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Manipulation of milk composition by transgenesis has mainlyfocused on the use of the mammary gland as a bioreactor to pro-duce pharmaceuticals, primarily for economic reasons. However,genetic manipulation of milk for nutritional, physiochemical, ortechnological purposes is another important aim of this research.Thus, several companies are now working on the production ofnutraceuticals. One such product, first suggested in 1986, is a low-lactose milk1. Milk is rich in lactose, proteins, and lipids, and alsocontains hormones, vitamins, growth factors and minerals; it is theonly dietary source for neonates, and continues to be an importantingredient in adult food2. Lactose is the major sugar present in milkand is an important osmotic regulator of milk secretion3. It is syn-thesized in the mammary gland by the lactose synthetase complexcomposed of uridine diphosphate–galactosyltransferase (EC2.4.1.22) and of the mammary-specific a-lactalbumin (aLc)1.More than 70% of the adult population suffers from intestinal dis-orders after milk ingestion, as a consequence of lactose maldiges-tion that results from the normal drop at weaning of the intestinallactose-hydrolyzing enzyme, lactase–phlorizin hydrolase (LPH; EC3.2.1.62-108)4,5. Lactose maldigestion is also frequently associatedwith intestinal pathologies and with rare cases of congenital alacta-sia6,7. The undigested lactose in the small intestine accelerates theintestinal transit; reduces water absorption; and is metabolized bycolonic bacteria into volatile short-chain fatty acids, hydrogen, andcarbon dioxide. This provokes the typical clinical symptoms ofhypolactasia: abdominal pain, nausea, and diarrhea, which maysubsequently provoke severe dehydration8,9.

To circumvent the hypolactasia symptoms occurring after milkingestion, several expensive and time-consuming methods havebeen developed to get low-lactose milk in vitro (i.e., ultracentrifu-gation or treatment with soluble or immobilized fungal b-galac-tosidases)5,9. We have investigated a way to produce low-lactosemilk in vivo instead of in vitro, by expressing a lactose-hydrolyzing

enzyme in the mammary gland. To this end, we constructed ahybrid gene in which the rat cDNA encoding the precursor ofintestinal LPH10 was placed downstream of the murine mammary-specific a-lactalbumin promoter11. In the intestine, the 220 kDaLPH precursor is processed into the 130 kDa mature enzyme12,13.We and others, however, have reported that in nonintestinal cellsthe precursor is not proteolytically processed into the intestinal-type mature form. In addition, the LPH precursor is transport-competent and fully active; it shows a similar specific activity andKM toward lactose as the mature intestinal enzyme14–16. We demon-strate that transgenic mice expressing the LPH precursor in themammary gland during lactation produce milk with a significantreduction in lactose content.

ResultsEctopic expression of intestinal lactase in the mammary gland oftransgenic mice. The cDNA encoding the full-length transport-competent and enzymatically active rat intestinal LPH precursorwas placed downstream of the mammary-specific aLc promoter toobtain the aLc/LPHp hybrid gene (Fig. 1A). Following microinjec-tion into mouse eggs, four transgenic founders carrying theaLc/LPHp gene were obtained. The number of transgenic mice outof microinjected eggs and newborn G0 animals (4/42) was not dif-ferent from mean values obtained with other constructs in ourhands. Transgene transmission in the four lines followed theexpected Mendelian ratios. Furthermore, the number of newbornsand their viability were not different between transgenic and non-transgenic animals, suggesting no toxicity of the transgene.

The expression of the transgene in the mammary gland wasinvestigated in lactating females from the four lines, heterozygous atthe transgenic locus, during the G1 to G3 generations at the first lac-tation. Figure 1B shows that transcripts similar in size to the typicalintestinal LPH mRNA were expressed in the mammary gland dur-

Production of low-lactose milk by ectopicexpression of intestinal lactase in the

mouse mammary glandBernard Jost, Jean-Luc Vilotte1, Isabelle Duluc, Jean-Luc Rodeau2, and Jean-Noël Freund*

Institut National de la Santé et de la Recherche Médicale, Unité 381, Strasbourg, France. 1Institut National de la Recherche Agronomique, CIJ, Jouy-en-Josas, France.2Centre National de la Recherche Scientifique, UPR 9009, Strasbourg, France. *Corresponding author (e-mail: [email protected]).

