(Received for publication, August 25, 1994; and in revised form, November 22, 1994)
From the
In order to gain better understanding of the function of hepatic
lipase (HL) in vivo, we have generated mice that lack HL using
gene targeting in embryonic stem cells. No mRNA for HL was detected in
the liver of homozygous mutants, and no HL activity was detected in
their plasma. Total cholesterol levels in plasma of mutant mice were
increased by about 30% compared with wild type animals. Plasma
phospholipids and high density lipoprotein (HDL) cholesterol were also
increased, but plasma levels of triglycerides were not altered.
Analysis of density fractions of plasma lipoproteins revealed that
HDL (d = 1.02-1.04) was increased in
homozygous mutants fed regular chow. In response to a diet containing
high fat and high cholesterol, HDL cholesterol was doubled in the
mutants, but was slightly decreased in the wild type mice. These
results clearly demonstrate the importance of HL in HDL remodeling and
metabolism in vivo. Various earlier studies suggested a role
of HL in metabolism of triglyceride-rich particles, but the mutant mice
appear to have no impairment in clearing them; the mutants clear
exogenously introduced chylomicrons from plasma at a normal rate, and
they tolerate acute fat loading as well as normal animals unless the
loading is extreme. These differences may reflect species differences.
However, it is also possible that the consequence of absence of HL as in our mutants is different from the consequence when nonfunctional HL protein is present as in the human
HL-deficient patients and in rats treated with HL antibodies. We
hypothesize that absence of HL in mutant mice allows other lipases to
bind to the sites in the liver normally occupied by HL and facilitate
the clearance of triglyceride-rich particles in these mice.
Hepatic lipase (HL) ()is synthesized by hepatocytes
and following secretion it binds to the surface of the sinusoidal
endothelium of the liver(1) . Several lines of evidence suggest
that HL plays an important role in high density lipoprotein (HDL)
metabolism. Specifically, HL is thought to be responsible for the
conversion of HDL
to HDL
. HL activity reduces
the concentration of phospholipids and triglycerides in the HDL
subfraction and increases the phospholipid concentration in the
HDL
subfraction(2) , and Kuusi et al.(3) have reported that HL activity is negatively
correlated with the level of HDL
cholesterol, HDL
phospholipid, HDL
protein, and total triglyceride.
Hepatic lipase deficiency in humans results in the accumulation of HDL
particles containing elevated levels of triglyceride and/or
phospholipid(4, 5, 6, 7) . In
experimental animals treated with antibodies blocking the function of
HL, the HDL
subfraction increases in triglyceride and/or
phospholipid content, whereas the HDL
subfraction is
unaffected(1, 8, 9, 10) . The
observation that HDL
phospholipids are substrate for HL in vitro(11, 12) further supports the
suggestion that HL is involved in the hydrolysis of phospholipids in
HDL
.
Studies have shown that hepatic lipase enhances the uptake of cholesterol by hepatocytes in vitro(13, 14) . In vivo, inhibition of HL activity by HL antibody administration significantly decreases the rate of chylomicron remnant cholesterol uptake by the liver(15, 16) . HL has been implicated in the metabolism of VLDLs, intermediate density lipoproteins (IDL), and low density lipoproteins (LDL); administration of HL antiserum can increase the triglyceride, phospholipid and cholesterol content of these lipoproteins(8, 9, 17, 18) . Similarly, in some human individuals with HL deficiency, triglycerides and phospholipids accumulate in LDL and VLDL(5, 19, 20) .
Although HL is clearly involved in the metabolism of circulating lipoproteins, inconsistencies among various investigations prevent a definite conclusion as to its specific physiological role. Some of these inconsistencies may be due to differences in experimental procedures. Others may be due to inadequacies in the experimental model. For example, results from humans naturally deficient in HL may be confounded by the co-existence of other abnormalities that contribute to the observed phenotype(20, 7) . Results of HL inactivation by antibody treatment are variable partly because antibody treatment does not always completely inhibit the enzyme and partly because function of HL as a facilitator of remnant uptake may well be independent of catalytic activity of HL. Specific and complete inactivation of HL in mice by genetic means should supply an useful model for enhancing our understanding of the function of HL in vivo. Accordingly, we have used gene targeting in mouse embryonic stem cells to generate mice that specifically lack HL, and we have described the consequences of HL deficiency on the lipoprotein metabolism in the resulting animals.
