(Received for publication, April 5, 1995)
From the
The mouse -macroglobulin gene was inactivated
in embryonic stem cells by homologous recombination. Liver
-macroglobulin mRNA and plasma protein was absent in
homozygotes and reduced to 50% in heterozygotes.
-Macroglobulin-deficient mice were viable and produced
normally sized litters with normal sex ratio over 3 generations.
Characterization of adult homozygotes included diets with different fat
content, treatments with endotoxin, bleomycin, carbon tetrachloride,
and ethionine to test for immune system, lung, liver, and pancreas
toxicity, respectively. Knock-out mice were more resistant to endotoxin
but more sensitive to a choline-free diet supplemented with ethionine.
Regulation of murinoglobulin mRNA expression during pregnancy was
analyzed as a possible back-up mechanism for the deficiency in
-macroglobulin. In addition, expression of mRNA was
studied, coding for
-macroglobulin
receptor/lipoprotein receptor-related protein, low density lipoprotein
receptor, and very low density lipoprotein receptor and for some common
ligands, i.e. apolipoprotein E, lipoprotein lipase, and the
44-kDa heparin binding protein. Their differential regulation in the
knock-out mice relative to C57Bl mice was evident and is discussed. The
impressive 15-fold increase in maternal liver murinoglobulin mRNA at
partum in the knock-out mice indicated increased consumption, compared
to only 4-fold in normal mice. Thus, murinoglobulin appears as the
major proteinase inhibitor around partum, obviously solicited to a much
greater extend in
-macroglobulin-deficient mice.
Mouse -macroglobulin (MAM) (
)is a
typical member of the proteinase inhibitors of the
-macroglobulin (A2M) family, capable of inhibiting
proteinases from all classes by a steric trapping
mechanism(1, 2, 3) . Upon cleavage of a
peptide bond in the bait region by the proteinase, A2M traps the
proteinase by a major conformational change of the tetrameric A2M
structure. Recognition sites for the
-macroglobulin
receptor thereby become exposed, allowing for the specific elimination
of A2M-proteinase complexes(2) .
The precise role of A2M in vivo is far from clear. In contrast to some species, human A2M is not an acute phase protein, as changes in plasma levels are moderate and never diagnostic for any disease(4) . Decreased A2M concentrations, resulting probably from enhanced consumption and clearance of A2M-proteinase complexes, might occur in states associated with proteolytic problems, for example, pancreatitis(5) . On the other hand, the interaction of A2M with a wide range of cytokines and growth factors is not understood, although it might explain some of the immunochemical and growth promoting properties ascribed to A2M(6, 7) .
In humans, no individual with a complete A2M deficiency has ever been reported. This could mean that such a deficiency is either phenotypically silent or prohibitive for full-term development and lethal in utero. Evidence for either possibility can be obtained in an experimental, transgenic model. To define the function of the A2M system including the murinoglobulins and their receptor experimentally in vivo, we have embarked on characterizing this in the mouse in molecular detail. Previously, we have reported the cloning of the cDNA and the genes coding for mouse A2M(8, 9) , of three murinoglobulins, the single-chain proteinase inhibitors of the A2M type (10, 11) and the mouse A2MR/LRP cDNA (12) and its coding gene(13) .
The gene coding for MAM, cloned from the 129/J mouse strain was used in a construct to target the MAM gene successfully in ES cells by homologous recombination(8) . We now report that the disrupted MAM gene was introduced into the germline and resulted in heterozygous and homozygous MAM-deficient offspring. The mice are viable, produced normally sized litters and showed no phenotypic abnormalities at the age of well over 1 year. Experiments with the MAM-/- mice demonstrated several potentially important differences in susceptibility to toxic agents known to cause malfunction of specified organs or systems. Tests of endotoxin(14) , carbon tetrachloride(15) , and bleomycin (16) are presented as well as the effect of diets with different fat contents (17) and a choline-deficient diet containing ethionine instead of methionine known to induce acute pancreatitis (18) .
