Copyright 0 1993 by the Genetics Society of America
Effects of Multiple Retrovirus Insertionson Quantitative Traitsof Mice
Peter D. Keightley,* MartinJ. Evanst and William G. Hill*
*Institute of Cell, Animal and Population Biology, University of Edinburgh, Edinburgh EH9 3JT, Scotland and tWellcome/CRC
Institute of Cancer and Developmental Biology and Department of Genetics, University of Cambridge, Cambridge CB2 la,England
Manuscript received April 23, 1993
Accepted for publicationJuly 26, 1993
ABSTRACT
To assess the potential to generate quantitative genetic variation by insertional mutagenesis in a
vertebrate, lines of mice in which many provirus vector inserts segregated at a low initial frequency
to divergent artificial selection on
body weight
on an inbred background (insert lines) were subjected
at 6 weeks and responsesandheritabilityestimatescompared
to control lineslackinginserts.
Heritability estimates were more than 1.5 times greater in the insert lines than in the controls, but
because the phenotypic variance was substantially higher in the insert lines the genetic variance was
about 3 times greater. Realized heritability estimates tended to be lower than heritabilities estimated
by an animal model which utilizes information in covariances between all relatives in the data set. A
surprisingly large response to selection occurred in the inbred control line. Insert lines were about
20%less fertile than controls.
Division of the selection linesinto inbred sublines
in the later generations
of the experiment revealed substantially greater variation among sublines of the insert lines than
similar to typical estimates for the trait in outbred populations.
among the controls. Heritabilities were
In conclusion, there was clear evidence of extra variation deriving from inserts, which has yet to be
attributed to individual genes.
P
ROGRESS in the understanding of the maintenance and nature of quantitative genetic variation requires knowledge of the effects of the individual trait loci. T h e effects ofquantitativetrait
loci
(QTL) are usually small, the environmental variation
large, and effects of tightly linked polygenes difficult
to distinguish from one another, so specific information on the genes causing quantitative genetic variation is hard to obtain. Insertional
mutagenesis, the
random insertion of foreign DNAinto the genome,is
a powerful method for generating mutations. Selection on adesiredphenotype
is often employed to
isolate specific classes of mutations. Recently, T. F.
C.MACKAYand co-workershave developed insertional mutagenesis strategiesfor theisolation of genes
controlling quantitative traitsin Drosophila. T h e first
such experiments involved dysgenic crosses in which
a high rate of uncontrolled transposition Pofelements
occurred. Artificial selection on quantitative traits in
populations derivedfrom such crosses led to increased
responses relative to “non-dysgenic”
controls
(MACKAY 1984, 1985).The amount of new genetic
variation generated in a dysgenic cross is of the order
of 100 times greater than that produced by one generationofspontaneousmutation
(MACKAY 1987).
Later, by using systems in which transposition can be
controlled (ROBERTSON
and ENCELS1989),inbred
lines containing different subsets of small numbers of
stable P elementinsertswere
generatedandtheir
effects on various traits measured (MACKAY, LYMAN
Genetics 135: 1099-1 106 (December, 1993)
and JACKSON 1992). Although most inserts had little
or no effect on any trait, about 10% of inserts had
detectable quantitative effects, some of several phenotypic standard deviations. These would be classed
as “major genes” if present in a segregating population. Mutations of large effect tend to be recessive,
and most though not all, are highly deleterious with
respect to fitness.
Many of the common laboratory mutations of Drosophila are caused by transposable element insertions
(RUBIN1983), but in mice it is unclear whether this is
also the case, as few previously known mutations have
been associated with insertions. Two exceptions are
the dilute coat color mutation, caused by the insertion
of an ecotropic murine leukemia virus (JENKINS et al.
198l), and the hairless mutation which is associated
with the insertion of a non-ecotropicmurine leukemia
virus (STOYEet al. 1988).
Artificial insertion of foreign DNA into the mouse
genome by injection or electroporation into eggs or
embryonic stem (ES) cells, or by retrovirus infection
generates visible or recessive lethal mutations at the
rate of about 0.05 per insertion UAENISCH1988).
