The Expression Vector Construction And Site-directed Mutation In Vitro Of RET Oncogene

In 1985, RET proto-oncogene was found by Takahashi when he transfected the lymphoma DNA into NIH3T3 cell. He found that RET can Rearrange during transfection and be activated as well. According the research, we find that the RET gene isocated in the centromeric region of chromosome 10q11.2, and consists of 20 exons . The length of the first exon is about 24kb, and the length of others is about 31kb in the total.The transcription productions of RET is primary transcripts, Which can turn into 5 kinds of RNA molecule after being modified. The protein coming from every mRNA can combine each other and we will obtain 3 kinds of protein molecules at last. By the study, We conclude that Ret is high expression in central neural system, peripheral neural system, excretion system and so on at the early embryo stage. And during the middle stage, we can find the Ret high espressive activity in the neural crest-derived cell lineages, kidney tube cell, pheochromocytoma cell and in the progenitors of parathyroid gland et al.The Ret is one of the receptor tyrosine kinases with a cadherin-related motif and a cysteine-rich domain in the extracellular domain. Its structure is typical Tk structure. RET is the signaling component of multisubunit receptor complexes for glial cell line-derived neurotrophic factor (GDNF) family ligands (GFLs), which belong to the TGF-P superfamily. The main function of Ret is transmit informations coming from GDNF into the cell. The interaction between the GFLs and RETis mediated by glycosylphosphatidylinositol (GPI)-linked cell-surface glycoproteins, called GFRctl-4. when the complex of GDNF-GFRas-Ret is formed, tyrosine (Tyr) residues in RET will be phosphorylated. As a result, different signaling pathways are activated, informations is transmitted into the cell. So, outer signal can control the differential, multiplication and transferring to enteric wall of neural crest-derived cell. The conservation and expression situation of Ret is the early affair for enteric ganglia system, ureteric bud and kidney embryogenesis. According to the study, RET proto-oncogene will be activated when it happen to mutate , lack and rearrange. All these changes play an important role in the occurring of multiple endocrine neoplasia ,mastoid thyroid carcinoma and Hirschsprung' s disease.Among these conditions , MEN2B, which has an extremely low rate of incidence, is the most severe form. Site-mutation is the early affair for such disease. And its clinical presentation includes C-cell hyperplasia or medullary thyroid carcinoma , pheochromocytoma, ganglioneuromatosis, accompanied with marfanoid body habitus, over 95% of MEN-2B patients have a specific point mutation at codon 918 in exon 16 of RET. That is to say , methionine is replaced by threonine. [918Met(ATG) -*Thr(ACG)]. Because the mutation point is located at the activated center of tyrosine kinase domain . Intracellular structure of Ret protein will change with mutation, resulting in activating by itself through dimerization. After being phosphorylated, Ret can activate the downstream signal molecuLes and transmit the information into the cell. At last, the cell accepting signals will grow excessly and form a tumor.Nowdays, scientists coming from china and other countries incline to clone the RET gene by RT-PCR from all kinds of tissues, they detect the mutation point through the DNA sequence analyse, and show the relation between the mutation and diseases.This research model from unit tomolecular level belongs to the positive research model and is limited to disclose the real gene function during the progress of disease occurring. In order to solve the problem and prepare for transfecting cell or making transgenic animals, we study the RET gene on the base of reverse demonstration. We construct RET expression vector, fulfil a site directed mutation at 2753 bp of the RET cDNA. And we analyse the products of RET oncogene by 0MIGA2. 0 protein analyzing software.Methods:(1)Using DNA reconstruction technology, such as restrict enzyme digestion(Handlll and Not I), T4 DNA ligase ligation, et al.we digest PSK-RET and obtain the cDNA fragment of RET. we insert the cDNA fragment of RET into pcDNA3. 0 and we obtain espression vector pcDNA3. 0-RET, select positive strain by Amp resistant, restrict enzyme digestion, and DNA sequencing.(2)Using a PCR-based site-directed mutagenesis method, we make a mutation at 2753 bp of the RET cDNA. After Dpn I digestion of the anplification products, we introduce the products into E. coli TGI. select positive strain by Amp resistant, polymorase chain reaction, and DNA sequencing.(3)Using 0MIGA2. 0 protein analyzing software, we compare the cDNA sequence of RET with the cDNA sequence of mutative RET. We analyse the protein structure , domain and some specialties of Ret.Results:(1) By Amp resistant, restrict enzyme digestion, and DNA sequencing, we find that the pcDNA3. 0-RET plasmid was successfully constructed.(2)The optimal anneal temperature is 61°C, and the result of DNA sequencing show that mutation has been finished successfully.(3)Contrary to Ret protein,the mutative protein has changed at secondary structure and a new phosphorylated site is found.Conclusion:We have constructed RET expression vector and made a site-directed mutation successfully, and we find that there are differences between the Ret and imitative Ret. All these work may provide a clue to study the function of RET oncogene in the process of tumor occurring.