122
Brain Development
Construction of the gene targeting vector
Gene casette for negative
selection
(e.g. Thymidine kinase gene)
Gene casette for positive selection
(e.g. Neomycin resistance gene)
Homologous regions
for the gene of interest
Electroporate the
targeting vector
into ES cells
Random insertion into the genome
Homologous recombination
Positive/negative selection
(e.g. Neomycin + Ganciclovir)
ES cells heterozygous for
the targeted allele by
homologous recombination
Thymidine kinase converts
ganciclovir into a toxic material,
killing ES cells with random insertions
of the targeting vector
Chimeric mice with ES cells'
contribution into the germ line
+/+
+/+
+/+
+/-
+/-
-/-
+/+
+/-
+/-
+/-
Fig. 11
How to generate gene targeting (knockout) mice.
it is now possible to drive gene expres-
sions in any tissue at any developmental
stage. Compared to the virus mediated
gene transfer method, EP mediated gene
transfer is less toxic to the relevant tissue
as well as for researchers. Furthermore,
EP is superior in its capability to con-
trol the area of gene transfer because
DNA constructs with a negative charge
will only migrate to the tissue facing the
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