Ratesofmutationfrominsertionalmutagenesis
in
Drosophila and mice are therefore similar in spite of
the largedifference in genome size. Insertion by
retroviruses, which can be made to occur
in “dysgenic”
crosses (JENKINS and
COPELAND
1985), or by infection
of ES cells (ROBERTSON
et al. 1986) appears tobe the
only practical approach presently available for gener-
1100
D. P.
Keightley, M. J. Evans and Hill
W. G.
ating large number of insertions in mice.
In this study, we investigate the effects on quantitative traits of the insertion of many retroviral vectors
in a population of mice derivedOperation
from ES cells. Lines
derived from this population were artificially selected
for increased and decreased body size. The genetic
background was inbred and the inserts were initially
at low frequencies in the population, so any selection
response could be attributed to the selection of insert(s) which had disrupted a gene affecting the trait.
The genetic variance estimated from informationcontained in the observed responseto selection and from
covariances between all relatives is compared to that
of controls lacking inserts.
MATERIALS AND METHODS
Base population: Insert lines: The base population for
lines containing provirus vector insertions (PVIs) was a
cohort of animals bredatthe
Department of Genetics,
University of Cambridge, which was derived from an embryonic stem cell line multiply
infected with mos"neo retrovirus vectors (ROBERTSON
et al. 1986). After infection, stem
cells were injected into blastocysts (BRADLEY
et al. 1984) in
order to generate germ line chimeras. The stem cell line
was from the inbred strain 129/Sv which had been made
congenic for the Gpi-1' allele by backcrossing for 8 generations (the Gpi-l" allele is normally present in strain 129/Sv).
Animals derived from chimeras were therefore initially 99%
inbred on the 129/Sv background, but were subsequently
crossed for 2-3 generations to inbred 129/Sv homozygous
for the Gpi-1" allele. Segregation therefore occurred at the
Gpi-1 locus. The base population comprised 53 individuals
from 10 different stemcelllines. The overall number of
PVIs present in these individuals was about 130, andalmost
all werein the heterozygous state. Individuals from the same
line varied with respect to their complement of PVIs.
Three pairs of divergent selection lines (insert lines R V l ,
RV2, RV3)were established from mice containing different
subsets of PVIs. The base population for R V l was 4 stem
cell lines and contained a total of about 60 PVIs. RV2 was
derived from a different subset of 4 stem cell lines from
R V l and contained about 50 PVIs. Line RV3 was derived
from a total of 10 different stem cell-derived lines, and
contained a total of about 130 PVIs, most of which were
common to RVl or RV2. Because the base populations for
the different selection lines were crosses of several sublines
in which PVIs were segregating, the initial frequencies of
the PVIs were low, typically inthe range 0.05-0.1.
Control lines: The base population for the controls was
inbred strain 129/Sv congenic for the Gpi-1"allele. No PVIs
were present, and no manipulation via the embryonic stem
cell route had been carriedout.One
pair of divergent
selection control lines was established. An unselected control
inbred line of 6 pairs maintained by full-sib mating was also
maintained andrecorded. Control and insert lines were
housed in cages in adjacent racks.
Selection regime: Insert and control lines were maintained under the same selection regime for the duration of
the experiment. Single pair matings had at most two litters.
Lines were kept in synchrony as far as possible; high and
low lines of the same base population were always so, but
for management reasons the time of mating of different
lines varied by up to 3 weeks.
T o maximize local inbreeding and thereby increase the
TABLE 1
Scheme of the experiment
Generation
0
1-6
7-9
10-12
Lines established.
Circular
mating.
Within
family selection in high
and low lines.
Highand low lines subdivided, full sib mating.
Selection mostly within families.
Full sib mating. Selection relaxed.
fixation probability of recessive mutations, in generations
1-6 matings were made between full-sibs in odd numbered
generations, and followed a circular scheme (KIMURAand
CROW 1963) in even numbered generations. The selection
criterion during this period was within litter within sex
deviation of body weight at 6 weeks, and one male and one
female were selected from each family where possible. Substitutes were taken from related families. Becausebreeding
success was variable and sometimes poor during this period,
the number of matings per selection linevaried, but in most
generations was between 20 and 30.