The positive-negative selection system of Mansour et al.(22) was used to disrupt the HL gene. HL targeting constructs were made by inserting an XhoI/HindIII fragment containing the bacterial neomycin resistance (neo) gene driven by the herpes simplex thymidine kinase (TK) promoter and polyoma virus enhancer (pMC1neopoly(A), Stratagene, La Jolla, CA) into an NcoI site of exon 4 within the 7.3-kb EcoRI fragment by blunt end ligation (see Fig. 1). Two constructs, HL1 and HL2, were made that differ in the orientation of the neo gene with respect to the HL gene. The neo gene is in the opposite transcriptional orientation in HL1 and in the same orientation in HL2. In addition, the TK gene with a polyoma virus enhancer was inserted both 5` and 3` of the region of homology of the HL gene, that are 0.25 and 7 kb in length, respectively (see Fig. 1). The targeting constructs were linearized at the unique SacII site prior to introduction into ES cells.
Figure 1: Targeted disruption of the mouse hepatic lipase gene. a, the mouse hepatic lipase gene. Exons 3-6 are indicated by black boxes. b, the targeting construct. The neomycin resistant gene (neo) was inserted into an NcoI (N) site of exon 4. The herpes simplex thymidine kinase gene (TK) sequence was attached to both ends of the region of homology, and the plasmid was linearized at the SacII site (S) prior to electroporation. c, modified locus can be detected by Southern blot analysis using the probe shown in a. Restriction enzyme sites used for the analyses are shown: X, XbaI; Bg, BglII; B, BamHI; H, HindIII; E, EcoRI. The 5.5-kb BglII fragment of the endogenous locus altered to 6.6 kb in the targeted locus is shown as an example.
Mice were fed ad libitum either regular chow (containing 4.5% (w/w) fat and 0.022% (w/w) cholesterol (number 5012, Ralston Purina, St. Louis, MO)) or an atherogenic diet (containing 15.8% (w/w) fat, 1.25% (w/w) cholesterol, and 0.5% (w/w) sodium cholate (TD88051, Teklad Premier, Madison, WI)). The animals were handled following the National Institutes of Health guidelines for the care and use of experimental animals.
For lipoprotein analysis, blood was also collected for lipoprotein analysis in the morning from nonfasted mice by heart puncture under anesthesia with a lethal dose of avertin (2,2,2,-tribromoethanol). Female mice of mixed genetic background between C57BL/6J and 129, and of similar age (8-10 months old), were fed regular chow (six each genotype) or fed an atherogenic diet for two weeks (four each genotype). Lipoproteins were fractionated from 1 ml of plasma combined from two to three mice by sequential density ultracentrifugation at 100,000 rpm at 4 °C(30) . Lipoproteins in the density ranges of d < 1.006, d = 1.006-1.02, d = 1.02-1.04, d = 1.04-1.06, d = 1.06-1.08, and d = 1.08-1.10 g/ml were obtained following successive centrifugation for 2.5 h. The d = 1.10-1.21 g/ml fraction was isolated following centrifugation for 4 h. Lipoproteins from each fraction were recovered by tube slicing and dialyzed in 10 mM Tris buffer, pH 7.4, 150 mM NaCl, and 1 mM EDTA. Lipoproteins were separated by agarose gel electrophoresis and detected by Fat Red 7B (Ciba Corning, Palo Alto, CA). The apolipoproteins on agarose gels were transferred to nitrocellulose by blotting and were detected by immunoreaction with specific antibodies against human apoB, human apoA-I, and rat apoE (30) . The apolipoprotein composition of each fraction was determined by SDS-polyacrylamide gel electrophoresis of each fraction followed by Coomassie Brilliant Blue staining of the proteins. Lipid analyses of total plasma and of fractions were carried out as described previously (30) using reagents for cholesterol and triglycerides from Boehringer Mannheim and for phospholipids from Wako Chemical USA Inc. (Richmond, VA).
Figure 2:
Northern blot analysis of liver mRNA.
Poly(A) RNA (10 µg) isolated from liver of wild
type and homozygous mutant mice were electrophoresed, blotted, and
hybridized to a rat cDNA for HL as a probe. Judging from the
rehybridization of the filter to an
actin probe, the amount of
total mRNA in the +/+ lane was approximately twice of that in
the -/- lane.
