Construction And Functional Analysis Of The Lentiviral Expression Vector For Over Express And RNA Interference Of CIRP Gene

CIRP gene function is required for the initiation of DNA replication and is a key regulatory protein duringcell cycle progression. The CIRP gene is basice expressed in most normal tissues, in contrast it is overexpressed during cold and many other stresses. An effective way to explore the gene functions of CIRP is toknock-down the CIRP messenger RNA （mRNA） or over express CIRP in nammal condition,and then examinethe phenotypic consequences.In presented work, new approach for the control of CIRP gene expression in mammal cells has beendeveloped. The technique is based on using the RNA-interference, over express and lentiviral transductionmethodology. The aim of this study is to construct a expression vector pLenti6/V5-CIRP encoding CIRP gene,to achieve the long, efficient and stable expression in mammal cells, and a lentiviral expression vectorpLenti6/V5-GW/EmGFP-miR-CIRP for RNA interference of CIRP gene to provide a basis for investigatingthe role of CIRP gene in the signaling pathway involved in resist apoptosis. We have used RNA interferenceand gene expression system based on Gateway Technology. The system contains a series of entry anddestination vectors that enables easy transfer of miRNA or cDNA into lentiviral expression systems with avariety of selection or marker genes.（1） The CIRP cDNA was amplified from plasmid rat by RT-PCR. ThecDNA was subcloned into pENTR-11to generate recombinant plasmid pENTR-CIRP. Then, pENTR-CIRPwas homologously recombinated with pLenti6/V5-DEST. The recombinant was named as pLenti6/V5-CIRPand confirmed by PCR and DNA sequencing.（2） Five CIRP gene miRNA sequence was designed using asoftware available on-line. After synthesis and annealing, the double-stranded oligonucleotides （dsOligoe）were cloned into the pcDNA^TM6.2-GW/EmGFP-miR plasmid,and achived five RNAi plasmids aspcDNA^TM6.2-GW/EmGFP-CIRP-131/264/405/645/707.（3）To create a simple and effective primary culturemethod for rat’s hippocampal neurons. Use a syringe device instead of pasteurpipette method to make cellsuspension of hippocample. Comparing with traditional methods, the method with syringe device can savemuch time, and simpler, the cells with higher activity and purity. This approach is a more convenient andeffective method that can meet various neural tests, providing a basis for researching the related disorders ofthe hippocampal neurons.RESULTS:（1） The recombinant plasmid of pLenti6/V5-CIRP was constructed. The sequence of amplified CIRP genewas consistent with that reported in GenBank.（2） Achive five RNAi plasmids as pcDNA^TM6.2-GW/EmGFP-CIRP-131/264/405/645/707.（3） Create a simple and effective primary culture method for rat’s hippocampal neurons.