From generations 7 to 12, to further increase local inbreeding, the selection lines were divided into sublines of
three mating pairs per subline. Within a subline, matings
were between full sibs and, where possible, allthree matings
were from the samefamily. From generations 7to10,
between family selectionwas practized within sublines based
on family mean 6 week weight corrected for litter size, sex
and parity. In large families,selection pressure was also
applied within families. In order to reduce theinfluence of
maternal effects on body size,the selection on 6-week weight
was relaxed in generations 1 1 and 12, but
inbreeding within
sublines was continued. Up to 7 sublines were maintained
per selection line. The scheme of the experiment is shown
in Table 1.
Analysis of retrovirus vectors: The retrovirus vector
inserts were analyzed in Southern blots with a neo probe
which recognizes a single junction fragment per provirus
insert (ROBERTSON
et al. 1986).
T o assess the purity of the genetic background of the
inbred lines Southern blots were carried out with a probe
specific to a broadclass ofendogenous non-ecotropic murine
proviruses. Such proviruses are stable and highly polymorphic between inbred strains (FRANKEL
et al. 1990), and
therefore serve as a good indicator of strain purity (BLATT
et al. 1983). The probe was the 622-base pair BamHI-EcoR1
fragment of murine retrovirus MCF 247 (HOLLAND,
WOZNEY and HOPKINS
1983), which recognizes a single junction
per provirus in a BamHI restriction digest of genomic DNA.
Southern blotting: Genomic DNA was extracted from tail
or from spleen tissue by standard methods. A sample of 10
r g ofDNAwas
digested to completion with BamHI and
separated at 2.3 V/cm for 24 hr in a 20-cm 0.7% agaroseTBE gel containing 1 pg/mlof ethidium bromide. The
DNA was transferred to a charged nylon membrane (Hybond-N+, Amersham Ltd., England) by capillary blotting
with 0.4 M NaOH for 18 hr. About 20 ng of probe DNA in
a low melting point agarose gel fragment were labeled with
"P by random oligonucleotide primer extension (FEINBERG
and V~CELSTEIN
1983) to a specific activity of about 1.5 X
10' dpm/pg and hybridized to the membrane-bound DNA
in 1 mM EDTA, 0.5 M NaHP04, pH 7.2,7% SDS (CHURCH
and GILBERT1984)for18 hr at 65".
Membranes were
washed three times at 65" for 30 min in 1 mM EDTA, 40
mM NaHP04, pH 7.2, 1 % SDS, and autoradiographed for
1101
Retroviruses and QuantitativeTraits
1-5 days at -70" using an intensifying screen.
Statistical analysis of quantitative data: Realized heritability estimation: The realized heritability (FALCONER1989,
Ch. 1 1 ) canbe obtainedfrom the observedresponseto
selection, but becauseof the structure of the experiment, a
decline in the genetic variance from inbreeding needs be
to
accounted for. The heritability in the base population, h&
was computed by iterating the following equationsin order
to finda value of the base population heritability which best
predicts R , the observed selection response:
O
,l
b1
14
0
1
s
m
3
6
9 1 2
1
Generation
2ol
where &,*is the intensityof within family selection, a;, is the
phenotypic variance; Vg,(is the genetic variance; VE is the
total environmental variance;V, is the variance of environmental effects common to littermates; Ne,*is the effective
population size for thewhole selection lineor, where appropriate, for sublines;t is the generation number; andn is the
number of individuals of the same sex recorded per family.
The genetic variance at generation t refers to the variance
in a selection line,or after division into sublines, the variance
in a subline.
Animal model REML estimation of variance components:
Genetic and environmental components of variation of 6weekweightwere estimated using an animal model
with
Restricted MaximumLikelihood(REML) (MEYER 1989).
Information from the response to selection and from covarin the data setis thereby utilized.
iances between all relatives
The genetic variationin the populationis assumed tobe the
result of the segregationof a large numberof genes of small
additive effect("the infinitesimal model"). The variance of
environmentaleffectscommontomembersofthesame
litter, V,, and fixed effects ofsex, generation number, parity
and litter size (6 categories) were also estimated. In cases
where data from more than one line were analyzed simultaneously, a line X generation effect rather than a generation effect was fitted in order to account for some lines not
being strictly contemporary.
