Generation of chimeras by microinjection.

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.

Generation of chimeras by morula aggregation.

This chapter describes the tools and the experimental route of targeted manipulation by aggregation in the mouse using targeted embryonic stem cells (ES cells). Instead of injecting ES cells into the blastocoel of a diploid blastocyst-stage embryo (3.5 dpc) ES cells can be brought together with diploid morula-stage embryos (2.5 dpc). The zona pellucida of the embryo needs to be removed and one or two embryos (sandwich aggregation) are put together with ES cells into an indentation well of a cell culture grade dish overnight for aggregation. This can be performed manually using a stereomicroscope and does not require any special training or expensive instrumentation.The next day, the embryo would have developed into a blastocyst in vitro and can be transferred to a pseudopregnant female mouse (see Chapter 15 ).The use of tetraploid embryos generated by electrofusion will lead to entirely ES cell-derived fetuses.

Surgical techniques for the generation of mutant mice.

The previous two chapters have described the generation of chimeric embryos by blastocyst microinjection and morula aggregation. This chapter describes the reimplantation of these embryos into pseudopregnant female recipient mice (foster mice) in order for the embryos to develop to term. Four different surgical techniques will be described in detail.The transfer of blastocyst-stage embryos back into the uterus is most commonly used for chimeric embryos generated by microinjection or aggregation, or for rederivation purposes when strains are transferred to a clean environment. Fertilized oocytes manipulated by pronuclear microinjection or two-cell-stage embryos are reintroduced to the infundibulum of the oviduct of pseudopregnant female mice.To generate pseudopregnant females, they need to be mated to sterile male mice. These males can be either generated by vasectomy of stud males or naturally sterile males can be used. Two different techniques for the vasectomy are shown: the vas deferens is accessed through an incision in the scrotal sac or through an abdominal incision.

One step generation of fully chimeric antibodies using Cgamma1- and Ckappa mutant mice.

Humanized antibodies (Abs) are effective drugs against a variety of diseases such as cancer, autoimmune diseases, transplant rejection and others. The most powerful technology to develop humanized Abs is the use of mice that produce humanized Abs. By modifying the genetic background of F004 mice a new mouse substrain was developed for optimized "one step" generation of chimeric humanized monoclonal Abs. The new mice (F004-Jen) demonstrated improved fertility still expressing the human locus at the same level as the parental F004 mouse. The value of these mice for the generation of chimeric Abs was exemplified for a panel of chimeric Abs against the human neural cell adhesion molecule (NCAM): The fully chimeric human IgG1/kappa Ab Ch.MK1 bound to NCAM expressing cells with a K(D)=4.3-8.7 x 10(-8) M and was functionally active as demonstrated by depleting NCAM expressing cells. We also demonstrated that chimeric IgG1/kappa Abs can be induced by hybridoma class switching of IgM producing hybridoma cells, providing an alternative way to chimeric Abs. The present data highlight F004-Jen mice as an efficient tool for "one step" generation of chimeric Abs.