Screening Lentiviral Vector With Carrying Effectively SiRNA Of ROCK2Gene

Objective：To screen lentiviral vector carrying siRNA thatcould significantly suppress the ROCK2gene expression in the corpuscavernosum smooth muscle cells of the spontaneously hypertensive rat(SHR). Method:1.We used the organizational adherence method toisolate and culture the SHR corpus cavernosum smooth muscle cells.2.To design the small interference RNA (siRNA) specific to SHRROCK2gene by RNA interfering technique, construct its recombinantlentiviral expression vectors, and identify these vectors by DNAsequencing.①RNAi target sequences were designed, then the targetsequences of Oligo DNA were synthesized and annealed todouble-stranded DNA, which were connected with GP-SupersilencingVector digested by Hpa I、XhoI Short hairpin RNA lentiviral vectors wereconstructed．②The recombinant plasmid was transformed into competentDH5α cells. The positive recombinant colony was selected by ampicillinmedium agar and identified by DNA sequencing.③The recombinedvectors were identified by restriction enzyme analysis and sequencing.Lentivirus was produced after GP-Supersilencing Vector and threepackage plasmids were cotransfected to293T cells.3. Five male SHR cell samples were randomly divided into six groups（each group n=5）,each group for each sample were3×104cells. Group A（control groupwhich untransfected）；Group B（GFP lentiviral transfected group）；GroupC～F（transfected group with lentiviral vector respectively carryingsiRNA1~4targeted to the ROCK2gene）.Transfect SHR corpuscavernosum smooth muscle cells as MOI=80. GFP expression wasobserved under fluorescent microscope after transfection48hours,and themRNA expressions of ROCK2gene in the transfected corpus cavernosumsmooth muscle cells were detected by RT-PCR. Results:1.PCR andDNA sequencing demonstrated that the recombination plasmids targetingthe ROCK2gene were successfully constructed.2.Infection efficiency ofcorpus cavernosum smooth muscle cells of SHR observed underfluorescence microscope was more than50%.3.RT-PCR to detect thetransfected cells ROCK2mRNA expression:The expression of ROCK2mRNA in the corpus cavernosum smooth muscle cells of SHR were verysignificantly decreased in group C、D、F as compared with group A（P<0.01）, resulted in suppression of ROCK2mRNA expression by43.91±8.19℅、47.15±6.64℅、25.7±6.03℅， respectively； and theexpression were significantly decreased in group E as compared withgroup A（P<0.05）, resulted in suppression by16.81±5.94℅. But theexperimental result did not show any significant difference betweengroup B and group A (P＞0.05). Conclusion:1.A lentiviral vector of ROCK2gene RNAi was constructed successsfully by the geneticengineering technology, and it provides a condition forfurther researchED in vitro and vivo of SHR.2.The study has been successfullyconstructed four kinds of lentiviral vector carrying siRNA againstROCK2gene, all of them could suppress the SHR corpus cavernosumsmooth muscle cells ROCK2gene expression, while three kinds oflentiviral vector have stronger inhibition.