RESULTS
Body size: Phenotypic means and realized heritabilities: Mean 6-week weights of high and low selection
lines, correctedfor sex,litter size andparity,are
plotted against generation number in Figure 1. T h e
pattern of response to selection varied markedly along
the lines. T h e control lines diverged immediately, but
the response at generation 2 was barely exceeded in
the last 6 generations. T h e unselected control inbred
line mean was similar tothe low line. Immediate
selection responses did not occur in the insert lines,
but RVI and RV2 showed a consistenthigh-low divergence from generation 6 onwards, andRV2 exceeded
the divergence of the control
in the later generations.
RV3 showed little evidence of a
selection response.
T h e overall high-low divergence for all three insert
lines increased slowly, and the control divergencewas
exceeded in the last 2 generations. A drop in 6-week
0
3
6
9 1 2
.Generation
Generatlon
201
1
4
1
0 3 6 9 1 2
Generation
FIGURE1 .-Mean 6-week weight (g) corrected for litter size, sex
and parity in high and low selection lines plotted against generation
number. (a) Control,(b) R V l , (c) RV2, (d) RV3, (e) all RV.
weight, particularly noticeable in the insert lines, occurred at generations 3-6, and may correspond to a
decline in the health status of the animals.
Because of thechange in the selection regime,
which occurred midway through the experiment, and
the likely divergence between high and low lines because of a positive maternal environmental covariance
between motherand
offspring (FALCONER1965;
KEICHTLEYand HILL
KIRKPATRICK
and LANDE 1989;
1992),inferenceofthe
realized heritability is not
straightforward. Because selection was relaxed, it can
be assumed that thephenotypic means of generations
1 1-1 2 are relatively free from theinfluence of maternal effects, so the mean divergence between high and
low lines in these generationswas taken as an estimate
of the cumulativedivergencefrom
selection. Estimates of cumulative divergences, cumulative
selection
differentials and realized heritabilities are given in
Table 2. T h e genetic variance is underestimated because linkage disequilibrium is generated by selection
(BULMER1971), but trivially so in this case because
selection was very weak due to the poor breeding
success of the inbred. T h e realized heritability estimatefor all threeinsert lines combined is rather
higher than for the control, and the estimate of the
P. D. Keightley, M. J. Evans and W. G . Hill
1102
TABLE 2
Estimates of cumulative divergences, cumulative selection differentials and realized heritabilities
High
Line
R (g)
Low
Control 16.7 17.3
RVl
18.30.75
RV2
18.4
RV3
18.2
16.9 All RV
18.3
(SE)
i.1-6
(0.26)0.68
1.08
17.5
0.98 (0.29) 16.4
2.5
16.7
1.67
(0.22)
1.15
16.8
1.34
(0.48)
0.89
1.34
(0.17)
i.7-IO
0.53
0.48
0.78
0.59
8.7
n
N e w
VE
Vg.0
h:
2.6
14.0
2.4
2.5
21.1
0.20 1.19
0.17
2.92
4.09
4.23
4.65
0.38
0.49
1.04
0.11
0.11
0.20
0.91
Line means and accumulated divergences ( R ) of 6-week weight for generations 1 1 and 12; and realised heritabilities (hi) and base
population genetic variances V,,Ocalculated from the between line divergence. Line means were computed by REML with generation number,
sex, parity, litter size and selection line as fixed effects, and litter as a random effect. Standard errors (SE) of line means are about 0.7 times
the SE of the accumulated divergence. The intensity of within family selection (L)is with respect to the high-low divergence averaged over
generations ( t ) , and is given separately for t = 1-6 and t = 7-10 because of the change in the breeding regime. The between family selection
pressure for t = 7- 10 was negligible and was ignored. The effective pouplation size (Ne)within lines was computed from the pedigree for t
= 1-6, and was assumed to be 2 for t = 7-10 because sublines were maintained by full-sib mating.
