The Design And Selection Of Interference RNA Sequences Against Mouse Somatostatin

Somatostatin(SS),a growth hormone secreting pituitary inhibition of neuropeptides,widely distributed in the central and peripheral nervous system of vertebrates,pancreas and gastrointestinal tract,such as the structure of a class-related peptide,including making 14 peptide,28 peptides,peptide and SS original form.Animal Growth controled by growth hormone(GH),including growth hormone releasing factor(GRF)and somatostatin(SS).SS inhibited expression in vivo and reduced the secretion,then GH concentrations can be increased,to serve the purpose of promoting animal growth.Endogenous or exogenous to the double-stranded RNA into cells,and the homologous mRNA by the RNA degradation.so that the corresponding genes have been restrained,This is caused by RNA interference gene silencing.The method has become so specific gene inactivation in the latest in an effective manner.The application of RNA interference technology to explore gene induced SS silence.First,the sequence of mouse somatostatin(SS)mRNA was obtained from GenBank(serial number:NM 009215).So this mRNA sequence was used as the target to design siRNA against SS.Begin with the AUG start codon and scan the length of the gene for AA dinucleotide sequence.Record the AA and its 3'adjacent 19 nt as potential siRNA target sites.Finally,three siRNA with 40 %~50 % G/C content and so similarity to other mRNA were selected from the CDS of this mRNA.Meanwhile,the local structure of siRNA target sequences was predictd and analyzed.It is probably that the complex structure of siRNA target sequence plays an important role in determining the inhibition efficacy of siRNA.A hairpin structure of siRNA transcript template targeting SS gene was synthesized and cloned to pSilencerTM 2.1- U6.A siRNA expression vector targeting SS,pSilencerTM 2.1- U6-SS,was constructed successfully.The constructed siRNA vector was co-transfected into cells with SS expression vector(pcDNA3.1-SS or pEGFP-SS).Then three plasmids expressing hairpin siRNA were constructed and used to transfect cells.The levels of SS mRNA and protein in the cells were quantitated by RT-PCR,real time PCR,EFGP fluorescence observation,Western blot and RIA.The results showed that all the three kinds of siRNA could effectively decrease SS mRNA and protein level in these cells. In our study,three siRNAs targeting SS were designed,and their inhibition efficacies were evaluated.And the closely related the secondary structure of siRNA target sequence. SS inhibit the expression inhibited GH secretion of factors reduce to improve animal performance with new ideas and methods.