectively reduces the expression of ESR1 in the ARN following the injection of gRNA and Cas9, and that transgenic mice can selectively target Cas9 to GABAergic neurons. For the first aim, bilateral stereotaxic surgery was used to introduce AAVs encoding Cas9 and gRNA directed at ESR1 into the ARN of wildtype mice, which were analysed with immunocytochemistry for ESR1. For the second aim, a Cre-dependent Cas9 knock-in mouse line was crossed with a vesicular GABA transporter (VGAT)-Cre mouse line to generate mice with Cas9 and a green fluorescent protein (GFP) reporter selectively expressed in GABAergic neurons. The proportion of neurons that co-express ESR1 and GABA were then assessed using immunocytochemistry for ESR1 and GFP on transgenic Cas9-expressing tissue. Immunocytochemistry for ESR1 revealed that the RP3V, the ARN and the posterodorsal medial amygdala (MEApd) had robust populations of ESR1-expressing neurons. Mice injected with AAV-gRNA and AAV-Cas9 showed a qualitative reduction in ESR1-immunoreactive cell numbers at the injection site, providing evidence of CRISPR-Cas9 mediated knockdown in vivo. The transgenic mice generated in this study were found to express Cas9 and GFP exclusively in GABAergic neurons by comparison of GFP immunoreactivity with a well-characterised VGAT-Cre x GFP reporter mouse line. Further characterisation of these transgenic mice with immunocytochemistry for GFP and ESR1 revealed regions containing robust populations of GABA-ESR1 co-expressing neurons. These regions were identified as the RP3V, caudal medial preoptic nucleus (cMPN), ARN and MEApd. The proportion of GABAergic neurons that co-express ESR1 in male mice compared to female mice was greater in the AVPV (24.4% compared to 6.9%, p=0.0286, Mann-Whitney U test) and the PVpo (27.7% compared to 9.8%, p=0.0286).The localised reduction in ESR1-immunoreactive cell numbers in the AAV-injected brain regions suggests that CRISPR-Cas9 can be adapted for the knockdown of ESR1 in vivo. The transgenic mice expressing Cas9 in GABAergic neurons that were developed and characterised in this study will be used to assess CRISPR-Cas9 for brain regional and phenotype specific knockdown of ESR1. This may elucidate the role of precise populations of ESR1-expressing GABAergic afferents to GnRH neurons in estrogen positive feedback.
Date:
2018
Advisor:
Herbison, Allan
Degree Name:
Bachelor of Biomedical Sciences with Honours
Degree Discipline:
Department of Physiology
Publisher:
University of Otago
Keywords:
GnRH; Estrogen; Feedback; CRISPR; CRISPR-Cas9; GABA; Positive; Reproduction
Research Type:
Thesis
Languages:
English
Collections
- Physiology [154]
- Thesis - Honours [340]