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[Expression of human betaIVS- II -nt 654 C --> T allele in HeLa cell]
1Shanghai Institute of Medical Genetics, Shanghai Children's Hospital, Shanghai 200040.
Objective:
To establish a cell model expressing human betaIVS- II -nt 654C --> T allele (beta654 mutant).
Methods:
DNA fragment of entire beta-globin gene encompassing the codon region and poly(A) signal of the allele was amplified by long PCR from the genomic DNA of a homozygote with beta65.4 mutation. The amplified fragments were cloned into the Hind III and Xba I sites of the pcDNA3.1 vector. After reidentification of the clone with beta654 mutant by DNA sequencing, the recombinant plasmid was transfected into HeLa cell using liposome method. The expression of the beta654 mutant gene in the transfected cells was detected by RT-PCR.
Results:
Both the normally processed beta globin mRNA (183bp) and aberrant processed beta globin mRNA (256bp) were identified in the transfected HeLa cells, while no RT-PCR product was detected in the controls.
Conclusion:
The transfected HeLa cells can express human beta654 allele.
