Characterization Of The Regulatory Element Of The Porcine Adipose Tissue-specific Expressed Gene Adiponectin

The definition of Gene expression is structural gene transcribe into mRNA by the action of regulatory sequence, then processed and translated into corresponding protein along with ribosome. The core of Gene Engineering is technique for gene expression. As segment, promoter provides recognition and combination sequences for RNA polymerase for activating gene translation. At present, in the research of Animal Gene Engineering, promoter as an essential element to decide the destiny of exogenous gene. So it requires that the promoter needs rigorous temporal and spatial specificity; and sometimes for strengthen the temporal and spatial specificity and single enhancer or composite cnhancer should be designed to match the promoter. This research sees the promoter-1671—+263 effective segment of porcine adipose tissue-specific expressed gene adiponectin as an object. A 640bp sequence is selected and named adi-pro. CRE binding site is contained and reported in porcine adiponectin only. Next processing an analysis for adi-pro regulatory element and assemble two types of hybrid promoters. Finally tissue and cell specificity of adi-pro and the hybrid promoter which is of higher activity are determined. The main research findings are as follows:1. The DNA template from Mei Shan and Large White and adi-pro is amplified by PCR and analyse the 640bp fragment on NCBI. The adi-pro with Kpn I and Mlu I is amplified and the promoter luciferase reporter gene vector pGL3-adi-pro is constructed, then transfect it into differentiated 3T3-L1 and C2C12 and through identifying the ratio of fluorescence.The ratio of fluorescence is demonstrated. pGL3-Basic as a nagetive control and the vector system takes adi-pro display activity. After transfected into differentiated 3T3-L1 and C2C12, the result is shown obviously. In contrast, pGL3-adi-pro which transfected into differentiated 3T3-L1 have a higher activity and adi-pro performs an activity enhancement during the adipogenic conversion of 3T3-L1 preadipocytes.2. For confirming whether adi-pro serves as an effective element in the vector systems which take SV40 promoter and SV40 enhancer, the adi-pro is cloned into these systems. The adi-pro with Kpn I and Mlu I is cloned into pGL3-Promoter and pGL3-Enhancer, then the transient cotransfection experiment is proceeded.TK acts as a reference and nagetive control is pGL3-Promoter and pGL3-Enhancer. After 7 days of differentiation, the unassembled systems which contain adi-pro are transfected into differentiated 3T3-L1 show a higher activity.3. Two types of hybrid promoter are assembled. The CMV-adi-pro contains CMV promoter and the SVenh contains SV40 enhancer. The CMV with Kpn I and Mlu I is amplified from pIRES2-EGFP, then subcloned into pGL3-Basic. In order to assemble CMV-adi-pro regulatory element, the adi-pro with Mlu I and Xho I is amplified and cloned into pGL3-CMV. The SVenh with Kpn I and Mlu I is amplified from pGL3-Enhancer. Instead of CMV, the SVenh is cloned into pGL3-adi-pro by digesting pGL3-CMV-adi-pro under the Kpn I and Mlu I enzyme digestion. These constructed vectors are divided into two groups and transfect the 3T3-L1 and C2C12 under the undifferedtiation and differentiation status. Testing the luciferase activity and contrasting the activity of these assembled regulatory elements. The hybrid promoters CMV-adi-pro and SVenh-adi-pro show a higher activity than unassembled ones; and the cell and tissue specificity of adi-pro is still alive in these hybrid promoters.4. The adi-pro-Red binding gene is assembled. The Red with Mlu I and Xho I is amplified from pHcRedl-N1/1 and subcloned into pGL3-adi-pro. As a result, the adi-pro-Red system is constructed. The Cla I and EcoR I are chose by judging from lentivirus vector pLenti7.3V5-GWlacZ, then the adi-pro-Red with Cla I and EcoR I is amplified and subcloned into pGL3-Basic. Through enzyme digestion and directed sequencing, the adi-pro-Red is constructed successfully.