Construction And Screening Subtractive CDNA Libraries Related To Visual Cortex Plasticity In Rats

Objective:To construct subtractive cDNA libraries which associated with visual cortex plasticity in the rat by using suppression subtractive hybridization technique and screening the related genes which associated with termination of the critical period of the visual cortex plasticity or associated with visual cortex plasticity.And to investigate the molecular mechanism involved.Methods:1,Designed and made the dark rearing box for rats and established animal models of dark rearing rats and dark-reared for 60 days postnatal and light-exposed for 7 days rats.2,A subtracted cDNA library was constructed with the visual cortex of rats that were dark-reared for 67 days postnatal(D67) and of those that were dark-reared for 60 days postnatal and light-exposed for 7 days(D60+L7) by using suppression subtractive hybridization technique.Differentially expressed genes were screened by polymerase chain reaction(PCR),reverse Northern hybridization,sequencing and homology analysis to screening genes which related to termination the critical period.3,To construct subtractive cDNA libraries using juvenile(P28) and adult(P60) rats' visual cortex with suppression subtractive hybridization technique.Using PCR technique,reverse Northern hybridization, sequencing and homology search to analysis the differential expression genes fragments to screening genes which associated with plasticity.4,Used semiquantitative RT-PCR to measure the expression level of several genes which screened by SSH in the visual cortex of different groups to further verify the reliability of the SSH.Results:1,The dark rearing box was made successfully and established animal models of dark rearing rats and light-exposed after dark-reared rats.2,The subtracted cDNA library associated with termination of the critical period of visual cortex plasticity in rats was constructed successfully.After screening,14 sequences which were up-regulated in the visual cortex of D60+L7 rats were obtained.There were 13 known genes fragments and a novel one.Of the known genes,we found Znf9,Eno-1,Hsp8,30 kDa adipocyte complement-related protein,Ppia,Non-Neuronal Enolase and ftp-3 gene participation the termination course of the critical period of visual cortex plasticity.And we also found those genes which encodingβ-Tubulin,myelin basic protein,and Cyclophilin participation the termination of the critical period which's function had been reported to be associated with visual cortex plasticity.3,Juvenile and adult rats' visual cortex differential expression genes' subtractive cDNA libraries were set up successfully.11 genes were obtained by sequencing and homology search which were up-regulation expressed in the visual cortex of P28 and 4 genes were up-regulation expressed in P60.We found Scd2,Hba-al,Glu-Pro dipeptide repeat protein,mDj4,hnRNP C1/ hnRNP C2 and strain BN/SsNHsdMCW mitochondrion gene which were up-regulation expressed in the visual cortex of P28 rats, which participate the critical period of plasticity and were the genes which related to plasticity,Hsp8,strain F344 x BN F1 mitochondrion,BHE/Cdb tRNA-Lys gene,complete mitochondrial genome participate the termination of the critical period of visual cortex plasticity of adult rats.And we also found those genes encoding calmodulin 2,cytochrome b and proteolipid protein were up-regulation expressed in the visual cortex of P28 rats, whose function had been reported to be associated with visual cortex plasticity.4,The results of semiquantitative RT-PCR indicating that the level of Hsp8 mRNA expression was significant higher in the visual cortex of P60 rats than that of P28 rats,the level of Plp mRNA expression was significant higher in the visual cortex of P28 rats than that of P60 rats,the level of Mbp mRNA expression was significant higher in the visual cortex of D60+L7 rats than that of D67 rats,which were in agreement with the SSH results and further verifies the reliability of the subtractive cDNA library we constructed.The results confirmed that Hsp8 gene and Mbp gene were the genes which termination the critical period,and Plp gene was the plasticity gene.Conclusions:The successfully construction of the subtracted cDNA libraries laying an important basis for elucidating the molecular mechanism of the critical period of visual cortex plasticity.A set of candidate genes association with termination of the critical period and association with visual cortex plasticity were screened from the subtracted cDNA libraries.Further screening and verification these genes maybe found the most important genes which determine whether or not termination of the critical period.It will make it possible to use gene therapy to regulation the critical period of visual cortex plastitity and...