21 Transgenie Animals
321
Lastly, transgenic techniques can be used to establish permanent cell
lines derived from transgenic animals by fusing a tissue specific promoter
to a gene that has oncogenic potential. The transgenic animals then develop
tumors in the tissue where expression occurs. The tumors can be used to
isolate celllines which express the desired gene product in perpetuity ( 6, 7).
Methods for generating transgenic animals
Several methods are used to generate transgenic animals. The most common are:
• Direct microinjection of foreign DNA into the pronucleus of fertilized
oocytes (8-9)
• Use of embryonie stem cells previously transfected with the DNA of interest to produce chimeras via transfer into blastocysts (1 0-12)
• Retroviral infection of pre-implantation blastocysts (13-15). Infection
can be attained either by direct exposure ofblastocysts to viral particles
or by cocultivation with virus-producing cells.
A common disadvantage of direct microinjection and retroviral infection is
that these methods do not permit the delivery of the gene to a specific location of the chromosome, so that integration occurs at random. Therefore,
transfer ofDNA by direct microinjection into the pronucleus can cause insertional mutations which mayinterfere with the phenotype. In this context,
a wide range of mutations have been decribed previously (16-18). Furthermore, random positioning in the vicinity of other transciptional regulatory
sequences may lead to a position effect, resulting in higher or lower expression levels (18).
The microinjection of foreign DNA is widely used and is considered the
most efficient method for construction of transgenic animals. Whereas this
method has become a routine procedure in mice during the last ten years (9,
13 ), its application for the generation of transgenic rats has been developed
only recently. An approach to obtain a reliable positioning and copy number of the transgene, to regulate the level of expression, and to make the
transgene independent of the neighboring chromosome may be the use
of recombinant yeast artificial chromosomes (Y AC), which allow the introduction of large gene constructs into the germ line (19).
321
Lastly, transgenic techniques can be used to establish permanent cell
lines derived from transgenic animals by fusing a tissue specific promoter
to a gene that has oncogenic potential. The transgenic animals then develop
tumors in the tissue where expression occurs. The tumors can be used to
isolate celllines which express the desired gene product in perpetuity ( 6, 7).
Methods for generating transgenic animals
Several methods are used to generate transgenic animals. The most common are:
• Direct microinjection of foreign DNA into the pronucleus of fertilized
oocytes (8-9)
• Use of embryonie stem cells previously transfected with the DNA of interest to produce chimeras via transfer into blastocysts (1 0-12)
• Retroviral infection of pre-implantation blastocysts (13-15). Infection
can be attained either by direct exposure ofblastocysts to viral particles
or by cocultivation with virus-producing cells.
A common disadvantage of direct microinjection and retroviral infection is
that these methods do not permit the delivery of the gene to a specific location of the chromosome, so that integration occurs at random. Therefore,
transfer ofDNA by direct microinjection into the pronucleus can cause insertional mutations which mayinterfere with the phenotype. In this context,
a wide range of mutations have been decribed previously (16-18). Furthermore, random positioning in the vicinity of other transciptional regulatory
sequences may lead to a position effect, resulting in higher or lower expression levels (18).
The microinjection of foreign DNA is widely used and is considered the
most efficient method for construction of transgenic animals. Whereas this
method has become a routine procedure in mice during the last ten years (9,
13 ), its application for the generation of transgenic rats has been developed
only recently. An approach to obtain a reliable positioning and copy number of the transgene, to regulate the level of expression, and to make the
transgene independent of the neighboring chromosome may be the use
of recombinant yeast artificial chromosomes (Y AC), which allow the introduction of large gene constructs into the germ line (19).