Milk composition and lactation of beta-casein-deficient mice.
ABSTRACT beta-Casein is a major protein component of milk and, in conjunction with the other caseins, it is assembled into micelles. The casein micelles determine many of the physical characteristics of milk, which are important for stability during storage and for milk-processing properties. There is evidence that suggests that beta-casein may also possess other, nonnutritional functions. To address the function of beta-casein, the mouse beta-casein gene was disrupted by gene targeting in embryonic stem cells. Homozygous beta-casein mutant mice are viable and fertile; females can lactate and successfully rear young. beta-Casein was expressed at a reduced level in heterozygotes and was completely absent from the milk of homozygous mutant mice. Despite the deficiency of beta-casein, casein micelles were assembled in heterozygous and homozygous mutants, albeit with reduced diameters. The absence of beta-casein expression was reflected in a reduced total protein concentration in milk, although this was partially compensated for by an increased concentration of other proteins. The growth of pups feeding on the milk of homozygous mutants was reduced relative to those feeding on the milk of wild-type mice. Various genetic manipulations of caseins have been proposed for the qualitative improvement of cow's milk composition. The results presented here demonstrate that beta-casein has no essential function and that the casein micelle is remarkably tolerant of changes in composition.
Article: Rabbit beta-casein promoter directs secretion of human interleukin-2 into the milk of transgenic rabbits.[show abstract] [hide abstract]
ABSTRACT: To test the potential usefulness of transgenic rabbits as production systems for human proteins of pharmaceutical value, we cloned the rabbit beta-casein promoter and fused it to the genomic sequence of the human interleukin-2 (hIL2) gene. Four transgenic female rabbits were tested for expression and biological activity of the foreign protein in their milk. The milk of all four females proved to contain biologically active hIL2. The results show that transgenic rabbits may represent a convenient and economic system for the rapid production of biologically active protein in milk.Bio/Technolgy 03/1990; 8(2):140-3.
Proc. Natl. Acad. Sci. USA
Vol. 91, pp. 6138-6142, June 1994
Milk composition and lactation of f3-casein-deficient mice
(gene targeting/casein micelles)
SATISH KUMAR*, ALAN R. CLARKEt, MARTIN L. HOOPERtt, DAVID S. HORNE§, ANDREW J. R. LAW§,
JEFFREY LEAVER§, ANTHEA SPRINGBETT*, ELIZABETH STEVENSON§, AND J. PAUL SIMONS*1¶
*Agricultural and Food Research Council Roslin Institute (Edinburgh), Roslin, Midlothian, EH25 9PS, United Kingdom; tCancer Research Campaign
Laboratories, Department of Pathology, University of Edinburgh, Edinburgh EH8 9AG, United Kingdom; *Agricultural and Food Research Council Centre
for Genome Research, University of Edinburgh, Edinburgh EH9 3JQ, United Kingdom; §Hannah Research Institute, Ayr, KA6 5HL, United Kingdom; and
IDepartment of Anatomy and Developmental Biology, Royal Free Hospital School of Medicine, Rowland Hill Street, London NW3 2PF, United Kingdom
Communicated by Neal L. First, December 6, 1993 (receivedfor review October 7, 1993)
and, in conjunction with the other caseins, it is assembled into
micelles. The casein miceiles determine many of the physical
characteristics ofmilk, which are important for stability during
storage and for milk-processing properties. There is evidence
that suggests that j-casein may also possess other, nonnutri-
tional functions. To address the function of(-casein, the mouse
cells. Homozygous i-casein mutant mice are viable and fertile;
females can lactate and successfully rear young.
expressed atareduced level in heterozygotes andwascompletely
absent from the milk of homozygous mutant mice. Despite the
deficiency of 1-casein, casein micelles were assembled in het-
erozygous and homozygous mutants, albeit with reduced diam-
eters. The absence of R-casein expression was reflected in a
reduced total protein concentration in milk, although this was
partiay compensatedforbyanincreased concentration ofother
proteins. Thegrowth ofpups feeding on the milk ofhomozygous
mutants was reduced relative to those feeding on the milk of
wild-type mice. Various genetic manipulations of caseins have
been proposed for the qualitative improvement of cow's milk
composition. The results presented here demonstrate that
f-casein has no essential function and that the casein micelle is
remarkably tolerant of changes in composition.
