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  • Title: Generation of chimeras by microinjection.
    Author: Plück A, Klasen C.
    Journal: Methods Mol Biol; 2009; 561():199-217. PubMed ID: 19504073.
    Abstract:
    Since the technique of introducing a targeted mutation ('gene targeting') into the mouse genome was published almost 20 years ago (Cell 51:503-512, 1987), the number of mouse mutants (mouse models) is increasing, especially after the advent of the full mouse genomic sequence in 2002 and the human genomic sequences in 2003 that reveals more and more large stretches of similarity between the two species at the genomic level. This chapter describes the tools and the experimental route of targeted manipulation by microinjection in the mouse using targeted embryonic stem cells (ES cells).The techniques have become standardized over recent years (Nature 309:255-256, 1984; Practical Approach. IRL Press, Oxford, 254 pp, 1987; Science 240:1468-1475, 1988; Practical Approach. IRL Press, Oxford, New York, 1993; Transgenic Animal Technology: A Laboratory Handbook, 2nd edition. Academic Press, San Deigo, 2002; Manipulating the Mouse Embryo - A Laboratory Manual, 3rd edition. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 2003) and basically two methods have been used to generate chimeric mice that transmit the mutation of interest via the ES cell genome to the offspring:Microinjection of ES cells into blastocyst or morula stage embryos (this chapter) or aggregation of ES cells with morula stage embryos (see Chapter 14 ).Microinjection of ES cells into the blastocoel (cavity) of the blastocyst stage embryo and also morula injections using micropipettes driven by micromanipulators require sophisticated manual skills and an expensive phase contrast inverted microscope. Although most commonly used, it is quite expensive to establish this technique in a laboratory, in particular, if piezo- or laser- supported routes come into play. Although the establishment of germ-line potent ES cells was first published in 1981 (Proc Natl Acad Sci U S A 78:7634-7636, 1981; Nature 292:154-156, 1981), up to now it has not been possible to establish germ-line transmitting ES cells from any other mammalian species, not even from rat which is closely related, nor was it possible to introduce targeted mutations by different means to the germ-line of mammals. After 20 years, the mouse is still the only mammalian species where mutations can be introduced in a targeted manner and therefore it is very important to many fields in biology, like immunology, neurobiology, and developmental biology to study gene function and disease. Through means of introducing even point mutations to single relevant molecules of a signal transduction pathway in order to study regulation of cellular and physiological processes in complex organisms in a tissue specific or inducible manner (conditional gene targeting, (Cell 73:1155-1164, 1993; Science 265:103-106, 1994)), more recently the field has expanded exponentially.
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