TABLE 3
Animal model REML estimates of components of variance of 6week weight
Line
Control
RVl
RV2
RV3
All RV
V,@)
VE@)
d@)
0.59
2.66
0.86
3.69
1.89
2.93
4.09
4.23
4.64
4.34
3.52
6.76
5.08
8.34
6.24
c2
h2
Support
limits
0.30
0.29
0.31
0.27
0.29
0.17
0.39
0.17
0.44
0.30
0.10-0.25
0.28-0.51
0.09-0.28
0.32-0.54
0.24-0.37
The environmental intraclass correlation of litter mates is cz.
Values of h2 outside the support limits are more than e' = 7.4 times
lesslikely than the ML estimate. These limits are approximately
equivalent to 95% confidence limits.
genetic variance is greater by a factor of about 2.5.
The average performance of the low insert lines was
similar to thecontrol low line, and theaverages of the
high insert lines were greater than the control high
line (Table 2), suggesting asymmetry of response.
Animal model REML estimates of components of variance of body size: The data from the selection lines
were analyzed by animal model REML as described
above. Estimates of genetic and environmental components of variance and heritability estimates along
with support limits are shown in Table 3. REML
heritability estimates are higher than realized heritabilities, particularly for the insert lines. T h e heritability estimate from all three insert lines combined is
significantly higher than that from the control (0.01
C P < 0.05), and is comparable with heritability estimates for 6 week weight from many outbred populations (FALCONER1989). An interesting feature of the
estimates of variance components is the inflated phenotypic variance in the insert lines. Some of this
increase may have been due to poor health status,
particularly in the early generations. The differences
in health problems largely disappeared by generation
10, but an increase in phenotypic variance for the
insert lines is still apparent in these generations (next
9 10 1 1 12
Generot~on
g/lO
"2
Generot ion
FIGURE2.-Mean 6-week weight (9) corrected for litter size, sex
and parity in inbred sublines at generations 9-12. (a) Control, (b)
RVI,(c)RV2,(d)RV3.
section). The combined animal model REML estimate
of the genetic variance for the insert lines is more
than threefold higher than for the control (Table3).
Variability
among
inbred
sublines:
Mean 6-week
weight corrected for sex, litter size and parity in the
insert and control sublines in generations 9-1 2 are
shown in Figure 2. Much of the high-low divergence
in the control appears to be associated with one low
line. All of the insert lines have high sublines which
are consistently heavier than the high sublines of the
control. An increased variability of subline averages
in the insert lines is apparent, particularly for RV3.
This was further analyzed using a REML model (PAT-
TraitsQuantitative and Retroviruses
TABLE 4
REML estimates of components of variation of &week weightin
relaxed selection generations(1 1 and 12) in control and insert
lines
Line
Control
RVl
RV2
RV3
AllRV
u%gP)
VI(.)
3.59
3.91
4.37
7.42
4.97
1.32
1.43
0.85
1.19
1.15
(SE)
V, (g')
(SE)
P
sp
(0.21)
(0.28)
(0.20)
(0.33)
(0.15)
0.36
0.43
0.74
3.23
1.18
(0.21)
(0.28)
(0.36)
(1.85)
(0.34)
0.37
0.37
0.19
0.16
0.23
0.10
0.11
0.17
0.44
0.24
Vi is the estimate of the variance among litters within sublines,
and 1' is the intraclass correlation of litter mates. V, is the estimate
of variance among sublines, and sp is the intraclass correlation of
subline members. Fixed effects of generation number, sex, litter
size and parity were alsofitted.
TABLE 5
Numbers of individuals and litters in each selection
line
(including the unselected inbred control), the proportion
of
mating which failed to produce anypups (alive or dead), and
the mean numberof live pups born per litter
Line
Control
RVl
RV2
RV3
All RV
Proportion of
infertile matIndividuals Litters
ings
3595
2462
2814
1794
6902
664
490
572
365
1397
0.06
0.21
0.14
0.37
0.24
(SE)
Live born
per litter
5.62
5.49
(0.01) 5.57
5.45
(0.02)
5.50
(0.01)
(0.01)
(0.02)
(SE)
(0.07)
(0.08)
(0.14)
(0.09)
(0.06)
and THOMPSON
197 1) in which sublines and
litters were fitted as random effects (Table 4). The
proportion of variance associated with subline means
in the combined insert lines is more than twice that
of the controls.
