Establishment Of Galanin Gene For Microinjection And The Study Of The Optimal Time Of Male-pronucleus Of Fertilized Ovums For Microinjection

Objective1. The study was performed to clone galanin gene and construct the transgenic expression vector of galanin gene which can be specifically expressed in the nervous tissues.The galanin gene fragment for microinjection was procured. This work has laid foundations for the construction of galanin transgenic animal models.2. For obtaining the higher quantity and quality of male-pronucleus of fertilized ovums, the study was performed to obtain the optimal time of male-pronucleus of fertilized ovums for microinjection.MethdosFirst part:Total RNA and genome DNA was extracted from the brain of the mouse.The galanin 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 chain reaction(PCR).The objective galanin gene was amplified by overlap-extension PCR and cloned into pMD19-T plasmid.After it was confirmed by restriction digestion and automatic sequencing.lt was named pMD19T-GAL.The objective galanin gene was amplified by restriction digestion of recombinant plasmid pMD19T-GAL.It was linked plasmid pUC18 and was named pUC18-GAL. The transgenic vector was constructed by inserting PDGF B-chain promoter on the upstream of the galanin gene.It was named PDGF-GAL by identification. The transgenic vector with galanin specially expressed in nervous tissues was successfμlly constructed. The objective galanin gene was amplified by restriction digestion of recombinate plasmid PDGF-GAL.The objective DNA fragment was recovered by comparison with the staining gel.It can be used for microinjection.Second part:To induce the superovμlation with 4-6 week old female mouse by pregnant mare'serum gonadotropin(PMSG) and human chorionic gonadotrophin (HCG).The fertilized ovums was harvested at different time. The condition of male-pronucleus of fertilized ovums was overviewed and the optimal time of male-pronucleus of fertilized ovums for microinjection was chose.ResμItsAnalysis of the sequence resμlts showed that the fμll length of the fragment cloned from brain tissue of mouse was correctly amplified. This sequence is coincident with the galanin cDNA sequence in the GenBank.The transgenic vector with galanin specially expressed in nervous tissues was successfully constructed by enzymatic digestion, The objective DNA fragment was recovered by the staining gel.It can be used for microinjection.Quantity and quality of male-pronucleus of fertilized ovums harvested at 20-25h after injecting HCG was much higher than that at other period. The male-pronucleus was decreased after injecting HCG 26h. The male-pronucleus was smaller after injecting HCG 20-23h. The male-pronucleus was bigger after injecting HCG 23-25h. The two pronucleus of fertilized ovums was too bigger and was fusioning after injecting HCG 26-28h.Conelusions1.The transgenic vector with galanin specially expressed in nervous tissues was successfμlly constructed. The galanin gene fragment for microinjection was recovered. This work has laid foundations for the construction of galanin transgenic animal models.2. The optimal time of male-pronucleus of fertilized ovums for microinjection is at 25h after inject of HCG At this period, the male-pronucleus of fertilized ovums was bigger and limpid than the other periods.lt was located in the center of fertilized ovums and was suitable for microinjection.