The cDNA
clone mLDLRc90 for mouse LDLR was kindly provided by M. Hofker (Sylvius
Laboratory, Leiden, The Netherlands). The 700-base pair EcoRI
insert was used as a probe(19) . The mouse ApoE cDNA clone,
pmEUC18 was obtained from S. Tajima (Osaka, Japan) (25) . To
detect A2MR/LRP mRNA, a 1.4-kb EcoRI cDNA fragment was used as
a probe corresponding to positions 776-2197 of the mouse
cDNA(12) . The cDNA probe used to detect LPL mRNA was a 1-kb PstI restriction fragment isolated from the coding region of
rat LPL (provided by J. Auwerx, Lille, France). A 2.0-kb human
-actin cDNA probe (Clontech) was used as a control in Northern
blotting to allow for normalization of mRNA loading. Labeling of the
probes was performed by hexanucleotide mediated incorporation of
[
P]dCTP, as described
before(10, 22) .
Figure 1: Recombinant DNA construct used to target the MAM gene in ES cells. Top, the hatched box (PGK-HYG) represents the hygromycin marker gene which was embedded in intron 17. The construct was linearized at the unique BamHI (B) site in exon 18. StuI restriction sites are marked (S), pUC denotes the bacterial cloning vector. Middle, partial structure of the wild type MAM gene with the region used in the construct represented as a box. Restriction sites recognized by StuI (S) are 11.5 kb apart. R and L represent genomic DNA probes, located at the right and left relative to and externally of the genomic region used in the construct. Bottom, predicted structure of the targeted MAM gene. Insertion of the construct by homologous recombination will result in the loss of the wild type 11.5-kb StuI fragment and the generation of StuI fragments of 10.5 (L probe), 3.8 kb (R probe) and 8.5 kb (HYG probe). Inset, Southern blotting of one of the selected ES cell lines, digested with StuI and hybridized with the three different probes (R, L, and HYG).
Figure 2: Screening by Southern blotting of offspring of heterozygous matings. Genomic DNA, isolated from the tail tip, was digested with StuI and hybridized with the 0.5-kb MluI-EcoRI fragment (R probe). Among 18 pups, 4 were homozygous, indicated by the diagnostic 3.8-kb fragment, 9 were heterozygous (3.8- and 11.5-kb fragment), and 5 were wild type (11.5-kb fragment only).
At this moment 3 generations of MAM-/- mice have been obtained with the oldest homozygous MAM-deficient mice now 15 months old. They are kept in an open animal house on standard mouse chow, available ad libitum, and appear healthy without overt health problems.
Figure 3: Northern blotting of poly(A) mRNA isolated from the liver of wild type C57Bl, heterozygous, and homozygous MAM-deficient mice. The blot was hybridized with a cDNA probe specific for MAM (9, 22) and subsequently with a cDNA probe specific for MUG(10, 22, 27) . Exposure was for 6 and 4 h at -70 °C, respectively, and the size of both mRNA species identified was 5 kb as indicated on the right. Overexposure for 5 days after hybridization with the MAM cDNA probe did not reveal the 5-kb band in the MAM-/- mice. Note the very low expression levels of MUG in certain females, depending on their hormonal status as reported recently (see text and (22) and (27) for details).
Rocket immunoelectrophoresis of plasma of the same mice and of many other MAM-/- mice confirmed the results obtained by Northern blotting. No MAM protein was present in the plasma of MAM-/- mice, while plasma levels were between 45 and 60% in MAM+/- mice, relative to wild type mice (Fig. 4A).
Figure 4: Rocket immunoelectrophoresis of mouse plasma. Plasma samples of wild type C57Bl (+/+), heterozygous (+/-), and homozygous (-/-) MAM-deficient female mice were run into antisera directed against MAM (panel A) and MUG (panel B). Plasma (2.5 µl) was applied undiluted in 3-mm wells punched in 0.8% agarose gels on glass plates. Some cross-reaction is evident with both antisera with unidentified plasma proteins. The most dense rocket in panel B corresponds to MUG as determined in separate experiments by addition of purified MUG1 protein, isolated from mouse plasma and authenticated by N-terminal amino acid sequencing (9, 10) .
The plasma levels of murinoglobulins in normal mice are more variable than those of MAM and, in female mice, are also dependent on the hormonal status of the animal. Nevertheless, comparing the MUG plasma levels of adult nonpregnant mice demonstrated no systematic difference with C57Bl control mice, in all MAM-/- and MAM+/- mice analyzed to date (Fig. 4B).
