Biomedical Engineering Reference
In-Depth Information
Artificial chromosomes can be used to clone significantly larger
stretches of DNA, as much as two million bp. The use of artificial chromo-
somes in constructing transgenic mice can be used for studies of linked
genes, as well as for studies in which the correct spatial and temporal
regulation of a gene(s) is required.
E.
Targeted transgenes: “knockout” and
“knockin” mice
Targeted transgenic mice are special types of transgenics in which an
endogenous gene is functionally or completely eliminated (knockout) or
altered (knockin). The process for creating targeted transgenic mice is
quite different than the method for creating transgenic mice. Instead of
beginning with a fertilized ovum, targeted transgenes begin with em-
bryonic stem cells (ES cells) and a modified genomic DNA construct
called a targeting vector. The targeting vector is used to alter or replace
the endogenous gene using the process of homologous recombination
within the ES cell line. Homologous recombination is the process that re-
sults from the exchange of genetic material between two DNA molecules
(usually paired chromosomes) at sites of DNA identity (homology). In
the case of making targeted transgenics, one of the two “chromosomes”
is replaced by the targeting vector carrying the alteration of interest.
ES cells are cells that have the capacity to give rise to all cells of the
body. Human ES cells have recently received considerable attention in
the press and in government due to their potential use for cloning human
beings. The isolation and description of the first ES cells in vitro was
reported by Martin Evans and Matt Kaufman in 1981 (44). Evans shared
the Lasker Award for Biomedical Research in 2001 with Mario Capecchi
(45,46) and Oliver Smithies (47,48) for their work on developing knockout
technology. A number of Lasker Award winners have gone on to win
Nobel Prizes of their own.
Knockout mice
Knockout mice are used to create models of human diseases in which
genes have been silenced, such as Alzheimer's disease, cystic fibrosis,
heart disease, and diseases caused by immunological defects. For gen-
erating knockout mice, the targeting vectors contain the gene of interest
with variable amounts of flanking sequences (usually 1 to 10 kb). One
or more exons of the gene are replaced by a selectable marker under
the control of a constitutively active promoter. The selectable marker
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