Milk is usually the sole source of nourishment of young
mammals, and the milk of domestic animals is an extremely
important food source for much of the world's population,
both as liquid milk and in a very wide variety of processed
forms. Most of the protein in milk is found in the caseins,
which are aggregated into large micellar structures that are in
colloidal suspension in native milk. Although the detailed
structure of casein micelles is not yet established, there are
a number ofmodels of micelle structure (for review, see ref.
1). The "calcium-sensitive" caseins (asi-, a52-, and 3-caseins
in cow's milk) are generally thought to be located predomi-
nantly within the micelles, and K-casein is thought to coat the
micelle, serving to stabilize the structure. The calcium-
sensitive caseins are highly phosphorylated, and calcium
phosphate interacts with them via their phosphate groups. In
this way the casein micelles carry large amounts of(normally
highly insoluble) calcium phosphate into milk and retain it in
suspension. A further consequence of the assembly of the
caseins into micelles is that the viscosity ofmilk remains low
despite the high protein concentration. Thus, casein micelles
are offunctional importance forprotein and mineral nutrition
of the young and in determining the physical properties of
Selective breeding has been very successful in increasing
milk yields ofcattle, butthis approachhasbeenfound to have
limited potential for the alteration of milk composition. We
and others have previously demonstrated the profound al-
teration of the protein composition of milk by expression of
milk protein genes in transgenic mice (2-4), and the expres-
sion of human pharmaceutical proteins in the milk of trans-
genic mice, rabbits, goats, pigs and sheep has been demon-
strated (5-13). In some casesvery high yieldsofactive human
proteins have been obtained (6, 8, 11-13), and this approach
is being commercialized. In addition to pharmaceutical ap-
plications, genetic manipulation may be of use for the pro-
duction of milk with altered nutritional, allergenic, or pro-
cessing properties. The storage and processing properties of
milk are, in large measure, determined by the caseins, and
these proteins thus represent a potentially important target
for genetic manipulation (14, 15).
There is some evidence that suggests that caseins may
possess additional functions. A number of peptides derived
by proteolytic cleavage of bovine /-casein and known as
/3-casomorphins have been shown topossess potent opioid
activity and have been suggested to be natural agonists for
opioid receptors in the gut (16). In addition to expression in
the mammary glands, casein expression has been detected at
the RNA level in mouse cytotoxic T-lymphocyte cell lines
and in thymus (17). The authors suggested that casein mi-
celles may serve to sequester perforin in cytotoxic T lym-
phocytes in conditions that prevent its polymerization. The
sequestration of perforin may protect the cytotoxic T lym-
phocytes against perforin-mediated lysis, and casein micelles
may function to deliver perforin to the target cells.
To investigate the function of 3-casein in milk and to
determine whether this protein possesses any other essential
function, we generated mice deficient for /3-casein by gene
targeting in embryonic stem cells. The effects ofthe mutation
on gene expression, milk protein content, and casein micelle
structure have been characterized.
MATERIALS AND METHODS
Gene Targeting. The gene targeting vector(pP3MClneo/
TK) includes a 4.7-kb Sca I-EcoRI fragment of the (3-casein
gene (18) from a C57BL/6 mouse, with the neomycin-
resistance gene from pMClneo(C) (19) inserted into the
Asp700I site in exon 2 ofthe gene in the same orientation. A
herpes simplex virus thymidine kinase expression cassette
was placed at the 3' end of the region of homology, for use
in positive-negative selection (20). Embryonic day-14 stem
cells (21) were electroporated withp.3MClneo/TKat 31.25,
93.75, or 187.5 pg ml-1. After 24 hr, the cells were selected
withGeneticin (0.3 or 0.5 mgml-1), and afterafurther4 days,
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Proc. Nati. Acad. Sci. USA 91 (1994)
ganciclovir (2 ,uM) selection was applied. The enrichment
obtained with ganciclovir was 4.4- to 15-fold.