Extracted mRNA of intact
MAM-/- embryos of 15 days post-coitus was analyzed by
Northern blotting to confirm the absence of MAM mRNA. Further
hybridization failed to demonstrate any expression of murinoglobulin at
this embryological age, identical to our previous observations in
normal mice(22) . Thus, murinoglobulins, which are
normally expressed only in the second week postnatally, do not
substitute for embryonic expression of MAM in the MAM-/-
mice.
The maternal hepatic MUG mRNA levels in control
C57Bl mice were at parturition about 4-fold higher relative to the
levels at day 12 postcoitus (Fig. 5). This increase is much more
pronounced in the MAM-/- mice, since the maternal MUG mRNA
levels are more than 14-fold higher at partum relative to day 12
postcoitus (Fig. 5). This impressive rise in maternal liver MUG
mRNA levels is only partially reflected in the circulating MUG protein
levels: at birth very similar concentrations of about 0.4-0.45
mg/ml were measured in the C57Bl and in MAM-/- mice (Fig. 5), pointing to extensive consumption of maternal MUG
around birth in C57Bl mice, which is clearly much more
evident in the MAM-/- mice.
Figure 5:
Maternal plasma MUG protein levels and
hepatic MUG mRNA levels in pregnant C57Bl and MAM-/- mice. Panel A, histogram representation of quantitative
densitometric scanning of Northern blots of liver mRNA probed for MUG
and normalized to the -actin signal. Liver mRNA was extracted from
pregnant mice at 12 and 19 days postcoitus (pc) and at 1 day
postpartum (pp). Each time point represents the calculated
mean of determinations of three mice at each time point. The values are
given in arbitrary units and are normalized to the signal obtained by
hybridization with an actin probe of the same Northern blot (see
``Materials and Methods''). Panel B, histogram
representation of quantitative rocket immunoelectrophoresis of MUG in
maternal plasma of pregnant mice. Plasma was isolated from pregnant
females at 12 and 19 days and from females 1 day postpartum. Each time
point represents the calculated mean of measurement on three separate
mice.
Figure 6:
Histograms of levels of mRNA coding for
A2MR/LRP, LDLR, VLDLR, HBP-44, ApoE, and LPL in liver, placenta, and
uterus in MAM-/- and C57Bl mice. Extraction of mRNA from
liver (A and D), placenta (B and E), and uterus (C and F) of
MAM-/- (A, B, and C) and C57Bl (D, E, and F) mice was consecutively hybridized with cDNA probes
specific for A2MR/LRP, LDLR, VLDLR, HBP44, ApoE, LPL, and -actin.
The histograms represent, in arbitrary units, the quantitation by
densitometric scanning of the Northern blots, normalized to the
-actin signal on each blot. Each time point, i.e. 12 and
19 days postcoitus (pc) during pregnancy and 1 day postpartum (pp), as indicated, represents the calculated mean of
determinations of three individual mice at each time point that were
analyzed on different filters. Abbreviations of each of the mRNA
species probed for is indicated, eventually with each mRNA species
detected and identified by its size.
In placenta,
A2MR/LRP and LDLR are regulated oppositely in C57Bl mice (Fig. 6) confirming that the placenta shifts toward A2MR/LRP
mediated uptake of ApoE-VLDL lipoproteins with progressing
pregnancy. This is much less marked in the
MAM-/- mice, which in conjunction with the different
hepatic expression pattern of these receptors, indicated that
lipoprotein metabolism is regulated differently in the
MAM-/- mice. The possible involvement of or even
compensation by another receptor of this family, i.e. the
VLDL-receptor (VLDLR), was supported by the results: from day 12 to day
19 postcoitus placental VLDLR mRNA levels increased in C57Bl mice but
not or hardly in MAM-/- mice (Fig. 6).
In the uterus of both C57Bl and MAM-/- mice, the constant expression levels of A2MR/LRP mRNA contrasted sharply with the 8-fold down-regulation of LDLR mRNA at parturition (Fig. 6). In C57Bl mice this is eventually compensated for by increased expression, at least of the 3.9 kb, the smaller of the two transcripts coding for VLDLR, an increase which is not observed in either mRNA transcript of the VLDLR in the uterus of MAM-/- mice (Fig. 6).
