5.6.3 Gene Therap y
The ability to introduce new or altered genes into a mammalian genome has tremendous implications.
For example, it may prove possible to cure some genetic diseases by introducing a healthy copy of a gene
into an afflicted individual. It is already possible to introduce into mammals genes that encode economic-
ally or medically important polypeptides such as insulin growth hormones and interferon. The intention is
that the animal either grows faster or produces large amounts of protein, which can be harvested. We will
address the ways in which this can be carried out, leaving the ethical questions raised by this issue
to others.
There are three major ways of introducing DNA into mammalian germ tissue such that the progeny of
the recipient will carry the gene. The first involves microinjection of DNA solutions into the nucleus of an
egg by means of an extremely fine capillary. Such a technique works very well with a mouse egg but is more
difficult with other mammals, such as sheep, where it is extremely hard to see the nucleus. In this way,
transgenicanimals have been created which carry functioning genes from another organism.
The second method involves the use of retrovirus-based vectors (Figure 5.20). As described in Section
6.4.6, retroviruses can infect a cell and then insert their DNA into its chromosomes. The gene to be intro-
duced into the host is ligated into the genome of the retrovirus. The retroviral DNA is then introduced into
a cultured cell line, which is capable of producing all components of a retrovirus except for the viral RNA
(such a cell culture is called a helper cellline). This cell line will then package the recombinant virus stock
into virus particles that can be harvested from the culture medium. Helper cells are necessary because the
presence of the insert in the retroviral genome disrupts some of the normal retroviral genes needed for
viral production. The harvested recombinant virus stock is then used to infect an early embryo, which is
then replaced into a donor mother. During growth, some cells of the embryo become infected by the virus
and the retroviral gene, including the gene insert, becomes stably inserted into the DNA of these cells.
Because not all cells become infected, the animal is a chimera. However, if the germ cells of this animal
contain proviral DNA then its offspring will retain the recombinant in every cell of its body.
In addition to introduction of a new gene coding for a protein, it is also possible to introduce viaa retro-
viral vector a gene that codes for a specialised RNA (e.g.antisense RNA (Section 5.7.1), short interference
RNA (Section 5.7.2) or an RNA that folds into ribozyme or an aptameric structure (Section 5.7.3) that can
act in transto interact with and block the function of another RNA.
The third method for introducing DNA into the mammalian germ line relies upon the existence of cul-
tured cell lines, which can become germ cells if injected into early embryos. This approach is particularly
useful in the mouse, where such cells, embryonal carcinoma cells, can be grown in dishes. The gene of
interest can be introduced into these cells, which are then injected into embryos.
192 Chapter 5
Figure 5.19 Nucleoside analogues used in error-prone PCR mutagenesis