Genetic manipulation of animals - Principles of Gene Manipulation - page 210

Box 11.1 Position effects

Independently derived transgenic animals and plants
carrying the same expression construct often show
variable levels and patterns of transgene expression.
In many cases, such variation is dependent on the
site of transgene integration, and this phenomenon
has been termed the position effect (reviewed by
Wilson et al . 1990). Position effects result from
the influence of local regulatory elements on the
transgene, as well as the architecture of the
surrounding chromatin. For example, an integrated
transgene may come under the influence of a local
enhancer, resulting in the alteration of its expression
profile to match that of the corresponding
endogenous gene. The position dependence of
the phenomenon has been demonstrated in mice
by isolating the entire transgenic locus from such
an anomalous line and microinjecting it into the
pronuclei of wild-type eggs, resulting in 'secondary'
transgenic lines with normal transgene expression
profiles (Al-Shawi et al . 1990). Position effects are
also revealed by enhancer-trap constructs, which
contain a minimal promoter linked to a reporter
gene (O'Kane & Gehring 1987; see Chapter 13).
Unlike the specific influences of nearby regulatory
elements, chromatin-mediated position effects are
generally non-specific and repressive. They reflect
the integration of the transgene into a chromosomal
region containing repressed chromatin
(heterochromatin). The molecular features of
heterochromatin, including its characteristic
nucleosome structure, deacetylated histones and,
in many cases, hypermethylated DNA, spread into
the transgene, causing it to be inactivated (Huber
et al . 1996, Pikaart et al . 1998). In some cases,
variegated transgene expression has been reported
due to cell-autonomous variations in the extent of
this spreading process (reviewed by Heinkoff 1990).
Negative chromosomal position effects can be
troublesome in terms of achieving desirable
transgene expression levels and patterns; thus
a number of different strategies have been used
to combat them.

Incorporating dominantly acting
transcriptional control elements
Certain regulatory elements are thought to act as
master-switches, regulating the expression of genes
or gene clusters by helping to establish an open
chromatin domain. The locus control region (LCR)
of the human b -globin gene cluster is one example
(Forrester et al . 1987). Transgenic mice carrying
a human b -globin transgene driven by its own
promoter show a low frequency of expression and, in
those mice that do express the transgene, only a low
level of the mRNA is produced (e.g. Magram et al .
1985, Townes et al . 1985). However, inclusion of the
LCR in the expression construct confers high-level
and position-independent expression (Grosveld et al .
1987). There is evidence that LCRs induce chromatin
remodelling over large distances. For example, the
murine immunoglobulin heavy-chain LCR has been
shown to induce histone deacetylation in a linked
c- myc gene (Madisen et al . 1998). This suggests
that LCRs could protect against position effects by
converting heterochromatin to open euchromatin at
the site of transgene integration (Festenstein et al .
1996, Milot et al . 1996). The interested reader can
consult several comprehensive reviews of LCR
research (Bonifer 1999, Grosveld 1999, Li et al .
1999).

Using boundary elements/matrix
attachment regions
Boundary elements (insulators) are sequences that
can block the activity of enhancers when placed
between the enhancer and a test transgene driven by
a minimal promoter. For example, an 'A element' with
insulator activity is found upstream of the chicken
lysozyme gene. This inhibits the activity of a reporter
gene when interposed between the promoter and an
upstream enhancer, but not when placed elsewhere
in the construct (Stief et al . 1989). However,
by flanking the entire construct with a pair of

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