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Knockout mice: a paradigm shift in modern immunology

Nature Reviews Immunology volume 1, pages 11–19 (2001)Cite this article

Key Points

  • The development of genetic engineering techniques to functionally 'knock out' a single gene of interest in a living animal has revolutionized our study of mammalian physiology in general and immunology in particular.

  • Single gene knockouts, multiple gene knockouts, recombinase-activating gene (Rag) complementation mutants and knock-in transgenic animals have all contributed to our increased understanding of gene functions in lymphocytes and other haematopoietic cells.

  • Several factors, including genetic background and targeting strategy, must be borne in mind when analysing the phenotypes of gene-deficient mice.

  • Gene targeting has contributed greatly to the establishment of basic models of lymphocyte biology. Thymocyte development and survival, positive and negative thymocyte selection, activation pathways in immature versus mature T cells, intracellular signalling downstream of T-cell-receptor engagement, co-stimulation, and regulatory pathways have been examined using knockout technology.

  • Future advances in gene-knockout technology will probably include the ability to turn a specific gene on and off at will, and mutational systems that examine gene function and interaction more holistically. Insights into mechanisms underlying cancer and autoimmunity can be expected.

Abstract

In the past decade, advances in genetic engineering and mouse knockout technology have transformed our understanding of the immune system. In particular, new perspectives on T-cell development, co-stimulation and activation have emerged from the study of single and multiple gene-knockout animals, as well as from conditional knockout and 'knock-in' mutants. Analysis of these animals has clarified important intracellular signalling pathways and has shed light on the regulatory mechanisms that govern normal immune responses and autoimmunity.

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Figure 1: The classical approach to gene-targeting in embryonic stem cells.
Figure 2: RAG-deficient blastocyst complementation.
Figure 3: Cre/loxP-mediated gene targeting in embryonic stem cells.
Figure 4: Schematic of thymocyte development as shown by gene deletion.
Figure 5: Schematic of TCR signalling.

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Acknowledgements

We thank I. Ng for administrative assistance, D. Bouchard for graphic design of the Figures and M. Saunders for scientific editing. This work is supported by grants from Amgen, Inc., the Canadian Institute for Health Research and the National Cancer Institute of Canada. J.M.P. is also supported by the Premier's Research Excellence Award and holds a Canadian Research Chair in Cell Biology. P.S.O. is also supported by the G.H. Wood Foundation and holds a Canadian Research Chair in Infection and Immunity.

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Authors and Affiliations

  1. Departments of Medical Biophysics and Immunology, Amgen Research Institute, University of Toronto, 610 University Avenue, Toronto, M5G 2M9, Ontario, Canada

    Tak W. Mak & Josef M. Penninger

  2. Ontario Cancer Institute and Departments of Medical Biophysics and Immunology, University of Toronto, 610 University Avenue, Toronto, M5G 2M9, Ontario, Canada

    Tak W. Mak, Josef M. Penninger & Pamela S. Ohashi

Authors
  1. Tak W. Mak
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  2. Josef M. Penninger
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  3. Pamela S. Ohashi
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Corresponding author

Correspondence to Tak W. Mak.

Related links

Related links

DATABASES

LocusLink:

 β2-microglobulin

Bcl10

Bcl-xL

CD8

DNA polymerase-β

immunoglobulin μ-chain

NFAT4

RasGRP

ROR-γ

Slp76

Vav1

 MGI:

 aly

Akt

beige

Brca1

c-Cbl

CD3

CD28

CD40

CD45

CD80

CD86

CD134

CD152

CD154

Csk

GADS

gld

Hprt

ICOS

ID3

IL-2

Il4

IL-15

IL-21

IRF1

ITK

LAT

lpr

Mog

motheaten

nude

OX40L

p53

PD1

Pten

RAG1

RAG2

RLK

scid

Syk

WASP

ZAP70

FURTHER INFORMATION

BioMedNet mouse knockout and mutation database

Bioscience mouse knockout database

Harvard mouse knockout database

TBASE

Glossary

HOMOLOGOUS RECOMBINATION

Recombination of an engineered version of a gene into its normal locus in a genome, as opposed to random integration. The use of 'arms' of DNA that are homologous to regions surrounding the gene of interest, facilitates the pReferential insertion of genetically manipulated targeting vectors into the desired locus.

KNOCKOUT TECHNOLOGY

This technology allows for the deletion of a gene, or its function, by gene targeting. A targeting vector in which the gene of interest has been disrupted is introduced into the genome of a mouse embryonic stem cell by homologous recombination.

KNOCK-IN MUTATION

Introduction of a transgene into a precise location in the genome, rather than random integration. Knocking-in uses the same technique of homologous recombination as a knockout strategy, but the targeting vector is designed to preserve or enhance the function and control of the gene of interest, rather than disrupt it.

POSITIVE THYMOCYTE SELECTION

The process in the thymus that selects thymocytes expressing T-cell receptors (TCRs) that have the ability to interact weakly with self-MHC. This weak interaction generates differentiation and survival signals in these lymphocytes, the TCRs of which later recognize foreign peptides bound to self-MHC. Positive selection establishes the MHC-restricted T-cell repertoire.

NEGATIVE THYMOCYTE SELECTION

The deletion of self-reactive thymocytes in the thymus. Thymocytes that express T-cell receptors that strongly recognize self-peptide bound to self-MHC undergo apoptosis in response to the signalling generated by high-affinity binding.

ADOPTIVE TRANSFER

An experimental method in which lymphocytes from an antigen-primed donor mouse are introduced into a recipient mouse that lacks lymphocyte function.

CO-STIMULATION

Receptor-mediated signals required in addition to antigen-receptor engagement to achieve complete lymphocyte activation.

IMMUNOLOGICAL SYNAPSE

The structure formed on the cell surface between a T cell and an antigen-presenting cell; also known as the supra-molecular activation cluster. Important molecules involved in T-cell activation, including the T-cell receptor, numerous signal transduction molecules and molecular adaptors, accumulate at this site. Mobilization of the actin cytoskeleton of the cell is required for immunological synapse formation.

E3 UBIQUITIN LIGASE

Enzyme required to attach the molecular tag ubiquitin to proteins destined for degradation in the proteosomal complex.

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Mak, T., Penninger, J. & Ohashi, P. Knockout mice: a paradigm shift in modern immunology. Nat Rev Immunol 1, 11–19 (2001). https://doi.org/10.1038/3509551

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  • DOI: https://doi.org/10.1038/3509551

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