Plasma HL activity was assayed using a procedure specific for HL. Activities (nanomoles of free fatty acid released per min/ml ± S.D.) in wild type, heterozygous, and homozygous mice were 86.3 ± 19.4, 44.2 ± 6.6, and 1.8 ± 0.8, respectively (n = 6). All pairwise comparisons are significant (p < 0.0005). Thus, our targeted modification of the HL gene successfully eliminates HL activity in the mice.
In order to characterize the alterations in lipoprotein particles, plasma lipoprotein of different densities were separated by sequential ultracentrifugation. Plasma was collected in the morning from nonfasted normal and homozygous mutant mice fed regular chow. Analyses of the density fractions by agarose gel electrophoresis revealed two important differences in the plasma of the homozygous mutants compared with that of wild type mice (Fig. 3).
Figure 3:
Agarose gel electrophoresis of lipoprotein
fractions. Equal volumes of plasma were pooled from six wild type mice
and from six homozygous mutant mice fed regular chow, and plasma
lipoproteins were separated by sequential density ultracentrifugation
using the density ranges shown. Equal volumes of each fraction were
loaded on a 1% agarose gel. Gels were stained with Fat Red 7B (a) or blotted to nitrocellulose and reacted with antibodies
against human apoB (b), rat apoE (c), and mouse
apoA-I (d). Left panels represent lipoproteins from
wild type mice, and right panels represent those from mutant
mice. The gel origins (arrow) and the positions of - and
-migrating particles are indicated on the right.
The first and most striking
difference is an increase in the d = 1.02-1.04
density fraction in the homozygous mutants. This fraction contains
-migrating LDL, slow
-migrating apoB48-containing remnants,
and
-migrating HDL (HDL
). The lipoprotein particles
migrating in this band react strongly with an antibody against apoE (Fig. 3c), but moderately with an antibody against
apoA-I (Fig. 3d). They do not react at all with
anti-apoB (Fig. 3b). We conclude that these HDL
particles are apoE rich and lack apoB. The increase of
immunoreactive apoE-rich HDL
in the d =
1.02-1.04 g/ml fraction is consistent with an increase in the
mass of apoE in this fraction that we observed in SDS-polyacrylamide
gel electrophoresis analysis of the same lipoprotein fractions (Fig. 4; note twice as much sample of each density fraction from
normal animals as that from mutants was loaded on this gel).
Accumulation of these large HDL particles in the plasma of the mutants
is consistent with previous findings that HL plays a role in the
conversion of larger HDL into smaller HDL particles.
Figure 4: SDS-polyacrylamide gel electrophoresis of isolated lipoprotein fractions from chow-fed mice. Aliquot of 30 µl for each density fraction from normal mice and 15 µl from mutants were applied to electrophoresis in denaturing 3-15% polyacrylamide gradient gels. Gels were stained with Coomassie Blue.
The second
difference is in the amount of -migrating particles in density
range 1.10-1.21 that do not stain with Fat Red 7B (Fig. 3a), but react strongly with antibody against
apoE (Fig. 3c) and moderately with antibody against
apoA-I (Fig. 3d). These particles are decreased in the
mutants compared in the wild type mice. This lipid-poor and
-migrating HDL in normal mouse plasma was described previously by
de Silva et al.(30) as a potential precursors for
larger HDL particles similarly to human pre-
-HDL particles,
although its precise origin and function remain to be determined.
The third notable difference is in the size of apoB containing particles. Although the plasma of chow fed normal animals contains small particles carrying apoB of density higher than d = 1.04, these small apoB-containing particles are reduced and not detectable in the plasma of chow fed homozygotes (Fig. 3b). The presence of smaller apoB containing particles in normal mouse plasma and their co-elution with HDL particles in Superose 6 FPLC have been described(30) . We infer from these results that in mice conversion of IDL to LDL is not affected by the lack of HL, although further processing to smaller LDL may be affected. On the other hand, there is no indication that LDL particles accumulate preferentially in the mutant mice, since the VLDL, IDL, and LDL fractions are all equally elevated.
