Methods

The following sections describe methods for injection of DNA into zebrafish and subsequent analysis of the embryos for transient transgene expression. Methods for identifying founder fish with mosaic germ lines and F1 fish that are hemizygous for transgene constructs are also described. A recent improvement in screening for transgenic F1 fish has been developed by Kawakami and Hopkins (36). Their strategy is to identify founder fish with mosaic germlines by raising injected embryos to adulthood and mating them with a wildtype fish. DNA is them extracted from 100 24-h-old F1 embryos and a PCR amplification performed. Only 2-20% of the F1 progeny will carry the transgene so DNA from a large number must be tested. If the PCR is positive for a transgene insertion, then new embryos must be raised and rescreened individually to identify transgenic F1 fish. Previously this was done by taking a fin biopsy, but this requires raising the F1 fish to adulthood. If only 2% carry the transgene then this is uneconomical and labor intensive. The improved strategy is to treat 7275 h postfertilization F1 embryos with proteinase K (20 pg/mL) for a short time (12 min at room temperature). This is sufficient to release enough DNA into solution for PCR without killing the embryo. Use of 96-well culture plates and a multichannel pipet can facilitate performing 96 PCR amplifications. Analysis of the amplification products by gel electrophoresis means that the positive transgenic F1 fish are identified when still young and the nontransgenic can be disposed.

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