Overview of Techniques

Transgenic / Gene Targeting Facility

Genetically Engineered Mice

It is now technically feasible to systematically modify and express virtually any gene in vivo in a mouse (Capecchi, 1989). The avenue for such modification is a recombination machinery capable of exchanging or inserting genetic information from DNA supplied by the investigator into the host cell chromosome. If the exogenous DNA has been experimentally altered, homologous recombination permits transfer of the modified information to specific sites in the cellular genome. This process, referred to as gene targeting, allows genetic alterations designed by the investigator to be fixed at defined genetic loci and can be used both as a locus-specific mutagen as well as a means to correct endogenous genetic lesions. Gene targeting has been successfully used in cultured cells to modify genes involved in cellular metabolism and has been used to alter, at the germ cell level, genes of the experimental mammal model, the mouse. Exogenous genes can also be introduced and expressed in mice via nonhomologous recombination, thus permitting the generation of transgenic mice harboring virtually any gene of interest from any organism (Hogan et al., 1986). This ability to manipulate the germ line of the mouse is not only furthering the genetic dissection of mammalian growth and differentiation, but is advancing the use of the mouse as a model system in the study of human genetic disorders.

To date, all targeted gene modifications at the whole animal level have been performed in mice (Cappechi, 1989; Joyner, 1993). Although transgenes can be introduced into a variety of animals, mice are also the preferred species for this methodology (Hogan et al. 1986; Murphy and Carter, 1993; Pinkert, 1994). There are two avenues for introducing cloned genes into every cell in the animal. The first of these is to directly microinject a cloned gene into the pro-nucleus of a single-cell embryo (Fig. 1). Subsequent microsurgical implantation of this injected embryo into pseudopregnant surrogate mothers results in founder offspring containing the exogenous gene in all cells of the body.

 

 

 

 

 

 

 

 

 

 

 

 

 

Figure 1.  Outline of procedure for generating transgenic mice by microinjection of DNA into the pronuclei of single-cell embryos (Wasserman and DePamphilis, 1993).

 This method relies on non-homologous integration of the cloned gene at apparent random sites in the mouse cell genome. Because non-homologous recombination is a relatively efficient process, transgenic mice result from a high proportion of single-cell embryos that are injected (~5-25% assuming that the injected gene is not lethal). This procedure of pronuclear microinjection is well-suited for generating transgenic mice harboring virtually any gene. It is particularly advantageous for studies of oncogenes as well as other genes with dominant phenotypes.

An alternative method for introducing foreign genes or targeted gene sequences into mice is to make use of mouse embryo-derived stem (ES) cells (Fig. 2). These cells are pluripotent cells isolated from the inner cell mass of mouse blastocysts. They can be passaged in great number in culture, subjected to selection and screening protocols and returned to blastocysts, which are in turn reimplanted into pseudopregnant females and allowed to develop to term. The pluripotential nature of these cells enables them to colonize any tissue of the resulting chimeric animal, including cells of the germ line (Bradley et. al., 1984). Germ cells, derived from the ES cells, thus serve as founders for perpetual colonies of animals containing the designed targeted modifications. Because most if not all genes in ES cells are substrates for targeted recombination, the design of the genotype is left to the imagination of the investigator. Recessive lethal mutations can be passed in the heterozygote state, and their effects monitored after breeding of carrier adults. This procedure is well-suited for studies of both recessive and dominant alleles and for developing mouse lines that model human genetic diseases.

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

Figure 2. Generation of germline chimeric mice following gene targeting in ES cells (from Capecchl, 1989). 

The transfer of genetically modified ES lines to mouse embryos entails the microinjection of ES cells into the blastocoel of 3 1/2 day old blastocysts, or aggregation of ES cells with 2.5 day old morulae which are subsequently cultured to blastocysts. The male ES lines, derived from mouse line 129/Sv, carry an agouti ("A" locus) coat color marker and the host embryos are derived from a Bl6, black coat ("a" locus) line of mice. Following surgical implantation of the potential chimeric blastocysts into pseudopregnant females, the embryos are allowed to grow to term. Approximately 2 weeks following birth the coat color of the chimeras has developed sufficiently that an accurate estimate of the extent of ES cell contribution to the mouse can be made. Those male mice showing greater than 30 percent coat color chimerism are retained for mating with Bl6 tester mice. Because of the dominant transmission pattern of the Agouti (A) marker, sperm originating from the ES-cells in the chimera will transmit a detectable coat color marker to the offspring. Offspring with agouti coats are retained and DNA from tail biopsies analyzed for the presence of the modified gene of interest. Unless the modified gene is tightly linked to the A locus, one expects 50% of the agouti offspring to serve as founders for the new mouse line.

