Biomedical Engineering Reference
In-Depth Information
the way for animal cloning and the rapid production
of élite transgenic herds.
Pronuclear microinjection
Direct microinjection of DNA was the first strategy
used to generate transgenic mice. Simian virus 40
(SV40) DNA was injected into the blastocoele
cavities of preimplantation embryos by Jaenisch and
Mintz (1974). The embryos were then implanted
into the uteri of foster-mothers and allowed to
develop. The DNA was taken up by some of the
embryonic cells and occasionally contributed to the
germ line, resulting in transgenic mice containing
integrated SV40 DNA in the following generation.
Transgenic mice have also been recovered following
the injection of viral DNA into the cytoplasm of the
fertilized egg (Harbers
et al
. 1981).
The technique that has become established is the
injection of DNA into one of the pronuclei of the egg
(reviewed by Palmiter & Brinster 1986). The tech-
nique is shown in Fig. 11.2. Just after fertilizaton,
the small egg nucleus (female pronucleus) and the
large sperm nucleus (male pronucleus) are discrete.
Genetic manipulation of mammals
Methods for producing transgenic mice
The ability to introduce DNA into the germ line of
mice is one of the greatest achievements of the
twentieth century and has paved the way for
the transformation of other mammals. Genetically
modified mammals have been used not only to study
gene function and regulation, but also as bioreactors
producing valuable recombinant proteins, e.g. in
their milk. Several methods for germ-line transforma-
tion have been developed, all of which require the
removal of fertilized eggs or early embryos from
donor mothers, brief culture
in vitro
and then their
return to foster-mothers, where development con-
tinues to term. These methods are discussed below
and summarized in Fig. 11.1.
Attach DNA
to sperm
Unfertilized
mouse egg
DNA transfection
Adult cells
Nuclear
replacement
Embryonic stem
(ES) cells
Fertilized egg
Recombinant
retroviral infection
DNA
microinjection
Cell transfer
Early cleavage
Blastocyst
Embryonic
development
Fig. 11.1
Summary of methods for producing
transgenic mammals.