The distribution of
cholesterol, phospholipids, and triglyceride among lipoproteins in the
various density fractions of chow-fed, nonfasted animals used in this
experiment (n = 6) are shown in Fig. 5a. Plasma levels of cholesterol and triglyceride
in the wild type mice were 75 ± 12 and 90 ± 9 mg/dl
± S.D. and in the mutant animals were 114 ± 25 and 110
± 59 mg/dl ± S.D. Phospholipids recovered in the total
lipoprotein fraction from 1 ml of plasma were 1.29 mg for normal mice
and 2.99 mg for mutant mice. Although these data were derived from
nonfasted animals and were using different reagent, the effect of
genotype is the same as in the fasted animals described above (Table 1). The distribution of cholesterol in the homozygous
mutants was similar to that of wild type animals, except that in the
mutants the fraction in the density range 1.02-1.04 contained 4.5
times more cholesterol compared with wild type. Since this fraction
usually includes apoE-containing HDL particles, this result supports
our observation of an increase in HDL band seen in the
agarose gel electrophoresis (Fig. 3). The phospholipid
concentration in all fractions is increased in the mutants compared
with the wild type animals. Triglycerides were mainly in particles of
density range d < 1.006 in both normal and mutant mice.
Figure 5: Lipid distribution among the lipoprotein fractions. Total amount of cholesterol, phospholipids, and triglycerides in each density fraction from animals fed regular chow (a) or high fat diet (b) are shown. -/-, homozygotes; +/+, wild type animals. Values are average of two separate experiments and are adjusted for the total recovery.
In order to assess the effect of HL deficiency on the handling of high fat/high cholesterol diets, we fed animals an atherogenic diet containing 15.8% fat, 1.25% cholesterol, and 0.5% sodium cholate for 2 weeks. Normal mice and homozygous mutants both responded to the diet. Total plasma cholesterol increased by about 60 mg/dl; 159 ± 20 and 196 ± 5 (mg/dl ± S.D.), respectively, for normal mice (n = 4) and for homozygous mutant mice (n = 4). Plasma triglyceride levels in both normal animals and homozygotes are decreased slightly to 45 ± 14 and 67 ± 20 (mg/dl ± S.D.), respectively. Total phospholipid recovery in lipoprotein fractions was 2.26 and 5.00 mg/ml plasma from normal and mutant mice, respectively.
The distribution of lipids in the lipoprotein fractions from wild type animals and homozygous mutants fed the atherogenic diet are shown in Fig. 5b to compare with the distribution in chow-fed mice (Fig. 5a). Wild type animals responded to the high fat diet with an increase to about nine times normal cholesterol in the particles of density range d < 1.02 and with a decrease of cholesterol in particles of density range 1.06-1.21 to 60% of the level in chow-fed animals. The marked increase in the d < 1.02 fraction which contains VLDL and remnants, and the decrease in the d = 1.06-1.21 fraction which contains HDL, in response to the atherogenic diet of wild type mice is consistent with the earlier observations made by us and by others(37, 38) . The changes in cholesterol distribution in the homozygous mutants in response to the atherogenic diet were quite different. Lipoprotein in the VLDL density range increased modestly to about 1.7 times normal. Cholesterol in d = 1.06-1.21 g/ml fractions increased to about twice that in chow-fed mutant animals (the cholesterol-carrying particles in these fractions are HDL). The distribution of phospholipids in mutants was very similar to that of cholesterol. No significant change in the distribution of triglycerides was seen in either normal or mutant animals in response to the dietary change. Overall these results again suggest that HL plays a major role in HDL metabolism.
Figure 6:
Clearance of exogenous mouse chylomicron
by control and HL-deficient mice. Chylomicrons labeled with
[H]cholesterol were harvested from normal mice
and injected into the femoral vein of control and homozygous mutant
mice that had been anesthetized with nembutal. At the indicated times,
50 µl of blood was taken from the tail vein and radioactivity
remaining in plasma was measured (a). Livers were perfused for
30 s with saline through the inferior vena cava and processed for
determination of incorporated radioactivity (b). Each point
corresponds to the measurement of a single
animal.
