Cloning The Genes Differentially Expressed In Tremor Rat By Suppression Subtractive Hybridization

ObjectiveThe relationship between the genovariation and disfunction of voltage - gate sodium channel and genetic epilepsy is very closely. This report used genetic epileptic rat, (tremor) as experimental animal ,to clone genes differentially expressed in epileptic rat by means of the suppression subtractive hybridization (SSH). To screening original and specific genes about mutation of voltage - gated sodium channel of epilepsy, and provide theoretical foundation for gene therapy of epilepsy .MethodsTen tremor and Wistar rats were in each group. They were killed by decapitation, and the brain was removed rapidly, hippocampus tissue was separated, extracting total RNA. Then, formaldehyde denaturing gels electrophoresis tested total RNA quality. MRNA was isolated from tremor and Wistar rat hippocampus tissue for reverse transcription to ds cDNA, respectively used PCR - Select cD-NA subtraction kit. Tester and Driver respectively digested with Rsa I, a four -base - cutting restriction enzyme that yields blunt ends. The tester cDNA is then subdivided into two portions, and each was ligated with different cDNA adaptor (1or 2R) . Adaptor outer side and the first PCR primer had same sequences, left with the second PCR (nested - PCR) primer, which are the same series, to screening PCR expansion products subsequently. Moreover adaptor containingT7 promotor and a series of important enzymes identification spaces points to facilitate subsequent cloning and sequencing. 2% gels electrophoresis to identify efficiency of ligation, efficiency was more than 25% . Two hybridization: excessive driver cDNA samples have respectively mixed with two Tester cDNA samples. Then mixed the two hybrid samples, while adding a new degenerative Driver cDNA for second hybrid, which further enriched by differences expressed cDNA. Two PCR reaction : For the first PCR products: which have two different adaptors ,can be amplified exponetially. A secondary PCR amplification is performed using nested primers to further reduce any background PCR products and to enrich for differentially expressed sequences. 2% gels electrophoresis identified two PCR products. The second PCR products were cutted in ultraviolet lamp for recycling, purified by PCR Purification kit. Purified PCR products ligated with pMD18 - T vector to set up the subtractive cDNA library. Transforming bacteria;amplification of the library was carried out with E. coli strain JM109. Then coaenobium were screened through the blue - white screening system. Co-enobium PCR to identify efficency of ligation with vector, 2% gel electrophoresis to identify ligation efficiency. The clones, which were selected randomly, were amplified by PCR, and then they were sequenced and analyzed in GenBank with Blast search. Hybrid efficient detection;second PCR products of subtractive and unsubtractive as templates, G3PDH 5 and 3'primers as primer carried out PCR amplification to evaluate subtractive efficiency.ResultsThe subtracted cDNAs of differentially expressed genes in tremor look like smears from 300bp to 800bp. Twenty - three positive clones were obtained from subtracted cDNA library through white - blue visual analysis and enzyme digestion. One different cDNA sequences were detected by sequencing analysis of ten clones . Sequence analysis showed that the .subtractive cDNAs had 98% similarities with rat mitochondria.ConclusionMutation of mitochondria possible cause trembling of genetic epileptic rat (tremor). Suppression subtractive hybridization is a highly efficient method for cloning differentially expressed genes, and by which the subtracted cDNA fragments of epileptic rat could establish a basis for screening novel and specific genes of epilepsy.