As a means of generating linked shRNAs. doi:10.1371/journal.pone.0056110.gpc/pgRNA and HBeAg expression dose-dependently (Fig. S2 and S3). We next connected these shRNAs by sub-cloning and compared different combination knockdown efficiencies both in vitro and in vivo. As illustrated in Fig. 6B, the combination of AS139, AS1819 and AS3172 resulted in the efficient suppression of HBV antigen expression in vitro and the best inhibition rate in vivo (above 90 , Fig. 6C and 6D).DiscussionIn this study, we have constructed a shRNA vector with several characteristics for its better and easier use. First, we Elbasvir site employed the H1 RNA polymerase III promoter instead of U6 whose toxicity has been previously identified [13]. The Pol III H1 promoter has a well-defined transcription start site proven to be more flexible than the U6 promoter with regard to +1 sequence changes. Second, proper isocaudomers (Bam HI and Bgl II) were used for easier cascade connected shRNAs construction. Third, the CMVemGFP Nazartinib site cassette was used to track shRNA transfection. This cassette could be replaced easily by other therapeutic genes as a means of overexpressing one gene while concomitantly knocking down another gene. The resulting vector was named pshOK-basic (overexpression and knockdown). For efficient and lower-cost shRNA construction, several routes was reported previously including the one-oligonucleotide method combined with PCR [14] or the four short oligonucleotides based strategy [15]. The most significant advantage to the method described in this study is that only single long (without PCR) or two short oligonucleotides were required for cloning shRNAs. To accomplish this goal, a unique palindromic shRNA scaffold was screened and optimized. The terminal sequence of the Hpromoter was first changed to AAA for shRNA clone with restriction enzyme Sap I. Next, several shRNAs with different palindromic loops were cloned, compared and carefully selected based on their respective knockdown efficiencies. Previously, various loop sequences were used and systematically investigated by different investigators, but these results were inconsistent [16,17]. In general, the shRNAs with a relative long open loop sequence would have greater silencing activity than the corresponding shRNA with a shorter complementary loop sequence. But the shRNAs with only a 3-nt loop were also reported for their good knockdown efficiency, and the well-known pLKO.1-puro vector possessed a unique 6-nt palindromic loop (CTCGAG). In this study, we demonstrated that most of shRNAs with a more than 4-nt palindromic loop would have a relative good silencing activity. The inferior performance of shRNAs with a 4-nt loop may be due to their inefficient processing by Dicer [17]. Due to its palindromic nature, there may be some contribution of this loop sequence to the stem portion of the shRNA molecule. Thus if the sense-loop-antisense shRNAs were designed by our method, the 5′-terminal of the antisense sequence would be changed by Dicer procession and possibly interfere with the target recognition. In addition, according to the latest research progress, the imperfect specificity of Dicer itself was also contributed to the miRNA and siRNA length heterogeneity [18,19]. This was part of the reasons why the antisense-loop-sense shRNA scaffold was chosen in this paper. In addition, this shRNA system was also compared with the popular used 1407003 vector pSuper and the results indicated that a relative long and open loop w.As a means of generating linked shRNAs. doi:10.1371/journal.pone.0056110.gpc/pgRNA and HBeAg expression dose-dependently (Fig. S2 and S3). We next connected these shRNAs by sub-cloning and compared different combination knockdown efficiencies both in vitro and in vivo. As illustrated in Fig. 6B, the combination of AS139, AS1819 and AS3172 resulted in the efficient suppression of HBV antigen expression in vitro and the best inhibition rate in vivo (above 90 , Fig. 6C and 6D).DiscussionIn this study, we have constructed a shRNA vector with several characteristics for its better and easier use. First, we employed the H1 RNA polymerase III promoter instead of U6 whose toxicity has been previously identified [13]. The Pol III H1 promoter has a well-defined transcription start site proven to be more flexible than the U6 promoter with regard to +1 sequence changes. Second, proper isocaudomers (Bam HI and Bgl II) were used for easier cascade connected shRNAs construction. Third, the CMVemGFP cassette was used to track shRNA transfection. This cassette could be replaced easily by other therapeutic genes as a means of overexpressing one gene while concomitantly knocking down another gene. The resulting vector was named pshOK-basic (overexpression and knockdown). For efficient and lower-cost shRNA construction, several routes was reported previously including the one-oligonucleotide method combined with PCR [14] or the four short oligonucleotides based strategy [15]. The most significant advantage to the method described in this study is that only single long (without PCR) or two short oligonucleotides were required for cloning shRNAs. To accomplish this goal, a unique palindromic shRNA scaffold was screened and optimized. The terminal sequence of the Hpromoter was first changed to AAA for shRNA clone with restriction enzyme Sap I. Next, several shRNAs with different palindromic loops were cloned, compared and carefully selected based on their respective knockdown efficiencies. Previously, various loop sequences were used and systematically investigated by different investigators, but these results were inconsistent [16,17]. In general, the shRNAs with a relative long open loop sequence would have greater silencing activity than the corresponding shRNA with a shorter complementary loop sequence. But the shRNAs with only a 3-nt loop were also reported for their good knockdown efficiency, and the well-known pLKO.1-puro vector possessed a unique 6-nt palindromic loop (CTCGAG). In this study, we demonstrated that most of shRNAs with a more than 4-nt palindromic loop would have a relative good silencing activity. The inferior performance of shRNAs with a 4-nt loop may be due to their inefficient processing by Dicer [17]. Due to its palindromic nature, there may be some contribution of this loop sequence to the stem portion of the shRNA molecule. Thus if the sense-loop-antisense shRNAs were designed by our method, the 5′-terminal of the antisense sequence would be changed by Dicer procession and possibly interfere with the target recognition. In addition, according to the latest research progress, the imperfect specificity of Dicer itself was also contributed to the miRNA and siRNA length heterogeneity [18,19]. This was part of the reasons why the antisense-loop-sense shRNA scaffold was chosen in this paper. In addition, this shRNA system was also compared with the popular used 1407003 vector pSuper and the results indicated that a relative long and open loop w.
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