DNAandRNA Analysis. GenomicDNA was prepared from
embryonic stem cells and digested by the method ofLaird et
al. (22) and from mouse tails as described (23). RNA was
isolated by the method of Chomczynski and Sacchi (24) and
redissolved in 100% formamide (25). After electrophoresis,
DNA or RNA was blotted onto Hybond-N (Amersham) and
hybridized with probes labeled by random priming (26, 27)
using the method of Church and Gilbert (28).
Production of Chimeras. Chimeras were produced essen-
tially as described (29) by microinjection oftargeted cells into
the blastocoel cavity of 3.5-day C57BL/6 x CBA F2 em-
bryos. Microinjected blastocysts were reimplanted into the
uteri of 2.5-day pseudopregnant mice. Chimeras were iden-
tified on the basis ofthe presence ofpale patches offur on an
agouti or nonagouti background.
Milk Protein Analysis. Collection of milk and electropho-
retic analysis of milk proteins were done essentially as
described (2). For quantitative protein analysis, pools were
made of equal volumes of milk from 18 wild-type and 18
homozygous mutant mice. Total protein and whey protein
concentrations were estimatedbythe micro-Kjeldahl method
as described (30), except that protein concentration was
obtained by multiplying the nitrogen value by 6.38 (to take
account of the amino acid composition and nonprotein ni-
trogen content of milk). The factor 6.38 was derived from
calibrations with cow milk and may not be entirely accurate
for mouse milk. By SDS/PAGE, no casein contamination of
the whey fractions was detected. The (-casein concentration
was determined by measuring A280 of eluates from ion-
exchange fast-protein liquid chromatography (31); the ex-
tinction coefficient ofpure (-casein was found to be 1.4 [1%
(wt/vol) solution, 1-cm path length]. Casein micelle sizes
were estimated by the dynamic light-scattering method (32,
AnalysisofPupGrowth. Homozygous mutantfemales were
mated with wild-type males, and homozygous mutant males
were mated with wild-type females. All the pups were thus
heterozygous for theP-caseinmutation. Entire litters were
weighed three times per week (Monday, Wednesday, and
Friday) between birth and 11 days of age. Litter weight was
found to be linearly dependent upon age over this period;
correlation coefficients between weight and age were greater
than orequal to0.% for all individuallitters. Thegrowth rates
(slopes) obtained from individual litter weight vs. age regres-
sions were compared across genotypes and litter sizes by
using regression analysis. The model included effects for
genotype, litter size, and a quadratic effect of litter size with
interactions between genotype and litter size terms. Pre-
dicted growth rates for each combination of genotype and
litter size were then obtained.
A positive-negative selection replacement vector was con-
structed to target the disruption of the mouse f-casein gene
(Fig. 1A). The neo gene was inserted immediately down-
stream ofthe (3-casein translation initiation codon, within the
secretion signal-coding sequence; translation from this AUG
would be predicted to result in expression of a short peptide
bearing no resemblance to mature 13-casein. This vector was
introduced into embryonic day-14 stem cells by electropora-
tion followed by selection with Geneticin and ganciclovir. At
the 5' end of the vector, targeted recombination was pre-
dicted to give a 3.8-kb Sac I fragment in addition to a 2.7-kb
3.9 kb HindIlH
2.7 kb Sac
.... ~ ~~~5.
restriction fragments and the probes used to detect them are shown. TK, thymidine kinase. (B) Southern blot showing the fragments diagnostic
of homologous recombination via the 5' end of the targeting vector. (C) Southern blot showing the fragments diagnostic of homologous
recombination via the 3' end of the targeting vector. WT, wild type.
Targeting of theP-caseingene. (A) Structures of the target locus, the targeting vector, and the targetedP-caseingene. Diagnostic
7A n rh :Real
5.0 kb HindIII
8.1 kb Scal
6.4 kb BhtEll
AgriculturalSciences: Kumar et al.
Agricultural Sciences: Kumar et al.
fragmentfrom the wild-type allele, whenprobedwith a0.9-kb
HindIl-Sca I fragment that lies outside the targeting vector
and that encompasses exon 1 of the (-casein gene (Fig. 1A).
