Xenbase: a Xenopus web resource

Genetics: transgenics

1. Introduction
2. preparation of high speed extract
3. preparation of sperm nuclei
4. preparation of equipment
5. transfer
6. full protocol as pdf

1. Introduction

This collection of protocols describes the Kroll and Amaya (1996) technique for generating transgenic frogs.

Briefly the protocol involves the following steps:

• Sperm nuclei are incubated with linearised plasmid DNA.
• After a short incubation, a high-speed interphase egg extract and a small amount of the restriction enzyme are added to the sperm nuclei and plasmid mixture. The extract partially decondenses the sperm chromatin but does not promote replication and the restriction enzyme stimulate recombination and integration by creating double stranded breaks in the sperm chromatin.
• After the plasmid-treated nuclei are incubated for a brief period in the interphase extract, the mixture is diluted and the nuclei are transplanted into unfertilised eggs, resulting in the production of transgenic embryos.

This technique permits large scale transgenesis in Xenopus embryos. Unlike embryos injected with plasmids, the transgenic embryos show correct spatial and temporal regulation of integrated promoter constructs. One of the great advantages of this system over transgenesis in mice or zebrafish is that the transgene is integrated into the male genome prior to fertilisation, therefore the resulting embryos are not chimeric and breeding of animals is not required. In other words, transgenic embryos can be generated one day and analysed the next. This new technology can be used to

1. missexpress genes during development with much better spatial and temporal control
2. label specific structures in vivo, such as the musculature or nervous system
3. study the regulation of promoters of genes from amphibia, zebrafish and mouse
4. study the molecular basis of organogenesis
5. generate mutations in genes through gene trap approaches

In the following pages the details of the protocol for generating transgenic frog embryos are described. It includes a section on how to prepare high speed interphase extracts from eggs, a section on how to prepare sperm nuclei, and a section on how to integrate DNA into isolated sperm nuclei and transplant thenuclei into eggs, thus generating transgenic embryos. For up to date information on the procedure, see the Amaya Lab website (http://www.welc.cam.ac.uk/~ea3/).

Next: High Speed Extract Preparation

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