Construction And Expression Of Rat 5-HT_5A Receptor SiRNA Recombinant Adenovirus Expression Vector

Background and Objective5-HT5A receptor is a subtype of 5-HT receptors'family, which is a kindof G-protein-coupled receptor located in central nervous system. mRNAlocalisation studies in mouse, rat and human brain have revealed that5-HT5A receptor mRNA is widely expressed. In the periphery 5-HT5Areceptor mRNA expression appears to be very low or absent. Thedistribution of 5-HT5A receptor mRNA have provided important pointers tothe potential functional roles of the receptor in central nervous system, suchas cognition regulation, anxiety regulation, formation of sensation,neuroendocrine regulation, locomotion integration, circadian regulationand pain regulation. 5-HT5A receptor has been found more than ten years, butthere are few studies about the function, which is due to the lack of selectiveligands and antagons, so it was difficult to define the function of 5-HT5Areceptor in physiological and pathological process. So we constructed rat5-HT5A receptor siRNA recombinant adenovirus expression vector, andobserved its expression in rat never cells to provide technique sustain to the studies about the function of 5-HT5A receptor in vivo.Methods1. Three 5-HT5A receptor siRNA plasmid expression vector ofpGenesil-2 Htr5a1, pGenesil-2 Htr5a2, pGenesil-2 Htr5a3, and onescrambled sequence of pGenesil-2 HK were designed and constructed. Atthe same time, another plasmid expression vector pDsRed-EGFP-Htr5awhich could express 5-HT5A receptor, EGFP and AFP was constructed either.In order to find out the most effective siRNA plasmid expression vector, therecombinant vectors of pGenesil-2 Htr5a1, pGenesil-2 Htr5a2, pGenesil-2Htr5a3 and pGenesil-2 HK were cotransfected respectively withpDsRed-EGFP-Htr5a into the 293 cells. Forty-eight hours aftercotransfection, fluorescence intensities of GFP and AFP were detected byflow cytometry on 293 cells. The weaker of the GFP intensity the moreeffective was the siRNA plasmid expression vector.2. The most effective siRNA expression frame Htr5a2 was subclonedinto pGenesil1.2 to construct pGenesil1.2 Htr5a2 plasmid expression vector.Then pGenesil1.2 Htr5a2 was homologous recombinated with pGSadenoexpression vector to construct recombinant adenovirus expression vector.Then the expression vector of pGSadeno-Htr5a2 was introduced into 293cells to product recombinant adenovirus. Subsequently, the recombinantadenovirus was transfected into 293 to generate a higher titer viral stock.Then the high titer recombinant adenovirus was transfected into primary cultured rat nerve cells. Forty-eight hours after transfection, the expressionof 5-HT5A at the level of mRNA was detected by RT-PCR.Results1. The siRNA expression vectors were successfully constructed, whichwas identificated by restriction endonuclease and DNA sequencinganalyzing. After cotransfection with pDsRed-EGFP-Htr5a into 293 cells,the most effective siRNA plasmid expression vector was ascertained byflow cytometry which could detect the fluorescence intensities of GFP andAFP. The ratio of the average fluorescence intensity of GFP to AFP was0.39 in 293 cells transfected with pGenesil-2 Htr5a2. The ratios were 0.539,0.417, and 1.626 in 293 cells transfected with pGenesil-2 Htr5a1,pGenesil-2 Htr5a3 and pGenesil-2 HK, respectively. So pGenesil-2 Htr5a2expression vector can suppress actively the expression of 5-HT5A.2. The recombinant adenovirus expression vector of pGSadeno Htr5a2was successfully constructed, which was identificated by restrictionendonuclease. Because there was a GFP expression frame in pGSadenoHtr5a2, GFP expression could be observed after that the vector wastransfected successfully into the 293 cells. The appearance of GFPexpression in 293 cells meant that recombinant adenovirus have beenpackaged successfully. And the titer of recombinant adenovirus was 109pfu/ml detected by TCID50. Then, pGSadeno Htr5a2 recombinantadenovirus was transfected into primary cultured rat nerve cells. Twenty-four hours after transfection, GFP expression could be observed innerve cells. And pGSadeno Htr5a2 recombinant adenovirus could suppressactively the expression of 5-HT5A in nerve cells, which was detected byRT-PCR after 48hrs.Conclusions1. 5-HT5A receptor siRNA plasmid expression vectors of pGenesil-2Htr5a1, pGenesil-2 Htr5a2 and pGenesil-2 Htr5a3 were constructedsuccessfully. And pGenesil-2 Htr5a2 was the most effective recombinantplasmid expression vector that could suppress actively the expression ofexogenous 5-HT5A in 293 cells, which was prepared for constructing5-HT5A siRNA recombinant adenovirus expression vector.2. 5-HT5A receptor siRNA recombinant adenovirus expression vectorof pGSadeno Htr5a2 was constructed and packaged successfully. AndpGSadeno Htr5a2 recombinant adenovirus could suppress actively theexpression of endogenous 5-HT5A in nerve cells, which provided techniquesustain to the studies about the function of 5-HT5A receptor in vivo.