Tissue Localization Of GHR, IGF-1, IGF-1R In Antler Tip And Cloning Of CDNA For GHR

Subject: Through tissue localization of GHR,IGF-1 and IGF-1R gene and cDNA cloning of GHR gene initially illuminate secretion mechanism of growth factors and regulation role in antler rapid growth phase. During antler growth phase GH controlled the level of IGF-1, IGF-1 directly act to target cells and mediated biological effects of GH. IGF-1 effect GH binding to its receptor(GHR) can initiate GH-dependent signal transduction pathways which will generate many biological effects. This reveals that regulation laws of some growth factors in antler rapid growth stage, for further study to provide certain data and methods.Method: (1) A pair of primers were designed according to the homology region of GHR among other species. Total RNA was isolated from antler tip, the cDNA sequence of GHR gene was cloned by RT-PCR technology, and the cloned fragment was recovery, sequencing then was carried out homologous alignment analysis. (2) To isolate the genomic DNA from deer as template, amplified GHR,IGF-1 and IGF-1R gene, amplification products were purified, then ligated into promoter, transcribed using T7 transcriptase and labeled using digoxin, purified labled products as probes. (3) To prepare frozen sections, GHR,IGF-1 and IGF-1R were localization using in situ hybridization in antler tip. (4) Total RNA was extracted from the epidermis/dermis, reserve mesenchyme/ precartilaginous, and cartilaginous, then RT-PCR was performed to detect whether IGF-1 and GHR express in the antlers.Result: (1) The cDNA sequence of deer(Cervus nippon Temminck) GHR gene was cloned by RT-PCR firstly (Genbank accepted number is EU381142). Thereafter, It was inserted into PMD19-T vector by T/A cloning, transformed into DH-5α, tested by PCR and sequenced. The fragment was 1967bp, including complete coding sequence of GHR gene. Deer GHR precursor is 634 amino acids in length, Compared with others, the homology of GHR to bovine, ovine and human was 97%, 97% and 80%, respectively, but the homology of protein was 97.9%, 97.6% and 77.3%, respectively. (2) Examined concentration of labeling probes of GHR, IGF-1 and IGF-1R, these probes can be achieved the concentration which in situ hybridization requested. (3) The results of in situ hybridization showed positive signal, that indicated all regions had expression of GHR, IGF-1 and IGF-1R in antler tip. (4) The relative expression level of GHR and IGF-1 in epidermis/dermis, reserve mesenchyme/precartilage and Cartilaginous zone was determined by Semi-Quantitative RT-PCR, both IGF-1 and GHR had higher expression in epidermis/dermis and Cartilaginous zone than in reserve mesenchyme/precartilage (P<0.05 or P<0.01).Brief summary: Synthetic all above the resultes, it has demonstrated that GHR, IGF-1 and IGF-1 are expressed in antler tip.