Transgenic mouse derived from ES cells with a disrupted gene

Embryonic stem cells (ES) from a mouse blastocyst
are isolated (1) after 3.5 days of gestation
(of a total of 19.5 days) and transferred to a cell
culture (2). Here they will grow on a layer of irradiated
cells that are themselves unable to
grow (feeder layer). Target DNA (see B) from a
mouse homozygous for a marker coat color, e.g.,
dominant black, is added to the ES cell culture.
Very few cells or perhaps just one may take up
target DNA by recombination with the homologous
gene in the ES cell (3). This disrupts
the normal gene. These recombinant ES cells
can be grown in a selective culture medium
(nonrecombinant cells will not grow, see B). Recombinant
ES cells containing a copy of the disrupted
gene are injected into a recipient mouse
blastocyst (4). These cells will be integrated into
the early mouse embryo (5). The blastocyst
partly containing recombinant ES cells is transferred
into a pseudopregnant mouse (6). After
birth (6), mice derived fromnormal and recombinant
cells (chimeric mice) can easily be identified
by spots of black coat color (7). When
adult chimeric mice are mated to normal mice
homozygous for another coat color allele (e.g.,
white, 8), the birth of black progeny indicates
that the targeted gene is present in the germ
line (9). The mating of such heterozygous mice
(not shown) will produce mice that are homozygous
for the disrupted gene