Received 22 July 1998; accepted 4 December 1998

We have investigated, in mice, an in vivo method for producing low-lactose milk, based on the creationof transgenic animals carrying a hybrid gene in which the intestinal lactase–phlorizin hydrolase cDNAwas placed under the control of the mammary-specific a-lactalbumin promoter. Transgenic femalesexpressed lactase protein and activity during lactation at the apical side of mammary alveolar cells.Active lactase was also secreted into milk, anchored in the outer membrane of fat globules. Lactase syn-thesis in the mammary gland caused a significant decrease in milk lactose (50–85%) without obviouschanges in fat and protein concentrations. Sucklings nourished with low-lactose milk developed normal-ly. Hence, these data validate the use of transgenic animals expressing lactase in the mammary gland toproduce low-lactose milk in vivo, and they demonstrate that the secretion of an intestinal digestiveenzyme into milk can selectively modify its composition.

Keywords: lactose maldigestion, transgenic mice, fat globules

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ing lactation in two lines, aLc/LPHp-1 and aLc/LPHp-2, whereasno expression was detected in the two other lines, aLc/LPHp-3 andaLc/LPHp-4, or in control nontransgenic littermates. This suggeststhat the expression of the transgene was dependent on the integra-tion site in the genome. Accordingly, we found no correlationbetween the level of mRNA and the copy number of the transgene(Table 1). On western blots using antirat-LPH antibody, lactaseantigen was present in the Triton-X114-solubilized membrane frac-tion of the mammary glands of lactating aLc/LPHp-1 andaLc/LPHp-2 females but not in the aLc/LPHp-3, aLc/LPHp-4 andcontrol mice (Fig. 1C). The molecular form corresponded to the220 kDa LPH precursor instead of the 130 kDa mature LPH presentin the intestine. Immunodetection of lactase antigen in mammarycryosections of lactating aLc/LPHp-1 females revealed a sharpstaining at the apical side of the alveolar cells as well as a faint intra-cellular signal (Fig. 2A). This staining was very similar to the cellularstaining of aLc in the mammary alveoli (Fig. 2C). Incubation ofmammary cryosections with a specific chromogenic substrate oflactase (X-Fuc) revealed enzyme activity in the alveoli, in close cor-relation with the immunostaining of lactase protein (Fig. 2B com-pared with Fig. 2A). Intracellular rounded vesicular-like structureswere also labeled by X-Fuc (Fig. 2D). Taken together, these datademonstrate that enzymatically active lactase is expressed in themammary gland of lactating transgenic mice from the aLc/LPHphybrid gene. In contrast to findings in the mammary gland, thetransgene was not expressed in the liver or the stomach, suggesting acorrect tissue-specific pattern (data not shown).

Decrease in milk lactose in mice expressing lactase in the mam-mary gland. Milk collected from the aLc/LPHp-1 and aLc/LPHp-

2 transgenic females at day 10 of lactation contained lactase proteinand lactase activity in contrast to the milk produced by theaLc/LPHp-3 and aLc/LPHp-4 mice (Table 1). After fractionationinto cream and skim milk, western blot analysis demonstrated thepresence of the 220 kDa LPH precursor in the cream produced bythe aLc/LPHp-1 and aLc/LPHp-2 females, but not by theaLc/LPHp-3, aLc/LPHp-4, or control mice (Fig. 3A); no lactaseantigen was detected in the skim milk (Fig. 3A). Direct enzymaticmeasurements confirmed the presence of lactase activity in thecream of the aLc/LPHp-1 and aLc/LPHp-2 females (Fig. 3B). Tofurther address the presence of lactase in the cream fraction, mam-mary cryosections of aLc/LPHp-1 females, or cream dropletstransferred to glass slides, were analyzed at high magnification afterincubation with X-Fuc or anti-LPH antibody. X-Fuc staining oflactase activity revealed rounded fat globules in alveolar ducts, con-

