Sitting in one of the many waiting rooms of Diamniadio Children's Hospital (DCH),Aissatou Cisse patiently waits for her turn.That morning,she b.rought her two sick children from the Senegalese capital of Dakar all...Sitting in one of the many waiting rooms of Diamniadio Children's Hospital (DCH),Aissatou Cisse patiently waits for her turn.That morning,she b.rought her two sick children from the Senegalese capital of Dakar all the way to Diamniadio,a new city being built at the other end of the Dakar Region.展开更多
Objective To investigate the reversal of aberrant splicing of β thalassemia allele (IVS 2 654 C→T, β 654 ) by antisense RNA in vitro and in vivo.Methods The vector expressing antisense RNA which targeted a...Objective To investigate the reversal of aberrant splicing of β thalassemia allele (IVS 2 654 C→T, β 654 ) by antisense RNA in vitro and in vivo.Methods The vector expressing antisense RNA which targeted against the aberrant splice sites of β 654 pre mRNA was constructed in pcDNA3, and then used to repair the defective splicing of the mutant pre mRNA in an in vitro transcription and splicing system, as well as in HeLa β 654 cells and cultured β 654 erythroid cells by lipid mediated DNA transfection method. The effect of the antisense RNA was identified by RT PCR mediated mRNA quantitative assay as well as globin chain micro biosynthesis.Results The antisense RNA decreased the aberrant splicing product and restored the correct splicing pattern in vitro and in vivo efficiently. In the in vitro transcription and splicing system, the level of normally spliced mRNA [β/(β+β *)] increased from 0.25 to 0.60. In cultured HeLa β 654 cells, the level of β/(β+β *) increased from 0.07 to 0.43 on the 15th day after transfection. In cultured β 654 erythroid cells, the level of mRNA [β/(β+β *)] increased from 0.19 to 0.58 on the 8th day after transfection in β 654 /β 654 erythroid cells, from 0.02 to 0.38 in β 654 /β 41 42 erythroid cells, and from 0.45 to 0.83 in β 654 /βA erythroid cells, respectively. Correspondingly, the ratios of globin chain (β/α) biosynthesis increased from 0.16 to 0.52 on the 8th day after transfection in β 654 /β 654 erythroid cells, 0.05 to 0.36 in β 654 /β 41 42 erythroid cells, and 0.42 to 0.81 in β 654 /β A erythroid cells, respectively. The splicing pattern did not show significant changes as compared to the untreated, as well as to the control antisense fragment.Conclusions Antisense RNA transcribed from the expression vector described here could efficiently suppress the aberrant splicing pattern of β 654 mutant mRNA and restore the correct splicing pathway in vitro and in vivo, leading to the improvement of globin chain biosynthesis in thalassemic cells. Our antisense strategy provides an alternative approach to the gene therapy of β thalassemia.展开更多
The Hematology Oncology Center of Beijing Children's Hospital, affiliated to Capital Medical University is a unique center that integrates clinical care, scientific research, teaching, and training. It is staffed ...The Hematology Oncology Center of Beijing Children's Hospital, affiliated to Capital Medical University is a unique center that integrates clinical care, scientific research, teaching, and training. It is staffed by 269 doctors, nurses, and other health professionals, including one academician and 31 senior medical staff. In addition, 26 staff members are doctoral degree holders and 56 are master's degree holders. Dr. Yamei Hu and her team were the first to carry out clinical research on childhood leukemia in China from 1976.展开更多
World Journal of peer-reviewed journal published by Children's Hospital,Zhejiang University School of Medicine,Hangzhou,China.The joumal gives priority to reports of outstanding clinical and experimental works as ...World Journal of peer-reviewed journal published by Children's Hospital,Zhejiang University School of Medicine,Hangzhou,China.The joumal gives priority to reports of outstanding clinical and experimental works as weU as important contributions related to common problems of infants,children and adolescents worldwide.Submission ManuscriptS should be submi仕ed Via the submission system at,https://www.editorialmanager.com/wjop.The address of the editorial office is as follows:Children’s Hospital,Zhejiang UniVersity school of Medicine,3333 Binsheng Rd,Birljiang District,Hallgzhou 31 0052,Chilla.Email:wjpch@zju.edu.cn.展开更多
文摘Sitting in one of the many waiting rooms of Diamniadio Children's Hospital (DCH),Aissatou Cisse patiently waits for her turn.That morning,she b.rought her two sick children from the Senegalese capital of Dakar all the way to Diamniadio,a new city being built at the other end of the Dakar Region.
文摘Objective To investigate the reversal of aberrant splicing of β thalassemia allele (IVS 2 654 C→T, β 654 ) by antisense RNA in vitro and in vivo.Methods The vector expressing antisense RNA which targeted against the aberrant splice sites of β 654 pre mRNA was constructed in pcDNA3, and then used to repair the defective splicing of the mutant pre mRNA in an in vitro transcription and splicing system, as well as in HeLa β 654 cells and cultured β 654 erythroid cells by lipid mediated DNA transfection method. The effect of the antisense RNA was identified by RT PCR mediated mRNA quantitative assay as well as globin chain micro biosynthesis.Results The antisense RNA decreased the aberrant splicing product and restored the correct splicing pattern in vitro and in vivo efficiently. In the in vitro transcription and splicing system, the level of normally spliced mRNA [β/(β+β *)] increased from 0.25 to 0.60. In cultured HeLa β 654 cells, the level of β/(β+β *) increased from 0.07 to 0.43 on the 15th day after transfection. In cultured β 654 erythroid cells, the level of mRNA [β/(β+β *)] increased from 0.19 to 0.58 on the 8th day after transfection in β 654 /β 654 erythroid cells, from 0.02 to 0.38 in β 654 /β 41 42 erythroid cells, and from 0.45 to 0.83 in β 654 /βA erythroid cells, respectively. Correspondingly, the ratios of globin chain (β/α) biosynthesis increased from 0.16 to 0.52 on the 8th day after transfection in β 654 /β 654 erythroid cells, 0.05 to 0.36 in β 654 /β 41 42 erythroid cells, and 0.42 to 0.81 in β 654 /β A erythroid cells, respectively. The splicing pattern did not show significant changes as compared to the untreated, as well as to the control antisense fragment.Conclusions Antisense RNA transcribed from the expression vector described here could efficiently suppress the aberrant splicing pattern of β 654 mutant mRNA and restore the correct splicing pathway in vitro and in vivo, leading to the improvement of globin chain biosynthesis in thalassemic cells. Our antisense strategy provides an alternative approach to the gene therapy of β thalassemia.
文摘The Hematology Oncology Center of Beijing Children's Hospital, affiliated to Capital Medical University is a unique center that integrates clinical care, scientific research, teaching, and training. It is staffed by 269 doctors, nurses, and other health professionals, including one academician and 31 senior medical staff. In addition, 26 staff members are doctoral degree holders and 56 are master's degree holders. Dr. Yamei Hu and her team were the first to carry out clinical research on childhood leukemia in China from 1976.
文摘World Journal of peer-reviewed journal published by Children's Hospital,Zhejiang University School of Medicine,Hangzhou,China.The joumal gives priority to reports of outstanding clinical and experimental works as weU as important contributions related to common problems of infants,children and adolescents worldwide.Submission ManuscriptS should be submi仕ed Via the submission system at,https://www.editorialmanager.com/wjop.The address of the editorial office is as follows:Children’s Hospital,Zhejiang UniVersity school of Medicine,3333 Binsheng Rd,Birljiang District,Hallgzhou 31 0052,Chilla.Email:wjpch@zju.edu.cn.
