Chromosome, Microdissection and Microcloning
47
myelogenous leukemia. Microdissection
of a ring chromosome in another AML-
M5 case disclosed a (8 : 15) translocation
with overrepresentation of 8q22. The ori-
gin of a ring chromosome in a myxoid
malignant Fbrous histiocytoma (M±H)
was also determined by microdissection
and ±ISH analyses. Hybridization of the
microdissected labeled DNA to normal
metaphase cells revealed that the signal
localized only to 20q. Three signals were
seen in the tumor cells using either the
microdissected 20q probe or the chromo-
some 20 centromeric probe, indicating the
involvement of both the long arm and
the centromere in the ring chromosome.
Small accessory chromosomes (SACs) are
often associated with the centromeres of
other chromosomes. The occurrence of
more than two SACs in a single individual
is extremely rare. Recently, a case of mul-
tiple SACs was reported in two different
tissues of a moderately mentally retarded
male. Microdissection combined with reg-
ular ±ISH demonstrated that SACs were
derived from the centromeres of different
chromosomes.
6.6
Gene Transfer Using Chromosome
Fragments
Gene transfer into tissue culture cells or
embryos has become an exceptionally use-
ful method for the functional analysis of
the regulatory and coding regions of genes.
These methods are also used to induce
phenotypic changes in cells as well as
in whole organisms. The preferred vehi-
cle for gene transfer and expression is
a cloned gene (usually a cDNA with the
appropriate flanking regulatory regions,
promoters, and enhancers). Alternatively,
whole genomic DNA has been trans-
ferred, usually with dominant selective
markers. Gene-transfer experiments using
microdissected fragments from a speciFc
chromosome region would have several
potential advantages. Transfer of large
contiguous DNA stretches from microdis-
sected chromosome fragments allows the
gene to be presented ‘‘in context’’ – that
is, with its own promoters, enhancers,
control regions, and introns. These DNA
sequences may be critical to the appro-
priate cell-type-speciFc expression of the
transgene. The gene or locus of interest
does not have to be identiFed, isolated, and
cloned prior to gene transfer. This abil-
ity would be particularly advantageous in
cases of a disease-related locus that was
mapped genetically to a chromosome band
by linkage analysis but was not further
characterized. The transfer of genes into
embryos through the mediation of chro-
mosome fragments may be a useful tool
in generating animal disease models and
in conFrming genetic linkage studies that
map disease-causing genes to speciFc re-
gions. To be transferred to an animal,
however, the disease must behave as a pos-
itive dominant-acting genetic trait; it may
not be associated with a structural deletion.
An obvious disadvantage of chromosome
fragment–mediated gene transfer is that
only single-copy gene transfer is techni-
cally feasible. There are several reasons
for this limitation. One would be the size
and amount of DNA accepted by the re-
cipient cell and the labor-intensiveness of
the methodology. Two, physical manipu-
lation of large stretches of microdissected
DNA and microinjection would undoubt-
edly result in shearing of DNA into smaller
fragments. Although this method will re-
sult in position-independent integration,
the transferred DNA will be subject to
DNA methylation, controlled by genotype-
speciFc modiFers, as occurs with other
transgenes. Richa and Lo reported the Frst
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