1. Relationship between Genotype and Phenotype
Molecular Basis for
Relationship between Genotype and Phenotype
genotype
DNA
DNA sequence
transcription
RNA
translation
amino acid
sequence
protein
function
phenotype
organism

2. Method of Introducing Transgene in Mouse
Refer to Figure 10-28, Griffiths et al., 2015.

3. Determining Gene Function
Isolate normal gene DNA.
Generate mutant allele*.
Introduce mutant DNA* into host.
Score phenotype.
* knockout allele

4. knockout mutation:
disruption of a coding sequence of a gene by insertion of a selectable marker.
Resulting insertional mutation generates a knockout mutant allele of a gene.
Targeting vector is created by adding another selectable marker.
thymidine
kinase
gene
Herpes thymidine kinase gene
Add
tk+ gene.
Insert neoR
into exon 2.
neomycin
resistance gene
normal DNA sequence including complete gene
Refer to Figure 10-29, Griffiths et al., 2015.

5. herpes thymidine kinase gene: Confers sensitivity to ganciclovir
neomycin resistance gene:
Confers resistance to neomycin and its analogs
Refer to Figure 10-29, Griffiths et al., 2015.

6. herpes thymidine kinase gene: Confers sensitivity to ganciclovir
neomycin resistance gene:
Confers resistance to neomycin and its analogs
Refer to Figure 10-29, Griffiths et al., 2015.

7. Production of ES Cells with Gene Knockout
targeting vector
tk+
neoR
transformation
targeted insertion
ES Cell
allele replacement

8. Embryoblast or inner cell mass of blastocyst
Embryonic Stem Cells:
Embryoblast or inner cell mass of blastocyst
Pluripotent
Potential for replication indefinitely
Herpes thymidine kinase gene
Refer to Figure 10-29, Griffiths et al., 2015.

9. 1. ES cells contain normal DNA sequence for the gene. 2. ES cells are
transformed
with vector to
knockout the
normal DNA
sequence.
Herpes thymidine kinase gene
Refer to Figure 10-29, Griffiths et al., 2015.

10. tk+ not inserted – ganciclovir resistant
Refer to Figure 10-29, Griffiths et al., 2015.
NO VECTOR INSERTION:
tk+ not inserted – ganciclovir resistant
neoR not inserted – neomycin sensitive

11. tk+ inserted – ganciclovir sensitive
Refer to Figure 10-29, Griffiths et al., 2015.
ECTOPIC INSERTION:
tk+ inserted – ganciclovir sensitive
neoR inserted – neomycin resistant

12. Replacement of normal gene
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

13. 3 possible fates = 3 different results
Refer to Figure 10-29, Griffiths et al., 2015.
3 possible fates = 3 different results
Targeted insertion results in cells that confer neomycin-resistance and do not harbor the tk+ gene (confers ganciclovir-resistance).
Cells can be isolated selectively.

14. 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.

15. 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.

16. 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.

17. Generation of Knockout Mice
Chimeric male
is mated to
a/a female.
Refer to Figure 10-30, Griffiths et al., 2015.

18. 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.

19. Generation of Knockout Mice
1. Progeny mice are screened for targeted insertion.
Refer to Figure 10-30, Griffiths et al., 2015.
2. Phenotype(s) of mice that are homozygous for the
targeted insertion are scored.

20. Germ-line Therapy vs Somatic Therapy
Gene Therapy
Germ-line Therapy vs Somatic Therapy

21. Gene Therapy in Mammals

22. Approaches to treatment of diseases
Human Gene Therapy
Approaches to treatment of diseases
DNA transfer into afflicted individual