Figure 1. Ectopic expression of lactase in the mammary glandof transgenic aLc/LPHp mice. (A) Restriction map of theaLc/LPHp transgene. The black box corresponds to the a-lactalbumin promoter, the white box to the LPH cDNA, and thegray box to the 3´ region of the b-globin gene. (B) RNAextracted from the mammary glands of lactating mice of theaLc/LPHp-1, aLc/LPHp-2, aLc/LPHp-3, and aLc/LPHp-4 linesat day 15 of lactation (respectively, lanes 1–4; 20 µg/lane),from the mammary glands of a control nontransgeniclittermate (lane 5), and small-intestinal RNA extracted from15-day-old suckling mice (lane 6; 2 µg) were hybridized to therat LPH cDNA. (C) Membrane proteins prepared from themammary glands of aLc/LPHp-1 to aLc/LPHp-4 females atday 15 of lactation (lanes 1–4; 70 µg/lane), of a controlnontransgenic littermate (lane 5) and from the intestinal brushborder membrane of suckling rats (lane 6; 10 µg/lane) wereseparated by 7% SDS-PAGE and revealed using anti-LPHantiserum. The 220 kDa form detected in the mammary glandcorresponds to the LPH precursor, and the 130 kDa form isthe mature enzyme present in the small intestine.

A

B

Figure 2. Histologic detection of lactase and a-lactalbumin in the mammarygland of aLc/LPHp-1 mice. Lactase protein (A) and activity (B) were detectedby incubation of mammary cryosections at day 15 of lactation with anti-LPHmonoclonal antibody or with the chromogenic substrate of lactase: X-Fuc. (C)a-Lactalbumin immunostaining of the mammary alveoli. (D) High-magnification X-Fuc staining of the mammary gland showing intracellularvesicular-like structure (open arrows). ac, alveolar cells; fg, fat globule; L,lumen. (E) X-Fuc staining of fat globules within the alveolar ducts. The slidewas focused on the surface of fat globules instead of the alveolar cellscorresponding to the surrounding diffuse signal. (F) Immunostaining of milk fatglobules with anti-LPH monoclonal; the slide illustrates an upper view (bottom,right) and a transverse view (top, left) of fat globules. Bars represent 100 µm in(A–C), 10 µm in (D), and 25 µm in (E and F).

A B C

D E F

Table 1. Characteristics of the mouse line.

Transgenic Transgene copy Lactase Lactaseline number protein activity

(µg/ml milk) (mU/ml milk)

aLc/LPH-1 1 14 312aLc/LPH-2 3 8 184aLc/LPH-3 7 n.d. n.d.aLc/LPH-4 17 n.d. n.d.

The copy number of the transgene in the four transgenic mouse lines wasdetermined on Southern blot. The level of lactase protein present in milk at day10 of lactation was established by comparison with calibrated amounts ofintestinal lactase. n.d.: not detectable.

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sistent with the immunostaining of lactase protein in the outermembrane of these globules (Fig. 2E and F).

Milk carbohydrates were analyzed by thin-layer chromatogra-phy (TLC). Milk produced by the two lines that did not display lac-tase expression in the mammary gland, aLc/LPHp-3 andaLc/LPHp-4, contained a similar amount of lactose to controlnontransgenic littermates, whereas milk produced by theaLc/LPHp-1 and aLc/LPHp-2 females showed a significant reduc-tion in lactose content (Fig. 3C and Table 2). To provide direct evi-dence that the decay in lactose was related to the presence of lactasein milk, we tested milk for lactase activity in vitro. For this purpose,milk was incubated for 4 h at 37°C and then analyzed by TLC. Thisincubation provoked the almost complete disappearance of theresidual amount of lactose present in the milk of aLc/LPHp-1 andaLc/LPHp-2 females, whereas the same treatment applied to themilk of control or aLc/LPHp-3 and aLc/LPHp-4 females was inef-fective (data not shown). In addition, exogenous lactose was alsohydrolyzed in the presence of milk collected from aLc/LPHp-1females, but not by milk from control mice (Fig. 3D). These datademonstrate that the synthesis of intestinal lactase in the mamma-ry gland and its secretion into milk result in the production of low-lactose milk in vivo.

