Module 3: Knockout Resources
Barry Rosen WTSI
• Overview of High Throughput Mutant Mouse
Vector and ES Cell Resources
• Survey of IKMC Alleles
• Brief Overview of EUCOMM/KOMP pipeline
• IKMC Database Interface
• EUCOMMTOOLS Alleles, Modular Vectors and
Cre Alleles
OBJECTIVE:
-Increase Accessibility and Use of Mouse
Models Through the Creation of a
Comprehensive Resource of Modular
Vector Resources and Gene Targeted
ES Cells
-Shift
Emphasis from Vector/Targeted ES Cell
Production to Phenotyping Mutants
(in both Mice and ES Cells)
IKMC Consolidates Mutant Mouse Resources
International KO programs
21,000 genes
gene trapping
gene targeting
lacZ-tagged null
C57BL/6
Mutant ES cell
resource
Mutant mice
IKMC Partners
EUCOMM
EUTOOLS
KOMP
NorCOMM
mirKO
TIGM
Phenotyping
programs
International Mouse Knockout Consortium. (2007). Cell
128:9-13.
EUCOMM/KOMP CSD Products:
A dual resource of targeted ES cells
and modular targeting constructs
16,000 genes
EUCOMM
KOMP
LacZ-tagged mutant ES
cell resource
KOMP
mutant mice
8,000 genes (EUCOMM)
5,000 genes (KOMP CSD)
3,000 EUCOMMTOOLS (new program)
Modular targeting
constructs & cassettes
additional alleles
Production Considerations versus User Considerations
Common web portal for KO resources
www.knockoutmouse.org
International Mouse
Phenotyping Consortium
(IMPC)
• Mouse models for the elucidation of
mammalian gene function, insights into
human biology and disease mechanisms
• Production of thousands of mutant mice
from IKMC ES cell resource
• Systematic broad-based phenotyping
• Common data archive and web portal
• www.mousephenotype.org
IKMC PROGRESS
June 2012
http://www.knockoutmouse.org
IKMC Progress to Goals
IKMC PROGRESS
IKMC PROGRESS
Large Scale Mouse KO Projects
KOMP CSD vs. RGN targeting
(798 genes)
# genes
454
276
49
19
CSD
REGN


x
x

x

x
98% of genes targeted
IKMC alleles
Conditional
Deletion
`````
Random Gene Traps
Targeted Non-Conditional
http://www.knockoutmouse.org/about/
Mouse developmental genetics and ES cell mutagenesis
Principle of Random Gene Trapping
A public library of gene trap ES cells
NIH-funded (1999 – 2004)
WT- funded (2003 – 2005)
www.genetrap.org
International Gene Trap Consortium
(IGTC) Website
http://www.genetrap.org/
Factors Enabling high-throughput gene targeting
 Mouse genome sequence
• Reference genome sequence (C57Bl/6)
• Manual/automatic annotation of gene
structures
•Informatics of the genomic
infrastructure
•BAC recombineering
• Homologous recombination in E. Coli
• Indexed BAC libraries
• Manipulate genomic clones with
nucleotide precision
 High throughput methods
•Sequencing
•Robotics
KOMP Regeneron Deletion Allele
EUCOMM/KOMP(CSD)
Conditional Alleles
Designing: Identification of Critical Exon(s)
CE
critical exons
2
2 1
1 0
0 0
0 0
0
phase
5’-most exon common to all mRNA isoforms
which when deleted induces a frameshift mutation.
