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An in vitro system for expression analysis of mutations of the β‐globin gene: validation and application to two mutations in the 5′ UTR
Author(s) -
Ho P. Joy,
SloaneStanley Jackie,
Athanassiadou Aglaia,
Wood William G.,
Thein Swee Lay
Publication year - 1999
Publication title -
british journal of haematology
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.907
H-Index - 186
eISSN - 1365-2141
pISSN - 0007-1048
DOI - 10.1046/j.1365-2141.1999.01659.x
Subject(s) - biology , gene , microbiology and biotechnology , untranslated region , gene expression , enhancer , globin , rna splicing , transcription (linguistics) , transfection , messenger rna , regulation of gene expression , genetics , rna , linguistics , philosophy
We describe the setting up of an in vitro expression system for the analysis of mutations of the β‐globin gene. The system is based on the stable transfection of a normal or mutated β‐globin gene into mouse erythroleukaemia (MEL) cells. The expression construct contains an A γ gene as an internal control and both globin genes are under the control of the HS2 element of the β LCR. The system enables analysis of transcription, RNA processing and transport, as well as mRNA stability. With non‐mutant genes, high‐level expression of both β and A γ genes is seen and both mRNAs are stable. The system was validated by comparing the expression of the β 654 thalassaemia splicing mutation in MEL cells with its well‐characterized expression in vivo . The level of the initial transcript, the proportion of abnormally spliced mRNA and its instability during erythroid cell maturation were all faithfully reproduced. The system was used to examine the mechanism by which two mutations in the β‐globin 5′ untranslated region (5′ UTR) result in β thalassaemia. Surprisingly, the mechanism appeared to differ in the two cases, with the C‐G substitution at position +33 affecting transcription, whereas the −T deletion at position +10 resulted in a translational defect. The stably transfected MEL cells, with an internal control and an endogenous enhancer, appear to be a valid and realistic experimental model, superior to transient expression studies. This system should find wide application in the analysis of the effects and mechanisms of gene inactivation in mutations affecting the β‐globin as well as other genes.