University of Utah Transgenic/Gene Targeting Facility

 

PURPOSE

The objective of the Transgenic/Gene Targeting Mouse Facility is to provide ready access to mouse genetic technologies and thus enhance the research productivity of the University of Utah. Methods of manipulating the mouse embryo and its genome are demanding and costly and cannot be practically sustained in the laboratories of multiple individual investigators. Thus, the Transgenic/Gene Targeting Facility brings these technologies to research groups within the University who otherwise would not have access to these powerful genetic methods. The Transgenic/Gene Targeting Facility also provides a focus for scientific interaction and consultation, facilitates new research, and further strengthens scientific and organizational cohesion within the University animal research community.

DESCRIPTION

The Transgenic/Gene Targeting Mouse Laboratory is organized as a central core unit to serve the University research community. Researchers provide purified gene constructs, and the Laboratory generates mice harboring these constructs as transgenes or gene-knockout recombinants. The Laboratory maintains all necessary nontransgenic breeding colonies and is fully equipped and staffed with technical experts who conduct all microinjections, transfections, surgical procedures and manipulations of mice, embryos and ES cells.  The Laboratory will provide ES cell DNA for analysis of targeting events,  and tails for analysis of transgenic mice.  The Laboratory will house transgenic animals while they are being analyzed, and will breed chimeras to check for germ line transmission.  Tails will be given to the investigator and positive mice will be transferred to the individual researcher.  Laboratory staff provide guidance in the design of transgenic and gene targeting experiments and work closely with each investigator to optimize success in generating positive mice. The Laboratory is also available for hands-on teaching of transgenic and gene targeting techniques.

 

Literature

Babinet, C., Morello, D. and Renard, J.P. (1989). Transgenic mice. Genome 31:938-949.

Bradley, A., Evans, M., Kaufman, M. H. and Robertson, E. (1984). Formation of germ-line chimaeras from embryo-derived teratocarcinoma cell lines. Nature 309: 255-256.

Bronson, S.K. and Smithies, O. (1994). Altering mice by homologous recombination using embryonic stem cells. J. Biol. Chem. 269:27155-27158.

Capecchi, M.R. (1989). The new mouse genetics: altering the genome by gene targeting. Trends Genet. 5:70-76.

Capecchi, M.R. (1989). Altering the genome by homologous recombination. Science 244: 1288-1292.

Capecchi, M. (1994). Targeted gene replacement. Scientific American, March 1994, pp. 52-59.

Hanahan, D. (1989). Transgenic mice as probes into complex systems. Science 246:1265-1274.

Hogan, B., Costantini, F. and Lacy, E. (1986). Manipulating the Mouse Embryo. A Laboratory Manual. (Cold Spring Harbor: Cold Spring Harbor Labortory Press).

Iannaccone, P.M. and Scarpelli, D.G. (1993). Exploring pathogenetic mechanisms using transgenic animals. Ann. Med. 25:131-138.

Jaenisch, R. (1988). Transgenic animals. Science 240:1468-1474.

Joyner, A.L., eds. (1993). Gene Targeting. A Practical Approach. (New York: Oxford University Press).

Kappel, C.A., Bieberich, C.J. and Jay, G. (1994). Evolving concepts in molecular pathology. FASEB J. 8:583-592.

Murphy, D. and Carter, D.A., eds. (1993). Transgenesis Techniques. Principles and Protocols. (Totawa: Humana Press).

Palmiter, R.D. and Brinster, R.L. (1986). Germ-line transformation of mice. Ann. Rev. Genet. 20:465-499.

Pinkert, C.A., eds. (1994). Transgenic Animal Technology. A Laboratory Handbook. (San Diego: Academic Press).

Robertson, E.J. (1991). Using embryonic stem cells to introduce mutations into the mouse germ line. Biol. Reprod. 44:238-245.

Wasserman, P.M. and DePamphilis, M.L., eds. (1993). Guide to Techniques in Mouse Development. (San Diego: Academic Press).

Westphal, H. (1989). Transgenic mammals and biotechnology. FASEB Journal 3:117-120.

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