An Inducible and Reversible Mouse Genetic Rescue System - Genetic Engineering: Recent Developments in Applications - page 261

PCR analysis of the ES DNA using two vector-specific and gene-specific primer
pairs. The ApoE-specific primers are antisense and located in the fourth exon.
Several independent retroviral insertions in the ApoE gene were identified. PCR
fragments were sequenced to confirm insertion into the gene. A library tube with
a clone of interest identified by the PCR also contains a few hundred other ES
cell clones. The sole desired clone was isolated from the mixture by three rounds
of cell sorting and growing followed by PCR using the same pair of primers to
identify positive clones. Additional PCRs using ApoE primers located in the third
intron flanking the viral insertion site were conducted to confirm the precision of
the insertion and integrity of the genomic sequence of ApoE.
Generation of tiGre es clones containing target Genes
Full length cDNAs for the coding sequences of target genes were cloned into
the TIGRE-targeting vector containing TRE, insulators, a PGK promoter and a
pair of loxP sites (Figure 5A lower panel). The TIGRE-targeting vectors were co-
transfected with a Cre-expressing plasmid into the neo-sensitive ES cells carrying
the minimal TIGRE locus with a single loxP site and the promoterless, ATGless
loxneo marker. When the TIGRE-targeting vector is integrated into the TIGRE
locus through Cre/lox-mediated recombination, neo-resistance is restored to the
ES cells by the addition of PGK promoter and in-frame fusion of ATG to the
loxneo marker. Correctly integrated ES clones were identified and confirmed by
PCR screening and southern blot analysis.
Animal Production and Maintenance
ES cell clones were injected into blastocysts of C57BL/6J mice following stan-
dard techniques. Chimeric mice were bred with C57BL/6J mice to test germline
transmission and generate heterozygous mice. Mice from the corresponding KO
lines and TIGRE lines were crossed with each other to produce inducible KO
mice according to the scheme shown in Figure 1B. All mice used for studies in
this paper were in a mixed genetic background of 50% 129S1/SvImJ and 50%
C57BL/6. For the ApoE KO line, southern blot using rtTA coding sequence as
probe confirmed the correct insertion of the retroviral vector into the endog-
enous ApoE gene. It also revealed an additional retroviral insertion somewhere
else in the genome. The additional insertion was selectively bred out, and the
ApoE iKO colony was maintained with a single retroviral insertion at the ApoE
locus. MMTV-tTA, PCAMKII-tTA and PNSE-tTA mice were purchased from
The Jackson Laboratory (Bar Harbor, ME). All experimental procedures were ap-
proved by the Institutional Animal Care and Use Committee of NCI and Nura,
Inc. in accordance with NIH guidelines.