Efficiency of introns from various origins in fish cells.
Several vectors containing (1) regulatory regions from Rous sarcoma virus (RSV), human cytomegalovirus (CMV), and herpes simplex thymidine kinase (TK); (2) introns from early or late SV40 genes and from trout growth hormone gene (tGH); (3) chloramphenicol acetyltransferase gene (CAT); and (4) transcription terminators from SV40 were transfected into carp EPC cells, salmon CHSE cells, tilapia TO2 cells, quail QT6 cells, and hamster CHO cells. CAT activity was measured in extracts from several cell lines 3 days after transfection and in the fish EPC stable clones. The CMV and RSV promoters were the most potent in all cell types. The intron from late SV40 genes (VP1 intron) worked properly in QT6 and CHO cells but not in EPC and very weakly in TO2 cells. The tGH intron was efficient in all cell types but preferentially in fish cells. The small t intron from SV40 was processed in all cell types. The small t and, to a lesser extent, the tGH introns amplified expression of cat gene in stable clones, in comparison to the transiently transfected cells. These results indicate that elements from mammalian genes may not be properly recognized by the fish cellular machinery and in an unpredictable manner. This finding suggests that vectors prepared to express foreign genes in transfected cultured fish cells and transgenic fish should preferably contain DNA sequences from fish genes or, alternatively, those sequences from mammalian genes that have been previously proved to be compatible with the fish cellular machinery.
Unité de Différenciation Cellulaire, Institut National de la Recherche Agronomique, Jouy-en-Josas, France.
O H
OH
Bétancourt
J
J
Attal
M C
MC
Théron
C
C
Puissant
L M
LM
Houdebine
D000818:Animals; D001358:Avian Sarcoma Viruses / Q000235:genetics; D001483:Base Sequence; D015152:Blotting, Northern; D002478:Cells, Cultured; D015500:Chloramphenicol O-Acetyltransferase / Q000096:biosynthesis / Q000235:genetics; D003587:Cytomegalovirus / Q000235:genetics; D005399:Fishes / Q000235:genetics*; D005786:Gene Expression Regulation / Q000502:physiology*; D005814:Genes, Viral; D005822:Genetic Vectors / Q000502:physiology*; D007438:Introns / Q000502:physiology*; D008969:Molecular Sequence Data; D016133:Polymerase Chain Reaction; D011401:Promoter Regions, Genetic / Q000502:physiology; D013539:Simian virus 40 / Q000235:genetics; D013696:Temperature; D014162:Transfection / Q000379:methods*
D015500:Chloramphenicol O-Acetyltransferase