[The possible mechanisms underlying low expression of human beta-globin gene cloned in a retroviral vector].
Murine erythroleukemia (MEL) cell line was used as a model to evaluate the potential value of retroviral construct containing human beta-globin express io n cassette in gene therapy of beta-thalassemia and to explore possible mechanisms underlying low expression of retrovirally cloned human beta-globin gene. A recombinant retroviral vector was constructed, which harbored 2.0 kb beta-globin gene w it h a 374 bp deletion in intervening sequence II coupled with a mini locus control region (miniLCR) composed of DNaseI hypersensitive sites (HS) 2 and 3 from human beta-LCR. The recombinant retroviruses were generated from an established psi-2 producer cell line, and by transient transfection of amphotropic packaging cell l ine psi-A, respectively. The integrity of provirus in transduced MEL cells was determined using Southern blot, and the expression of transferred human beta-globin gene was detected using RNase protection assay. The structure of provirus was further analyzed by sequence analysis of PCR products from genomic DNA of MEL individual clone as template. The results demonstrated that the average expression of human beta-globin gene was (52.4-/+11.2)% (n=12) and (73.8-/+14.3)% (n=12, without copy-number determination), compared with that of endogenous murine alpha-globin ge ne, in MEL cells transduced with the recombinant retrovirus from transient transfection of psi-A and MEL cells transfected with the construct, respectively. In M EL cells transduced with virus from psi-2 producer cell line, however, the average expression was less than 3%. A point mutation was detected in HS2 of provirus i n MEL cell clone with low expression of human beta-globin gene. The possible mechanisms involved in low expression, including position effect, DNA methylation a nd RNA interference are discussed in addition to the point mutation.
Dong
Wen-Ji
WJ
National Laboratory of Medical Molecular Biology, Institute of Basic Medical Sciences, Chinese Academy of Medical Sciences and Peking Union Medical College, Beijing 100005, China. liudp@95777.com
Zu
Zhen-Xiang
ZX
Liu
De-Pei
DP
Hao
De-Long
DL
Guo
Zhi-Chen
ZC
Liang
Chih-Chuan
CC
D001483:Base Sequence; D015870:Gene Expression; D005822:Genetic Vectors / Q000235:genetics; D005914:Globins / Q000235:genetics*; D006801:Humans; D017354:Point Mutation; D012190:Retroviridae / Q000235:genetics*; D017384:Sequence Deletion; D014162:Transfection; D014407:Tumor Cells, Cultured
D005914:Globins