Research On Conditional Expression Of A30Pα-synuclein Transgenic Mice By Tet-on System

PartⅠEstabilishment of conditional expression of A30Pα-synuclein in transgenic mice by Tet-on systemObjective To construct the conditional expression of A30Pα-synuclein in transgenic mice by Tet-on systemMethods Human wild type SNCA gene was cloned from fetus brain by using RT-PCR. The recombinant pTZ57R/T-SNCA vectors containing wild type SNCA synonymous mutations involving EcoRⅠand BamHⅠsites within code region of SNCA gene were constructed by site-directed mutagenesis using primer variance in mononucleotide, respectively. Based on these synonymous mutations, its Ala30Pro pathogenic mutation within code region of SNCA gene,was further constructed. The Ala30Pro pathogenic mutation was subcloned into the expressing vector pTRE2 of Tet-on system. Based on the tetracycline regulatory Tet-on system, rtTA gene was amplified by polymerase chain reaction and coned into pBC-vector, pBC-rtTA was obtained. Transgenic mice were established by microinjection genes of pBC-rtTA and pTRE2-humanA30P into male nuclei at the same time. Then ova were transplanted into pseudopregnant mouse recipients.Offsprings born from the ova were tested by PCR.Results The objective band of 481 bp was obtained by PCR. The objective band of 460 bp was obtained by the BamHⅠand HindⅢdouble digestion of recombinant pTRE2-humanA30P vectors and ultimately the E.coli DH5αtransformed with corresponding recombinant pTRE2-humanA30P vectors had been comfirmed successfully by gene sequencing. By PCR screening, 1 transgenic mice were detected binary positive for pBC-rtTA and pTRE2-humanA30P and 13 transgenic mice were detected positive for pTRE2-humanA30P.Conclusion The conditional expression of A30Pα-synuclein in transgenic mice by the tet-on system had been constructed successfullyPartⅡBehavioral study of condition expression of A30Pα-synuclein in transgenic mice by Tet-on systemObjective To investigate the influence of expression ofα-synuclein on motor function of binary positive mice.Methods The double-transgenic FVB mice was divided into three groups (n=15 in each group) and nine normal mice were construced a control group. All mice were treated with continuous doxycycline (1.5mg/ml, water). Rolling bar tests, spontaneous activity tests and pole climbing tests were used to evaluate the motor function of mice.Results The double-transgenic mice of continuous doxycycline for 2 weeks had no significant difference in Rolling bar tests, spontaneous activity tests and pole climbing tests with the normal mice(P>0.05). But the group of doxycycline treated for 4 weeks had signficant less spontaneous activity,shorter rolling bar test latency and longer pole-climbing time than the control group. The group of doxycycline treated for 8 weeks had the least spontaneous activity,the shortest rolling bar test latency and the longest pole-climbing time.Conclusion The double-transgenic mice of continuous doxycycline treated for 4 weeks had influence on their motor functions. T he more longer we treated ,the more serious their motor fuunction were after 4 weeks.PartⅢEstablishment of condition expression of A30Pα-synuclein in transgenic mice and PD model by Tet-on systemObjective To investigate the expression of A30Pα-synuclein and the pathologic basements of the transgenic mice.Methods The double-transgenic FVB mice was divided into 3 groups (n=15 in each group) and 15 normal mice were construced a control group. All mice were treated with continuous doxycycline (1.5mg/ml, water). Western blotting was used to evaluate the protein level ofα-synuclein in normal FVB mice and continuous doxycycline treated groups for weeks(2weeks 4weeks and 8weeks). RT-PCR was performed to evaluate the mRNA level ofα-synuclein. Quantitative morphology was performed to evaluate the number of tyrosine hydroxylase (TH) positive cells of substantia nigra pars compacta (SNc).Resultsα-synuclein expressed in cerebellum,brainstem,hippocampus and cerebral cortex. More expressed in hippocampus and cerebral cortex. The expressed ofα-synuclein in brainstem was significant increase in double-transgenic mice of continuous doxycycline for 4 weeks than nomal ones. The RNA lever ofα-synuclein in transgenic mice were express in brain,lung,liver,spleen,kidney,stomach, intestine and thymus. There was no express in heart. The RNA lever ofα-synuclein in brain were significant in double-transgenic mice of continuous doxycycline for 4 weeks than control group. There was a significant decrease of SNc TH positive cells in continuous doxycycline for 4 weeks.Conclusionα-synuclein were overexpress in brainstem of the transgenic mice of A30Pα-synuclein through continuous doxycycline treated for 4 weeks. There was a significant decrease of SNc TH positive cells of tansgenic mice of continuous doxycycline for 4 weeks.