Biology Reference
In-Depth Information
7.
Transfer nylon filters to the paper soaked in neutralizing solution and incubate
for 5 min. Repeat this step transferring filters to the second neutralizing solution
wet 3M paper.
8.
Repeat from
step 5
for each Petri dish.
9.
Prehybridize all nylon filters in Church's buffer for at least 1 h at 65
C. Hybrid-
ize filters at the suitable temperature (
see
Note 1
) overnight in fresh Church's
buffer containing about 10
6
cpm/mL of radioactively labeled probe.
°
10.
Wash with the required stringency (
see
Note 1
) and expose filters. Mark the
developed film with the position of the holes on the filters.
11.
To isolate positive colonies, superimpose the marks on the film to the marks on
the Petri dish. With a cut blue tip, excise a cylinder of agarose corresponding to
the positive colony. Incubate the piece of agarose in 1 mL of LB-Amp to resus-
pend cells. Plate serial dilution of this aliquot to obtain single colonies. Repeat
from
step 4
to
10
until positive clones can be isolated as single colonies.
3.2. Generation of Constructs
The next step, after isolation of a genomic clone is to generate, by general
DNA cloning means
(27)
, a construct bearing the mutation that has to be intro-
duced into ES cells DNA.
First, a detailed restriction map of the genomic clone must be obtained. To
accomplish this task it is advisable to isolate subclones of the cosmid DNA and
analyze their restriction patterns separately. Each subclone should be digested,
following standard molecular biology protocols
(27)
, with as many as possible
restriction enzymes. Contigs of subclones should be generated and DNA frag-
ments that are supposed to contain interesting exons sequenced.
Once the structure of the isolated locus is determined, the clone must be
ideally divided into two nonoverlapping regions. The first must contain the
probe to isolate mutant clones by Southern blot and can be as short as few
hundred bp (
see
Note 2
). The second must comprise the sequences required to
generate the construct. It has been shown that gene-targeting frequency strongly
depends on the length of the ES DNA used
(28)
. Thus, a general strategy con-
sists of finding a DNA fragment containing the region that has to be mutated
(e.g., an exon or a promoter) flanked by at least 8 kb (
see
Note 3
). Because ES
cells are transfected with linear DNA, it is important to keep a single restric-
tion site at the junction between either one of the arms and the vector.
3.2.1. Definition of the Probe
To initially establish a gene-targeting strategy, a probe that permits the iden-
tification of the mutants by Southern blot analysis must be isolated. Genomic
DNA is interspersed by repetitive elements, which give high backgrounds in
hybridization analysis. It is therefore essential to test several fragments of the
cosmid clone to isolate a probe encoding a single genomic sequence.
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