The impact of postpubertal overexpression of Makorin ring finger protein 3 (MKRN3) on arcuate kisspeptin neurons in a novel mouse model
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Abstract
BACKGROUND: Makorin ring finger protein 3 (MKRN3) is thought to function as a ‘brake’ in the initiation of puberty, inhibiting gonadotropin-releasing hormone secretion. Loss-of-function mutations in MKRN3 are the most common genetic cause of central precocious puberty. In female mice, Mkrn3 deficiency has been demonstrated to advance the onset of puberty and Mkrn3 overexpression results in delayed puberty. Mkrn3 has been demonstrated to inhibit activators of GnRH secretion, including neurokinin B and kisspeptin in the arcuate nucleus. OBJECTIVE: This project aims to investigate the effects of Mkrn3 overexpression in the arcuate nucleus of postpubertal female mice. This novel mouse model will be used specifically study the inhibitory effects of Mkrn3 on kisspeptin neurons and explore the developmental windows in which Mkrn3 acts on the hypothalamic-pituitary-gonadal axis.
METHODS: A novel adeno-associated virus (AAV) to selectively overexpress Mkrn3 in co-secreting kisspeptin, neurokinin B and dynorphin neurons will be introduced in the arcuate nucleus of postpubertal Kiss1-Cre female mice via bilateral stereotaxic injection. To generate the selective AAV-injected mouse model, this project first focused on generating the plasmid with the appropriate constructs for AAV packaging. To create AAV-DIO-Mkrn3, pAAV-DIO-mCherry was modified to include Mkrn3 and IRES, which were inserted via oligonucleotide synthesis. Plasmid prep kits were used to isolate and purify DNA. Plasmid constructs were validated using restriction enzyme digestion and Sanger sequencing. Prior to AAV packaging, DNA was isolated using EndoFree plasmid prep kits and validation was reperformed.
RESULTS: From the standard plasmid prep kit derived DNA, the digestion of pAAV-DIO-mCherry, EcoRI and BamHI created linearized fragments at approximately 6.0 Kb, as expected. pAAV-DIO-Mkrn3, EcoRI created fragments at 1.4 Kb and 7.0 Kb and SmaI created visible fragments at approximately 2.2 Kb, 2.6 Kb, and 3.6 Kb, as anticipated. Subsequently, the digestion with the EndoFree plasmid prep kit derived DNA with EcoRI and SmaI, pAAV-DIO-mCherry had more bands by gel electrophoresis than expected, suggesting remaining undigested DNA. For pAAV-DIO-Mkrn3, the digested fragments were the anticipated sizes. Sanger sequencing of pAAV-DIO-mCherry showed annealing and sequence verification of one out of two primers. All five primers for the pAAV-DIO-Mkrn3 plasmid annealed and verified the plasmid sequence.
CONCLUSION: This model of selective Mkrn3 overexpression will provide insights to the mechanism of action of Mkrn3 in the hypothalamus and its potential to induce hypogonadism. The results of this study that this novel pAAV-DIO-Mkrn3 plasmid has the appropriate coding sequence and is now ready to be packaged into an AAV for stereotaxic injection into the ARC of female postpubertal Kiss1-Cre mice.
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2024