Normally, cellular responses to modified siRNAs or new siRNA delivery systems have been studied in group cell behavior by PCR, western blotting and fluorescence microscopy. In this study, we present a novel high-conte...Normally, cellular responses to modified siRNAs or new siRNA delivery systems have been studied in group cell behavior by PCR, western blotting and fluorescence microscopy. In this study, we present a novel high-content screening （HCS） strategy to evaluate a novel delivery system （named CLD） of siRNA therapeutics, with which both the content of intracellular siRNAs and changes in protein expressing levels have been quantified in group cells and cellular population. We also observed that with the better cell uptake, CLD provided siRNA therapeutics （siBraf） better antitumor capability. This novel strategy was proved to be with efficiency, accuracy and high competency to adherent cell lines, thus making siRNA research more simplified.展开更多
The objective of this study was to investigate the relationship between gene expression of nutrient（amino acid, peptide, sodium and proton） transporters in the small intestine and embryonic growth in domestic pigeon...The objective of this study was to investigate the relationship between gene expression of nutrient（amino acid, peptide, sodium and proton） transporters in the small intestine and embryonic growth in domestic pigeons（Columba livia）. One hundred and twenty-five fertilized eggs were randomly assigned into five groups and were incubated under optimal conditions（temperature of 38.1 °C and relative humidity of 55%）. Twenty embryos/birds from each group were sacrificed by cervical dislocation on embryonic day（E） 9, 11, 13, 15 and day of hatch（DOH）. The eggs, embryos（without yolk sac）, and organs（head, brain, heart, liver, lungs, kidney, gizzard, small intestine, legs, and thorax） were dissected, cleaned, and weighed. Small intestine samples were collected for RNA isolation. The m RNA abundance of intestinal nutrient transporters was evaluated by real-time reverse transcription-polymerase chain reaction（RT-PCR）. We classified these ten organs into four types according to the changes in relative weight during embryonic development. In addition, the gene expression of nutrient transporters was differentially regulated by embryonic day. The m RNA abundances of b^0,＋AT, EAAT3, y^＋LAT2, Pep T1, LAT4, NHE2, and NHE3 increased linearly with age, whereas m RNA abundances of CAT1, CAT2, LAT1, EAAT2, SNAT1, and SNAT2 were increased to higher levels on E9 or E11 and then decreased to lower levels until DOH. The results of correlation analysis showed that the gene expressions of b^0,＋AT, EAAT3, Pep T1, LAT4, NHE2, NHE3, and y^＋LAT2 had positive correlations with body weight（0.71〈correlation coefficient（CC）〈0.82, P〈0.0001）, while CAT1, CAT2, EAAT2, SNAT1, and SNAT2 had negative correlations with body weight（-0.86〈CC〈-0.64, P〈0.0001）. The gene expressions of b^0,＋AT, EAAT3, LAT4, Pep T1, NHE2, NHE3, and y^＋LAT2 showed positive correlations with intestinal weight（0.80〈CC〈0.91, P〈0.0001）, while CAT1, CAT2, and EAAT2 showed negative correlations with intestinal weight（-0.84〈CC〈-0.67, P〈0.0001）. It was concluded that the differences between growth trajectories of organs and gene expression of nutrient transporters in small intestine were due to their functional and physiological properties, which provided a comprehensive study of amino acid and peptide transporter m RNA in the small intestine during embryonic growth of pigeons.展开更多
To provide a highly efficient adenov iral vector Ad CMV hTGFβ1 for the study of gene therapy for reversion of the intervertebral disc degeneration.Methods: A newly developed recombinant adenoviral vector constr uctio...To provide a highly efficient adenov iral vector Ad CMV hTGFβ1 for the study of gene therapy for reversion of the intervertebral disc degeneration.Methods: A newly developed recombinant adenoviral vector constr uction system was used in the study. The cDNA of hTGFβ1 was first subcloned into a shuttle plasmid pShuttle CMV. The resultant plasmid was linearized by d ig esting with restriction endonuclease PmeI, and subsequently transformed into E .coli. BJ5183 cells with an adenoviral backbone plasmid pAdEasy 1. Recombinants were selected by kanamycin resistance and confirmed by restriction endonuclease analysis. Finally, the recombinant plasmid linearized by PmeI was transfected in to 293 cells. Recombinant adenoviruses were generated within 2 weeks. Results: The recombinant adenoviral plasmids were cut by BamHI and PacI respectively, and the diagnostic fragments appeared in 0.8 % agarose electrophoresis. The infected 293 cells showed evident cytopathic effect (CPE). The productions of PCR confirmed the presence of recombinant adenovirus. The exp ression of hTGFβ1 was verified by immunohistochemical staining. Conclusions: The successful generation of the adenoviral vector Ad CMV hTGFβ1 and the confirmation of the interest gene expression make it p ossible for the experimental study of the reversion of the intervertebral disc d egeneration by gene therapy.展开更多
基金Ministry of Science and Technology of China(Grant No.2012CB720604)NSFC(Grant No.20932001)
文摘Normally, cellular responses to modified siRNAs or new siRNA delivery systems have been studied in group cell behavior by PCR, western blotting and fluorescence microscopy. In this study, we present a novel high-content screening （HCS） strategy to evaluate a novel delivery system （named CLD） of siRNA therapeutics, with which both the content of intracellular siRNAs and changes in protein expressing levels have been quantified in group cells and cellular population. We also observed that with the better cell uptake, CLD provided siRNA therapeutics （siBraf） better antitumor capability. This novel strategy was proved to be with efficiency, accuracy and high competency to adherent cell lines, thus making siRNA research more simplified.
