Expression of caprine â-lactoglobulin in the milk oftransgenic mice

ELENA IBANEZ 1�, JOSEP M. FOLCH2, FRANCESCA VIDAL1 ,AGUSTINA COLL2 , JOSEP SANTALO 1 , JOSEP EGOZCUE 1 andARMAND SANCHEZ2

1Unitat de Biologia Cel·lular, Departament de Biologia Cel·lular i Fisiologia, Facultat de Ciencies,2Unitat de Genetica i Millora, Departament de Patologia i de Produccio Animals, Facultat de Veterinaria,Universitat Autonoma de Barcelona, 08193 Bellaterra, Spain (Fax: 34-3-581 22 95)

Received 20 March 1996; revised 15 July 1996; accepted 2 August 1996

A 14.5 kb-long transgene containing the complete caprine â-lactoglobulin gene transcription unit as well as 6.1 kband 3.7 kb of the 59- and 39-flanking regions, respectively, was microinjected into pronuclear stage mouse embryos.Four lines of transgenic mice were obtained, three of them expressing the transgene in their mammary glandsduring lactation. Western blot analysis of caprine â-lactoglobulin in the milk of hemizygous transgenic animalsdemonstrated the presence of the exogenous protein at concentrations up to 0.5 mg mlÿ1 of mouse milk.

Keywords: â-lactoglobulin; goat; transgenic mice; milk

Introduction

Transgenic animals offer numerous biotechnologicalapplications and expression of foreign proteins in themilk of transgenic animals is considered as a promisingalternative method to expensive cell culture systems forthe production of recombinant proteins (Clark et al., 1987;Hennighausen, 1992). A number of milk protein genesfrom a variety of species have been expressed in themammary gland of transgenic mice in order to study milkprotein gene regulation and to test the ability of theirpromoter elements to direct the synthesis of foreignproteins to the mammary gland (reviewed by Groenen andvan der Poel, 1994; Maga and Murray, 1995). Unfortu-nately, in most cases a position-dependent and copynumber-independent expression of the transgenes in themammary gland of the transgenic animal was obtained.However, ovine â-lactoglobulin transgenes (Whitelaw etal., 1992) and rat WAP transgenes (Dale et al., 1992)represent an exception, as they are expressed at high levelsand in a position-independent manner in transgenic mice.

â-lactoglobulin (BLG) is the major whey protein in themilk of ruminants and it is present in the milk of almostall mammalian species, except for rodents and primates

(Pervaiz and Brew, 1985). The entire caprine BLG genehas previously been cloned and sequenced (Folch et al.,1994). In the present study, we have produced transgenicmice for the caprine BLG gene to determine whetherhigh level position-independent expression (as found withthe ovine gene) could be achieved in the mammary glandof transgenic mice.

Materials and methods

Construction of the transgene

The BLG transgene was prepared from a caprine genomicDNA clone containing the BLG gene (Folch et al., 1994).The 14.5 kb Sal I insert, containing 6.1 kb of the 59-flanking region, the entire transcription unit and 3.7 kb ofthe 39-flanking region (Fig. 1), was separated on a 0.4%agarose gel and purified using the GeneClean II kit (Bio101 Inc). The resulting DNA was diluted in 10 mM Tris/HCl pH 7.6, 0.25 mM EDTA to a final concentration of 2and 5 ìg mlÿ1.

Generation and screening of transgenic mice

One-cell pronuclear stage mouse embryos were obtainedfrom mature B6CBA F1 female mice superovulated andmated with B6CBA F1 stud males. The DNA solution

Transgenic Research 6, 69–74 (1997)

0962–8819 # 1997 Chapman & Hall

�To whom correspondence should be addressed.