DNA from each clone was analyzed by Southern blotting,
and the presence of the predicted 3.8-kb Sac I fragment
indicated that targeted clones were obtained in each of four
experiments; 21 targeted clones were obtainedfrom a total of
492 clones analyzed. A number ofthe clones may have been
mixed populations of targeted and wild-type cells or aneu-
ploid because the diagnostic 3.8-kb Sac I fragment was of
lower intensity than the 2.7-kb fragment. The majority of
cells in two clones (A77 and B256) possessed 40 chromo-
somes, and in these clones the two Sac I fragments were of
equal intensity. Further analysis of DNA from clones A77
and B256 confirmed the 5' recombination (Fig. 1B) and
demonstrated that the 3' end of the vector had also recom-
bined in the predicted manner (Fig. 1C). Chimeras were
generated with both ofthese targeted clones and were mated
with MF1 mice. Germ-line transmission of the embryonic
stem cell-derived component was obtained from nine chi-
meric males and one chimeric female, all derived from clone
B256. Heterozygous mutant mice were identified by South-
ern blotting of tail DNA and were interbred. The number of
progeny from the intercrosses of csnb+/csnb- mice were as
follows: csnb+/csnb+, 57; csnb+/csnb-, 152; and csnb-/
csnb-, 70; the genotypes of these offspring were determined
by Southern blot analysis ofDNA from tail biopsies taken at
U-5 weeks of age. Segregation did not deviate significantly
from a 1:2:1 ratio (Pearson's goodness of fit test statistic =
3.45 with 2 df, P > 0.1).
Female mice of the three genotypes were mated for anal-
ysis of mammary gene expression and of milk composition.
Northern blot analysis was performed on RNA isolated from
mammary glands at midlactation (11 days postparturition).
Heterozygous mice consistently expressed
at a lower level than wild-type mice, and in homozygous
mutant mouse mammary glands there was no detectable
(-casein mRNA (Fig. 2A). Hybridization of Northern blots
with a neo probe, to detect transcripts from the targeted
allele, gave some diffuse signal but no discrete bands (data
not shown); control experiments eliminate the possibility that
this signal was due to degradation of the RNA or to contam-
ination of the RNA with genomic DNA (data not shown).
Milk was collected from 11-day lactating mice and ana-
lyzed by SDS/PAGE: f-casein was found to be absent from
the milk of homozygous mutant mice and to be expressed at
a lower level by heterozygotes than by wild-type animals
(Fig. 2B). No other differences were evident from this
analysis. To investigate the effects of the mutation on the
total protein content of milk, micro-Kjeldahl analysis was
performed on milk from wild-type and homozygous mutant
mice. Milk from (3casein-deficient mice contained signifi-
cantly less protein than milk from wild-type mice (Table 1).
Similar measurements on whey revealed an increase in the
whey protein content of milk from /3-casein-deficient mice
We followed the growth between birth and 11 days after
birth, of litters that were being nursed by wild-type and
homozygous mutant mice. The rates ofgrowth of litters that
werefeeding onwild-type milk were significantly greaterthan
those oflitters feeding on (3-casein-deficient milk (Fig. 3). No
other differences were noted in the pups feeding on (-casein-
Perturbing the casein content ofmilk might be expected to
influence its physical properties. We therefore investigated
the effects ofthe mutation on one such property: the sizes of
casein micelles. The casein micelles in the milk of homozy-
gous mutant mice were significantly smaller than those in
wild-type mouse milk, and micelles from heterozygous mice
were intermediate in size (Table 2).
FIG. 2. RNA and protein expression. (A) Northern blot analysis
of mammary gland RNA from wild-type (+/+), heterozygous (+/
-), and homozygous (-/-)P-casein-deficientmice, hybridized with
a(3-caseincDNA probe (exons 7-9). (B) SDS/PAGE analysis ofmilk
proteins. Milk from wild-type (+/+), heterozygous (+/-), and
homozygous (-/-) (3-casein-deficient mice was diluted, and fat was
removed. The equivalent of75 n1 ofmilk was loaded ineach lane and
was electrophoresed together with molecular weight markers (M);
the gel was stained with Coomassie blue.
