The specific experimental inactivation of a gene
affords an opportunity to study its normal function
by comparing it with that of the inactive
state. This will yield information about the role
of a particular gene in development or other
functions that would not be available otherwise.
In the approach described here, a normal
gene is replaced by a mutant allele by disrupting
the normal gene (targeted gene disruption).
The effects can be studied in different embryonic
stages of mouse development and after
birth. Subsequently the results can be compared
to the effects ofmutations in homologous
human genes as seen in human genetic diseases.
The method requires the use of mouse
embryonic stem (ES) cells. ES cells are
pluripotential, i.e., they are capable of giving
rise to different kinds of cells but not to an entire
organism. These cells can be grown in culture
through many generations and yet retain
the potential to be integrated into a mouse blastocyst.
Here they participate in embryonic
development allowing mice to produce that are
homozygous for a mutation introduced into a
specific gene (knockout mice).
Sunday, April 12, 2009
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
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
Double selection for ES cells
containing the disrupted gene
The isolation of mouse ES cells with a gene-targeted
disruption requires positive and a negative
selection. A bacterial gene conferring resistance
to neomycin (neoR) is added (2) into
DNA cloned from the target gene (1). DNA containing
the thymidine kinase gene (tk+) from
herpes simplex virus is then also added to the
vector outside the region of homology. These
two selectable modifications (neoR and tk+) are
part of the replacement vector (3). The vector is
introduced into ES cells, which are grownin culture.
The isolation of mouse ES cells with a gene-targeted
disruption requires positive and a negative
selection. A bacterial gene conferring resistance
to neomycin (neoR) is added (2) into
DNA cloned from the target gene (1). DNA containing
the thymidine kinase gene (tk+) from
herpes simplex virus is then also added to the
vector outside the region of homology. These
two selectable modifications (neoR and tk+) are
part of the replacement vector (3). The vector is
introduced into ES cells, which are grownin culture.
ES cells
All ES cells that do not take up the vector
(the majority) are sensitive to neomycin and
will not grow in a medium containing the antibiotic
(positive selection, not shown). Cells that
take up the vector (about 1%) do so either by
nonhomologous insertion at random sites (4) or
by homologous recombination at the target site
(5). These cells can be distinguished when
grown in a selective medium containing gancyclovir,
a nucleotide analogue toxic to cells containing
tk+. Only cells containing the gene-targeted
insertion (through homologous recombination)
will grow because they do not contain
tk+ (negative selection).
(the majority) are sensitive to neomycin and
will not grow in a medium containing the antibiotic
(positive selection, not shown). Cells that
take up the vector (about 1%) do so either by
nonhomologous insertion at random sites (4) or
by homologous recombination at the target site
(5). These cells can be distinguished when
grown in a selective medium containing gancyclovir,
a nucleotide analogue toxic to cells containing
tk+. Only cells containing the gene-targeted
insertion (through homologous recombination)
will grow because they do not contain
tk+ (negative selection).
Genomics, the Study of the Organization of Genomes
A genome contains all biological information
required for life and/or reproduction.
The term genomics for the study of genomes
was introduced in 1987 by V. A.McKusick and F.
H. Ruddle at the suggestion of T. H. Roderick of
the Jackson Laboratory, Bar Harbor,Maine, USA.
The term genomics extends beyond genetics.
While the latter mainly deals with heredity and
its mechanism and consequences, the term
genomics encompasses many aspects relating
to molecular and cell biology: the different
types of genomic maps; nucleic acid sequencing;
assembly, storage, and management of
data; gene identification; functional analysis
(functional genomics), evolution of genomes;
and other interdisciplinary areas relating to the
wide variety of genomes in different organisms.
A eukaryotic genome, contained in the chromosomes,
is many times larger than a prokaryote
genome. A prokaryotic genome consists of a
circular chromosome with compactly arranged
genes.
required for life and/or reproduction.
The term genomics for the study of genomes
was introduced in 1987 by V. A.McKusick and F.
H. Ruddle at the suggestion of T. H. Roderick of
the Jackson Laboratory, Bar Harbor,Maine, USA.
The term genomics extends beyond genetics.
While the latter mainly deals with heredity and
its mechanism and consequences, the term
genomics encompasses many aspects relating
to molecular and cell biology: the different
types of genomic maps; nucleic acid sequencing;
assembly, storage, and management of
data; gene identification; functional analysis
(functional genomics), evolution of genomes;
and other interdisciplinary areas relating to the
wide variety of genomes in different organisms.
A eukaryotic genome, contained in the chromosomes,
is many times larger than a prokaryote
genome. A prokaryotic genome consists of a
circular chromosome with compactly arranged
genes.
Important insights
Important insights about the functions, evolutionary
relationships, antibiotic resistance, and
other metabolic aspects required for the
development of new strategies for therapy are
gained from sequencing the genome of microorganisms
relationships, antibiotic resistance, and
other metabolic aspects required for the
development of new strategies for therapy are
gained from sequencing the genome of microorganisms
The genome of a small bacteriophage
The genome of a bacteriophage usually consists
of double-stranded DNA, although some phage
genomes consist of single-stranded DNA or of
RNA. The size of the genome of phages ranges
from 1.6 kb to over 150 kb, representing anywhere
from a few to over 200 genes. One of the
smallest phages is !X174. F. Sanger and coworkers
sequenced the genome completely and
found that several of the ten genes of !X174
overlap.
of double-stranded DNA, although some phage
genomes consist of single-stranded DNA or of
RNA. The size of the genome of phages ranges
from 1.6 kb to over 150 kb, representing anywhere
from a few to over 200 genes. One of the
smallest phages is !X174. F. Sanger and coworkers
sequenced the genome completely and
found that several of the ten genes of !X174
overlap.
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