NMD(nonsense mediated decay)
should degrade mRNA
Knockout-first allele: Promoterless selection cassette
Type of Allele/State of “Critical Exon”
tm1a
Conditional
Ready
FRT
1
lacZ
Flp
WT/C
tm1c
1
2
3
Cre
1
RN/TT
loxP
N/D
3
neo
2
3
Cre
tm1b
1
lacZ
neo
RN/D
3
RN: Reporter Null
TT: Targeted Trap
C: Conditional
N: Null
D: Deletion
Knockout-first allele with
Promoter
RN/TT+Pr
tm1a
Conditional
Ready
FRT
lacZ
1
neo
loxP
2
Flp
WT/C
3
Cre
tm1b
tm1c
1
N/D
FRT loxP
loxP
2
3
Cre
tm1d
1
3
1
lacZ
RN/D
3
RN: Reporter Null
TT: Targeted Trap
C: Conditional
N: Null
D: Deletion
+P: Contains Promoter
Knockout-first allele*
EUCOMM/KOMP-CSD
40%
40%clones
clones
*based on Testa et.al, Genesis, 2004
Conditional targeting strategy for single
exon genes-Artificial Intron
Artificial Intron
2
1
IKMC ALLELE POTENTIALS
=Frt
3
Conditional
Pipeline Potential None
=LoxP
CE
SSR Exposure
Cre
Flp
Flp Cre
-
WT allele
-
Deletion (lacZ replacement):
1
ATG
lacZ
neo
pA
no
RN/D+Pr RN/D
EUCOMM
KOMP-CSD
yes
RN/TT
EUCOMM
KOMP-CSD
yes
KOMP-CSD
no
no
KOMP-REGN
pA
Knockout-first (promoterless)
lacZ
1
2
neo
pA
SA
3
CE
RN/D
WT/C
N/D
RN/TT+Pr RN/D
WT/C
N/D
RN/D+Pr
RN/D
N/D
RN/TT
-
Knockout-first (promoter)
1
lacZ
SA
2
neo
pA
pA
3
CE
Deletion (lacZ-tagged)
1
lacZ
SA
3
neo
pA
pA
-
Targeted, non-conditional (promoterless)
1
lacZ
SA
EUCOMM
KOMP-CSD
3
2
neo
CE
pA
WT/Rev
-
WT/Rev
-
Targeted, non-conditional (promoter)
1
lacZ
SA
neo
pA
pA
2
CE
3
EUCOMM
KOMP-CSD
no
RN/TT+Pr RN/TT
June 2011
High Throughput Targeting Pipeline
I) In silico Phase
II)Vector
Construction
Phase
III)ES cell
Gene Targeting
Phase
Gene Annotation and Design
• Design on automatically annotated genes risky
• Original designs were on genes with full manual
annotation
– Slow, becomes a bottleneck!
• “Design First” Strategy
– Automated scripts run on genome(Vega/Ensembl)
– Confirmation of design by annotator
– Higher througput, >95% accuracy
Automatic Selection of Recombineering oligos
CE
Oligo postions define a DESIGN
loxP
Gateway Cassette
LacZ_neo_pA
>0.1kb
0.3kb
U5
G5
U3
0.1kb
>0.3kb
D5
D3
intron size
>0.7 kb
>0.7 kb
deletion size
~1 kb
~5 kb
BAC clone
G3
homology arms
~5 kb
- pre-defined distances from CE
- candidate regions (U, D and G)
- 50mers
- unique hit on the BAC (100kb either site of CE)
-avoid deletion of conserved elements
ARRAY OLIGO SELECTOR PROGRAM(AOS)
View vector designs on genome browser
96-well serial recombineering
3-way Gateway reaction
L3L4_DTA
C57BL/6N ES cells
JM8
JM8.F6
JM8.N4
Kent Lloyd
UC Davis
SNL feeders
GIBCO KO medium
10%-15% serum
+ Glutamine
+ BME
+ LIF
feeder-free
JM8.F6
SNP
panel
CGH
JM8.N4
1.7 Mb gain
Chr 10
Pettit et al., 2009, Nature Methods
C57BL/6N ES cell validation:
Establishment of a JM8 Agouti cell line
Microinjections
#
Cell line
JM8A3 (A/a)
Male chimeras
% of
Testcrosses
% of
# clones
% clones
clones
# clones
clones
# clones at
clones
# clones
with
injected with
injected
at birth
injected
weaning
injected
set up
GLT*
GLT*
11
11
100%
10
100%
10
9
82%
*GLT, germline transmission
Non-agouti (a)
A B
CD
2
3
4
VL30
Testcross:
JM8.F6
(A/a)
chimera
X
C57BL/6N
(a/a)
C57BL/6N
(A/a)
Pettitt S. Et al., 2009, Nature Methods
Genotyping Strategy: LRPCR-Seq
3’ homology arm
5’ homology arm
FRT
1
GF
FRT loxP
lacZ
5’U
2
neo
3’U
lox
P
3
LX
GR
LR-PCR
primers
3’ arm
LR-PCR
products
GR
Sequencing
primers
cassette
5’ arm
GF
5’Us
3’Us
LR
Skarnes et.al., 2011 Nature
Loss of Allele Genotyping(LOA) Can Also be Employed
WHAT HAVE WE LEARNED ABOUT GENE
TARGETING?
