Biomedical Engineering Reference
In-Depth Information
phenotype, allowing a BAC clone corresponding to
the functional
Shaker-2
gene to be identified. The
gene was shown to encode a cytoskeletal myosin
protein. This was then used to screen a human
genomic library, resulting in the identification of the
equivalent human gene. Note that no sequence
information was required for this screening proced-
ure, and without the functional assay there would
have been no way to identify either the mouse or
human gene except through a laborious chromo-
some walk from a linked marker. The recent devel-
opment of high-capacity transformation vectors for
plants (p. 236) has allowed similar methods to be
used to identify plant genes (e.g. Sawa
et al
. 1999,
Kubo & Kakimoto 2001).
of quiescent mouse 3T3 fibroblast cells either in
culture or when transplanted into 'nude mice' (e.g.
Brady
et al
. 1985). Many different specific assays
have also been developed for the functional cloning
of cDNAs encoding particular types of gene product.
For example,
Xenopus
melanophores have been used
for the functional cloning of G-protein-coupled
receptors. Melanophores are dark cells containing
many pigment organelles, called melanosomes. A
useful characteristic of these organelles is that they
disperse when adenyl cyclase or phospholipase C
are active and aggregate when these enzymes are
inhibited. Therefore, the expression of cDNAs encod-
ing G-protein-coupled receptors and many types of
receptor tyrosine kinases leads to redistribution of
pigmentation within the cell, which can be used
as an assay for the identification of receptor cDNAs
(reviewed be Lerner 1994).
Screening by 'gain of function'
Complementation analysis can be used only if an
appropriate mutant expression host is available. In
many cases, however, the function of the target
gene is too specialized for such a technique to work
in a bacterial or yeast expression host and, even in
a higher eukaryotic system, loss of function in the
host may be fully or partially compensated by one or
more other genes. As an alternative, it may be possi-
ble to identify clones on the basis that they confer a
gain of function on the host cell. In some cases, this
gain of function is a selectable phenotype that allows
cells containing the corresponding clone to be posi-
tively selected. For example, in an early example
of the expression of a mammalian gene in
E. coli
,
Chang
et al
. (1978) constructed a population of
recombinant plasmids containing cDNA derived
from unfractionated mouse mRNA. This population
of mRNA molecules was expected to contain the
transcript for dihydrofolate reductase (DHFR). Mouse
DHFR is much less sensitive to inhibition by the drug
trimethoprim than
E. coli
DHFR, so growing trans-
formants in medium containing the drug allowed
selection for those cells containing the mouse
Dhfr
cDNA.
In other cases, the phenotype conferred by the
clone of interest is not selectable, but can be detected
because it causes a visible change in phenotype. In
mammalian cells, for example, clones corresponding
to cellular oncogenes have been identified on the
basis of their ability to stimulate the proliferation
Difference cloning
Difference cloning refers to a range of techniques
used to isolate sequences that are represented in one
source of DNA but not another. Normally this
means differentially expressed cDNAs, representing
genes that are active in one tissue but inactive in
another, but the technique can also be applied to
genomic DNA to identify genes corresponding to
deletion mutants. There are a number of cell-based
differential cloning methods and also a range of PCR
techniques. Each method follows one of two prin-
ciples: either the differences between two sources are
displayed, allowing differentially expressed clones to
be visually identified, or the differences are exploited
to generate a collection of clones that are enriched
for differentially expressed sequences. The analysis
of differential gene expression has taken on new
importance recently with the advent of high-
throughput techniques allowing the monitoring of
many and, in some cases, all genes simultaneously.
Difference cloning with DNA libraries
Displaying differences - differential screening
An early approach to difference cloning was
differential
screening
, a simple variation on normal hybridization-
based library screening protocols that is useful for
