Construction And Identification Of Rat NeuroD Gene Eukaryotic Expression Vector

NeuroD (otherwise known as BETA2) is a basic helix-loop-helix (bHLH) transcription factor that is capable of converting embryonic epidermal cells into fully differentiated neurons in Xenopus embryos. In insulinoma cells, NeuroD can bind and activate the insulin promoter. When NeuroD is deleted in mice, the early differentiating pancreatic endocrine cells and a subset of the neurons in the central and peripheral nervous systems die, resulting in cellular deficits in the pancreatic islets, cerebellum, hippocampus and inner ear sensory ganglia. As a consequence, mice become diabetic and display neurological defects including ataxia and deafness. These gain-of-function and loss-of-function phenotypes suggest that NeuroD controls both common and distinct sets of molecules involved in cell survival and differentiation in different tissue types. Understanding the primary function of NeuroD will be extremely valuable in the diagnosis and cure of the diseases that involve this transcription factor, which plays essential roles in the development and function of the pancreas and the nervous system. We constructed the plasmid expression vector of rat NeuroD gene.Methods:1. Cloning of NeuroD cDNAThe total RNA was extracted from the newly born rat cerebellum. Reverse transcript reaction and PCR were used to obtain the entire NeuroD cDNA. The fragment of neurod cDNA and pMD19-T were ligated by Ligation Mix. After transferring them into the JM109 competent cells, the recombinant clones were selected and identified through blue and white screening and PCR. Extracted the recombinant plasmids DNA and digested it by restriction endonuclease NheI and XhoI for further identification.2. Construction the palsmid expressing vector of NeuroD and sequencing analysisThe DNA of pMD19-T-NeuroD and pcDNA3.1 A with restriction enzymes XhoI and NheI was digested and purified respectively, then the fragements with sticky ends of NeuroD gene and pcDNA3.1A were obtained. Ligated them with T4DNA ligase and transformed them into the DH5a competent cells. Extracted the recombinant DNA and identified the positive clones with XhoI and NheI. Then the positive recombinant pcDNA3.1 A-NeuroD was sequenced. Blast the gene in gene bank in the Internet. Results:1 .Cloning of NeuroD cDNAThe full length of NeuroD cDNA amplified by RT-PCR was 1.0kb, which was corresponded with its length provided by GenBank. After transformation of the ligated product as well as blue and white screening, many colonies were obtained. Through PCR with the primers and certain restricton endonuclease digestion, the positive transformed clones were identified.2. Construction of the plasmid expressing vector of neuroDAfter restriction endonuclease digestion of recombinant plasmids, two segments of about 5.5kb and 1.0kb appeared clear and bright in agarose gel electrophoresis. The sequence of cloned NeuroD gene was conformity with the sequence deposited in GenBank. Conclusions:Rat NeuroD cDNA was correctly cloned and the recombinant plasmid expression vector pcDNA3.1A-NeuroD was constructed successfully. This research has provided one of the most practical tools to study the role of NeuroD during the neural differentiation and application of gene therapy for the diseases of nerve injury and nerve degeneration.