Fitnesstraits: The mean proportion of infertile
matings and the mean number of live born per litter
averaged over generations in control and insert lines
are shown in Table 5 . The insert lines were substantiallyless fertile than the controls (P < 0.001), and
there is evidenceof variation in the proportion of
infertile matings among the insert lines. The small
difference in litter size between the insert lines and
the controls does not differ significantly from zero
( P > 0.2).
Distribution of provirus vector inserts:The insert
selection line RV2 showed the greatest response to
selection. In later generations the average 6-week
weight among sublines of its high
line approached half
a standard deviation heavier than the control's. Any
association between a response to selection and a PVI
is most likely therefore to be found in this line. To
investigate this possibility,the frequencies of PVIs in
RV2 between and within sublinesat generation 9 were
assessed by Southern blotanalysisusing
the PVIspecific ne0 probe (Figure 3). A summary ofthe results
is shown in Table 6. Several PVIs show divergences
TERSON
1103
in frequency between the high and the low line, but
divergences are expected because of genetic drift. It
is not straightforward to test whether any of these
divergences are associated with variation in6-week
weight among the sublines because the observations
are correlated, but the estimated frequencies of PVIs
within sublines were fitted as covariables in a REML
analysis of data for the relaxed selection generations
(Table 6). Some PVIsare associated withlarge effects
on size, but the estimates should be treated with
caution because of the non-independence of the sublines.
Genetic background and genetic monitoring:Single locus probes: Founder mice for the control lines
and oneindividual from each stem cell line were typed
at the Gpi-1 locus, and for hemoglobin phenotypes by
electrofocusing (BULFIELD
and BANTIN1981). As expected, the control founders werehomozygous for
the Gpi-1" allele, whereas Gpi-I" and Gpi-I" alleles
segregated in the insert line founders. No variation in
hemoglobin phenotype was observed, implyinghomozygosity at the Hba and Hbb loci (data not shown).
In addition, individuals were typed with a probe specific to ecotropic proviruses by Southern blotting
(PANTHIER,
CONDAMINE
and JACOB 1988). No such
loci were detected, as is expected for strain 129 UENKINS et al. 1982) (data not shown).
Multiple locus probe: At various times during the
experiment, the genetic purity of the lines was monitored by Southern blotting with the non-ecotropic
provirus probe as described above. Figure 4 shows an
autoradiograph from such a Southern analysis of control individuals from generation 6, and alsoshows
individuals from other inbred lines maintained in the
laboratory (C57BL/6J, C3H/He, RF/J). A large number of genetic differences among these inbred strains
can berecognized, whereas in comparison to the other
inbreds the experimental mice are very homogeneous
withrespect to these markers. In three cases (e.g.,
Figure 4),segregation at a non-ecotropic provirus loci
was observed, which suggests residual genetic variation, presumably associated withthe backcross of the
Gpi-1"allele into the 129/Sv founder strain.
DISCUSSION
Artificial selection on body weight at 6 weeks was
carried out in lines of mice in which a total of about
130 retroviral vector inserts segregated in an inbred
background. The genetic variance of the trait was
estimated from the response to selection, and from
covariancesofallrelativesin
the data set(animal
model REML). Both methods give very much higher
estimates of the genetic variance of 6-week weight in
the insert lines than in control lines lacking insertions.
A simple interpretation of this result is that the insertional mutagenesis has
generated a substantial amount
P. D. Keightley, M. J. Evans and W. G . Hill
1104
Ll
L2 H4 H4 H6
L2
L5 H1 L6
H3 H6 L3 H7WI
29
-
I
.
I
94’
6.6,
-
w
D
-- -
11
-“
4I O
4~
-w
17
- v
=
4.4b
-
-*.I
w
FIGURE3.-Autoradiograph
showing junction
fragments corresponding to provirus vector inserts
segregating in high and low sublines of insert line
RV2.