• Gene Targeting Can Be Highly Efficient
– 40% Average Targeting Frequency
• 35% Using Promoter Driven Selection
• >70% Using Promoterless Selection
– Gene Restrictions for Promoterless Vectors
• Factors Contributing to High Targeting
Frequencies:
– Long(10 kb total) homology arms
– Virtually isogenic DNA
– Strong Negative Selection(DTA)
– Optimal Cell Handling
ALL GENES CAN BE TARGETED,
MOST AT HIGH FREQUENCY
80% of time, re-prepping vector and repeating
electroporation rescues failed project
>50% of time, re-designing vector around new
critical exon|(s) rescues failed project
Common web portal for KO resources
www.knockoutmouse.org
Looking up a gene
Visualization of Vector Maps
Genbank File
Synthetic Vector Map
EUCOMM/KOMP CSD Project Statuses
EUCOMMTOOLs
• Expansion of Conditional Clone Resource
– 3,000 genes
– Mainly Artificial Intron Conditionals
• Previously deletions in KOMP
• 250 New Inducible Cre Driver Mouse Strains on
C57Bl/6 Background
– BAC Knock-ins
– Targeted Knock-ins
– Adult Disease Relevant Tissues Priorities
EUCOMMTOOLs
• Modular Vector Cassette Toolbox (Sanger)
– Modules for fluorescent reporters, recombinases, etc
• Dual Use, Gateway and RMCE (Skarnes/Rosen)
• Technology Development
– New Ligand Inducible Recombinases (Stewart)
– Zinc Finger Nuclease mediated RMCE (Wurst/Kuhn)
(1)EUCOMMTOOLs genes mostly small transcription units
1400
1200
# genes
1000
800
600
400
200
0
0
1
2
3
4
5
6
7
8
# exons
9
10
11
12
13
14 or15
more
Conditional targeting strategy for single
exon genes-Artificial Intron
Artificial Intron
Artificial intron (Ifitm2 gene)
R1/Zeo-PheS/R2
attR1
1
attR2
EM
7
147 bp
Zeo
pheS
2
307 bp
CAT
R6K ori
pR6K_Ifitm2i_ZP
Barry Rosen
Validation of artificial intron
Atrx targeting
alleles
w.t.
Atrxtm1
Atrxtm1.1
Atrx
protein
loading
control
+ Flp
Manousos Koutsourakis
EUCOMMTOOLS
MODULAR
VECTORS
EUCOMMTOOLS MODULAR
VECTORS
• Re-engineering of Targeted Cell Lines by RMCE
• Re-targeting using gene specific intermediate
and novel cassettes
• Types of Alternative Alleles:
– Fluorescent reporters
– Recombinases
– Mutant/humanized cDNAs
EUCOMM: Tools for
Functional Annotation
of the Mouse Genome
EUCOMM Vector Assembly 3-Way Gateway Reaction
EUCOMM: Tools for
Functional Annotation
of the Mouse Genome
EUCOMM TOOLs Modular Vector Assembly
EUCOMM: Tools for
Functional Annotation of
the Mouse Genome
RMCE Mediated Knock-in of H2bVenus to Smad4 Locus
RMCE Vector
+
iCre/FlpO expression vector
Short Range PCR genotyping
Target Line
LacZ
Correct RMCE Clones
LacZ
YFP
YFP
Efficiency of RMCE:
73% promoterless(Smad4)
52% promoter(Zfp503)
Javier Lopes-Rios, Rolf Zeller
EUCOMM TOOLS Cre Driver Mouse Production
• Two Types of Alleles:
– Cre BAC Knock-ins to into acceptor loci (HPRT/ROSA26)
– Targeted Cre Knock-ins to endogenous loci
– C57Bl/6 background, 250 lines
• Preferred form of Cre: inducible CreERT2
– Associated nlsEGFP reporter
• Assembly of knock-ins from EUCOMM/KOMP
Intermediates
– New L1L2 Gateway CreERT2 Cassette
DUAL USE FLUORESCENT PROTEIN / CreERT2 CASSETTE
LF2A
attL1 FRT
SA
nlsEGFP
T2A
Rox
CreERT2
pA
GATEWAY
RMCE
PGK:puro
Rox loxP attL2
`
pA
`
Two proteins translated:
-Nuclear Localized EGFP
-CreERT2
CREATE for Conditional Resources
www.creline.org
EUCOMMTOOLS Cre Progress on
creline.org
IKMC knockout resources
lacZ-tagged null
C57BL/6
Mutant ES cell
resource
Mutant mice
Phenotyping
programs
www.knockoutmouse.org
Vector
resource
EUCOMM
EUTOOLs
KOMP
NorCOMM
mirKO
TIGM
New alleles
EUMODIC
Sanger MGP
KOMP2
www.europhenome.org
www.sanger.ac.uk/mouseportal
Dual RMCE
iCre
FlpO
Osterwalder et al, Nature Meth. 2010
EXERCISE –VECTOR/ES CELL RESOURCES
and CRITICAL EXON READING FRAME
DETERMINATION
•
•
•
•
•
•
Find a design for your favorite gene on knockoutmouse.org
Determine vector and targeted ES cell resources
Find Critical Exon (or first exon if multiple exons are floxed)
Determine Reading Phase(0,1,2) of Critical Exon(s)
Download and Visualize Genbank file of vector
If there aren’t the desired products (eg a conditional
allele), can you determine the reason?