The 1.2-kb ApoE transcript (25) is abundantly detected
in both control and in MAM-/- mice, in liver, placenta, and
uterus. With progressing pregnancy, the uterus and especially the
placenta are characterized by a vast increase in ApoE mRNA levels, more
than 30-fold in MAM-/- mice, while the liver ApoE mRNA
levels decreased (Fig. 6). This is in liver again an opposite
regulation as seen in the C57Bl strain, in which liver ApoE mRNA levels
increased from mid pregnancy toward parturition (Fig. 6).
The 4-kb LPL mRNA (30, 31) was up-regulated
about 10-fold in placenta and uterus as pregnancy progresses from day
12 to 19 postcoitus in MAM-/- mice (Fig. 6) very
similar to observations in C57Bl mice. The regulation of
expression of liver LPL mRNA was again the exception, since in
MAM-/- mice it increased very importantly by about 13-fold,
while it was somewhat decreased in C57Bl mice (Fig. 6).
Figure 7:
Survival of mice following an
intraperitoneal injection of endotoxin. Wild type C57Bl and
MAM-/- mice (10 each) were injected intraperitoneal with
doses of endotoxin corresponding to the LD or to 5 times
that dose (as reported by the manufacturer, see ``Materials and
Methods''). The graph represents the survival curves for each
group as indicated.
, C57Bl 5
LD
;
,
C57Bl 1
LD
;
, MAM-/- 5
LD
;
, MAM-/- 1
LD
.
Figure 8:
Evolution of body weight of wild type
C57Bl and MAM-/- mice given a fat-rich or a regular chow
diet during 9 weeks. Symbols used are for the fat-rich diet: (bar 1 of inset), 28 female, and
(bar 4 of inset) 18 male MAM-/- mice;
(bar
2 of inset) 12 female and
(bar 5 of inset) 12 male C57Bl; and for the regular chow diet:
(bar 3 of inset) 6 female and
(bar 6 of inset) 6 male MAM-/- mice. Data represent
the calculated mean weight of all mice in each experimental group,
except for the MAM-/- mice on the fat rich diet in which 7
female and 3 male mice died during the experiment as represented in the inset. All C57Bl mice survived.
One female and 3 male MAM-/- mice died spontaneously while 5 others were sacrificed for necropsy as they became terminally ill. The liver of all these mice was very much enlarged and fatty, the gall bladder was filled with stones and debris, and the bile fluid was green and turbid instead of the normal clear yellow aspect. These symptoms were, however, not the only cause of their bad condition since the liver of mice on the same diet but looking healthy was found to be similarly enlarged and fatty when examined at the end of the test period (results not shown).
Authentic gallstones of different size or a sandy precipitate was present in the gall bladder of all mice on the high fat diet, which is unlike and even contradictory to results reported for this diet(35) . Light microscopic histological examination of the livers revealed no difference in morphology between MAM-/- mice and C57Bl mice on the high fat diet. The cytoplasm of the liver cells contained fat, anisokaryosis was observed, and in some livers evidence for local inflammation with infiltrating lymphocytes and foam cells and local cell lysis (results not shown).
The circulating
blood levels of MUG were determined by rocket immunoelectrophoresis on
all mice subjected to these experiments. Mice on the high
fat/cholesterol diet showed elevated MUG levels with a larger relative
increase in females (60%) than in males (25%) compared to mice fed the
regular chow diet (results not shown). This is contradictory to results
reported in hamsters, in which a high fat and cholesterol diet
suppressed the expression almost 10-fold of -inhibitor 3, the
single chain proteinase inhibitor that is the homologue of MUG in
hamster(36) .
Figure 9: Survival of female C57Bl and MAM-/- mice on a choline/methionine-free diet supplemented with ethionine. Two groups of 6 female C57Bl and MAM-/- mice were given free access to this diet for 8 days. At the end of the experiment, the pancreas of the surviving mice were examined histologically (see text for details).
We have generated mice that are deficient in plasma
-macroglobulin by targeted inactivation of the MAM
gene. The MAM-/- mice produced litters of normal size with
a normal sex ratio among the pups over 3 generations. The oldest
MAM-/- homozygotes are at this moment 15 months old and do
not show any phenotypic abnormality. Apparently, the complete absence
of MAM does not adversely affect their viability, their fertility, or
their health.
The fact that in humans no complete A2M deficiency was
ever described has been interpreted to mean that such a deficiency is
either phenotypically silent or lethal in utero, as stated in
the Introduction(2) . The former hypothesis is supported by our
findings, proving that -macroglobulin is not vital to
nor required for the normal development of mice.
