Gene Cloning

The Production and Uses of Transgenic Organisms 399
neo gene
1
Vector
Gene X
Thymidine kinase gene
from H. simplex virus
Gene X
Chromosome
neo gene
2
Vector
Chromosome
Figure 12.17 Production of and selection for cells carrying homologous
recombination events leading to gene knockouts. The crosses show the
sites of cross-over events, which will lead to integration of the vector DNA
between the cross-overs into the chromosome. Event (1) (homologous
recombination) will lead to the insertional inactivation of the chromosomal copy
of gene X, leading to a neo R phenotype for the cells which contain this event.
The cells will be resistant to gancyclovir as the tk gene will not be integrated.
Event (2) (non-homologous recombination) will lead to cells which are neo R but
also gancyclovir-sensitive, and these will not grow on medium containing both
compounds.
Fortunately, cells containing the tk gene (which are not wanted in this
experiment) can be selected against by adding the chemical gancyclovir.
The thymidine kinase if present and expressed converts this into a
nucleotide analogue which is incorporated into replicating DNA, an event
which is lethal for the cell in which it occurs. Thus the combined presence
of neomycin and gancyclovir in the growth medium selects for all cells
where an insertion of novel DNA has occurred, and against those where the
insertion is non-homologous, leaving those where the insertion is homologous
and has thus disrupted the target gene.
There is much more work to do before a transgenic animal with the
specified gene knockout results. First, the transformed ES cells must be