基金Project supported by the Spark Program of Guangdong,China(No.2012A020603012)
文摘The objective of this study was to investigate the relationship between gene expression of nutrient（amino acid, peptide, sodium and proton） transporters in the small intestine and embryonic growth in domestic pigeons（Columba livia）. One hundred and twenty-five fertilized eggs were randomly assigned into five groups and were incubated under optimal conditions（temperature of 38.1 °C and relative humidity of 55%）. Twenty embryos/birds from each group were sacrificed by cervical dislocation on embryonic day（E） 9, 11, 13, 15 and day of hatch（DOH）. The eggs, embryos（without yolk sac）, and organs（head, brain, heart, liver, lungs, kidney, gizzard, small intestine, legs, and thorax） were dissected, cleaned, and weighed. Small intestine samples were collected for RNA isolation. The m RNA abundance of intestinal nutrient transporters was evaluated by real-time reverse transcription-polymerase chain reaction（RT-PCR）. We classified these ten organs into four types according to the changes in relative weight during embryonic development. In addition, the gene expression of nutrient transporters was differentially regulated by embryonic day. The m RNA abundances of b^0,＋AT, EAAT3, y^＋LAT2, Pep T1, LAT4, NHE2, and NHE3 increased linearly with age, whereas m RNA abundances of CAT1, CAT2, LAT1, EAAT2, SNAT1, and SNAT2 were increased to higher levels on E9 or E11 and then decreased to lower levels until DOH. The results of correlation analysis showed that the gene expressions of b^0,＋AT, EAAT3, Pep T1, LAT4, NHE2, NHE3, and y^＋LAT2 had positive correlations with body weight（0.71〈correlation coefficient（CC）〈0.82, P〈0.0001）, while CAT1, CAT2, EAAT2, SNAT1, and SNAT2 had negative correlations with body weight（-0.86〈CC〈-0.64, P〈0.0001）. The gene expressions of b^0,＋AT, EAAT3, LAT4, Pep T1, NHE2, NHE3, and y^＋LAT2 showed positive correlations with intestinal weight（0.80〈CC〈0.91, P〈0.0001）, while CAT1, CAT2, and EAAT2 showed negative correlations with intestinal weight（-0.84〈CC〈-0.67, P〈0.0001）. It was concluded that the differences between growth trajectories of organs and gene expression of nutrient transporters in small intestine were due to their functional and physiological properties, which provided a comprehensive study of amino acid and peptide transporter m RNA in the small intestine during embryonic growth of pigeons.
文摘To provide a highly efficient adenov iral vector Ad CMV hTGFβ1 for the study of gene therapy for reversion of the intervertebral disc degeneration.Methods: A newly developed recombinant adenoviral vector constr uction system was used in the study. The cDNA of hTGFβ1 was first subcloned into a shuttle plasmid pShuttle CMV. The resultant plasmid was linearized by d ig esting with restriction endonuclease PmeI, and subsequently transformed into E .coli. BJ5183 cells with an adenoviral backbone plasmid pAdEasy 1. Recombinants were selected by kanamycin resistance and confirmed by restriction endonuclease analysis. Finally, the recombinant plasmid linearized by PmeI was transfected in to 293 cells. Recombinant adenoviruses were generated within 2 weeks. Results: The recombinant adenoviral plasmids were cut by BamHI and PacI respectively, and the diagnostic fragments appeared in 0.8 % agarose electrophoresis. The infected 293 cells showed evident cytopathic effect (CPE). The productions of PCR confirmed the presence of recombinant adenovirus. The exp ression of hTGFβ1 was verified by immunohistochemical staining. Conclusions: The successful generation of the adenoviral vector Ad CMV hTGFβ1 and the confirmation of the interest gene expression make it p ossible for the experimental study of the reversion of the intervertebral disc d egeneration by gene therapy.