Choosing the mouse as the obvious transgenic model might, however, pose a typical problem in animal modelling. Mouse plasma, like all rodents but unlike mammals, contains two different types of these wide-spectrum proteinase inhibitors: the tetrameric A2M and the monomeric or single-chain murinoglobulins(3, 9, 10, 11, 37) . The former has many structural features in common with human A2M, including concentrations in plasma around 2-4 mg/ml, that are maintained rather constant in most physiologically different conditions. They are thought to function protectively, trapping and eliminating unwanted proteinases(2, 38, 39) and possibly cytokines and growth factors of different type and origin (7) . The precise target proteinases in vivo are unknown and can only be inferred from in vitro tests. Even less well understood is the function in vivo of the single chain inhibitors of the murinoglobulin type, which are typical for rodents. These act rather like complement components C3 and C4, using proteolytic activation and covalent trapping (instead of sterical trapping) to ``tag'' proteinases for disposal by receptor-mediated endocytosis via the A2MR/LRP receptor.
The
murinoglobulins might back up for and functionally take over from MAM
in the MAM-/- mice, which would not be without precedent in
this type of gene knock-out experiment. We cannot exclude at this
moment that this is indeed the case in adult MAM-/- mice.
During embryonal development and early postnatal life, however, it is
clear from previous and present data that embryonal expression of MAM
mRNA begins in the second week of pregnancy(22) , while MUG
mRNA and protein becomes detectable only in the third week postnatally,
just before weaning(22) . This separation in time,
which was confirmed to be maintained in the MAM knock-out mice, is
further proof for the important conclusion that proteinase inhibitors
of the A2M family, either tetrameric MAM or monomeric murinoglobulins,
are not essential for normal embryonal development and early postnatal
life of the mouse.
The implication for the involvement of proteinases during these most important developmental periods and for their control by proteinase inhibitors is still to be determined experimentally. It is not difficult to predict that these proteolytic processes could well be less important than has been inferred from in vitro or indirect experiments. On the other hand, sufficient overlap in the specificity and the availability of proteinase inhibitors might account equally well for these observations, a conclusion that was also evident from mice lacking the plasminogen activator inhibitor type 1(40) . On the other hand, inactivation of the gene coding for A2MR/LRP resulting in the functional deletion of the receptor by which A2M-proteinase complexes (and many other ligands) are cleared, does result in a lethal phenotype(41) . This combination of observations and, in each instance, their unexpected outcome illustrate vividly the importance of animal models and in vivo testing, even of hypotheses, based on irrefutable, in vitro data and logical deduction.
These
conclusions, inherent to the unexpected survival of the MAM-deficient
mice, have forced or allowed us, depending on the viewpoint taken, to
study the physiology of the proteinase inhibitors of the A2M family in
adult mice. Two parallel and complementary approaches have been
followed. On the one hand we have analyzed the normal condition of
pregnancy in female MAM-/- mice, extending from a recent
study aimed at understanding the role of the A2M proteinase inhibitors
in pregnancy. The analysis further included the expression
of necessary partners, operative in this system, i.e. A2MR/LRP
and HBP-44, the mouse equivalent of the 39-kDa receptor associated
protein. The expression of these receptor components was compared and
related to expression of other lipoprotein receptors and their common
ligands as described under ``Results.'' The results reported
here and elsewhere(22)
demonstrated that in
MAM-/- and C57Bl mice, pregnancy per se entails
differential regulation of this intricate network.
A major point in
case and relevant for the current problem, is the fact that around
partum a vast increase in maternal liver mRNA coding for murinoglobulin
is not reflected in the circulating MUG protein levels. This could be due to inefficient translation of this mRNA but
could also point to an increased consumption of murinoglobulin protein
as the result of an increased load of proteinases, originating from the
detaching placenta at birth. In the MAM-/- mice the
murinoglobulin mRNA overshoot effect is much more evident since hepatic
MUG mRNA levels are up-regulated to levels more than 14-fold relative
to day 12 of pregnancy, which is to be compared to only a
3-4-fold increase in the C57Bl strain.
The resulting
circulating MUG plasma levels are, nevertheless, in both strains
comparable. The increased consumption of the single chain proteinase
inhibitor MUG could compensate for the absent tetrameric inhibitor in
the MAM-/- strain and explain the discrepancy between high
hepatic mRNA levels and normal circulating protein levels. This point
will eventually be resolved in mice in which the MUG gene is
inactivated, currently in progress.
