| RNA interference (RNAi) is a new modality which can elicit down-regulationof gene expression. RNAi is a conserved system through which double strandedRNA (dsRNA) guides sequence specific mRNA degradation. The RNAi apparatusmay be artificially triggered by delivery of naked siRNA molecules or byplasmid-based expression of dsRNA. RNA interference is one of the most excitingdiscoveries of the past decade in functional genomics. Duplexes of 21 nt RNAs,known as short interfering RNAs (siRNAs), efficiently inhibit gene expression byRNA interference when introduced into mammalian cells. It was found thattransfection of mammalian cells with synthetic siRNA resulted in highly, sequencespecific RNA interference.From the find outs of Chronobiology, people know human beings also havetiming change life activities. Of all the timing change activities, year change, seasonchange, month change, day change, the one which has attract most attention is daychange. Circadian, a new created word is used to describe the pattern have 24 hoursperiod.The physiology and behavior of all living organisms from bacteria to humansare controlled by the circadian rhythms driven by internal oscillators, the circadian clock. It is composed of the positive and negtive loops that consist of coock genesand proteins. The period1 gene was found as a key gene of the circadian clock andplayed its function through protein-protein interaction. But it is still difficult todescribe the mechanism of the per1 in circadian rhythms. RNA interferencetechnology is emerging as a very potent tool to obtain a cellular knockdown of adesired gene. In this work we used the pTER-hper1 interference plasmid wasconstructed,vector-based RNA interference to inhibit hper1 expression in SH-SY5Ycell lines in vitro and investigated its interferencing effects, which established thebase for studying the function of hPER1. RT-PCR showed that the expression ofhper1 in SH-SY5Y cells transferred pTER-hper1-Ⅱwas decreased by 84.9%, whichdemonstrated that there was high interference efficiency of pTER-hper1-Ⅱinterference plasmid. Western blot technology showed that hPER1 inSH-SY5Y cells was decreased. Western blot technology showed that P—p44/42MAPK in SH-SY5Y cells was increased.In mammals,Circadian genes per1, per2, Bmall, Clock and cry1 have beendetected to be expressed during the spermatogenesis process, but in a stagespecificand circadian-independent manner. Most notably, the Clock gene expression hasexclusively been detected in the acrosome of round spermatids. These findingssuggest a potential role of the Clock gene in male reproductivity. Thus, in this studywe decided to determine the significance, if any, of the round spermatidspecificClock gene expression using a DNA-based RNAi strategy, which has been proven tobe very effective in silencing in vivo gene expression during spermatogenesis. Weharvested the mouse testes at different days after plasmid DNA injection andprepared tissue homogenates for Western blot analysis of mClock gene expressionCompared to the controls, mClock gene expression was downregulated by 55% atday 3 and 48% at day 6. |