Properties of the low-lactose milk. We compared the propertiesof the low-lactose milk produced by lactating females of theaLc/LPHp-1 line with normal milk produced by control nontrans-genic littermates. For these experiments, the sucklings were separat-ed from the dams 0 h, 4 h, or 8 h before milk was collected, to inves-tigate the effect of milk storage in the mammary gland on the lactosecontent. The results are given in Table 2 and in Fig. 4. Comparedwith control mice, the lactose concentration measured inaLc/LPHp-1 females was reduced by 50% when milk was collectedimmediately after the sucklings were removed from their mothers,and it was further reduced by 85% after milk storage in the mamma-


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ry gland for 8 h. It is noteworthy that there was a simultaneousincrease in glucose and galactose, although not in proportion withthe decay of lactose, suggesting that monosaccharides are reabsorbedby the alveolar cells. Unlike the carbohydrates, the percent of solid,the proportion of fat, and the protein content were unchanged in themilk of transgenic animals compared with controls. The general pro-tein pattern visualized by SDS-PAGE, as well as the specific level ofaLc were similar in low-lactose and normal milk (Fig. 4A and B).Osmolarity slightly increased in low-lactose milk, and the concentra-tion of osmotic ions (Na+, K+, and Cl–) was not significantly modi-fied in comparison with normal milk (Table 2).

The nutritional potential of the low-lactose milk was investigat-ed by testing its ability to sustain normal growth in suckling mice.

Figure 3. Lactase expression in the milk of aLc/LPHp females. (A)Western blot analysis of lactase in the cream (upper panel) and in theskim milk (lower panel) of transgenic females of the aLc/LPHp-1 toaLc/LPHp-4 lines at day 10 of lactation and of control nontransgeniclittermates (respectively, lanes 1–5; 50 µg/lane). (B) Lactase activitymeasured in the cream of the aLc/LPHp-1 to aLc/LPHp-4 mice and incontrol mice (respectively, lanes 1–5). (C) Thin-layer chromatographyanalysis of carbohydrates in the milk of aLc/LPHp-1 to aLc/LPHp-4females (lanes 1–4) and of a control nontransgenic littermate (lane 5);lane 6 corresponds to a mixture of glucose (Gluc), galactose (Gal),and lactose (Lact). (D) Exogenous lactose (50 mM, lane 1) was mixedwith 20 µL milk from a female of the aLc/LPHp-1 line (lane 2) or withmilk from a control mouse (lane 3), and incubated at 37°C for 4 hbefore thin-layer chromatography.

Table 2. Milk properties.

Control (4 h) aLc/LPH-1

0 h 4 h 8 h

Lactose (mg/ml) 22.5±1.1 12.1±1.1* 7.3±0.6* 3.4±0.4*Glucose (mg/ml) 0.05±0.05 1.2±0.4* 1.4±0.5* 0.5±0.4*Galactose (mg/ml) 0 2.2±0.6* 1.8± 0.2* 1.3±0.5*Solids 58.8±3.2 54.6±4.1 55.5±5.2 60.4±7.1Proteins in skim 99.0±6.1 96.3±5.2 103.6±13.2 107.3±6.6

milk (mg/ml)Fat (mg/ml) 175.3±24.2 165.0±14 185.7±17.2 159±22Na+ (mM) 76.9±6.0 n.d. 85.2±10.4 n.d.K+ (mM) 32.5±2.6 n.d. 30.3±5.8 n.d.Cl– (mM) 41.6±7.2 n.d. 46.8±4.2 n.d.Osmolarity (mOsm) 322.3±26.6 n.d. 375.5±14 n.d.

Sucklings were separated from their transgenic or nontransgenic control moth-ers 0 h, 4 h, or 8 h before milking. Analysis of milk from the nontransgenic con-trols did not reveal any obvious difference at the three time points, so that onlythe data at 4 h are given. Results are mean values from three to six measure-ments. *indicates a significant difference between transgenic and control miceby Student t-test analysis (p<0.05). n.d.: not determined.

