Target Discovery and Validation Reviews and Protocols

Keratin Transgenics and Knockouts 227
of Cre- and Flp-recombinases can be combined with double replacement to allow
a variety of options (Fig. 4D,E,F).
1.2.3. Large-Scale Genome Engineering
If it becomes necessary to delete larger genes or gene fragments from the
genome, the approaches described above become inefficient as the distance
between Lox P sites increases. To create deletions of a large segment of chromosomal
DNA, Bradley and colleagues developed the following scheme, based on
two consecutive homologous recombination events, one at each end of the target
locus. The 5′ vector consists of a neomycin resistance gene for gene targeting, a
Lox P site, a 5′ half HPRT minigene (nonfunctional HPRT fragment), and
approx 6- to 10-kb of genomic DNA homologous to one end of the target locus
(Fig. 4G). Similarly, the 3′ vector contains a puromycin resistance gene, a Lox
P site, a 3′ HPRT-minigene (nonfunctional HPRT fragment), and approx 6- to
10 kb of genomic DNA homologous to the other end of the target locus
(123).The Lox P site has been placed in an intron of the HPRT-minigene. These
vectors can be generated conventionally by sequential insertion of various
genetic components into a plasmid construct (123), or they can be isolated directly
from genomic libraries of premade targeting vectors (see http://www.ensembl.org/
and http://www.ensembl.org/Mus_musculus/). Depending on the orientation of
the LoxP sites, the location of the Lox P site on the two alleles, and whether the
recombination takes place between sister chromatids or between the nonsister
chromatids in the G 2 phase of cell cycle (see ref. 123), various recombination
events, such as deletion, inversion, and duplication of the region between the
LoxP sites, can occur. Upon Cre activity, recombination between the Lox P sites
reconstitutes a functional HPRT-minigene. This HRRT-minigene provides a
positive selection for this event when the ES cells are cultured in HAT medium.
1.3. Culture of ES Cells
ES cells are available from several laboratories and from commercial suppliers.
The majority of presently available lines has been derived from one of
several mouse 129 inbred strains and maintain good germline transmission
capabilities. C57BL/6 ES lines are also available, but they seem more susceptible
with respect to germline transmission. Recently, ES cells from F1 or mixed
hybrid genetic background strains were isolated, and they show very high
germline transmission frequencies because of hybrid vigor (124–126). These
cells were developed to shorten the time required for the generation of genetargeted
mice considerably. The concept is based on the use of tetraploid
embryos, generated by electrofusion of ES cells, for injection of genetically
engineered diploid ES cells. Tetraploid cells have been shown to give rise
exclusively to extraembryonic tissues, whereas diploid ES cells contribute to