Overview of Transgenesis Procedure

In the transgenesis approach described here, DNA is integrated into isolated sperm nuclei in vitro, followed by transplantation of the nuclei into unfertilized eggs, thus generating transgenic embryos. Nuclear transplantation of trans-fected cultured cells was previously used by one of us to produce transgenic Xenopus embryos, which expressed promoter-reporter plasmids nonmosaically (2). However, the cultured cells used as nuclear donors for these transplantations were aneuploid and rarely promoted development of the pseudo-triploid embryos to tadpole stages. To overcome these problems, we now use sperm nuclei to generate transgenic embryos. These nuclei offer many advantages over cultured cell lines. First, since sperm nuclei are haploid, there is no need to destroy the egg nucleus before transplantation to generate a normal diploid embryo. Second, sperm nuclei have been used for many years to investigate the processes of chromosome decondensation, nuclear assembly, and cell-cycle progression (3-5). These studies have provided us with valuable information regarding the manipulation of sperm nuclei in vitro. Indeed we have discovered that we can introduce DNA into sperm nuclei swelled and decondensed in cell-free egg extracts using restriction enzyme-mediated integration (REMI) (6,7). When these nuclei are transplanted into unfertilized eggs, we obtain large numbers of normal diploid tadpoles, which develop to advanced stages and express inserted genes at high frequency.

The protocol for Xenopus transgenesis described here involves the following steps:

1. Sperm nuclei are incubated with linearized plasmid DNA.

2. After a short incubation, a high-speed interphase egg extract and a small amount of the restriction enzyme used for plasmid linearization are added to the sperm nuclei/plasmid mixture. The extract partially decondenses sperm chromatin, but does not promote replication.

3. After the plasmid-treated nuclei are incubated for a brief period in the interphase extract, the mixture is diluted 50- to 100-fold (or 500- to 1000-fold total dilution of sperm nuclei).

4. Approximately one nucleus is transplanted into an unfertilized egg in a 5-15 nL volume. This procedure is schematically represented in Fig. 1.

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