Figure 4. (A) Milk proteins (70 µg per lane) from aLc/LPHp-1 toaLc/LPHp-4 females at day 10 of lactation (lanes 1–4) and from acontrol nontransgenic littermate (lane 5) were separated by 12%SDS-PAGE and visualized by Coomasie blue staining. (B)Immunodetection of a-lactalbumin in the same milk samples as in(A). (C) Growth curve of neonates nourished by aLc/LPHp-1 (blacksquares) or by control mice (open circles). The body weight is givenin grams. Data are mean values from nine animals; standarddeviation was omitted for clarity.


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Young animals continuously nourished by lactating females of theaLc/LPHp-1 line exhibited a similar growth curve to sucklingsnourished by control nontransgenic females (Fig. 4C).

DiscussionSince milk components are major ingredients of food, the very fre-quent maldigestion of lactose in humans represents an importantnutritional problem. Several expensive and time-consumingpostharvest methods have been developed to remove lactose frommilk in vitro, including the physical separation of lactose from othermilk components and the enzymatic treatment of milk with solubleor immobilized b-galactosidases5,9. Here, we validate the use of anoriginal preharvest in vivo method of production of low-lactosemilk using transgenic animals that ectopically express intestinal lac-tase in the mammary gland. Using mice, we demonstrate that thisapproach results in an up to 85% decrease in milk lactose. Hence, ifapplied to dairy cattle, this could allow the easy and efficient pro-duction of low-lactose milk to fulfil the requirements of lactosemaldigesters, because a 50–70% decrease in lactose is sufficient toprevent intestinal disorders after milk ingestion17,18.

The production of a b-glycosidase in the mammary gland tohydrolyze lactose in situ was proposed several years ago1, but nosuccess has been reported until now. Our choice of the aLc/LPHphybrid gene presented several advantages over previous approaches.First, the use of the aLc promoter instead of another mammary-specific promoter is thought to allow LPH synthesis in those cellsthat produce lactose by the lactose synthetase complex19; this prop-erty is important if the same approach is used in the case of dairycattle, as these animals exhibit a heterogenous cellular expression ofmilk proteins20. Second, LPH, which is synthesized as a fully activeand transport-competent precursor14–16, is the most efficientenzyme to hydrolyze lactose, as compared with other common b-glycosidases such as the product of the Escherichia coli lacZ gene.Third, LPH is membrane bound and is likely to be sorted via thesame route as lactose synthetase, so that lactose hydrolysis mayalready start intracellularly within the secretory vesicles in whichlactose is concentrated21. Indeed, the intracellular staining of round-ed structures by X-Fuc suggests that lactase is already active duringits vesicular transport toward the apical side of the alveolar cells.

The low-lactose milk produced by aLc/LPHp mice shows onlyslight modification in osmolarity and viscosity, in contrast to thehighly viscous lactose-free milk produced by aLc-deficient ani-mals22. This difference conceivably results from the distinct methodused to lower the lactose level in both types of mice. In aLc-defi-cient animals, lactose synthesis is abolished and free monosaccha-rides (glucose and galactose) are not secreted into the milk. Thedefinitive absence of lactose causes an important decrease in fluidswithin secretory vesicles due to the lack of lactose-dependentosmotic pressure and to the absence of compensatory effect bymonosaccharides22. On the contrary, in lactase-producing mice,lactose is synthesized and is expected to drive the flow of fluidstoward the secretory vesicles, even if lactose hydrolysis alreadybegins partially within the vesicles. Moreover, one would anticipatethat the low-lactose milk of aLc/LPH mice is diluted comparedwith normal milk, as after lactose synthesis by the lactose syn-thetase complex, the subsequent hydrolysis of this disaccharide bythe mammary lactase produces glucose and galactose, which exerta higher osmotic pressure than lactose. In fact, this is not the case,because the final concentrations in glucose and galactose in thelow-lactose milk are not in proportion to the decrease in lactosecontent. This indicates that monosaccharides are partially reab-sorbed within the alveolar cells after lactose hydrolysis. It should benoted that glucose is more efficiently reabsorbed than galactose,suggesting the involvement of an active process.

