Efficiency of Transgenesis Procedure

After transplantation with swelled sperm nuclei, 20-40% of the eggs cleave and develop normally. One person can transplant about 500 sperm nuclei/h to produce several hundred to one thousand embryos in a typical experiment. As with embryos produced by in vitro fertilization, the frequency of normal, advanced development varies somewhat, depending on the overall quality of the eggs; typically, 5-40% of the cleaving eggs develop normally beyond feeding tadpole stages. We commonly obtain 1-2 mo old tadpoles and are currently raising transplantation-derived metamorphosed froglets to sexual maturity.

Embryos from gastrula through tadpole stages derived from sperm nuclear transplantation express plasmids nonmosaically at high frequency. We have used transgenesis to introduce plasmids containing the simian cytomegalovi-rus (CMV) (8) or the X. borealis cytoskeletal actin (CSKA) (9,10) promoter into embryos (1). Transgenic embryos express genes from these promoters in every cell starting at the late blastula and early gastrula stages, respectively, as expected for these ubiquitously expressed promoters. When whole-mount in situ hybridization is used to detect plasmid expression, 20-50% of transplantation-derived embryos express CMV and CSK promoter-containing plasmids in every cell. In contrast, embryos injected with these plasmids never express reporter genes in all cells and typically express in only a small fraction (about 5-20%) of cells in the embryo.

We have also made transgenic embryos that express promoters that are spatially restricted (1). For example, we have used this method to introduce into embryos plasmids containing a muscle-specific actin promoter (11) linked to chloramphenicol acetyltransferase (pRLCAR) or green fluorescent protein (pCARGFP). We find that 40-60% of tadpoles derived from sperm nuclear transplantations with these plasmids show stable, nonmosaic expression. Expression is restricted to the somites and heart tissue, as expected for this regionally restricted promoter. We have also generated transgenic embryos with plasmid DNA that contains a neural specific P-tubulin promoter driving chloramphenicol acetyltransferase (CAT) (provided by Paul Krieg). These embryos express CAT in the primary neurons of the embryo as expected for this promoter. This correct expression is significant, since embryos injected with plasmids not only express CAT mosaically, but also ectopically, suggesting that integrating DNA into the genome is likely to give better regulation of cloned promoters than expression from nonintegrated DNA.

Fig. 1. Overview of transgenesis procedure. Sperm nuclei are incubated with linear DNA for a brief period of time. Interphase egg extracts and a restriction enzyme are then added. The egg extracts partially decondense the chromosomes, and the restriction enzyme very lightly cleaves them. These events facilitate the eventual integration of the linear DNA into the chromosomes. After incubation of nuclei in a mixture of extract, restriction enzyme, and plasmid DNA, the nuclei are diluted, and approximately one nucleus is transplanted per egg. Each activated egg requires a nucleus (or at least the centriole introduced with a nucleus) to divide; therefore, only eggs receiving a nucleus develop into embryos. Eggs that receive more than one nucleus (polyspermic eggs) divide abnormally into multiple cells at the first cleavage division. Embryos developing from monospermic eggs cleave normally during early divisions; only these embryos are isolated and analyzed. Generally, between 20 and 50% of these embryos will be transgenic.

Fig. 1. Overview of transgenesis procedure. Sperm nuclei are incubated with linear DNA for a brief period of time. Interphase egg extracts and a restriction enzyme are then added. The egg extracts partially decondense the chromosomes, and the restriction enzyme very lightly cleaves them. These events facilitate the eventual integration of the linear DNA into the chromosomes. After incubation of nuclei in a mixture of extract, restriction enzyme, and plasmid DNA, the nuclei are diluted, and approximately one nucleus is transplanted per egg. Each activated egg requires a nucleus (or at least the centriole introduced with a nucleus) to divide; therefore, only eggs receiving a nucleus develop into embryos. Eggs that receive more than one nucleus (polyspermic eggs) divide abnormally into multiple cells at the first cleavage division. Embryos developing from monospermic eggs cleave normally during early divisions; only these embryos are isolated and analyzed. Generally, between 20 and 50% of these embryos will be transgenic.

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