Creating mice with a pre-determined genetic makeup
A major aim of modern biology is to understand how normal gene activities
give rise to the structure and behaviour of complex organisms. In particular
it is important to study the function of genes and their derangement's
involved in human diseases. In most cases however, it is impossible
to achieve these studies directly in the human. It is easier therefore,
to carry out such studies in a more manipulable system such as the mouse.
However, natural mutations occur in a serendipitous manner, ie by chance.
To find a mutation that mimics a particular human disease is therefore
difficult. However, given a knowledge of the nucleotide sequence of
a gene, it is now possible to make changes to the corresponding endogenous
gene of an embryonal stem cell and to produce a mouse that is homozygous
for the desired mutation. This procedure is called gene targeting.
Gene targeting involves firstly the use of recombinant DNA technology
to modify a cloned gene (usually to stop the function of the gene).
At the same time a cultured cell line of embryonic stem (ES) cells is
generated by culturing cells from an early mouse embryo (a blastocyst).
The ES cells are totipotent and can be used to regenerate live normal
animals (ie it is possible to select a single ES cell and produce a
whole mouse from that cell, see below). Whilst in tissue culture the
modified gene is introduced into the ES cells and the normal gene is
replaced by the mutated (functionally inactive) gene. The modified ES
cells are then micro-injected into another mouse embryo and the ES cells
become integrated. These blastocysts are re-implanted into pseudo-pregnant
mice and give rise to live chimūric offspring that consist of the modified
injected cells as well as the normal cells. Since the injected cells
can also contribute to the testis of these mice, the breeding of a chimūra
with a normal mouse gives rise to an animal carrying the genes of the
modified stem cell (including the mutated gene). Interbreeding of the
heterozygous (F1) siblings finally yields transgenic animals homozygous
for the desired mutation (usually a deletion or a "gene knockout" mouse).
In this way co-isogenic animals can be generated, ie. animals which
are identical to the original mouse strain except that the function
of a single gene has been deleted thereby allowing the study of the
loss of this gene in vivo.
Gene targeting therefore allows the ability to study the function
of a cloned gene in the context of the whole mammal by creating mutant
mice defective in specific genes. This is particularly important since,
with gene targeting, mouse models can be created for studying human
genetic diseases and also provides a powerful approach to the development
of somatic gene therapy. Moreover, it is possible to also add genes
using similar processes resulting in "transgenic" mice. Our laboratory
has generated a number of different "knockout" and "transgenic" mouse
mutants using C57BL/6 or BALB/c ES cells that are at different stages
of investigation. These include mouse models of asthma, nerve re-generation,
xenograft rejection, parasite-host relationships, hypertension, drug
de-toxification and cancer.
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