Biology Reference
In-Depth Information
6.
Trypsin (
see
Subheading 2.3.
).
7.
Burker's chamber.
8.
9-cm Petri dish.
2.8.2. Induction of ES Cells-Derived Teratomas
1.
ES cells.
2.
ES medium (
see
Subheading 2.3.
).
3.
Feeder cells (
see
Subheading 2.3.
).
4.
Feeder medium (
see
Subheading 2.3.
).
5.
1X PBS (
see
Subheading 2.3.
).
6.
Trypsin (
see
Subheading 2.3.
).
7.
Sterile tubes (Falcon).
8.
Burker's chamber.
9.
1-mL syringe.
10.
Avertin (
see
Subheading 2.8.
).
3. Methods
3.1. Isolation of the Gene
The first step in a gene knock-out experiment is to isolate a genomic clone
spanning the locus of interest. To obtain high efficiency of homologous
recombination, the gene must be isolated from a genomic library made with
DNA from syngenic ES cells. Polymorphism present in DNA derived from
other mouse strains has been shown to greatly reduce the targeting rate
(25)
.
Using R1 ES cell line
(26)
, it is highly recommended that a cosmid library of
129sv mouse strain DNA is used.
1.
Plate serial dilutions of an aliquot of the library. After overnight incubation at
37
C, count the colonies and calculate the concentration of cells per mL of the
stock library.
°
10
5
cells in 1.5 mL of LB. Consider-
ing an average length of 45 Kb long inserts per cosmid, this number of cells
should cover the whole mouse genome approximately 3 times.
2.
Dilute the library stock to obtain about 2
×
3.
Plate 100
µ
L of the dilution onto 15 LB-Amp Petri dishes. Grow cells overnight.
4.
Cut round pieces of N+ nylon filters with a diameter shortly smaller than the
Petri dish itself.
5.
Overlay the colonies of one plate with a filter. Leave it in place for 1 min. In the
meantime, to allow a subsequent correct alignment of filters to the Petri dish,
make few punches with a syringe needle. Holes must not form a symmetric shape
and should mark the bottom of the dish perpendicularly to the surface of the
filter. Label marks with a black pen to ease their later detection.
6.
Prepare three trays containing a large piece of Whatmann 3M paper. Soak the
first with denaturing solution and the other two with neutralizing solution. Care-
fully peel off the nylon filter and overlay it upside down onto the first wet paper
(avoid formation of air bubbles). Incubate for 5 min.
Search WWH ::
Custom Search