Only a few examples have been reported in which a membrane

protein is produced in milk of transgenic animals: The cystic fibro-sis transductance regulator is associated with fat globules, but itsactivity was not investigated23. Active furin was shown to be associ-ated with fat globule membranes and was also released into the sol-uble fraction of milk24. Lactase is targeted to the apical side ofintestinal epithelial cells25 and to the plasma membrane in heterol-ogous Cos cells transfected with the full-length LPH cDNA14–16; themechanism of sorting involves the N-terminal signal peptide andthe C-terminal part of the molecule including the transmembraneanchor16,26. The present data indicate that in the mammary glandtoo, the LPH precursor is sorted in the membranous compartmentand is targeted to the apical cell surface. Subsequently, lactase isassociated with the membrane of fat globules, most likely duringthe process of apocrine secretion of fat. In this process, the outsideof the plasma membrane becomes the exposed surface on the fatglobule27, so that lactase is protruding from the globule into theaqueous phase of milk. Although without effect on lactase activity,the LPH precursor is not proteolyticaly processed in the mammarygland, as in transfected COS cells14–16 but unlike intestinalcells12,13,25. This is probably related to the absence in mammary cellsof an as-yet uncharacterized, intestinal-specific processing enzyme.Processing deficiency in the mammary gland of transgenic micehas already been reported for human protein C, but in this case,unlike lactase, this leads to the production of an inactive form ofthe molecule28.

In conclusion, we have demonstrated that milk compositioncan be modified by synthesis of an intestinal digestive enzyme inthe mammary gland and secretion at the surface of fat globules.Thus, we validate the use of transgenic animals producing lactasein the mammary gland to lower in vivo the lactose level in milk.aLc/LPHp mice represent a powerful model to investigate thephysiologic role of lactose as regulator of the osmotic pressure inmilk. Mammary cell lines derived from these mice, as well as fromaLc-deficient mice, may help to understand the mechanism ofsecretion of milk components and the metabolism of mono- anddisaccharides in the mammary gland. Finally, expression vectorscontaining the sequences encoding the N-terminal and C-terminalparts of the LPH precursor may represent efficient tools to targetrecombinant proteins to the membrane of milk fat globules.

Experimental protocolConstruction of the aLc/LPHp hybrid gene and generation of transgenic mice.The construction of the aLc/LPHp hybrid gene was as follows: The 1.2-kbPvuII-XhoI fragment of pSCT-Gal4-X556 including the second intron andpolyadenylation site of the rabbit b-globin gene29, and the 0.6-kb BamHI-SmaIfragment of prom-aLacMGF containing a mutated form of the murine aLCpromoter11,30, were respectively inserted in the PvuII/SalI sites and in theBamHI/EcoRV sites of ppolyIII-I (ref. 31). The plasmid was cut with XbaI/PvuIIto insert the 3.3-kb SpeI-EcoRV fragment of pS34T containing the 3´ region ofthe rat LPH cDNA, and then with SacI/SalI to insert the 3.7-kb SacI-SalI frag-ment of pS1234T corresponding to the 5´ cDNA region16. In the resulting plas-mid, named pPLc*S1234Tbg, the aLc/LPHp hybrid gene containing the murinea-lactalbumin promoter, the full-length rat LPH cDNA, and the 3´ part of therabbit b-globin gene, is included within a 7.6-kb XhoI fragment. Transgenicmice carrying the aLc/LPHp hybrid gene were generated by microinjection ofthe 7.6-kb XhoI fragment of pPLc*S1234Tbg into the pronuclei of C57BL/6 xCBA F2 hybrid eggs; they were bred according to established procedures32. Tailgenomic DNA of founder mice was analyzed by Southern blot using the 6.2-kbrat LPH cDNA probe10, or by PCR using the primers ALP-5 (dAAGGATC-CAAGTAGTAGTTGAGT) that hybridized to the aLc promoter11 and LPH-S3(dTTGCATGCATAAGCTTAAATTTCTGTCGGATTCCCAGTC) that hybrid-ized to the 5´-end of the LPH cDNA16. Throughout this study, control animalscorresponded to nontransgenic littermates. Transgenic and nontransgenicfemales were analyzed at the first lactation to minimize potential physiologicvariation of the characters measured.

