Materials and methods.(DOC)Author ContributionsConceived and designed the experiments: JJ Y-XQ Z-LJ. Performed the experiments: JJ Y-XQ PZ W-TG Z-QY B-RS Q-PY HK. Analyzed the data: JJ Y-XQ SC Z-LJ. Contributed reagents/materials/analysis tools: JJ Y-XQ Z-LJ. Wrote the paper: JJ Y-XQ SC Z-LJ.Supporting InformationFigure S1 Rab28 expression was elevated in the com-mon carotid arteries from the hypertensive rats.
RNA interference (RNAi), mediated by double-stranded RNA (dsRNA), is a natural cellular process associated with gene regulation[1]. Since the discovery of RNAi, scientists could use this instrument to deplete almost any of their interested genes not only as a tool in biological research but also as a therapeutic approach. RNAi uses a sequence-specific gene-silencing mechanism and is ultramost powerful. The general RNAi process could be artificially divided into 3 steps. First, short dsRNAs (,20?0 nucleotides) are generated by RNase III enzymes (either Dicer alone or a combination of Drosha and Dicer); second, these short dsRNAs are unwound and the strand with a thermodynamically less 3PO Indolactam V stable 59 end is preferentially loaded into the RNA-induced silencing complex (RISC) as the guide strand; third, RISC finds potential target RNAs and Argonaute (the key component of RISC) finally cleaves the target RNA containing a sequence homologous to the guide strand. There are many ways to induce RNAi for gene knockdown experiments [2], but the two most commonly used methods are chemically synthesized siRNAs or vector-based shRNAs. Compared to chemically synthesized siRNAs, vector-based shRNA expression achieves more sustained loss of function effect especially when it is embedded in the lentiviral vector. One of the widelyused shRNA expression vectors is pSuper described in 2002 [3]. It uses Pol III promoter H1 to transcribe a shRNA with a 21 bp (base pair) stem and 9 nt (nucleotide) loop structure. Furthermore, the first widely used genome-wide shRNA library utilized a lentiviral vector named pLKO.1-puro and this library has generated much of data that allowed for a better understanding of the diverse cellular processes associated with virology and cancer [4?]. We observed that the pLKO.1-puro vector possessed a unique palindromic loop (CTCGAG) different from other shRNA expression vectors such as pSuper [5]. This observation resulted in the hypothesis that a shRNA structure could be constructed using only a single long or two short oligonucleotides. We further describe a strategy for rapid cloning of multiple shRNAs which permits easier combination of the most efficient promoter-shRNA cassettes for the simultaneous knockdown of multiple genes or different targets of the same gene [8,9]. Here we gave proofs that our thought was feasible, and a shRNA could be constructed by only 1 long oligonucleotide or 2 short oligonucleotides with half the cost of conventional shRNA clone methods. Various parameters for the design of effective shRNAs based on our strategy were compared such as the palindromic loop sequences. Finally, the loop sequence “TTCTAGAA” was selected for shRNAs construction and then we gaveA Robust shRNA System Used for RNA Interferenceexamples that our method could apply to other genes such as the bacterial enzyme b-galactosidase (LacZ). This method was also used to successfully inhibit hepatitis B virus (HBV) antigen expression both in vitro and in vivo. 1407003 This approach is cost effective and more easily applied to many areas of basi.Materials and methods.(DOC)Author ContributionsConceived and designed the experiments: JJ Y-XQ Z-LJ. Performed the experiments: JJ Y-XQ PZ W-TG Z-QY B-RS Q-PY HK. Analyzed the data: JJ Y-XQ SC Z-LJ. Contributed reagents/materials/analysis tools: JJ Y-XQ Z-LJ. Wrote the paper: JJ Y-XQ SC Z-LJ.Supporting InformationFigure S1 Rab28 expression was elevated in the com-mon carotid arteries from the hypertensive rats.
RNA interference (RNAi), mediated by double-stranded RNA (dsRNA), is a natural cellular process associated with gene regulation[1]. Since the discovery of RNAi, scientists could use this instrument to deplete almost any of their interested genes not only as a tool in biological research but also as a therapeutic approach. RNAi uses a sequence-specific gene-silencing mechanism and is ultramost powerful. The general RNAi process could be artificially divided into 3 steps. First, short dsRNAs (,20?0 nucleotides) are generated by RNase III enzymes (either Dicer alone or a combination of Drosha and Dicer); second, these short dsRNAs are unwound and the strand with a thermodynamically less stable 59 end is preferentially loaded into the RNA-induced silencing complex (RISC) as the guide strand; third, RISC finds potential target RNAs and Argonaute (the key component of RISC) finally cleaves the target RNA containing a sequence homologous to the guide strand. There are many ways to induce RNAi for gene knockdown experiments [2], but the two most commonly used methods are chemically synthesized siRNAs or vector-based shRNAs. Compared to chemically synthesized siRNAs, vector-based shRNA expression achieves more sustained loss of function effect especially when it is embedded in the lentiviral vector. One of the widelyused shRNA expression vectors is pSuper described in 2002 [3]. It uses Pol III promoter H1 to transcribe a shRNA with a 21 bp (base pair) stem and 9 nt (nucleotide) loop structure. Furthermore, the first widely used genome-wide shRNA library utilized a lentiviral vector named pLKO.1-puro and this library has generated much of data that allowed for a better understanding of the diverse cellular processes associated with virology and cancer [4?]. We observed that the pLKO.1-puro vector possessed a unique palindromic loop (CTCGAG) different from other shRNA expression vectors such as pSuper [5]. This observation resulted in the hypothesis that a shRNA structure could be constructed using only a single long or two short oligonucleotides. We further describe a strategy for rapid cloning of multiple shRNAs which permits easier combination of the most efficient promoter-shRNA cassettes for the simultaneous knockdown of multiple genes or different targets of the same gene [8,9]. Here we gave proofs that our thought was feasible, and a shRNA could be constructed by only 1 long oligonucleotide or 2 short oligonucleotides with half the cost of conventional shRNA clone methods. Various parameters for the design of effective shRNAs based on our strategy were compared such as the palindromic loop sequences. Finally, the loop sequence “TTCTAGAA” was selected for shRNAs construction and then we gaveA Robust shRNA System Used for RNA Interferenceexamples that our method could apply to other genes such as the bacterial enzyme b-galactosidase (LacZ). This method was also used to successfully inhibit hepatitis B virus (HBV) antigen expression both in vitro and in vivo. 1407003 This approach is cost effective and more easily applied to many areas of basi.
