The 3'-end of the human beta-actin gene enhances activity of the beta-actin expression vector system: construction of improved vectors.
The human beta-actin promoter has been widely used to drive expression of genes of interest in mammalian cell lines and transgenic mice. The original form of the human beta-actin expression vector contains upstream sequences, 5'UTR (untranslated region) and intron 1 from the beta-actin gene linked to a three restriction site polylinker and SV40 (Simian Virus 40) 3'UTR. We have modified this vector now to contain the highly conserved beta-actin 3'UTR plus flanking region which replaces the SV40 sequences. An additional modification has removed the mRNA peripheral localization sequences present in the beta-actin 3'UTR. The new vectors also contain an improved polylinker. The activity of these two new vectors has been compared with that of the original vector and that of a vector using the popular cytomegalovirus (CMV) promoter. Mouse C2 myoblasts were transfected with each vector driving expression of enhanced green fluorescent protein (EGFP) and analyzed for EGFP mRNA levels. We find that both new vectors drive twice the level of mRNA accumulation of the original vector and over 30-times that of the CMV promoter. This suggests that these new vectors will provide a substantial elevation in levels of expression by virtue of inclusion of the beta-actin 3'UTR plus flanking region.
Qin
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Cell Biology Unit, Children's Medical Research Institute, Westmead, NSW, Australia.
Gunning
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D000199:Actins / Q000096:biosynthesis* / Q000235:genetics*; D000818:Animals; D001483:Base Sequence; D002460:Cell Line; D005822:Genetic Vectors; D049452:Green Fluorescent Proteins; D006801:Humans; D007438:Introns; D008164:Luminescent Proteins / Q000096:biosynthesis; D051379:Mice; D008969:Molecular Sequence Data; D018482:Muscle, Skeletal; D010957:Plasmids; D012333:RNA, Messenger / Q000032:analysis / Q000096:biosynthesis; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis; D012045:Regulatory Sequences, Nucleic Acid; D013539:Simian virus 40; D014162:Transfection / Q000379:methods
D000199:Actins; D008164:Luminescent Proteins; D012333:RNA, Messenger; D011993:Recombinant Fusion Proteins; D049452:Green Fluorescent Proteins