What is a Knockout Mouse

1
What is a Knockout Mouse?
- a mouse in which a gene has been
deleted/mutated - gene is inactivated -
specific gene is targeted - inactivate gene
missing or non-functional protein - loss of
function is assessed in the context of the
whole animal
2
Knockout vs. Transgenic Mouse?
Knockout - gene inactivated Transgenic -
gene(s) added - can be - a foreign gene
- extra copies of endogenous gene -
mutated endogenous gene  
3
Why Make a Knockout Mouse?
- Determine the function of a particular
gene/gene product. - eliminate a specific
protein - ask what cant that animal do?
4
What Kinds of Questions ?
- Examine a proteins role in a biological
process - protein X always increased during
process Y - Determine function of a newly
identified gene - genome sequencing projects
(gt30,000 genes) - many of unknown
function - some info gained from comparisons
5
What Kinds of Questions 2?
- Protein Families       - families of
similar yet distinct proteins       - have the
same biochemical function - all apparently do
the same thing - probably have different
physiological roles - Models for Human
Diseases - confirm mutation is responsible for
disease - studies not possible on humans -
experimental therapies
6
How to make a Knockout

• 1) Have 40,000
• 2) Alter gene of interest in
• Embryonic Stem (ES) Cells
• 3) Use ES cells to make a mouse
•     

7
Embryonic Stem (ES) Cells
- isolated from a pre-implantation embryos -
from inner cell mass of blastula stage
embryo - cells are undifferentiated - cells are
pluripotent - able to differentiate into many
(all?) different cell types in embryo -
most importantly germ cells - grown in
culture - need cells to divide but not
differentiate - longer time in culture
more differentation      
8
Embryonic Stem Cell Isolation
9
Gene Targeting Technology
- Cells take up exogenous DNA       - frequency
is very low       - can be induced to higher
frequency - chemical, electrical,
injection - In Dividing Cells - some DNA
incorporated into genome - Random
integration -rare event - Homologous
Recombination - even more rare
10
Random Integration
- DNA incorporated anywhere in genome (not
targeted)       - copy number can be very
high - disrupts the endogenous DNA at
insertion site - transgenic animals are
usually random integrations    
11
Random Integration
(
)
n
12
Homologous Recombination
- driven by the DNA sequences (targeted) -
rare (1000X less frequent than random)      
- increased efficiency - isogenic DNA -
longer stretches of homology - results in -
endogenous DNA replaced with exogenous -
specific - targeted - copy number 1
13
Homologous Recombination
14
Homologous Recombination Non-homologous DNA
Flanked by Homologous DNA
a'
b'
c'
d'
a'
b'
c'
d'
15
Targeting Construct for Positive/Negative
Selection
- To make targeting construct - a positive
selectable marker flanked by two arms of
homologous sequence - a negative selectable
marker outside one homologous arm
16
Gene Targeting using Positive/Negative
Selection Deletion Construct Homologous
Recombinant
NeomycinR
1
2
3
17
Gene Targeting using Positive/Negative
Selection Deletion Construct Random Integration
x
y
(
)
NeomycinR
HSV-TK
n
a
c
d
x
y
18
Selection Strategy
- Positive Selection G418 - Neomycin
Resistance gene       - confers resistance to
G418 - G418 selects for both - homologous
and random integrations - kills cells that
have not taken up DNA - Negative Selection -
Gancyclovir - Herpes Simplex Virus Thymidine
Kinase (HSV-TK) - sensitive to
gancyclovir - selects against random
integrants
19
Targeting
- Electroporate ES cells with Targeting
Construct - Select in G418 and
Gancyclovir - enriches for homologous
recombination - - Pick individual colonies of
resistant ES cells (100s) - Screen for
properly targeted cells - PCR and/or Southern
blot - Use targeted cells to make a mouse
20
Gene Targeting using Positive/Negative
Selection Insertion Construct
1
2
3
4
NeomycinR
HSV-TK
3
3
2
4
21
Analysis of MicePossible Outcomes
- Abnormal phenotype obvious. - due to -
loss of protein - compensation - abnormal
protein? - No phenotype obvious. - Why? -
functional redundancy - ability to detect -
No homozygous knockouts seen - embryonic lethal
22
From Gene to Protein
1
2
3
4
transcription
mRNA
AAAAAAAAAAAAAAAAAA
1
2
3
4
translation
protein
1
2
3
4
23
Molecular Consequences of Targeting
HSV-TK
a
d
b
c
c

insertions
AAAAAAAAAAAAAAAAAA
insertion/ frame shift
AAAAAAAAAAAAAAAAAA
truncation
AAAAAAAAAAAAAAAAAA
Unstable message degraded no protein
Stable message mutant protein
24
Embryonic Lethal - Now What?
- Conclusion - important for embryonic
development - Further studies - timed
matings - examine pups at different stages
25
Embryonic Lethal - Now What?
- Is protein important later in life? -
Phenotype in heterozygous animals - likely
50 protein - Conditional Knockouts -
tissue specific - temporally regulated -
Rescue - add back the gene
26
Cre/loxP System
- Cre Recombinase - site-specific
recombinase - P1 bacteriophage -
recombination between two loxP sites - no other
cofactors required - LoxP sites - 34bp
sequence - two inverted 13bp repeats
surrounding 8bp core
27
Cre Mediated Recombination
loxP
loxP
floxed
Cre

28
Floxing a Gene in ES Cells
1
2
3
loxP
loxP
loxP
HSV-TK
NeomycinR
1
2
3
NeomycinR
1
2
3
29
Cre Recombination in Targeted ES cells
loxP
loxP
loxP
NeomycinR
1
2
3
Transient Cre expression
OR
OR
NeomycinR
1
3
use to make mouse
1
2
3
30
Tissue Specific Cre-Expressing Mouse
Transgenic Mouse
Expresses Cre in a subset of tissues Note many
tissue-specific Cre-expressing mice have been
made, more are being made.
31
Tissue Specific Cre-Expressing Mouse
loxP
loxP
Targeted Mouse

mate
Transgenic Mouse
Mouse with knockout ONLY in tissues that
express Cre
32
Introduction of Point Mutations into Mouse Genome
1
2
3
loxP
loxP

HSV-TK
NeomycinR
1
2
3

NeomycinR
1
2
3
33
Cre Recombination in Targeted ES cells Mutation
Construct
loxP
loxP

NeomycinR
1
2
3
Transient Cre expression

1
2
3
34
Tissue Specific Rescue of Knockout Mouse
Targeted Mouse

mate
cDNA of KOed gene
Tissue Specific Promoter
Transgenic Mouse
Mouse missing protein ONLY in tissues that
dont express the cDNA
35
Temporal Regulation of Cre Steroid Hormone
Binding Domain Fusions
No hormone
Tamoxifen