RI
CEN S I S
U N IVE R S IT
Breeding of constitutive and conditional mouse mutants: a pragmatic approach
TU
AS
MDCCC
H.-P. Lipp, H. Welzl, R. Madani, D.P. Wolfer - University of Zurich, Institute of Anatomy, CH-8057 Zurich
NCCR Neural Plasticity and Repair, Swiss NF 31-54184.98 & 31.57139.99, Roche Research & Hartmann-Müller Foundations
XXXIII
2
Summary
An uncontrolled bias or shift of genetic background can severely
confound phenotypic characterization of genetically modified
mice. It has therefore repeatedly been suggested that co-isogenic
or at least congenic lines should be used exclusively. However,
generation of such a homogeneous genetic background is often
impossible because of limited resources and time. We provide
evidence that studying samples with a mixed genetic background,
typically a combination of C57BL/6 with a substrain of 129,
is a valid alternative, provided that essential rules are respected.
A set of breeding strategies is proposed which implement these
rules with a minimum of resources and can be used with
constitutive or conditional targeted mutations.
F2 to characterize a constitutive mutation,
co-isogenic line for its later maintenance
Essential rules according to 1997 Banbury Conference:
- use littermates as controls
- background must be documented and reproducible
- use few and commonly available strains
SD (m)
0.20
0.18
0.16
0.14
0.12
0.10
0.08
0.06
0.04
0.02
0.00
129 inbred
129 inbred
0.12
0.14
0.13
b
N1 +/-
N2 +/-
N3 +/-
a
129 chimera
N1 +/+
N2 +/+
F1 +/-
d
F2 +/-
N3 +/+
F3 +/-
F1 +/-
c
129 +/+
Cre founder
F1 +/+
F2 +/-
Cre recombinase
wildtype target
“floxed” target
recombined target
F3 +/-
F2 +/+
F3 -/-
a
maintenance
& re-seeding
B6 inbred
F2 -/-
inbred B6
>9 generations
129 lx/+
B6 inbred
129 chimera
c
F1 +/+
F1 lx/+
F2 +/+
F2 lx/+
d
B6
B6 Cre
b
F1 Cre;lx/+
F1 Cre;+/+
F1 lx/+
F1 +/+
F2 lx/lx
F2 Cre;+/+
F2 Cre;lx/+
F2 Cre;lx/lx
F3 +/+
first characterization
of mutation
(a) Rapid production of test samples for first phenotypic characterization: chimera crossed with
B6 females, heterozygous F1 offspring interbred to obtain F2. (b) Co-isogenic line for mutation
maintenance by backcrossing chimera to ES-cell donor strain. (c) Do not interbreed past F3!
(d) Later, new F2 should be generated using co-isogenic maintenance line.
IL/N
BALB/c
B6
M1
B10
D2
129/J
129/Sv
CBA
F1 B6VB
F1 B6Sv
F1 B6J
Nx D2Sv to B6
Fx B6D2Ola
Fx B6CBA
Sel MF2
Sel MFC
Fx B6OlaSv
Nx Sv to B6
M. oeconomus
Fx B6??
C. glareolus
Nx D2Ola to B6
Sel MF1
Fx B6C3
Nx ?? to B6
Rand
Fx B6Ola
Fx B6D2Sv
Fx B6J
Fx B6SJL
Nx C3 to B6
Fx B6Sv
Bx B6C
Fx B6M1
Swiss
Nx ILN to B6
129 chimera
>3 generations
maintenance
& re-seeding
2733 asymptomatic mice
Basic scheme with floxed allele kept in co-isogenic line
(ES-cell background) and Cre transgene on B6 background:
e
129 inbred
Reliable results and good base line
performance from outbred samples
Similar breeding strategy applied to conditional
models, for example using the Cre/LoxP system
129 inbred
Basic scheme with maintenance of mutation in
co-isogenic line (on ES-cell background):
wildtype target
mutated target
1
3
two-way analysis of Cre and Lx effects
(a) Cre transgene maintained in congenic line by backcrossing to C57BL/6, over as many generations as time allows. (b)
Chimera with floxed allele crossed with C57BL/6 congenic Cre transgenics and (c) with inbred C57BL/6. (d) F1 offspring
of first cross carrying both floxed allele and Cre mated to heterozygous F1 offspring of second cross. F2 then contains all
genotype combinations necessary for two-way analysis. (e) Floxed allele is maintained in co-isogenic line by backcrossing
chimera to ES-cell donor strain. Heterozygous mice of maintenance line replace chimera.
SD as measure of within sample variability varies over wide and similar ranges in outbred,
F1 hybrid as well as inbred samples. Inbred mice are not more reliable.
Alternative if mutation is in congenic (e.g. B6) line:
search error +SE (m*s)
60
***
30
***
50
crossing index +SE (m-1)
target zone +SE time (%)
.5
25
.4
40
20
.35
30
15
.25
**
***
20
0
.3
chance
.2
10
10
.15
.1
5
F1
Fx
B6
129
Escape performance
0
***
.45
.05
F1
Fx
B6
129
0
F1
Mix
B6
Spatial retention during probe trial
During swimming navigation, 129B6F1 mice outperform both parental strains. B6 beat
129 with respect to escape performance, but not on transfer tests. Outbred samples are
only marginally inferior to F1 and beat both parental strains as well.
129
(a,b) As above. (c) Mutation
maintained as congenic line after
backcrossing to C57BL/6. (d)
Creation of follow up samples
similar to original F2 by
outcrossing maintenance line
again to the ES-cell donor strain
(e.g. 129/SvEv). In resulting
reverse F1, heterozygous
animals are interbred to create
a reverse F2. The latter provides
test samples in which flanking
allele effects cannot occur,
because ES-cell donor alleles
make equal contributions to the
flanking region of the targeted
locus in all genotypes.
Alternative if Cre and LoxP Line are both on B6 background:
c
129 chimera
B6 inbred
a
B6 inbred
>9 generations
F1 +/129 inbred
B6 +/-
d
RF1 +/+
F1 +/-
F1 +/+
F2 +/-
F2 +/-
F2 -/-
F2 +/+
F3 +/-
F3 +/-
F3 -/-
F3 +/+
b
B6 +/+
maintenance
RF1 +/-
RF1 +/-
first characterization
of mutation
RF2 +/-
RF2 -/-
RF2 +/+
follow up and
flanking allele test
(a) Cre recombinase transgene and the
floxed allele only available in C57BL/6
congenic maintenance lines. (b) Reintroduce ES-cell donor background by
outcrossing heterozygous mice of both
congenic maintenance lines. (c) Cross of
F1 animals carrying floxed allele and Cre
recombinase transgene, respectively. (d)
Double heterozygous F2 is backcrossed
to F1 mice carrying floxed allele.
Resulting F3 has genetic background
close to the original F2, yet contains all
genotype combinations necessary for a
two-way analysis.
129 chimera
Cre recombinase
wildtype target
“floxed” target
recombined target
B6 inbred
>9 generations
Cre founder
B6 +/+
a
B6 lx/+
F1 +/+
F1 lx/+
wildtype target
mutated target
F3 +/+
d
F3 lx/+
B6 inbred
>9 generations
a
b
129 +/+
b
F1 lx/+
c
F1 Cre
F2 Cre;lx/+
F2 Cre;+/+
F3 lx/lx
B6 Cre
B6
F1
F2 lx/+
F3 Cre;+/+
two-way analysis of Cre and lx effects
F2 +/+
F3 Cre;lx/+
F3 Cre;lx/lx