Tissue-Specific KO of ECM Proteins - Methods in Molecular Biology: Extracellular Matrix Protocols - page 164

Fig. 4. Outline of the strategy to genotype litters by PCR. Arrows indicate the oli-
gonucleotide from 5' to 3'. The white arrow indicates an oligonucleotide that can bind
to both wild-type and mutant alleles. The black and hatched arrows correspond to the
downstream oligonucleotides that anneal to the wild-type and targeted allele, respec-
tively. Modulation of the distance from the common to the specific oligonucleotides
allows to distinguish the two amplified fragments. As shown in the framed panel,
amplification with the three oligonucleotides with homozygous and heterozygous
DNA produces one or two distinct bands, respectively.
potential of these double mutant cells to contribute to the formation of differ-
ent organs may give essential information on gene function in established
tissues (34) .
Homozygous ES cells can be generated by electoporation of a second tar-
geting construct that bears a different selection cassette (e.g., hygromycin in
place of neomycin resistance gene). Alternatively, cells bearing a null allele
generated by Cre -mediated excision of the selection marker gene ( Fig. 2 ) can
be transfected again with the same construct. In these two situations about 50%
of the recombinant clones should show by Southern blot hybridization a
homozygous-specific pattern of bands.
The simplest method, however, consists of growing heterozygous ES cells
in a medium containing very high levels of selection drug (35) ( see Note 16 ).