The effect of intron sequences on expression levels of Ig cDNAs.
Several cDNA expression vectors were constructed and tested by stable transfection into a murine lymphoid cell line in order to compare secretion rates of a human immunoglobulin (Ig) light chain (LC). When the cDNA was under transcriptional control of the SV40 promoter and enhancer and preceded by the SV40 19S late mRNA intron, a weak LC production was detected. Secretion rate was not improved by replacing the SV40 promoter and enhancer by a combination of a murine Ig heavy chain (HC) gene promoter and enhancer even with insertion of additional Ig enhancers. In contrast, replacement of the 19S intron by a large intron derived from a human Ig HC gene and containing the intronic enhancer dramatically increased the secretion rate. High-level production was also obtained with the same enhancer-containing intron placed downstream from the LC cDNA. Stable transfectants were obtained that secreted the human LC in amounts comparable to those obtained with Ig genes. Our results suggest that the SV40 19S late mRNA intron used in several expression vectors is not appropriate when the purpose is to produce large amounts of antibody molecules. By providing transcriptional, splicing and polyadenylation signals, the presently described vectors will be useful for easy cloning and high-level expression in lymphoid cells of cDNAs or PCR products encoding antibody molecules.
Khamlichi
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Laboratoire d'Immunologie et Génétique, CNRS URA 1172, IBMIG, Faculté des Sciences, Poitiers, France.
Rocca
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Cogné
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D000818:Animals; D016887:Cardiopulmonary Resuscitation; D002460:Cell Line; D018076:DNA, Complementary; D004742:Enhancer Elements, Genetic; D015870:Gene Expression; D005803:Genes, Immunoglobulin; D005822:Genetic Vectors; D006801:Humans; D007143:Immunoglobulin Heavy Chains / Q000235:genetics; D007147:Immunoglobulin Light Chains / Q000096:biosynthesis* / Q000235:genetics; D007438:Introns; D008214:Lymphocytes; D051379:Mice; D011401:Promoter Regions, Genetic; D015183:Restriction Mapping; D013539:Simian virus 40 / Q000235:genetics; D014158:Transcription, Genetic; D014162:Transfection
D018076:DNA, Complementary; D007143:Immunoglobulin Heavy Chains; D007147:Immunoglobulin Light Chains