(1–2 pl) was microinjected into the male pronucleus usingstandard techniques (Hogan et al., 1986) and injectedembryos were cultured overnight. Cleaved embryos weretransferred into the oviducts of pseudopregnant B6CBA F1

recipient mice.Transgenic founder mice were identified by PCR

analysis of genomic DNA prepared from tail biopsysamples taken at weaning. Primers BLG1 (59-CTGTCGA-CATGAAGTGCCTCCTGC-39) and BLG2 (59-TGGGAT-CCAGCTCCTCCACGTAC-39) were used to amplify a872 bp fragment spanning the first and second exons ofthe caprine BLG gene.

Transgenic offspring were produced by backcrossingthe founders to B6CBA F1 mice and transgenic lineswere established.

Southern analysis of transgenic mice

PCR-positive founder animals were confirmed by South-ern blot analysis and the presence and integrity of thetransgene were determined. Hemizygous offspring fromthe G1 generation of each transgenic line were alsoanalysed by Southern blotting to determine transgene copynumber and to detect possible somatic mosaicism in thefounder animals.

Ten ìg of genomic tail DNA were digested withHin dIII, fractionated on a 0.8% agarose gel, transferredto a nylon membrane and hybridized at 42 8C in 50%formamide with a digoxigenin labelled caprine BLGcDNA probe (25 ng mlÿ1). Washings were done at 50 8Cin 0.5 � SSC, 0.1% SDS.

Transgene copy number was determined by densito-metric scanning with respect to restricted goat genomicDNA and known amounts of restricted BLG clone.

Collection and fractionation of mouse milk

Milk was collected from nursing transgenic mice 15 daysafter parturition. Transgenic female mice were separatedfrom their litters and 3 hours later anaesthetized with0.5 ml avertin and injected intraperitoneally with 0.3 IUoxytocin (Syntocinon, Sandoz Pharma) to stimulate milklet-down. Milk was collected 10 min later by gentlemassage of the mammary gland and taken up in capillary

tubes (Simons et al., 1987). Milk samples were dispensedinto aliquots and stored at ÿ20 8C until use.

To prepare whey, milk samples were diluted 1/10 in10 mM Tris-HCl pH 8, 10 mM CaCl2 and centrifuged at14 000 g for 15 min.

Quantitative analysis of caprine BLG in mouse milk

For the detection of caprine BLG in transgenic mousemilk, whey samples were prepared and electrophoresed on14% SDS/PAGE gels as described in Grosclaude et al.(1987). Proteins were transferred onto nitrocellulosemembranes and caprine BLG was detected using I125-labelled protein A after incubation with an anti-BLGantiserum (Nordic Immunological Laboratories).

Concentration of goat BLG protein in mouse milk wasestimated by densitometric scanning using internalstandards of purified bovine BLG (Sigma).

Results

Production of transgenic mice for the caprine BLG gene

A total of 910 pronuclear stage embryos were injectedwith a 14.5 kb caprine BLG construct, containing theentire transcription unit and 6.1 kb and 3.7 kb of the 59and 39 flanking regions, respectively. Healthy microin-jected embryos were cultured overnight and 83.9%successfully divided. Transfer of 347 microinjected two-cell embryos into the oviducts of 33 pseudopregnantrecipient mice yielded 93 pups (26.8%), of which 3 diedaccidentally soon after birth.

PCR analysis of tail purified genomic DNA showedthat 4 out of 90 G0 mice analysed (4.4%) carried thecaprine BLG encoding transgene; two of these werefemales (23 and 58) and two were males (101 and 158).

All founder mice transmitted the transgene to theirprogeny and transgenic lines were propagated by matingwith B6CBA F1 mice. However, the low transmissionrates observed for founder mice 58 (11.1%), 101 (11.8%)and 158 (2%) were indicative of possible germlinemosaicism (Table 1).

Characterization of transgenic lines by Southern blot

The four G0 mice identified as transgenic by PCR werefurther analysed by Southern blot and transgene copynumber was determined (Fig. 2A). Founder mice 23 and58 contained between 1 and 3 copies of the transgene percell, while the other two (101 and 158) contained less thanone copy of the transgene per cell, reflecting somaticmosaicism.

