IMMUNOLOGY - Roitt I. - Mir 2000

Chapter 29. Immunological Methods

TRANSGENIC ANIMALS AND TARGETED GENE DELIVERY

Transgenic Animals

One approach to investigating the function of a specific molecule is the generation of transgenic animals in which the Gene for the molecule has either been deleted or is expressed at super-high levels or as an altered product. A classic method for producing transgenic animals involves microinjecting approximately one hundred copies of the gene of interest directly into the pronucleus of a fertilized oocyte. The oocyte is then transferred into the oviduct of a pseudopregnant female mouse, where the embryo develops in the Uterus. The resulting offspring exhibit considerable Variability. In a small number of embryos, one or more copies of the gene integrate into one of the Chromosomes prior to the first Cell Division, making these animals heterozygotes for the transgene. In another (likewise small) fraction of animals, transgene integration occurs after the First Division, resulting in chimeric mice that possess both normal Cells and cells containing the transgene. In the majority of mice, however, the transgene fails to integrate into the chromosome. The status of each mouse is determined by screening cell samples for the presence of the gene using Southern blot analysis. Based on these results, transgenic heterozygous animals are selected and subsequently bred to establish a homozygous transgenic line.

Typically, transgene integration occurs randomly and as a concatemer consisting of multiple copies. Importantly, the insertion of the transgene array does not disrupt other functionally important genes. The pattern of transgene expression depends on several factors. Sometimes these genes come under the control of ubiquitous promoters and are expressed in most Tissues. In other cases, they are linked to tissue-specific promoters, restricting their expression to only certain tissues and cell types (such as lymphocytes) or specific developmental stages. The phenotype of transgenic animals must be interpreted with caution, as high-level transgene expression in inappropriate tissues cannot be considered physiological.

Targeted gene delivery

This more sophisticated method involves, as a first step, The transfer of a gene that interacts or recombines with the endogenous gene of interest, resulting in its modification. For example, this endogenous gene may undergo a deletion (as is the case in so-called "knockout" mice), acquire point Mutations, or lose an exon. Such a modified gene is introduced into embryonic pluripotent stem cells, where it undergoes Homologous Recombination with the corresponding endogenous gene. These stem cells are then introduced into a blastocyst or implanted using the Procedure described above (Fig. 29.27).

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Fig. 29.27. Transgenic animals can be generated through targeted gene modification resulting from homologous recombination. This approach utilizes an exogenous gene segment (transgene) containing 1) a sequence homologous to the cellular gene but carrying a mutation, and 2) a selectable marker gene. The transgene is introduced into Embryonic Stem Cells by electroporation; cells that have successfully incorporated the exogenous gene are then selected based on the marker gene. Next, a secondary Selection isolates cells in which recombination between the exogenous and endogenous genes has occurred. These cells are introduced into blastocysts, which are subsequently implanted into the uteri of pseudopregnant mice. The resulting offspring comprise normal transgenic animals.

Questions for Structure/133.html">Discussion

■ Using newly generated Monoclonal Antibodies AND immunofluorescence, you have identified a novel molecule On the surface of T lymphocytes. Choose appropriate Methods to answer the following questions:

■ When T cells are cultured in the presence of the mitogen concanavalin A and varying concentrations of your antibodies, will these antibodies reduce the proliferative capacity of the cells? Will the number of IL-2-producing T cells decrease?

■ What is the molecular mass of the identified molecule?

■ Is this molecule present on CD4⁺ and CD8⁺ T cells?

■ What is the concentration of the resulting antibodies in the culture medium of the antibody-producing hybridoma?

Introduction/47.html">Further Reading

Coligan J.E., Kruisbeck А.М., Margulies D.H. et al. (eds.) 1991. Current Protocols in Immunology. New York: Greene Publishing Associates & Wileyinterscience, continually updated.

Hudson L., Hay F.C. 1989. Practical Immunology. 3rd. Oxford: Blackwell Scientific Publications.

Johnstone A., Thorpe R. 1987. Immunochemistry in Practice. 2nd edn. Oxford: Blackwell Scientific Publications.

Rose N.R. et al. (eds.) 1992. Manual of Clinical Laboratory Immunology. 4th edn. Washington: American Society of Microbiology.

Weir D.M. 1986. Handbook of Experimental Immunology. Vols I & II. 4th edn. Oxford: Blackwell Scientific Publications.

Winter F., Griffith A.D., Hawkins R.E. et al. 1994. Making antibodies by phage display technology. Annu. Rev. Immunol. 12: 433-55.