41,
a
41
41
TABLE 6
Phenotype frequenciesof segregating PVIs in insert
line RV2 at generation 9 (numbers homozygousor heterozygous for thePVI)
Low subline
phenotype
frequency
Over-
High subline phenotype frequency
PVI
I
2
3
4
5
6
7
13
12
2
0
1
0
30
3
0
3
2
2
3
0
0
2
1
0
0
1
0
4
0
10
0
1
2
11
1
9
8
7
6
5
4
3
2
1
1
0
2
2
0
0
0
2
0
2
2
0
0
0
0
0
2
2
2
2
0
0
2
2
3
3
0
0
3
2
0
0
0
2
0
0
0
0
1
0
0
0
3
3
3
0
0
33
0
0
0
0
0
1
3
0
1
3
5
0
0
6
0
0
12
0
0
0
2
0
1
2
0
0
03
Overall
freq.
1
2
3
4
5
6
7
all
freq.
0.56
1 0 2 0 2 2 0 0 . 2 9
0.10
3 1 3 1 0 0 1 0 . 4 0
0.61
3
1
3
1
0
0
1
0.40
0.67
3 2 3 3 2 0 2 0 . 7 7
0.03
0
0
0
0
0
2
0
0.14
0.00
0
1
3 3 12 0 0 . 5 0
0.14
0
1
1 0 0 0 0 0 . 0 7
0.69
0 2 2 1 2 0 0 0 . 2 9
0.14
0 1 0 0 0 0 0 0 . 0 4
0.64
3 1 0 0 0 0 0 0 . 1 8
0.77
2 1 0 3 0 0 0 0 . 2 5
0.00
0
1 3 0 0 0 0 0 . 1 8
0.03
0 0 2 0 0 0 0 0 . 0 6
Number of individuals typed
23
3
2
2
Effect (9)
*
(SE)
1.12 (0.39)
0.01 (0.66)
0.49 (0.42)
-0.65 (0.34)
-
-0.91 (0.42)
0.45 (0.38)
-
-0.63 (0.40)
1.13(0.31)
Overall insert frequencies expressed as averages of insert frequencies in the 7 sublines computed from the phenotype frequencies under
the assumption of Hardy-Weinberg equilibrium within sublines; estimates of effects associated with the inserts from a REML analysis with
the frequency of the inserts within subline fitted as a covariate, and otherfixed and random effects as in Table 4, except that a subline effect
was not fitted. Frequencies of inserts were simultaneously fitted in the model. PVls which segregated at low frequencies were not fitted in
the model as these provided little information for estimating the associated effect. The PVI numbers correspond to those in Figure 3; only
proviruses numbered 1-13 could be scored with confidence.
8
44A
FIGURE4.-Autoradiograph
showing non-ecotropic provirus
junction fragments in control individuals at generation 6 and in a
number of other inbred strains. The arrowhead indicates a segregating non-ecotropic provirus.
of new quantitative genetic variation. As in insertional
mutagenesis experiments in Drosophila, heritabilities
are similar to those typical of outbred populations.
This interpretation has to be further examined, however, because a selection response also occurred in the
inbred control line, and the heritability estimate was
about 15%, greater
than expected because the control
line was highly inbred. Heritability of 6-week weight
in “outbred” populations is typically 40%; in a cross
between the inbred strainsC57BL/6J and DBA/2J, it
was about 25% (G. BULFIELD
personal communication). There areseveral hypotheses that might explain
the high heritability estimate and selection response
in the control: (1) A gene of large effect on 6-week
weight segregated in the base population, which could
have been a spontaneous mutation or residual variation from the construction of the congenic line. There
is some evidence for this hypothesis: (a) The control
inbred line mean 6-weekweightclosely tracked the
mean of the low line, perhaps indicating thatone
Retroviruses and Quantitative Traits
allele became fixedin these lines. (b) An animal model
REML analysis including a term in the additive genetic variance-covariance matrixfor an accelerated
et
decline in genetic variance (WRAY1990; BENIWAL
al. 1992) indicated that the rateof decline of genetic
variance was faster than expected from drift alone.
