[Construction of recombinant vector expressing ALAS2 gene in X-linked sideroblastic anemia].
X-linked sideroblastic anemia (XLSA) is caused by mutations of erythroid-specific 5-aminolevulinate synthetase (ALAS2) gene. In this study a eukaryotic expression vector of ALAS2 was constructed and transfected into eukaryotic cells to observe the expression of ALAS2 gene. The full length cDNA of ALAS2 gene was inserted into plasmid pDs-red2-N1, named pDs-red2-N1/ALAS2. Then, the vector was transfected into K562 cells via electroporation. At 48 hours after transfection, total RNA from K562 cells was extracted, expressions of ALAS2 gene and protein with red fluorescence in the K562 cells were detected by RT-PCR and flow cytometry, respectively. The vector was also transfected into COS 7 cells via liposome. Both mRNA and protein expression in COS7 cells were detected by RT-PCR and fluorescence microscopy. The result showed that after the pDs-red2-N1/ALAS2 eukaryotic expression vector was digested by KpnI and BamHI, two fragments of 4 700 bp and 1 764 bp were displayed by electrophoresis on agarose gel. Sequence method confirmed that the sequence was correct. RT-PCR amplified the total RNA extracted from the transfected K562 and COS7 cells, and could find mRNA of ALAS2 gene that can't be found in K562 and COS7 cells usually. The expressions of both fluorescein and ALAS2 were significantly increased. The percentage of positive cells reached about 19.2% and 10.7%, respectively. ALAS2 expression lasted for 10 days in COS7 cells and the peak was at the third day. It is concluded that the eukaryotic expression vector of ALAS2 gene is successfully constructed; K562 and COS7 cells transfected with the vector via electroporation and liposome can express ALAS2 protein. So, the vector has the potential in gene replacement and can be used for patients with XLSA in future.
Wang
Yi-Qun
YQ
Department of Hematology, The First Hospital, Peking University, Beijing 100034, China.
Zhu
Ping
P
Shi
Yong-Jin
YJ
Gu
Jiang-Ying
JY
Bu
Ding-Fang
DF
Liu
Hui
H
Zhang
Ying
Y
D000624:5-Aminolevulinate Synthetase / Q000235:genetics*; D000756:Anemia, Sideroblastic / Q000235:genetics* / Q000628:therapy; D000818:Animals; D019556:COS Cells; D041321:Chromosomes, Human, X; D008040:Genetic Linkage; D015316:Genetic Therapy; D005822:Genetic Vectors; D006801:Humans; D020014:K562 Cells; D008856:Microscopy, Fluorescence; D020133:Reverse Transcriptase Polymerase Chain Reaction
D000624:5-Aminolevulinate Synthetase; C480079:ALAS2 protein, human