Cloning Recombinant Galanin Gene From C57 BL/6 Mice And Constructing Its Transgenic Overexpression Vector

Objective:To clone recombinant galanin gene and construct its overexpression transgenic vector,so as necessary to provide basis for the research of galanin overexpressing transgenic mice.Method:C57BL/6 mice recombinant galanin gene was cloned,which include its cDNA and second intron,using polymerae chain reaction(PCR).The recombinant galanin gene and promoter of human platelet-derived growth factor B chain were cloned into pSP73 vector to construct galanin transgenic overexpression vector plasmid pSP73-PDGFB-Gal-intron2.1.The total RNA and genomic DNA were extracted from C57BL/6 mice brain.C57BL/6 mice's galanin gene was found on PubMed and design the primers with Primer 5.0.The galanin cDNA was amplified by revers transcriptase-PCR(RT-PCR) technology from the total RNA.The 5' Non-coding Region(5'NCR) cDNA, 3'Non-coding Region(3'NCR) cDNA and second intron(itron2) of galanin gene was amplified by PCR technology from the genomic DNA.The exonl-2 of galanin gene was amplified by overlap extension PCR(OE-PCR),templated by 5'NCR and cDNA PCR products.The exon3-6 of galanin gene was amplified by OE-PCR,templated by 3'NCR and cDNA PCR products.Galanin-intron2 was amplified by OE-PCR,templated by exon1-2,exon3-6 and itron2 PCR products.2. Galanin-intron2 PCR product was purified from agarose gels using gel extraction kit.The Galanin-intron2 was cloned into pGEM-easyT vector through T-A cloning after A-tailing reaction,then it would be named as pGEMT-Gal-intron2.3.Galanin-intron2 was excised from pGEMT-Gal-intron2 with Hindâ…¢and Xhoâ… ,PDGF-B promotor was excised from psisCAT6a with Xbaâ… and Hindâ…¢,they were cloned into pSP73 vector after it was cut with Xbaâ… and Xhoâ… ,then it would be named as pSP73-PDGFB-Gal-intron2.Results:The recombinant galanin gene was obtained,and it was cloned into pGEM-easyT vector and pSP73 vector.The recombinant galanin gene in the recombinant plasmid pGEMT-Gal-intron2 and pSP73-PDGFB-Gal-intron2 was exactly confirmed by DNA electrophoresis after enzyme restriction and DNA sequencing.Conclusion: 1.The recombinant galanin gene was obtained by OE-PCR. 2.The pGEMT-Gal-intron2 plasmid with the recombinant galanin gene was was constructed through T-A cloning.3.The galanin transgenic overexpression vector plasmid pSP73-PDGFB-Gal-intron2 was constructed by molecular cloning technology.