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Molecular Basis for
Relationship between Genotype and Phenotype
genotype
DNA
DNA sequence
transcription
RNA
translation
protein
function
phenotype
organism
amino acid
sequence
Method of Introducing Transgene in Mouse
Refer to Figure 10-28, Griffiths et al., 2015.
Determining Gene Function
1.Isolate normal gene DNA.
1.Generate mutant allele*.
1.Introduce mutant DNA* into
host.
1.Score phenotype.
* knockout allele
knockout mutation:
thymidine
kinase
gene
Add
tk+ gene.
Insert neoR
into exon 2.
neomycin
resistance
gene
disruption of a coding sequence
Herpes
of a gene
by thymidine
insertionkinase
of a gene
selectable marker.
Resulting insertional mutation
generates a knockout mutant
allele of a gene.
Targeting vector is created by
adding another selectable
marker.
normal DNA sequence including complete gene
Refer to Figure 10-29, Griffiths et al., 2015.
herpes thymidine kinase gene:
Confers
sensitivity to ganciclovir
Herpes thymidine kinase gene
neomycin resistance gene:
Confers resistance to neomycin
and its analogs
Refer to Figure 10-29, Griffiths et al., 2015.
herpes thymidine kinase gene:
Confers
sensitivity to ganciclovir
Herpes thymidine kinase gene
neomycin resistance gene:
Confers resistance to neomycin
and its analogs
Refer to Figure 10-29, Griffiths et al., 2015.
Production of ES Cells with Gene Knockout
tk+
targeting vector
neoR
transformation
targeted insertion
allele replacement
ES Cell
Embryonic Stem Cells:
• Embryoblast or inner cell
mass of blastocyst
• Pluripotent
• Potential for replication
indefinitely
Refer to Figure 10-29, Griffiths et al., 2015.
Herpes thymidine kinase gene
2. ES cells are
transformed
with vector to
knockout the
normal DNA
sequence.
Refer to Figure 10-29, Griffiths et al., 2015.
1. ES cells contain normal
DNA sequence for the
gene.
Herpes thymidine kinase gene
Refer to Figure 10-29, Griffiths et al., 2015.
NO VECTOR INSERTION:
tk+ not inserted – ganciclovir resistant
neoR not inserted – neomycin sensitive
Refer to Figure 10-29, Griffiths et al., 2015.
ECTOPIC INSERTION:
tk+ inserted – ganciclovir sensitive
neoR inserted – neomycin resistant
double crossing over
Replacement of normal gene
with knockout allele by
homologous recombination
Refer to Figure 10-29, Griffiths et al., 2015.
HOMOLOGOUS RECOMBINATION:
tk+ not inserted – ganciclovir resistant
neoR inserted – neomycin resistant
Refer to Figure 10-29, Griffiths et al., 2015.
3 possible fates = 3 different results
Targeted insertion results in cells that confer neomycinresistance and do not harbor the tk+ gene (confers
ganciclovir-resistance).
Cells can be isolated selectively.
Refer to Figure 10-29, Griffiths et al., 2015.
In the presence of neomycin analog and ganciclovir,
only cells with targeted insertion will survive.
Such cells can be cultured and introduced into
embryos in the blastocyst stage.
Generation of Knockout Mice
Embryo is inserted into a
surrogate mother…
agouti
ES cells from agouti mouse (A/A) with
targeted insertion of a mutant allele
are inserted into blastocyst-stage
embryo of a/a genotype.
Refer to Figure 10-30, Griffiths et al., 2015.
Generation of Knockout Mice
Progeny that are chimeric
(black and agouti) for coat
color are selected.
Embryo is inserted into a
surrogate mother…
Refer to Figure 10-30, Griffiths et al., 2015.
Generation of Knockout Mice
Chimeric male
is mated to
a/a female.
Refer to Figure 10-30, Griffiths et al., 2015.
Generation of Knockout Mice
DNA from
progeny are
screened for
targeted
insertion.
A/a mice with
targeted
insertion are
crossed.
Refer to Figure 10-30, Griffiths et al., 2015.
Generation of Knockout Mice
Refer to Figure 10-30, Griffiths et al., 2015.
1. Progeny mice are screened for
targeted insertion.
2. Phenotype(s) of mice that are homozygous for the
targeted insertion are scored.
Gene Therapy
Germ-line Therapy vs Somatic Therapy
Gene Therapy in Mammals
Human Gene Therapy

Approaches
to treatment
of diseases
 DNA transfer
into afflicted
individual