Biomedical Engineering Reference
In-Depth Information
Examples of increased, decreased or even totally
extinguished expression were found. In some, but
not all, cases, the changes in expression correlated
with changes in methylation of the gene sequences
(Palmiter
et al
. 1982b). These results provided the
first examples of two complex phenomena,
position
effects
(Box 11.1) and
de novo transgene silencing
(Box 13.2), which often affect integrated transgenes.
Yeast artificial chromosome (YAC)
transgenic mice
Studies of the
MMT
promoter and others have
demonstrated the principle that transgenes with
minimal flanking sequences tend not to be expressed
in the same manner as the corresponding endogen-
ous gene. In many cases, it has also been shown
that authentic patterns and levels of protein expres-
sion occur only when the intact gene is used, and
this can span tens or hundreds of kilobase pairs
of DNA (Box 11.1). The transfer of large DNA
segments to the mouse genome has been achieved
by transformation with yeast artificial chromosome
(YAC) vectors. Jakobovits
et al
. (1993) were the first
to report transformation of ES cells with a YAC
vector, via fusion with yeast sphaeroplasts. The vec-
tor contained the entire human
HPRT
locus, nearly
700 kb in length. The disadvantage of this method is
that the endogenous yeast chromosomes were co-
introduced with the vector. Alternative strategies
involve isolation of the vector DNA by pulsed-field
gel electrophoresis (p. 10), followed by introduction
of the purified YAC DNA into mouse eggs by pro-
nuclear microinjection or transfection into ES cells.
The latter technique is more suitable because micro-
injection involves shear forces that break the DNA
into fragments. YAC transfer to ES cells has been
achieved by lipofection, as discussed in Chapter 10.
YAC transgenics have been used to study gene
regulation, particularly by long-range regulatory
elements, such as locus-control regions (reviewed
by Lamb & Gerhart 1995). They have also been used
to introduce the entire human immunoglobulin locus
into mice, for the production of fully humanized
antibodies (Mendez
et al
. 1997). It is also possible to
introduce chromosomes and chromosome fragments
into ES cells, using a technique called microcell-
mediated fusion. This involves the prolonged mitotic
Fig. 11.5
Transgenic mouse containing the mouse
metallothionein promoter fused to the rat growth-hormone
gene. The photograph shows two male mice at about
10 weeks old. The mouse on the left contains the
MGH
gene
and weighs 44 g; his sibling without the gene weighs 29 g.
In general, mice that express the gene grow two to three times
as fast as controls and reach a size up to twice the normal.
(Photograph by courtesy of Dr R.L. Brinster.)
high copy numbers of the
MGH
gene (20 - 40 copies
per cell) had very high concentrations of growth
hormone in their serum, some 100 - 800 times above
normal. Such mice grew to almost double the weight
of littermates at 74 days old (Fig. 11.5).
The similarities between the tissue distribution of
normal
MMT
expression and that of the hybrid
transgenes encouraged the hope that transgenic
mice would provide a general assay for functionally
dissecting DNA sequences responsible for tissue-
specific or developmental regulation of a variety of
genes. However, there were also some unexpected
findings. For example, independently derived trans-
genic mice carrying the
MK
transgene showed
significant variations in the levels and patterns of
transgene expression. Furthermore, while trans-
genic founders transmitted the construct to their
progeny as expected, when reporter activity was
assayed in these offspring the amount of expression
could be very different from that in the parent.
