Suppress HBsAg and HBeAg expression in the absence of detectable shRNA-related interferon responses (Fig. S5). However, we didn’t observe differences in knockdown efficiency enhancement following the combination of 2 or 3 Teriparatide shRNAs in vitro, suggesting that this phenomenon might be caused by promoter disturbances or competition. An indirect experiment using the 3′-UTR luciferase assay was used to demonstrate that the silencing activity of AS139-1819-3172 were indeed some lower comparing with AS139, AS1819 and 3172 respcetively (Fig. S6). We also found that the co-transfected multiple shRNA constructs had better silencing activity than the shRNA plasmid with multiple shRNAs when the same amount of each shRNA scaffold was used (Fig. S7). This verified our speculation that multiple H1 promoter in the same vector may interference with each other. Regardless, the three connected shRNA structure was shown to efficiently inhibit HBV antigens expression especially in vivo.shRNA system demonstrated here gave us an 11089-65-9 acceptable and more economical way to knockdown genes. At the last, two different shRNA clone methods were compared and the two short oligonucleotides based shRNA construction method was more efficient than the single long oligonucleotide based strategy. The single long oligo would be prone to form hairpin structure itself, and this could affect the double strands formation. We think this is the reason why the two short oligos method was superior to the single long oligo strategy. With shorter oligos, the error rate of synthesis was also decreased.ConclusionsWe describe a simple and robust shRNA construction system that will enable users to easily construct single or multiple shRNAs efficiently at a low cost. Using this method, we systemically screened the target sites for HBV knockdown and successfully depressed HBV antigen expression with connected multiple shRNAs both in vitro and in vivo. The method described here provides an inexpensive and powerful new tool with the potential of down regulating gene expression that can be applied to a varietyFigure 4. Suppression of two reporter genes by the shRNAs cloned with our methods. (A) HepG2 cells were seeded in 24-well plates and cotransfected with 200 ng of pAAV-LacZ, 200 ng shRNA plasmid and 100 ng pSEAP2-Control (used as a normalization control). LacZ was stained and photographed 48 h after cotransfection of HepG2 cells. Magnification 6200. The scale 18325633 bar represents 1 mm. (B) The same procedure described above was carried out using pCMV-Gluc in place of pAAV-LacZ. After 48 h, Gluc activity was determined. An shRNA scaffold (targeted to GUCUCCACGCGCAGUACAUUU) irrelevant to any known human or mouse gene sequence was designed as the negative control (“neg”) [23,24]. Means and standard deviations were generated from 3 independent experiments. doi:10.1371/journal.pone.0056110.gA Robust shRNA System Used for RNA InterferenceFigure 5. Screening of shRNAs for significant suppression of HBsAg and HBeAg. (A) shRNAs targeting to the conserved regions of HBV genome were designed and illustrated. The numbers represent nucleotide (nt) coordinates relative to the HBV (genotype B) pgRNA start site. (B) HepG2 cells were seeded in 24-well plates and cotransfected with 200 ng of pHBV1.31, 200 ng shRNA plasmid and 100 ng pSEAP2-Control per well. The HBsAg and HBeAg concentrations in cell supernatants were detected 48 h post transfection. Means and standard deviations were generated from 3 independent expe.Suppress HBsAg and HBeAg expression in the absence of detectable shRNA-related interferon responses (Fig. S5). However, we didn’t observe differences in knockdown efficiency enhancement following the combination of 2 or 3 shRNAs in vitro, suggesting that this phenomenon might be caused by promoter disturbances or competition. An indirect experiment using the 3′-UTR luciferase assay was used to demonstrate that the silencing activity of AS139-1819-3172 were indeed some lower comparing with AS139, AS1819 and 3172 respcetively (Fig. S6). We also found that the co-transfected multiple shRNA constructs had better silencing activity than the shRNA plasmid with multiple shRNAs when the same amount of each shRNA scaffold was used (Fig. S7). This verified our speculation that multiple H1 promoter in the same vector may interference with each other. Regardless, the three connected shRNA structure was shown to efficiently inhibit HBV antigens expression especially in vivo.shRNA system demonstrated here gave us an acceptable and more economical way to knockdown genes. At the last, two different shRNA clone methods were compared and the two short oligonucleotides based shRNA construction method was more efficient than the single long oligonucleotide based strategy. The single long oligo would be prone to form hairpin structure itself, and this could affect the double strands formation. We think this is the reason why the two short oligos method was superior to the single long oligo strategy. With shorter oligos, the error rate of synthesis was also decreased.ConclusionsWe describe a simple and robust shRNA construction system that will enable users to easily construct single or multiple shRNAs efficiently at a low cost. Using this method, we systemically screened the target sites for HBV knockdown and successfully depressed HBV antigen expression with connected multiple shRNAs both in vitro and in vivo. The method described here provides an inexpensive and powerful new tool with the potential of down regulating gene expression that can be applied to a varietyFigure 4. Suppression of two reporter genes by the shRNAs cloned with our methods. (A) HepG2 cells were seeded in 24-well plates and cotransfected with 200 ng of pAAV-LacZ, 200 ng shRNA plasmid and 100 ng pSEAP2-Control (used as a normalization control). LacZ was stained and photographed 48 h after cotransfection of HepG2 cells. Magnification 6200. The scale 18325633 bar represents 1 mm. (B) The same procedure described above was carried out using pCMV-Gluc in place of pAAV-LacZ. After 48 h, Gluc activity was determined. An shRNA scaffold (targeted to GUCUCCACGCGCAGUACAUUU) irrelevant to any known human or mouse gene sequence was designed as the negative control (“neg”) [23,24]. Means and standard deviations were generated from 3 independent experiments. doi:10.1371/journal.pone.0056110.gA Robust shRNA System Used for RNA InterferenceFigure 5. Screening of shRNAs for significant suppression of HBsAg and HBeAg. (A) shRNAs targeting to the conserved regions of HBV genome were designed and illustrated. The numbers represent nucleotide (nt) coordinates relative to the HBV (genotype B) pgRNA start site. (B) HepG2 cells were seeded in 24-well plates and cotransfected with 200 ng of pHBV1.31, 200 ng shRNA plasmid and 100 ng pSEAP2-Control per well. The HBsAg and HBeAg concentrations in cell supernatants were detected 48 h post transfection. Means and standard deviations were generated from 3 independent expe.
Related Posts
Wever, since the majority of subjects included in most previous studies
Wever, since the majority of subjects included in most previous studies were ESRD patients on hemodialysis (HD), little is known about the prevalence, natural history, and prognostic value of vascular calcification in peritoneal dialysis (PD) patients. In the present study, we investigated the prevalence of AoAC at PD initiation and the frequencies of AoAC progression […]
Mmp Vs Mvp
Doable modulation of NMDA receptors. A single oral administration of guanosine (0.05 5 mg/kg) in mice resulted in antidepressant-like activity in the forced swimming and tail suspension tests [111]. To date there are no research of chronic use of guanosine in depression. Increasing adult neurogenesis is a promising line of study against depression (to get […]
Ranch, 21 Jun 1885, C.R.Orcutt 1276 (DS, DS, US). 63 mi SE of
Ranch, 21 Jun 1885, C.R.Orcutt 1276 (DS, DS, US). 63 mi SE of Ensenada, 2? mi upstream of Disitertide site Rincon, 4.5 mi NE of Santa Catarina, canyon, 4300 ft [1310 m] 22 Apr 1962, R.E.Broder 772 (DS, US). 4 1/2 mi S of Portezuelo de Jamau, N of Cerro 1905, ca. 31?4’N, 115?6’W, 1775 […]