Milk carbohydrates analysis. Unless otherwise stated, the suckling animalswere separated from their lactating mothers 3 h before milk was collected.Milking was performed after 300 mU oxytocin injection from females at day


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10 of lactation33. Milk was diluted 10-fold in water and analyzed by TLC onTLC aluminium sheets silica gel 60 (Merck, Nogent, France) using an anilinediphenylamine spray to detect the carbohydrates34. In some experiments,exogenous lactose (50 mM in 0.1 M maleate buffer, pH 5.8, containing 1 mMp-chloromercuribenzoate) was added, and/or milk was incubated 4 h at 37°Cprior to TLC.

Protein preparation and western blot analysis. Milk collected at day 10 oflactation was fractionated into cream and skim milk by centrifugation at 20 Gfor 1 h. Membrane and soluble proteins from the mammary glands offemales at day 15 of lactation were separated by Triton X-114 phase-separa-tion35. Intestinal brush border membrane proteins were prepared from 15-day-old suckling rats36. Proteins were analyzed by conventional 7% or 12%SDS-PAGE and blotted on nitrocellulose filters. Lactase detection was per-formed using rabbit antirat-lactase antiserum (dilution 1:1500)37 andantirabbit IgG coupled to horseradish peroxidase (Amersham, Les Ulis,France; dilution 1:5000). aLc detection used antibovine aLc antiserum thatcross-reacts with mouse aLc (dilution 1:100)22. The level of lactase proteinpresent in the crude milk of transgenic animals was estimated by western blotanalysis compaired with calibrated amounts of intestinal brush border mem-brane lactase.

Histochemical detection of lactase and a-lactalbumin. Histoenzymaticdetection of lactase was carried out by overnight incubation of mammarycryosections in 0.1 M maleate buffer pH 6.5 containing 1 mM p-chloromer-curibenzoate, 3.4 mM potassium ferrocyanide, 3.4 mM potassium ferri-cyanide and 1 mM 5-bromo-4-chloro-3-indolyl-b-D-fucopyranoside (X-Fuc; Sigma, St. Quentin Fallavier, France), a specific chromogenic substrateof lactase16. Immunostaining of lactase antigen on mammary cryosectionsused the rabbit antirat-LPH antiserum (dilution 1:100)37 and fluoresceinisothyocyante-coupled antirabbit antibody (dilution 1:200; Institut Pasteur,Paris), as described previously38,39. aLc was detected with antibovine aLcantiserum (dilution 1:50)22.

Milk properties. Lactose, glucose, and galactose were measured on a 1:50dilution of milk using the Lactose/D-Glucose and the Lactose/D-Galactose kits(Boehringer Mannheim, Mannheim, Germany). Solids were determined bymilk lyophilization. Protein content was measured on a 1:50 dilution of skimmilk using the Bio-Rad (Ivry sur Seine, France) Protein Kit. Fat proportion inmilk was determined by creamatocrit40. Osmolarity was measured on 10 µlsamples using a Wescor (Logan, UT) 5500 vapor pressure osmometer. Na+ andK+ concentrations were measured on 5 µl skim-milk samples using microelec-trodes made with sodium ionophore I-cocktail A (Fluka, Buchs, Switzerland)and potassium ion exchanger (Corning, Medfield, MA)41. Electrode signalswere measured with respect to an indifferent 3 M KCl reference electrode. Cl–

concentration was determined using the Ag/AgCl earthing wire as a Cl– selec-tive electrode. Lactase specific activity (mU/mg protein) was determined oncream and skim milk, as described for intestinal samples42.

RNA analysis. RNA was extracted from the mammary glands of lactatingfemales and from the small intestine of suckling mice using TRIzolTM reagent(Gibco BRL, Cergy Pontoise, France). It was analyzed by Northern blot usingthe rat LPH cDNA probe, as previously described10,38,39.

AcknowledgmentsWe are most grateful to M.-G. Stinnakre and to S. Soulier (CIJ, INRA) for theirtechnical help in the generation and identification of the transgenic founder mice.

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2. National Dairy Council. 1985. Nutritional implications of lactose and lactaseactivity. Dairy Counc. Dig. 56:25–30.

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