What are "Transgenic Mice" ?
Although several steps in the evolution of transgenic technology had been performed in the 1970Zs, transgenesis was not widely recognized before Palmiter introduced the human growth hormone gene into mouse zygotes by pronuclear microinjection and transgenic offspring demonstrated dramatic growth. Since then transgenic techniques have been of immense importance, as they allow new approaches to life science research that general cell culture techniques cannot deliver. The manufacture of large quantities of complex, bioactive proteins like hormones or growth factors for therapeutic purposes (pharming) is only one example of a wide range of different applications for the study of genetic regulation, animal development or disease pathology. For this purpose, foreign DNA is introduced into fertilized oocytes or embryos (blastocysts) of mice, rats and other mammals.
Production of Transgenic Animals
Transgenic animals are frequently created by two different techniques: microinjection of DNA into the pronucleus of zygotes and injection of embryonic stem cells into blastocysts. The procedure is done with the help of a complete microinjection set-containing of a microscope, micromanipulators, microinjectors and micropipettes.
Microinjection of DNA into the pronucleus
The pronuclear microinjection method of producing a transgenic animal is based on the introduction of linear DNA sequences into the chromosomes of fertilized eggs. The foreign DNA must be integrated into the genome prior to the doubling of the genetic material that precedes the first cleavage in order for the animal to be born with a copy of this new information in every cell. For several hours following the entry of the sperm into the oocyte, the male and the female pronuclei can still be seen individually under a normal light microscope and they have not fused yet into a so called zygote. The foreign DNA may be injected into either pronuclei with no difference in results; however, the DNA is typically injected into the male pronucleus because it is slightly larger and closer to the oocyte surface. These oocytes are subsequently transferred into the uterus of pseudopregnant recipient animals. The offspring is screened to confirm a successful integration of the gene of interest for use in further studies.
Injection of ES cells into blastocyst-stage embryos
Embryonic stem cells (ES cells) are derived from the inner cell mass of blastocysts (early embryos). These cells are pluripotent, which means that they can develop into almost any type of tissue. ES cells are used for more precise modifications of the mouse genome. This technique makes it possible to insert as well as remove or modify DNA sequences. Knock-out, knock-in and conditional mutant mice can be produced with this method. The first step is the removal of ES cells from a blastocyst. After transfection of the ES cells, selection, cloning and screening methods make it possible to detect ES cell clones that demonstrate the desired, site-specific recombination. After microinjection of the genetically modified ES cells into blastocyst-stage embryos the ES cells divide and become part of the embryo. The following chimeric animals will subsequently transmit the recombinant genotype to their offspring, if the ES cells have contributed to their germ cells.