Target Discovery and Validation Reviews and Protocols

Keratin Transgenics and Knockouts 225
Fig. 4. (Continued) targeting vector. The short arm of homology carries the desired alteration
(upright dark gray bar in the 5′ open box) created by mutagenesis. If in addition, a new
restriction site has been generated without altering the properties of this sequence, presence
of the desired mutation can be directly screened by restriction analysis of PCR products.
Middle, target locus after homologous recombination. Presence or absence of desired alteration
is indicated by dotted lines before and after restriction of PCR products. Bottom,
target locus after deletion of marker gene by site-specific recombinase. One recognition site
resides in the genome. (C) Double replacement gene-targeting procedure by using
positive/negative selection. Top, target locus. Middle, gene-targeting vector. Middle, after the
first gene-targeting step, the gene or gene fragment has been replaced by an HPRT-minigene
(gray box), providing resistance against HAT-containing media. The target locus can be
replaced by modified gene sequences. Below, second step gene targeting to introduce a subtle
alteration (upright bar) in the left exon. Selection in media containing 6-TG (thioguanine)
indicates loss of HPRT-marker gene. (D) Conditional gene targeting by using three
Lox P recognition sites. Top, target locus. Middle, gene-targeting vector with lox P site
flanking the exon to be removed upon Cre activation (upright gray boxes, lox P recognition
sites). The lox P-flanked marker gene is placed in an intron. After Cre activity, several types
of deletions can occur. Type I, complete excision creating a null allele (type I), Type II
undesired backwardness of the marker gene. Type III, excision creating a conditional allele.
The outcome is determined by various parameters, e.g., the relative position of the lox P
sites and the extent of Cre activity. (E) Conditional gene targeting by using FRT and lox P
recognition sites. Top, target locus. Gene-targeting vector with FRT sites (light gray upright
boxes) flanking the marker gene (gray) and two lox P sites (gray upright boxes) flanking
the exon open box to be deleted. After homologous recombination and FLP activity, the
marker gene has been removed with one FRT site left behind (black). Upon Cre activity,
the “floxed” exon is deleted with one lox P site left behind. (F) Gene modifications by using
double replacement gene targeting. Top, target locus. Bottom, gene-targeting vector. Below,
after the first gene-targeting step, the gene or gene fragment has been replaced by an HPRTminigene,
providing resistance against HAT-containing media. The target locus can be
replaced by modified gene sequences as indicated below. (1) Replacement with a conditional
floxed allele. (2) Replacement with a related gene (open box). (3) Replacement with
a mutant gene copy (gray box). (4) Replacement with an inducible, promoterless Cre- or
FLP-recombinase to be driven by the target gene promoter. (5) Replacement with a conditional,
silent gene copy, followed by a wild-type copy of a related gene (gray box). (6) As
before, but followed by a mutant (bar) copy of a related gene. (G) Generation of large-scale
genomic deletions, based on a HPRT-deficient ES cell line. Top, target locus. Below, targeting
vector for 5′ flank of targeting vector, consisting of a nonfunctional half-HPRT-minigene
(small gray box, arrow indicates minigene promoter), followed by a lox P site (upright dark
gray box) and a neomycin resistance gene (dark gray framed box, arrow marking promoter).
After homologous recombination, G418-resistant ES cell clones are identified by
PCR. Below, targeting vector for 3′ end, consisting of a puromycin resistance gene
(light gray box) and the 3′ half of a nonfunctional HPRT-minigene (dark gray), preceded
by a lox P site (dark gray upright box). Below, after homologous recombination, puromycinresistant
ES cell clones are identified by PCR. Complex rearrangements can