IKMC Gene Pages
MartSearch interface
KO Design and Underlying Genome
Annotation
Automatic Annotation
(ENSEMBL)
Design
Rate:
Automated, not
limiting
Design
Reliability: Can be occasional
problems
Manual Annotation(VEGA)
15 genes/week/annotator
Bottleneck!
Close to 100% reliable,
but can change with evidence
All genes will eventually undergo full
manual annotation through
VEGA/HAVANA
Design First
Script run on Ensembl
& checked by Annotator
200 genes/week/designer
Removes Bottleneck
Estimate >95%
reliability
ES cell production statistics
EUCOMM/KOMP-CSD
17,700 electroporations
11,500 genes electroporated
500,500 colonies screened
10,200 gene targeted (90% success rate)
Average targeting efficiency = 40%
Average # colonies screened = 32
Average # targeted clones = 12
BEYOND KOMP and EUCOMM
Developing Modular
Gene Targeting Resources
2A Sequence Function in EUCOMMTOOLs Vectors
attL1
FRT
attL2
En2 SA
ORF
pA
PGK-puro
Rox
T2A Function
can be less
efficient in
the context
of signal sequence
containing genes
pA
Rox
loxP
aa 17
EGRGSLLTCGDVEENPGP
T2A peptide
+aa17
C
Endogenous
protein-En-2
fusion
N
+P
N ORF
O
C
PROTEIN OF INTEREST
N
C
puroR
LF2A Sequence Function in EUCOMM TOOLs Vectors
attL1
FRT
attL2
En2 SA
ORF
pA
PGK-puro
Rox
40 aa natural viral
protein 1D“leader”
N-term to F2A.
Results in more
efficient function of
2A sequence
on membrane bound
polysomes
(de Felipe et al
N
Biotech J 2010)
aa 40
1D Leader
+aa40 +aa17
C
Endogenous
protein-En-2
fusion
pA
Rox
loxP
O
F2A
+P
N ORF
C
PROTEIN OF INTEREST
N
C
puroR
EUCOMMTOOLS CASSETTES CAN BE USED TO TARGET HUMAN H9 ES CELLS
Brachyury-H2b-Venus knock-in
Tbx5-H2b_Venus knock-in
H9
Human
ES
Mesoder
m
Induction
H9 derived cardiomyocytes
Daniel Ortmann, Maria Ortiz, Roger Pedersen
Considerations of EUCOMM Tools Cre Knock-ins
• Simple to make with existing technology and resources
• Pro’s of Cre-Knock-ins
• Re-utilization of previously validated materials
• No new technology needed, only CreERT2 vector cassette
• Knocked-in Cre should faithfully recapitulate endogenous
expression
• Con’s of Cre Knock-ins
• Potential for haplo-insufficient phenotypes complicating
genetic analysis
Rosa-26 Knock-in of nlsGFP_T2A_CreERT2 in Arid1a LacZ ES Cells
Tamoxifen Inducibility of Cre Activity
-4OHT
+4OHT
G
F
P hours Tamoxifen
48
+
EUCOMM Farewell & EUCOMMTOOLS
Progress Meeting, Schloss
Hohenkammer, April 3-4, 2012
C
o
lAbove sub-cloned
o
n
i
e
s
Transient Dre Recombinase Treatment to Remove Roxed Selection Cassette
LF2A
attB1 FRT
SA
T2A
nlsEGFP
CreERT2
Rox
pA
CAGGS_Dre_IRES_bsd_pA plasmid
48 hour transient Bsd selection
LF2A
FRT
SA
nlsEGFP
EUCOMM Farewell & EUCOMMTOOLS
Progress Meeting, Schloss
Hohenkammer, April 3-4, 2012
66% Efficiency(n=3 genes)
Rox loxP
T2A
CreERT2
PGK:puro
Rox loxP attB2
`
pA
`
pA
`
`
Puro Copy Number qPCR
Puro sensitive
ALTERNATIVE CreERT2 ALLELE: 3’UTR IRES KNOCK-INs
Rox
attL1
IRES
nlsEGFP
T2A
CreERT2
Rox
pA
Bact::neo
attL2
pA
• Inserted after termination codon of endogenous gene’s
ORF
• Should preserve endogenous protein levels from modified
allele
• Uses endogenous 3’UTR and polyA after selection
cassette deletion
• Requires construction of new intermediate vector
EUCOMM Farewell & EUCOMMTOOLS Progress
Meeting, Schloss Hohenkammer, April 3-4, 2012
BEYOND KOMP and EUCOMM
Developing Modular Resources
and Additional Alleles