Establishment And Microinjection Of Overexpression Galanin Gene

ObjectiveIn order to investigate the function of Galanin in entirety,in the present study weattempted to constructe the vector of transgenic mice and generated transgenic mice modelwhich the Galanin was specific overexpressionin DNA neurons..GAL expresses lowly in normalphysiology,when body is in morbidity,GAL appears preternatural change. The transgenic micemodel can be uesd to investigate the function of endogenic GAL and provide the new thought ofdisease therapy .MethdosFirstly,total RNA and genome DNA was extracted from the brain of the mouse.Thegalanin coding region was obtained by reverse transcription-polymerase chain reaction(RT-PCR).The galanin 5′untranslated region and 3′untranslated region was amplified by polymerase chainreaction(PCR).The objective galanin gene was amplified by overlap-extension PCR.Thetransgenic vector was constructed by inserting PDGF B-chain promoter on the upstream of thegalanin gene.It was named PDGF-GAL by identification.The objective PDGF-GAL gene wasamplified by restriction digestion of recombinate plasmid PDGF-GAL with XbaⅠand HindIII.Secondly,by microinjection,the objective DNA fragment was injected into the Male-pronucleusof Fertilized ovums.Then the good Fertilized ovums was explantated into uterine tube ampulla ofpseudocyesis female mouse.ResultsAnalysis of the sequence resμlts showed that the fμll length of the fragment cloned frombrain tissue of mouse was correctly amplified. This sequence is coincident with the galanincDNA sequence in the GenBank.The transgenic vector with galanin specially expressed innervous tissues was successfμlly constructed by enzymatic digestion,The objective DNAfragment was recovered by the staining gel.It′s solution was diluted into 3 ng/μL with TE.Then it canbe used for microinjection.The objective DNA fragment was injected into the Male-pronucleus ofFertilized ovums successfully.The pseudocyesis female mouse was successful pregnant . ConclusionsThe transgenic vector with galanin specially expressed in nervous tissues was successfμllyconstructed. The galanin gene fragment for microinjection was recovered.The objective DNAfragment was injected into the Male-pronucleus of Fertilized ovums successfully.The pseudocyesisfemale mouse was successful pregnant .