Production techniques

Direct microinjection of DNA into one of the pronuclei of a zygote (Figure 1), has been the most rewarding technique for the production of transgenic mice, rats, rabbits and large domestic livestock such as sheep, pigs and cows. The technique (Figure 2) requires special equipment and technical skills but in comparison to other methods it remains the most straightforward and efficient way to produce transgenic animals. Briefly, the procedure as it applies in the mouse, consists of collecting a number of fertilized mouse eggs, visualizing their male and female pronuclei under appropriate microscope optics and injecting one of them with a DNA solution

(containing a few hundred copies of the gene of interest) using a fine glass needle prepared on specialized equipment (pipette puller). Injected eggs are then surgically transplanted into the oviducts of 'pseudo-pregnant' female mice (mice that have been mated with a vasectomized male) and left to develop to term. In general, it is expected that in skilled hands, as many as 90% of the injected eggs survive the microinjection, approximately 20% of these develop to term and approximately 20-50% of the latter are found to carry the injected DNA in their tissues. Integration of the transgene occurs apparently at a random site in the genome, either as a single copy or as a tandem head to rail array. In rarer cases, head to head arrangements and multiple integration sites should also be expected. In most cases transgene integration occurs in 100% of the animal cells, however, in as high as 30% of transgenic founder mice genetic mosaicism has been observed which means that only a subset of cells in the different tissues, including the germ line, carries the transgene. Subsequent derivation of transgenic progeny from such transgenic animals leads to the generation of fully transgenic animals that transmit their transgcncs in a Mendelian fashion.

In addition to the microinjection procedure, there have been several alternative methods for introducing foreign DNA into the germ line of mice. Infection of preimplantation embryos with recombinant retroviruses offers a highly efficient route of single copy-

Figure 2 Experimental scheme for the production of transgenic mice by the pronuclear microinjection technique. (Computer graphics by Katerina Akassoglou, Department of Molecular Genetics, Hellenic Pasteur Institute).

Embryo transfer

Analysis of DNA in tail biopsies

Microinjection of DNA into one of the pro nuclei of a fertilized oocyte

Offspring

Pseudop regnant mother

Analysis of DNA in tail biopsies

Offspring

Transgene detection by Southern or PCR

Transgene

Microinjection of DNA into one of the pro nuclei of a fertilized oocyte

Embryo transfer

Pseudop regnant mother integrations into the mammalian genome; however, a main disadvantage of this procedure is that retroviral vector DNA usually interferes with the expression patterns of the genes that it carries. Yet another route to transgenesis employs the introduction of exogenous DNA into totipotent mouse embryonic stem (ES) cells and their consequent incorporation into the blastocyst of a developing mouse embryo. This is technically a very demanding procedure and it does not offer advantages over the direct pronuclear microinjection for transgenesis; however it has been so far a uniquely applied technique for the targeted disruption ('knockout') or mutagenesis of endogenous genes in mice.

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