Localization Of Long Form Leptin Receptor MRNA In The Pig Brain Studied By In Situ Hybridization

1 Cloning of the pig long form leptin receptor (OB-Rb) targeted gene and development of the riboprobeAccording to the pig gene sequence published on Genebank, a pair of primers were designed and synthesized. Total RNA was isolated from the thypothalamus of a Landrace pig by Trizole method. 343 bp cDNA fragment was generated by reverse transcription-polymerase chain reaction (RT-PCR) and cloned into pGEM-T Easy Vector. The recombinant plasmid was identified by several kinds of specific methods and comfirmed that the cDNA fragment sequence is full consistent with the published pig OB-Rb gene sequence on Genebank. After the recombinant plasmid was linearized by Apa I and Sac I endonucleases, the antisense and sense riboprobes to the long form leptin receptor were generated by in vitro transcription. The dot blot method was used to estimate the concentration of the riboprobes, and the detection result showed that the concentration of the riboprobes were 100 ng/ul, meeting the requirement of the hybridization.2 Localization of OB-Rb mRNA in the hypothalamus in the pig studied by in situ hybridizationLocalization of OB-Rb mRNA in hypothalamus in 5 female Landrace pigs (2 to 3 months old) were investigated by in situ hybridization. OB-Rb mRNA was located in the arcute nucleus, ventromedial nucleus, dorsomedial nucleus, paraventricular nucleus, periventricular nucleus and lateral region of the hypothalamus in the pig.3 Localization of OB-Rb mRNA in the cerebral cortex, hippocampal formation, piriform lobe and amygdala in the pig studied by in situ hybridizationLocalization of OB-Rb mRNA in the cerebral cortex, hippocampal formation,piriform lobe and amygdala in 10 Landrace pigs (2 to 3 months bid) were studied by in situ hybridization. In the cerebral cortex and piriform lobe, OB-Rb mRNA were observed primarily in the II-VI layers and with a few in the I layer, Within the hippocampal formation, OB-Rb mRNA was found mainly in the stratum pyramidale of the hippocampus and the stratum granulosum of the dentate gyrus, with the minority in the stratum oriens of the hippocampus and the polymorphic layer of the dentate gyrus, and with a few in the stratum radiatum and stratum lacunosum of the hippocampus. In the amygdala, OB-Rb mRNA was found mainly in the corticomedial nucleus and with the minority in the basolateral nucleus and central nucleus.4 Localization of OB-Rb mRNA in the thalamus, cerebellum and medulla oblongata in the pig studied by in situ hybridizationLocalization of OB-Rb mRNA in thalamus, cerebellum and medulla oblongata in Landrace pigs (n = 5)(2to3 months old ) were studied by in situ hybridization. The results showed that OB-Rb mRNA was located in the thalamus, cerebellum and medulla oblongata in Landrace pigs. In the thalamus, OB-Rb mRNA were observed almost in all nuclei with most in the midline nuclear group, thalamic parayentricular nucleus and anterior nuclear group. Within the cerebellum, OB-Rb mRNA were found in all three layers, with the majority in the Purkinjie cell and granular layers. In the medulla oblongata, OB-Rb mRNA were observed in the inferior olivary nucleus, the nucleus of the solitary tract, dorsal motor nucleus of the vagus nerve and the hypoglossal nucleus.Our results were similar to those obtained in human, rodents and sheep, confirmed previous studied in other animals and enriched the data of OB-Rb mRNA gene expression in the brain. Our research work provided anatomic information tb understand the physiology and functional mechanism of leptin, and laid a foundation for a further study on co-localization of OB-Rb mRNA with others neuropeptides in the brain.