Biology Reference
In-Depth Information
The following two requirements should be met before undertaking the
construction of the vector. First, diagnostic restriction sites should be
identified that distinguish the targeted from the normal allele and that digest
DNA selected from ES cells fully and reproducibly. Second, hybridization
probes should be selected that yield little or no background with the same
DNA samples and that can “see” the recombination from the inside and the
outside, and from upstream and downstream of the region covered by the homology
arms of the vector.
Because the neo-cassette may occasionally affect proper expression of the
targeted gene
(4)
, one may wish to delete it after the ES clone selection. To this
end, short recognition sequences (
loxP
) for enzyme-mediated recombination
(
Cre
) can be placed 5' and 3' of the neo-cassette
(5)
. Expression of
Cre
results
in deletion of the DNA lying within the
loxP
sites arranged in a head-to-tail
orientation. The
Cre
enzyme can be expressed transiently in expanded cultures
of individual ES clones. It can also be expressed constitutively in siblings of
crosses between targeted ES-derived mice and
Cre
-recombinase-producing
transgenics. The less than optimal efficiency of
Cre
-mediated recombination
may yield a heterogenous population of ES cells in tissue culture, and germ-
line mosaicism in animals; both events can be readily monitored by careful
Southern blot analyses.
ES cells are derived from a mouse strain whose coat color (agouti; 129/sv) is
dominant over the one whose blastocysts they will eventually colonize (black;
C57BL/6J). ES cells are maintained and grown on a feeder layer of mitotically
inactivated and G418
r
embryonic fibroblasts (EF) prepared from embryos
homozygous for the neo-cassette
(6)
. The double selection scheme (G418/
Gancyclovir) eliminates most, but not all, of the nonhomologous recombinants.
Final validification rests on Southern analyses with probes 5' and 3' of, and
within and outside the chromosomal region being targeted. The efficiency of
homologous recombination varies greatly amongst different genes and within
different regions of the same gene.
2. Materials
2.1. ES Cell Culture and Storage
1.
Growth medium for ES and EF cells: Hepes-buffered 20 m
M
, pH 7.3 (Sigma, St.
Louis, MO; #H-0887) Dulbecco's modified Eagles medium (DMEM, high
glucose; Gibco, Gaithersburg, MD; #11995-040) supplemented with 15% heat-
inactivated Fetal Calf Serum (FCS; HyClone, Logan, UT; # SH30070.03), 0.1 m
M
nonessential amino acids (Gibco; #11140-050), 0.1
M
-mercaptoethanol (Sigma;
#M7522), 1X antibiotics (penicillin/streptomycin; Gibco; #15140-122) and 1000 U/mL
of Leukemia Inhibitory Factor (LIF; Gibco; #13275-029). Medium is sterilized
by filtering through a 0.22-
β
µ
m pore size filter system (Costar; #430769) and
stored at 4
°
C.
Search WWH ::
Custom Search