We have shown that (3-casein is not required for viability or
fertility ofeitherhomozygous mutant mice or ofmice feeding
on (3-casein-deficient milk. Because casein micelles are as-
sembled in the mammary glands of (3-casein mutant mice, it
is likely that any assembly of micelles elsewhere would not
be affected. For this reason, the analysis undertaken did not
address the proposed (17) function of casein micelles in
cytotoxic T lymphocytes. No increase in disease suscepti-
bilityofthe mutant mice was evident. The apparentreduction
in growth ofpups sucking milk ofcsnb-/csnb- mice may be
a consequence of the reduced protein content of the milk,
although we cannot exclude other possibilities-such as
alterations in calcium delivery to the pups or on the digest-
wild-type and mutant mice
Protein content of milk and of milk fractions of
Protein content, mgml-' ± SEM
97.1 ± 1.71
18.5 ± 0.81
*Both total protein and the whey protein content of milk were
affected by theP-caseingenotype (P < 0.001; Student's t test).
tSEMs were calculated from the variation among six mice of each
tSEMs were calculated byassuming the same coefficient ofvariation
for whey protein as for total protein.
§This value was calculated from the values for total protein andwhey
protein by subtraction.
87.3 ± 0.89
22.0 ± 0.54
Proc. Nad. Acad Sci. USA 91(1994)
Proc. Natd. Acad. Sci. USA 91 (1994)
8 9 1011 1213 141516
graph shows growth rates from birth to 11 days of age for litters
feeding on wild-type (o) and homozygous (3-casein mutant (ta) mouse
milk. All pups were heterozygous for the (-casein mutation. The
curves have common linear and quadratic coefficients, which are
0.80 (±0.06) and -0.030 (±0.004), respectively. The intercept for
wild type is -0.032 and for homozygous mutants is -0.61 (The SE
ofthe difference is 0.12, the intercepts are significantly different; P
< 0.01, Student's t test with 40 df). The litter sizes were 7.4 ± 0.8
(n= 23) and 8.3 ± 0.6 (a = 21) forwild-type andhomozygous groups,
The effectofmatenalgenotype on littergrowth rate. The
ibility of milk, or nonnutritional effects, such as loss of
During lactation, milk proteinmRNAs are extremely abun-
dant in the mammary gland. In the rat at mid-lactation,
P-caseinmRNA constitutes P20%o ofpoly(A)+ mRNA (34);
both transcription ofmilk protein genes and stability ofmilk
protein mRNAs are regulated (35). The absence ofabundant
transcripts fromthe mutated (-caseingene couldbedue to an
effect on transcription, stability, or both. From the data, we
cannot distinguish between these possibilities, although the
detection of neo transcripts demonstrates that the mutant
gene is transcribed. Antisense transcription of the endoge-
nous mouse (-casein gene has been detected in cultured
mammary cells (18), raising the possibility that the neoRNA
detected may not derive from transcription originating at the
1-casein promoter. The dosage effect seen in the heterozy-
gous mutant mice shows that the absolute level of (-casein
mRNA is not regulated, suggesting that the regulation ofmilk
protein gene expression is more general than specific.
We previously found that expression of sheep 3lactoglo-
bulin in transgenic mice did not result in any increase in total
milk protein concentration, despite the fact that 3-lactoglo-
bulin was estimated to constitute 29%6 of milk protein (36).
Thus, in the mouse, there is a physiological limitation on the
rate ofmilk protein synthesis and/or secretion that, underthe
conditions of the experiment, could not be overcome by
augmenting milk protein gene expression. In homozygous
(3-casein-deficient mice, the absence of 3-casein mRNA is
accompanied by an overall decrease in the total protein
content ofthe milk, suggesting thatmRNA availability is now
limiting the secretion of milk protein. Together, these data
Reduction of casein micelle sizes in 3-casein
Micelle diameters, nm ± SEM
280.5 ± 3.7 (12)
268.6 ± 3.6 (11)
Diameters of casein micelles in the milk of mice of the three
genotypes differ significantly from each other (P < 0.01, one-way
ANOVA and Student's t test).
255.8 ± 2.3 (14)
suggest that the mRNA levels are controlled such that there
is normally little or no mRNA in excess of the glands'
capacity to use it. Although -asein constitutes >200% ofthe
protein in the milk ofwild-type mice, the overall reduction in
total milk protein in homozygous mutant micewas only 10%.
