Clone Of The Recombinant Molecule GAL (IntronII) And Construction Of Mouse Noradrenergic Neurons-specific Expression Vectors

Objective:Galanin(GAL) is a 29 amino acid peptide (human30) that was isolated in 1983, it is widely distributed through the brain of various species. GAL has been associated with a variety of important functions and dysfunctions, including learning and memory, anxiety, epilepsy, Alzheimer's disease, depression, pain sensation. The human dopamine-beta-hydroxylase (hDBH) promoter is one of the best characterized of the neuronal subtype-specific promoters it can target transgene express into noradrenalin (NA) neurons in a cell type-specific manner. On the basis of the known control mechanisms of the hDBH promoter, in order to generate GAL transgenic mouse and investigate the function of GAL, in the present study we attempted to generate the vector of transgenic mice model which the GAL was specific expression in NA neurons.Methods:Firstly, by PCR, the 1.2kb hDBH promoter fragment was amplified, then it was cloned into pMD18-T vector. The recombinant vector pMD 18-T Vector/hDBH-promoter were obtained. Secondly, by RT-PCR, the coding part of GAL cDNA sequence was amplified, and by PCR, the 5'and 3'noncoding sequences of GAL cDNA were amplified. The full-length GAL cDNA sequence was obtained by overlap extention PCR (OE-PCR). Then the full-length GAL cDNA was cutted into two parts at the 15 bp s of the third exon and amplified them respectively. Using the two parts of the full-length cDNA and the second intron as a template by OE-PCR, the second intron was inserted into the galanin full-length cDNA, and the recombinant molecule GAL (intronll) was obtained. Then it was cloned into pMD19-T simple vector. Finally, the 1.2kb hDBH-promoter fragment from the pMD 18-T Vector/hDBH-promoter and the recombinant molecule GAL (intronll) were inserted into pSP73 vector in the correct order, to form the recombinant vector pSP73/hDBH-promoter/GAL (intronll).Results:The electrophoresis analysis illustrated a clear band of the recombinant vector pSP73/hDBH-promoter/GAL (intronll). The sequencings result of amplified sequence was blasted on NCBI with standard sequence, which has a very high nucleotide sequence identity (99%), and these results suggest that the construction of recombinant plasmid gets success.Conclusions:The transgenic vector pSP73/hDBH-promoter/GAL (intronll) was successfully constructed.