In order to test how mice lacking HL respond to acute fat loading, 5-month-old male mice were first fed 0.4 ml of olive oil as a bolus. Plasma triglyceride levels increased and peaked at about 2 h after loading in both mutants (n = 5) and control mice (n = 5) as shown in Fig. 7a; the area under the curves did not differ significantly between the mutants and controls. Thus there is no measurable difference in the response to this level of fat loading. When the mice (n = 4) were fed 1.0 ml of olive oil, initial clearance of triglyceride was scarcely impaired but mutants showed a second peak at 5 h that was not seen in the wild type animals (Fig. 7b). The majority of particles at 5 h after fat loading were in the fraction of d < 1.006, and the ratio of apoB100 to apoB48 (1:0.7) in these particles was similar to the ratio in the particles at 2 h. Our data do not allow us to decide the source of the 5-h particles. They could, for example, be chylomicrons made by delayed absorption of lipids by the intestine, or nascent VLDL particles packaged by the liver, or partially processed remnant particles released from the liver or other organs back to the circulation. Nevertheless, whatever the source of the 5-h particles, our results clearly show that there is a difference between mice lacking HL and normal mice in their handling of triglycerides when the system is stressed to the extreme. On the other hand, the mutant mice can tolerate a modest level of loading without a major disturbance in their clearance of triglycerides.
Figure 7: Response to acute fat loading in normal and HL-deficient mice. Four males of each genotype received an intragastric administration of 0.4 ml (a) or 1 ml (b) of olive oil. Mean plasma triglyceride levels and standard errors of means are shown at different time points. At some points, the error bars are too small to show.
Thus, although further detailed studies are necessary, results of our two experiments agree and suggest that the mutants do not have impairment in chylomicron and its remnant clearance.
We have generated mice lacking HL using gene targeting in mouse embryonic stem cells. Mutant mice lacking HL have elevated levels of plasma cholesterol, phospholipids, and HDL. These observations demonstrates in vivo the important role of HL in HDL metabolism and are consistent with the findings by Busch et al.(39) that transgenic mice expressing the human HL gene have reduced total plasma cholesterol and HDL cholesterol.
HDL in
circulation exists in heterogenous forms. In humans, HDL and HDL
are two discrete and stable classes of HDL
particles both rich in apoA-I molecules, whereas HDL
particles are larger than HDL
and are rich in apoE
but poor in apoA-I. In mice, HDL
and HDL
cannot
be separated as discrete populations, but are particles of continuous
sizes of density range d = 1.06-1.21. ApoE-rich
HDL
particles in mice, on the other hand, are quite
discrete in density range of d = 1.02-1.06, with
a clear separation from apoA-I-rich HDL particles. We found that the
mice lacking HL have elevated levels of HDL
particles
compared with wild type litter mates when both are maintained on
regular chow. The accumulation of HDL
in the mutants,
together with our observation of their elevated plasma cholesterol and
phospholipid levels, clearly demonstrate the importance of HL in the
conversion of larger HDL particles to smaller particles. Thuren et
al.(40) have shown that apoE-rich HDL are the preferred
substrate for HL in vitro. The increase in HDL
seen when HL is absent suggests that this is also the case in
vivo.
The heterogeneous HDL particles in circulation are
thought to be generated and interconverted by processes intimately
related to the metabolism of triglyceride-rich lipoproteins. Lipolysis
of triglyceride-rich VLDL and chylomicrons by lipoprotein lipase has
been shown to release excess surface coat, consisting of phospholipids,
cholesterol, and apolipoproteins(41) . These released
components as well as nascent HDL proteins produced by the liver (42) become precursors for mature HDL. They quickly acquire
cholesteryl esters and triglycerides through the function of lecithin
cholesterol acyltransferase and cholesteryl ester transfer protein and
eventually become larger HDL particles. At the same time, HL hydrolyzes
excess triglycerides and phospholipids in the HDL particles which
converts them into smaller size HDL particles. Our results showing that
the conversion of HDL to HDL
is prevented by
absence of HL confirm the suspected obligatory role of HL in this
process. Decrease of slow
-migrating HDL in d =
1.10-1.21 in mutant mice compared with wild type mice is
compatible with these particles being the precursors of larger HDL or
the products of hydrolysis.
We have shown that when mice lacking HL
are challenged with a high fat/high cholesterol diet, the amount of
cholesterol carried by particles in the density range d = 1.06-1.21 (mainly HDL and
HDL
) doubles. On the other hand the amount of cholesterol
carried by particles of d = 1.02-1.04 (mainly
HDL
) decreases in the mutant in response to the dietary
change. ApoE increases in response to atherogenic diets(43) ,
and apoE-rich HDL particles are cleared by LDL receptors and by
receptors that recognize apoE(44) . Possibly the clearance of
HDL
through receptors that recognize apoE is enhanced in
these mice. There is probably another pathway for the clearance of HDL
that preferentially clears smaller apoA-I-containing HDL
particles(45) . The increased level of HDL that we
observed in mice lacking HL could be the consequence of a lack of
surface phospholipid hydrolysis needed to remodel them into the smaller
HDL particles preferred for clearance.