Evidence has been accumulated
that the general endocytosis receptor A2MR/LRP and the more specific
lipoprotein receptors, LDLR and VLDLR, are differentially regulated in
normal mice during pregnancy. In addition, in the MAM
knock-out mice specific differences in regulation of expression were
seen for the hepatic A2MR/LRP and LDLR and for VLDLR in placenta and
uterus. Overall, the extent of the changes in mRNA levels, which are
considerable in C57Bl mice, are much more attenuated in the
MAM-/- mice, most typically illustrated by the data
obtained in placenta. Some of the differences might appear less
important, e.g. the absence of up-regulation of placental and
uterine VLDLR in MAM-/- mice, but further experimentation
is clearly needed to establish their precise impact.
Regarding the components, designated as ligands under ``Results,'' for which we have analyzed the evolution of relative mRNA expression in pregnancy, mention has to be made of the tremendous up-regulation in placenta of HBP-44, ApoE, and LPL mRNA. This effect is, however, not different in the MAM-/- mice and falls therefore outside the scope of this discussion. In the liver of MAM-/- mice, regulation of expression of ApoE and LPL mRNA is different from C57Bl mice: ApoE mRNA is decreased and LPL mRNA levels are dramatically increased at the end of pregnancy, although the latter is probably more directly related to the very low levels of LPL mRNA in the liver of MAM-/- mice.
By the second approach we wanted to explore the effect of external factors, i.e. diets with different fat contents (17) or a choline-methionine deficient diet supplemented with ethionine to induce pancreatitis(18) . Furthermore, MAM-/- mice were also challenged with endotoxin (bacterial lipopolysaccharide), bleomycin as an inducer of pulmonary fibrosis(16) , and carbon tetrachloride in an acute liver insult(15) . The effects resulting from these treatments demonstrated important differences in susceptibility of the MAM-/- mice relative to C57Bl mice.
Regarding the diets some caution is needed in interpretation of the results. The variation in susceptibility among inbred strains of mice to such treatments, especially in responses to atherogenic and other diets is well known(27, 42) . The fact remains that the MAM-/- mice are a genetic admixture of the 129 and C57Bl mouse strains, with the latter having the preponderant input. This caveat requires that further experiments will have to be carried out on mice with the silenced MAM gene in the homogenous and well studied C57Bl genetic background.
The results obtained and presented have, nevertheless, delineated those areas and directions in which the MAM-/- mice are likely to be functionally deficient or more susceptible. The endotoxin experiments demonstrated that the MAM-/- mice might be less vulnerable to lethal effects of these bacterial lipopolysaccharides. This should be tied in with an vast amount of literature, impossible to list here, but mostly indirect and on many occasions very contradictory, in which A2M has been implicated in very different aspects of the immune system(6, 7) . Although we are fully aware of the nature of the data and of the possible strain differences as outlined above, it is tempting to speculate that the MAM-/- mice, eventually combined with a murinoglobulin gene knock-out, offers an in vivo system in which some of the claims or hypotheses that have been put forward regarding the functioning of A2M in the immune system can be verified.
Very encouraging as a model, and not totally unexpected are the observations of acute pancreatitis in the MAM-/- mice subjected to the choline/methionine-deficient ethionine-supplemented diet. Careful adjusting of the different ingredients in this diet in combination with back-crossing the targeted MAM gene in the C57Bl background, is expected to provide an informative model for the role of proteinases and of the A2M proteinase inhibitors in the pathology and mortality of acute pancreatitis.
In conclusion, we have to accept the fact that a successful knock-out of the A2M gene in mice has not provided us the direct answer that could have been expected from the direct, and in retrospect naive, extrapolation from the human situation. On the other hand, the MAM-/- mice are expected to become informative for phenomena, such as shock and pancreatitis and others for which indications have been found or are being sought. Finally, to include in this study the contribution and the importance of the murinoglobulins in vivo, i.e. during pregnancy and adolescence as outlined above, we are generating mice in which the murinoglobulin 1 gene (11) is inactivated. Breeding the silenced MAM and MUG genes into the C57Bl background will trace the effect of both deletions in vivo, apart and combined and the latter is to be expected to mirror the human situation more closely.