This could have occurred as a consequence of selection changing gene frequencies. Arguments against
the hypothesis of a single gene of large effect in the
control are: (a) T h e selection response was extremely
rapid, but the mating regimewas such that the quick
fixation of a gene would be unlikely even if it had a
KEIGHTLEYand HILL
substantial effect (CABALLERO,
1991). (b) Much of the variation among inbred sublines appeared to be associated with a single low line
(Figure 2). (2) A non-geneticresponse to selection
resulting from the effect of maternal body size on size
of offspring (FALCONER1965; KIRKPATRICK and
LANDE1989). T h e inclusion of a term for a maternal
environmental covariance between mother and offspring removed little of the genetic variance in the
animal model REML analysis. We have found, however, evidence of such a non-genetic response in an
experiment involving selection on 6-week weight in a
different inbred (KEIGHTLEY
and HILL 1992). In the
present experiment, the divergence between
high and
low lines did not completely disappear after two generations of relaxed selection, suggesting that there
was a genetic difference between
the high and low
lines, but perhaps the rateof the decay of the maternal
effect is on a longer time scale than this (see below).
(3) A systematic upward bias in the animal model
REML resulting in inflated heritability estimates because environmental covariances arenot fully accounted for.T h e transmission of disease from parents
to offspring,forexample,could
lead to a positive
covariance of the trait between parents and offspring
and among members of the same family. Since such
“short range” covariance terms provide much of the
information for the animal model estimation, but not
for estimates based on the selection response alone,
the former would be more biased.
An interesting aspect of the results is the greatly
inflated total phenotypic variance of
6-week weight
compared to the controls. For example, the animal
model REML estimate of the phenotypic variance for
all insert lines combined is nearly twice as high as for
the controls. Thus, the heritabilityestimate, which
was 1.5-1.8 times higher (depending on the method
of analysis) in the insert lines implies a genetic component of variance 2.5-3.2 times higher. Ahypothesis
which could explainthe increased phenotypicvariance
is the segregation of deleterious recessive genes, Some
of the increased phenotypic variance may, however,
have been due to disease which affected the general
health and reproductive performance of the colony,
1105
although it is unclear why the controls should have
been less susceptible to such effects. It is also possible
thattheinsert
lines are subject to some unknown
effect of passing through ES cell culture (the control
lines were not treated in this way). The insert lines
had substantially reduced fertility compared to the
controls. T h e effect of P element insertion in Drosophila is also a substantial reduction in fitness [about
12% reduction in viability per homozygous insertion
(MACKAY,LYMANand JACKSON 1992)]. The insert
lines were known to contain a number of recessive
lethal mutations resulting from insertion, for example
bulgy eye (Be), which is lethal when homozygous (M.
B. CARLTONand M. J. EVANS,unpublished), and
nodal, a recessive lethal (CONLON,BARTHand ROBERTSON 1991; ZHOU et al. 1993). Moreover, inbreeding experiments suggested that approximately 2% of
insertions could not be
bred tohomozygosity, presumably on account of prenatal embryonic lethality (M. J.
EVANS,unpublished observations).
The results from division of the selection lines into
sublines in the later generations of the experiment
tend to confirm the results from earlier generations.
T h e phenotypic variance appeared to remaininflated
in the insert lines, and there was substantially more
variance in6-week weight associated with subline
means in insert lines. The maintenance of alarge
number of sublines is probably a better strategy than
artificial selection for detection of insertional mutants
because of the benefit of more rapid inbreeding of
mutations, most of which are likely to be recessive.
The amount of replication of the effect of any one
mutation is potentially less, however. In mice, artificial
selection is inefficient for selecting insertional mutants
in inbreds because their poor reproductive performance allows only weak selection on the quantitative
trait.
The analysis of the frequencies of the inserts among
sublines of one of the insert lines (RV2) shows divergences in the frequencies of a number of them, and
statistical analysis suggests that some may be associated
with the observed response to selection. Such inserts
are candidates for further genetic analysis based on
continued breeding of the most extreme insert sublines, firstly to determine if the extreme phenotype
has agenetic basis, thento associate theextreme
phenotype with PVIs by segregating them in an FS
population.
This work was funded by the Agricultural and Food Research
Council. P.D.K. wishes to acknowledge support from the Royal
Society. We aregrateful to LINDA MAY
for technical assistance.
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