A surprising finding in our study is that mice lacking HL do not appear to have impairment in clearance of triglyceride-rich particles. Thus we found that the plasma TG levels for the mutants are not significantly increased over those in wild type mice and that the mutants clear chylomicrons from plasma at a rate equal to that in wild type mice. In addition, the mutants can tolerate acute fat loading as well as normal mice, unless the loading is extreme. Our observations appear to contradict the observations that treatments of rat liver with anti-HL antibody slow down clearance of chylomicrons from plasma (15, 16) and that humans with S267F plus T383M mutation have HL deficiency and delayed clearance of chylomicrons(46) .
One of the likely explanations of these differences is that they reflect species differences. In mice, about two-thirds of HL function is in plasma, whereas in humans and rats HL is undetectable in plasma (47) . Furthermore, mouse HL has a lower affinity for heparin than does rat or human HL, and adult mouse liver has a significant amount of LPL activity, whereas there is little LPL activity in adult rat liver(47) . These observations suggest that mice under normal conditions may depend more on LPL for facilitating clearance of triglyceride-rich particles by the liver than do humans and rats. In addition, very low levels of cholesteryl ester transfer protein function may influence the metabolism of triglyceride-rich particles in mice differently from that in humans.
The differences may also reflect the problems associated with the various experimental models. For example, antibody moiety interferes with the association of lipoproteins with receptors or with apoE on the vascular wall that are important for their clearance. Human HL deficiency may also be confounded by additional abnormalities.
We should also consider another and very plausible explanation for the differences between our observations and those in rats and in human patients. We suggest that absence of the HL protein has different effects from those associated with the presence of nonfunctional HL protein. Indeed, our mouse data lead us to predict that HL-deficient patients who completely lack HL protein will have near-normal function in terms of triglyceride metabolism. The mutant mice make no detectable HL message. The binding sites for HL in the livers of the mouse mutants are, therefore, most likely available for binding LPL which in turn can facilitate the clearance of chylomicron remnants. In contrast, nonfunctional HL may still bind to the cell surfaces and other proteins even though it has no catalytic activity. The In vitro transfection experiments using of Durstenfeld et al.(48) support this idea; they have shown that the human S267F and T383M mutant proteins are stable but have an extremely reduced enzyme function and that secretion of the mutant protein from the transfected cells is reduced to 12 and 20% of normal, respectively. It will be of interest to determine whether these secreted, but nonfunctional, mutant HL proteins bind to liver endothelium and prevent LPL from facilitating clearance of triglyceride-rich particles.
Some comment is required on our finding that the mice lacking HL can handle triglycerides almost normally, but that they have increased levels of HDL cholesterol and plasma phospholipids. LPL or other lipases are likely to be sufficient to maintain clearance of triglyceride-rich chylomicrons and VLDL in mutant mice lacking HL under ordinary conditions, because HL only poorly hydrolyzes the lipids in these particles which are the preferred substrates for LPL(49) . We suggest that the clearance of triglyceride-rich particles occurs mainly because the lack of HL protein allows LPL to replace the function of HL in the liver. On the other hand, conversion of larger HDL into smaller HDL via phospholipid hydrolysis is unlikely to be efficiently compensated for in the mutants by LPL or other lipase activities in mutants, because LPL has much lower phospholipase activity than HL. This can explain why the mutants have elevated HDL cholesterol and phospholipids.
In conclusion, our mice lacking HL are useful for elucidating the complex pathways involved in the metabolism of HDL and triglyceride-rich particles and for investigating their interactions in vivo. Our suggested possibility that the consequence of total absence of the gene product could differ from the presence of nonfunctional gene product is not limited to the HL gene. This concept should be broadly considered when interpreting the results of various experiments where gene targeting is used to generate mutations in mouse genes.
Mutant mice lacking hepatic lipase have been deposited in and may be obtained from the Induced Mutant Resource, Jackson Laboratory, Bar Harbor, ME.