Target Discovery and Validation Reviews and Protocols

Keratin Transgenics and Knockouts 223
the latter case, HPRT-deficient ES cells are required. Only ES cells that harbor
an HPRT-minigene can grow in hypoxanthine, aminopterin, and thymidine
(HAT)-containing selective medium. The major advantage of the HPRT system
is that it allows for negative selection in medium containing 6-thioguanine
(6-TG) (103). In our experience, we have not observed any advantage for the
use of negative selectable marker genes outside the homology arms.
In most cases, selectable marker genes with their own promoters, usually
PGK-1, are used. In rare instances, the phosphoglycerate kinase (PGK)-1 promoter
becomes silenced because it carries a partial pCpG island and may have to
be replaced by other promoters (105). To minimize transcriptional interference
between the gene’s own promoter and the minigene promoter, we recommend
placing the selectable marker gene opposite the target gene’s transcriptional orientation.
To minimize potential effects of the selectable marker gene on neighboring
genes, and on the phenotype of mice, it is desirable to remove it. This removal
can be done by placing Lox P or FRT recognition sequences at both ends of the
selectable marker cassette. After transient expression of Cre or Flp-recombinase
in ES cells, the marker gene is removed and ES cells become sensitive to the
selective agent. This selectivity allows the use of the original targeting vector to
create ES cells homozygous at the targeting locus (106,107). If conditional gene
targeting is planned, it is advisable to use FRT sites to tag the selectable marker
gene (Fig. 4B).
For those IF genes that are transcribed in ES cells, very high gene targeting
frequencies can be achieved using promoterless selectable marker genes that
are placed in the first coding exon, either in frame with the endogenous ATG
codon or by positioning the initiation codon of the marker gene downstream
from the major transcription start site. If insertion of a promoterless marker
gene is required in an exon further 3′ into the gene, an IRES (108) can be incorporated
to enable translation of the marker gene. A slight modification of the
aforementioned design can be used to introduce a promoterless reporter gene
along with the selectable marker to follow the cells carrying the targeted allele.
The enhanced green fluorescent protein- or the more sensitive lacZ-reporter
genes are convenient but do not necessarily provide quantitative readouts
(109,110).
There are several ways to introduce subtle alterations, i.e., point mutations,
into genes. The mutation can be positioned in one of the homology arms of
the targeting vector and be cotransferred along with the selectable marker, provided
recombination does not separate the mutation from the selectable marker
(Fig. 4B). The selectable marker gene has to be positioned either in a noncoding
exon or in an intron, from where it can be removed using Flp or Cre-recombinases,
leaving one recombinase recognition site in the genome. In general,
cotransfer of mutations work well, provided the distance between the marker