Major Considerations in Performing Transgenic Experiments

When plasmid DNA is injected into zebrafish embryos, it may meet with several different fates. It may replicate and persist in the cell and its descendants for several cell divisions. It may integrate into the chromosomal DNA of

From: Methods in Molecular Biology, Vol. 97: Molecular Embryology: Methods and Protocols Edited by: P. T. Sharpe and I. Mason © Humana Press Inc., Totowa, NJ

the cell and generate a clone of transgenic somatic cells. It may integrate into the chromosomal DNA of a progenitor germ cell and generate a group of germ cells that will pass the transgene on to the F1 progeny. Alternatively, it may be lost from the embryo.

The first two fates lead to embryos and fish that are mosaic with respect to the presence of the plasmid DNA. If the plasmid carries a reporter gene fused to a promoter, which is active in zebrafish, then injected embryos can be assayed for the presence of transgene product. The promoter may be one that is active in all cells or one that will only drive expression of the reporter gene in a subset of cells in a spatially and temporally restricted manner. In either case, the pattern of transgene expression is transient and will be different in each injected embryo, since it depends on which cells in the embryo retain the plasmid DNA.

If the injected DNA is incorporated into the chromosomal DNA of the germ cells, the fish has the potential to pass the transgene on to its progeny. Such founder fish are expected to be mosaic in their germline, since not all germ cell precursors may have integrated the injected DNA. The fish may also be mosaic for somatic integration of the DNA. If all the germ cells carry a single insertion of the injected DNA, then 50% of the progeny are expected to inherit the transgene. However, if the germline is mosaic, the proportion of transgenic F1 progeny depends on the degree of mosaicism. Germline transmission may be detected by sampling DNA from either somatic or germ cells of F1 fish, since the fish should be hemizygous for the transgene in all cells. This is done by performing a polymerase chain reaction (PCR) amplification with DNA extracted from a fin biopsy using primers specific to the transgene construct. It is important to note that this alone does not confirm the DNA has integrated into a chromosome. Southern analysis will show the number of transgene copies present and also the presence of junction fragments derived from the point of insertion. Although the presence of junction fragments is indicative of integration into the genome and subsequent germline transmission, this must be confirmed by showing the Southern pattern of the integrated transgene is inherited by 50% of the offspring in the F2.

A transgene construct that expresses a reporter gene in a transient manner in the injected embryo may not show expression in the transgenic fish of the next generation. This may be because the transgene has undergone a rearrangement at some stage during integration into the genome, or the integrated DNA may be transcriptionally inactive.

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