Mendelian transmission, structure and expression of transgenes following their injection into the cytoplasm of trout eggs
- 62 Downloads
High frequencies of mosaic transgenic trout are generated when linear DNA fragments are injected into the egg cytoplasm. The fact that these animals transmit the foreign DNA to a minority of their offspring suggests that their germline is also mosaic, but it may also indicate that some transgenes are lost. We have produced seven F2 families from wild-type females mated with F1 transgenic males. In all of them, the ratio of transgenics was Mendelian, and classical Southern analyses clearly showed the segregation of one or two foreign loci. They also provide firm arguments for gene integration in families carrying single-copy transgenes. Images obtained by Southern blotting strongly suggest the presence of extrachromosomal linear concatemers in two other families. However, this hypothesis was contradicted by further analyses usingin situ hybridization on chromosomes and with pulse field gel electrophoresis (PFGE). The reconciliation of these apparently contradictory observations is allowed by a model in which integrated concatemers adopt hairpin loop structures at the junction of inversely oriented copies. Further experiments with denaturing electrophoresis, which opens such secondary structures, provides additional support for this model. In this stable gene transfer system accompanied by chromosomal integration, the CAT reporter gene was expressed from the promoter/enhancer of human cytomegalovirus early genes, in all tissues examined in F0 fishes, as well as in the F1 offsprings of nine males. This indicates that the palindromic structures of some of the transgenes do not prevent their expression.
Keywordstransformation integration expression DNA structure fish trout
Unable to display preview. Download preview PDF.
- Bearzotti, M., Perrot, E., Michard-Vanhée, C., Jolivet, G., Attal, J., Théron, M.C., Puissant, C., Grabowski, H., Dréano, M., Kopchick, J.J., Powell, R., Gannon, F., Houdebine, L.M. and Chourrout D. (1992) The expression of various gene constructs transfected into fish cells.J. Biotech (in press).Google Scholar
- Chourrout, D. (1988) Induction of gynogenesis, triploidy and tetraploidy in fish.ISI Atlas of Sciences: Animal and Plant Sciences Section 2, 65–70.Google Scholar
- Fletcher, G.L. and Davies, P.L. (1991) Transgenic fish for aquaculture. In: Setlow, J.K. ed.,Genetic Engineering; Principles and Techniques,13, 331–70.Google Scholar
- Kessler, C., Höltke H.J., Seibl, R., Burg, J. and Mühlegger, K. (1990) Non-radioactive labelling and detection of nucleic acids. I. A novel DNA labelling and detection system based on digoxigenin: anti-digoxigenin elisa principle (digoxigenin system).Biol. Chem. Hoppe-Seyler 371, 917–27.PubMedGoogle Scholar
- Sambrook, J., Fritsch, E.F. and Maniatis, T. (1989)Molecular Cloning: a Laboratory Manual. Cold Spring Harbor, NY: Cold Spring Harbor Laboratory Press.Google Scholar
- Seibl, R., Höltke, H.J., Rüger, R., Meindl, A., Zachau H.G., Rasshoffer, R., Roggendorf, M., Wolf, H., Arnold, N., Weinberg, J. and Kessler, C. (1990) Non radioactive labelling and detection of nucleic acids. III. Applications of the digoxigenin system.Biol. Chem. Hoppe-Seyler 371, 939–51.PubMedGoogle Scholar
- Shears, M.A., Fletcher, G.L., Hew, C.L., Gautheir, S. and Davies, P.L. (1991) Transfer, expression and stable inheritance of antifreeze protein genes in atlantic salmon (Salmo salar).Mol. Mar. Biol. Biotech. 1, 58–63.Google Scholar