High-copy expression vector based on amplification-promoting sequences.
We describe a new vector system that allows efficient expression of heterologous proteins in transformed mouse L fibroblasts. This is due to its persistence at high copy numbers, achieved by a 370-bp amplification promoting element (muNTS1) derived from the nontranscribed spacer of murine rDNA. Copy number determination showed that this sequence mediates a 40- to 800-fold amplification of the vector DNA in transfected L cells. High copy number was accompanied by increased expression levels of the reporter gene secreted alkaline phosphatase (SEAP). Analyzing the structural organization of multicopy plasmid DNA in mouse L cells revealed that plasmid DNA is integrated as reiterated head-to-tail concatamers into the chromosomal DNA. The vector described here can be used as a versatile high-copy expression system for heterologous proteins overcoming any limitation to enzyme-deficient cell lines.
Institute of Biochemistry, University of Würzburg, Germany.
C
C
Hemann
E
E
Gärtner
U H
UH
Weidle
F
F
Grummt
D000469:Alkaline Phosphatase / Q000235:genetics / Q000378:metabolism; D000818:Animals; D004275:DNA, Ribosomal / Q000235:genetics; D005784:Gene Amplification; D017930:Genes, Reporter; D005822:Genetic Vectors; D007739:L Cells; D051379:Mice; D010957:Plasmids; D013937:Thymidine Kinase / Q000235:genetics; D014162:Transfection; D014170:Transformation, Genetic
D004275:DNA, Ribosomal; D013937:Thymidine Kinase; D000469:Alkaline Phosphatase