The absence of(-casein expression is thus compensated for
byincreased secretionofothermilkproteins. Consistentwith
this is the finding that the whey protein concentration was
higher in the milk of mutant mice than in wild-type mice.
There are no major changes in the relative concentrations of
proteins other than (-casein (Fig. 2B), showing that the
compensation does not occur by a disproportionate increase
in the concentration of one component but is shared among
various proteins. This result suggests that the compensation
that occurs may not result from a specific regulatory mech-
anism but may simply reflect the metabolic consequences of
mRNA depletion. This result is consistent with the effect of
P-casein gene dosage.
Forthe dairy industry, milk with increasedprotein content
would be highly desirable. If the physiology of milk protein
secretion in livestock is similar to that of mice, it may be
difficult to obtain milk with an increased protein content by
genetic manipulation. There is some evidence that in cattle
the capacity for protein secretion is limited: the A allele of
(3-lactoglobulin is associated with a significantly increased
level of (-lactoglobulin but at the expense of a-lactalbumin
andcasein (37). Alterationofthebiochemicalandbiophysical
propertiesofmilk willbe more easily accomplished, although
ideally such improvements should not adversely affect yield
and gross composition. If in other species, compensation of
protein secretion occurs similar to that observed in the
mutant mice, it is possible that reduced expression of some
milk protein genes will not result in reduction of total milk
The finding that (-casein-deficient milk contains casein
micelles demonstrates that although (-casein is normally a
major component of the micelle, this protein is entirely
dispensible for micelle assembly and secretion. This is con-
sistent with the extremely high degree of sequence diver-
gence among (-caseins ofdifferent species. However,(- and
K-caseins are the only caseins that have been found in the
milk of all species analyzed (38), suggesting some specific
-casein. Although micelle stability was not inves-
tigated directly, any major destabilizing effects would have
been obvious because the caseins would precipitate. There
was no evidence of such instability of micelles in (-casein-
deficient milk, demonstrating that (-casein plays no major
role in stabilizing micelles at temperatures of 4P-3rC.
foundtobe smallerthanthatofmicellesfrom wild-type mouse
milk; the micelles of heterozygotes are intermediate in size.
The magnitude ofthe reduction in micelle diameter is consis-
tent with that expected if the volumes contributed to the
micelles by the caseins are in proportion to their respective
concentrations. This result suggests that the number of mi-
celles is not appreciably affected by the absence of(-casein;
again, this argues against any specific role of 3-casein in
micelle assembly. In cows' milk, small micelles have a higher
ratio of K-casein to (-casein than large micelles (30, 39),
consistent with K-casein being located on the surface. The
effects ofthe (-casein mutationon casein micelle size are thus
probably due to a change in the ratio of K-casein to calcium-
sensitive caseins. In bovine milk, small micelles have been
found to possess greaterheat stability than large micelles (40).
increased heat stability is not yet known.
The insensitivity of casein micelle assembly to major
with altered properties by genetic manipulation oflivestock,
although it remains possible that expression of modified
AgriculturalSciences: Kumar et aL
Agricultural Sciences: Kumar et al.
caseins [for example with increased phosphorylation (15)]
may interfere with micelle assembly ormay destabilize casein
micelles. Given the time scale, difficulty, and expense of
genetic manipulation of livestock, before undertaking such
manipulations it may be prudent to model them in mice. To
this end, it would be desirable to generate mice in which the
mouse casein genes are replaced by their bovine counter-
parts. Finally, targeting of transgenes designed for expres-
sion ofpharmaceutical proteins in milk into the casein locus
may enhance the levels ofexpression by providing a positive
position effect (41).
We thank Prof. Jeffrey Rosen for his kind gift ofP-caseinclones
and for communicating unpublished information. We thank Ray
Ansell, Lorraine Dobbie, Frances Thompson, Audrey Peter, John
Verth and his staff, and Roberta Wallace for excellent technical
assistance; Kenneth Dobie for help and advice; and John Clark for
support and discussions. This work was supported, in part, by Grant
TAP2B from the Agricultural and Food Research Council. S.K. was
in receipt of an Indian National Scholarship and an Overseas
Research Student Award from the Committee of Vice Chancellors
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