ABSTRACT
Objective To isolate and identify differential expression genes associated with multidrug resistance of leukemia . Methods Differential expression genes between leukemia cell line K 562 and resistant cell lines K562/DOX were isolated by using suppression subtractive hybridization (SSH) technique .Total RNA were extracted .cDNA were synthesized and digested by restric-tion enzyme Rsa Ⅰ ,then connected with adopter1 and adopter2R ,and linked with pMD19-T vector .Constructed vectors were trans-ferred into E .coli .Subtracted cDNA library was constructed ,and the positive clones were screened according to base sequences and homologous sequences .The differential expression genes were indentified by comparison analysis of Gene Bank database .Results A total of 220 differential expression genes were sequenced ,including hemoglobin ,ribosomes and mitochondria related genes ,and heat shock factor binding protein 1 (HSPB1) gene and other genes .Conclusion SSH method and molecular cloning technique could be used to construct subtracted cDNA library of differential expression genes between drug resistant and not -resistant leukemia cells , which might be useful for further screening and cloning of differential expression genes of multidrug resistant tumor cells .
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OBJECTIVE: To investigate differential gene expression profiling of symbiotic germinated seeds of Dendrobium officinale. METHODS: cDNAs from 5-week symbiotic germinated seeds and 5-week aseptic cultivated seeds, taken as the tester and driver respectively, were used to construct a suppressive subtractive hybridization (SSH) cDNA library. RESULTS: By sequencing positive clones and BLASTx analysis against GenBank database, 100 expressed sequence tags (EST) homologous to plant known genes were obtained. Functional annotation revealed that they were grouped into serials of cellular processes including cell and chromosome structure, RNA synthesis, signal transduction, energy metabolism, protein synthesis and degradation, and cell defense, etc. Real-time quantitative RT-PCR analyses showed that the five randomly selected genes were all up-regulated in symbiotic germinated seeds. CONCLUSION: The symbiotic seed germination of D. officinale is involved in multiple pathways, and the results of this study will lay a foundation for further molecular elucidation of seed germination in Orchidaceae.
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Objective:To find out the specific gene related to heroin dependence.Methods:Heroin-dependent models in mice were established by dose-increasing administration.After withdrawal syndrome was precipitated by naloxone,total RNA and mRNA were isolated from the brains of experimental and control groups respectively,then suppressive subtractive hybridization was proceeded.The hybridization sample was amplified by PCR and analyzed on a agarose/EB gel.Results:The mRNA level of the experimental group was significantly higher than that of control group and a differently expressing cDNA fragment was obtained from the brain of the experimental group.Conclusion:There is probably an expression of the specific gene in the brain of heroin-dependent mice.
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Objective To screen the Schistosoma japonicum female specific expressing genes. Methods{S.japonicum} adult worms were collected from the rabbits’ vein after six-week infection by affusing method. The adult worms were stabilized by RNA-later liquid, the male and female worms were carefully separated with nipper. The high quality total RNA was extracted and mRNA was obtained after purification. Double stranded cDNAs were synthesized after reverse transcription. Female subtractive (female as tester, male as driver) and male subtractive (male as tester, female as driver) cDNA libraries were constructed. The differentially expressed genes were further screened by dot-blot hybridization. The clones were selected and sequenced, which showed apparently higher signals when hybridizing with the female subtracting male probes, than those signals when hybridizing with the male subtracting female probes. The homology of these sequences was searched with BLAST program. The semi-quantitative PCR was applied to test the differential gene expression in female and male adult worms. Result Female subtracting male and male subtracting female cDNA libraries were constructed with SSH technique. After dot-blot hybridization, 50 clones were tested to be the potential female differentially expressed genes and were sequenced. 42 expressing sequence tags (ESTs) were received. After bioinformatics analysis, 17 fragments (about 40