Fig. 2. Southern blot and PCR analysis. (A) Southern blot analysis of targeted ES
cell lines. The positions of external probe and internal probe used for
screening targeted ES cell lines are indicated in
Fig. 1c. Targeted cell lines
were confirmed by two different restriction digestions (P) PstI, and
(N) NheI, generating a 6.1 kb wild-type band and 4.1 kb targeted
band, and 5.5 kb wild-type band and 7.3 kb targeted band, respectively. (B)
PCR identification of the swapped homeobox encoding the A13
homeodomain (HD) using primers a and b (indicated in
Fig. 1C). PCR amplified
fragments were digested with PstI, HincII (HcII) and
EcoRV (RV). Only the swapped A13 PCR fragment was cut by all
three enzymes to give distinguishable smaller sized bands. (PstI,
480, 354 and 126 bp; HincII, 480, 323 and 157 bp; EcoRV,
480, 260 and 220 bp). The presence of the precise homeobox swap was confirmed
by sequencing. (C) PCR identification of Cre recombination.
Cre-expressing transgenic mice were used to remove the Neo
gene in the targeted A11 locus. A11 specific primers c and d
(indicated in Fig. 1C) did not
amplify the 2 kb (loxpNeoloxp) fragment under the PCR conditions
used, but gave a 317 bp fragment with loxp, or a 285 bp fragment
without loxp (wild type). wt, wild type; M, pBR322 DNA-MspI
marker.
