Stable expression of antibiotic resistance genes using a promoter fragment of the U1 snRNA gene

• Published in: Molecular biology reports Vol. 17; no. 2; pp. 101 - 114
• Main Authors: ASSELBERGS, F. A. M, PRONK, R
• Format: Journal Article
• Language: English
• Published: Dordrecht Kluwer 01-02-1993
• Subjects: Analytical, structural and metabolic biochemistry; Animals; Base Sequence; Biological and medical sciences; Bleomycin - pharmacology; Cell Line; DNA - genetics; DNA Transposable Elements; Drug Resistance, Microbial - genetics; Enhancer Elements, Genetic; Fundamental and applied biological sciences. Psychology; Gene Expression; Genetic Vectors; Humans; Hygromycin B - pharmacology; Molecular Sequence Data; Nucleic acids; Plasmids; Promoter Regions, Genetic; Rna, ribonucleoproteins; RNA, Small Nuclear - genetics; Transformation, Genetic; Resistance; Ovary; Vertebrata; Antibiotic; Enhancer sequence; Mammalia; Transfection; Transcription promoter; Rodentia; Gene expression; Hamster; CHO cell line
• ISSN: 0301-4851; 1573-4978
• DOI: 10.1007/BF00996217

As U1 snRNA is produced in all mammalian cell types, antibiotic resistance genes driven by this promoter would be ideally suited as genetic selection markers. However, although the U1 snRNA gene is transcribed by RNA polymerase II, its native product is not a messenger RNA, but a splicing cofactor. To test whether this promoter could nevertheless produce a functional mRNA, sensitive reporter genes expressing resistance to the antibiotics hygromycin-B and bleomycin were constructed with either the U1 snRNA promoter or the SV40 early promoter. Resistant cell lines could only be obtained with constructs equipped with a functional polyadenylation signal. With the U1 snRNA promoter about three times fewer colonies were obtained than with the SV40 early promoter. Another potential advantage of the U1 snRNA promoter is that, in contrast to the promoters commonly used to express genetic selection markers, the enhancer-like element contained in the U1 snRNA promoter had only a minimal stimulative effect, only detectable with the most sensitive methods, on an adjacent mRNA-producing gene. The U1 snRNA promoter was also capable of expressing bleomycin resistance in the context of a self-inactivating retrovirus vector, whereby it was discovered that the mouse 3T3 cells used in this experiment were 10 times more sensitive to bleomycin than human or hamster cell lines.