Biomedical Engineering Reference
In-Depth Information
Production of transgenic mice
To produce a transgenic mouse, DNA is injected into the pronucleus
of a fertilized ovum (egg). In most transgenic models, DNA encoding
the gene of interest, including regulatory (promoter and enhancer) re-
gions surrounding the gene, is injected into the egg. The injected DNA
is randomly integrated into the host DNA in some of the eggs; frequently
multiple copies of the transgene are incorporated into a single integra-
tion site as a tandem array. The injected eggs are then re-implanted into
“pseudopregnant” female mice, which was prepared by hormone treat-
ment, and allowed to develop to full term. After birth, the DNA of the off-
spring is screened for the presence of the transgene, and positive mice
are then bred (usually to an inbred strain of mice) to produce a trans-
genic line. Because not all mice that have incorporated a transgene will
necessarily express it, mice are usually tested within 1 to 2 generations
for expression of the transgene. Transgenic lines that express the gene
will then be maintained for study.
In classic transgenic technology, transgenes are not integrated where
the gene would normally be encoded. Therefore, the ability of the trans-
gene to be expressed is often limited by a number of variables, includ-
ing whether all of the gene's regulatory regions have been successfully
cloned into the transgene. In addition, the site of integration can dramati-
cally affect both the level of expression and the correct tissue specificity
of expression of the transgene, since the site of integration may both
inhibit or permit (or amplify) expression. For this reason, a number of in-
dependently derived transgenic lines are often produced and examined
before selecting one or two for further studies.
BAC and YAC transgenics
Conventional transgenic mice are generated using purified DNA con-
taining a gene's coding sequences and its regulatory elements. Such
transgenes are generally limited to about 20,000 bp (20 kb) or less in
size. Conventional transgenic approaches, therefore, cannot be used
for genes that are encoded over larger regions of DNA, or have com-
plex regulatory regions that control their expression or tissue specificity.
Transgenic technology also cannot be used for the cloning and expres-
sion of linked genes that are encoded over larger stretches of DNA.
As already mentioned, the correct tissue specific expression of con-
ventional transgenes is subject to the site of integration. Some of
these limitations of transgenic technology are overcome by incorpo-
rating significantly larger segments surrounding genes using artificial
chromosomes, including BACs and YACs (described in Chapter 3).
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