Hemizygous G1 progeny of each founder animal werealso analysed by Southern blot (Fig. 2B) and comparisonof the G0 and G1 generation copy numbers showed thatfounder mouse 58, considered as germline mosaic byanalysis of transgene transmission rates, was also a

H HH H

1 Kb

1 2 3 4 56 7

Fig. 1. Structure and Hin dIII restriction sites of the caprine BLGtransgene (14.5 kb). H: Hin dIII. Black boxes correspond to theposition of the seven exons.

70 Ibanez et al.

somatic mosaic. While founder mouse 58 contained 1 or2 copies of the transgene per cell, animals in thesubsequent generation showed a more intense hybridiza-tion band corresponding to 2 or 4 copies of the transgeneper cell (Table 1).

Southern analysis was also used to investigate theintegrity of the transgene, showing the integration of atleast one complete copy of the transgene in lines 23, 58,and 158. Two bands of 7.6 kb and 5.2 kb were expectedfrom the Hin dIII digestion of an entire transgene, with amore intense hybridization signal on the 5.2 kb fragmentdue to its higher exon content (Fig. 1). However, inanimals from line 101 only the 7.6 kb fragment wasdetected, suggesting that the transgene had a deletion inits 39 end. In founder mouse 58 and its offspring, the7.6 kb and 5.2 kb bands showed equivalent hybridizationsignals and an additional 2.5 kb fragment was present, alltogether suggesting that one of the copies of thetransgene was also deleted in its 39 end (Fig. 2).

Additional fragments of smaller size were observed infounder mouse 23, most probably resulting from a secondsite of integration. These fragments were lost in G1

animals analysed presumably due either to segregation ormosaicism.

Expression of goat BLG in the milk of transgenic mice

Milk samples were obtained from two hemizygoustransgenic offspring of each founder mice at day 15 oflactation. Western blot analysis using a purified anti-BLGantiserum demonstrated expression of caprine BLG inthree of the four transgenic lines (Fig. 3). Concentrationsof the exogenous protein in mouse milk samples wereestimated by densitometry using internal standards ofpurified BLG and ranged from undetectable levels in line101 (less than 10 ìg mlÿ1) up to 0.5 mg mlÿ1 in line 23(Table 1). As expected, no signal was present in controlmouse milk, which does not naturally contain BLG.

The differences in the expression levels of caprineBLG observed between the four transgenic lines seem to

be related to the integration site rather than to transgenecopy number, as deduced from the comparison oftransgene copy number and level of expression. Inaddition, significant differences in the level of expressionwere also detected between individuals of the sametransgenic lines: 0.2–0.5 mg mlÿ1, 0–0.02 mg mlÿ1 and0.3–0.4 mg mlÿ1 for animals in lines 23, 58 and 158,respectively (Fig. 3).

Discussion

This study demonstrates that a 14.5 kb genomic fragmentcontaining the caprine BLG gene transcription unit and6.1 kb of the 59 and 3.7 kb of the 39 flanking DNAsequences is capable of driving the expression of caprineBLG to the mammary gland of transgenic mice, though atlower levels than those typically found in goat milk:1.4 mg mlÿ1 according to Hambling et al. (1992).

Expression of the BLG construct was detected in threeof the four transgenic lines obtained, at concentrationsranging from 0.02 to 0.5 mg mlÿ1 of mouse milk. Nocorrelation was found between transgene copy numberand the levels of expression, suggesting an influence ofthe site of integration. The lack of expression of thetransgene in animals from line 101 could be attributed tothe deletion in the 39 end of the only copy of thetransgene integrated in the genome of this line. However,the integration of the transgene in a chromatin domainnot permissive for expression cannot be ruled out (Clarket al., 1994).

Differences in the expression of caprine BLG weredetected, not only between animals from differenttransgenic lines, but also between animals from the sametransgenic line. This phenomenon has been seldomreported (Simons et al., 1987; Archibald et al., 1990;Persuy et al., 1992; Bleck and Bremel, 1994) andprobably reflects the variation in the total protein contentof individual milk samples (Simons et al., 1987) and/orthe genetic polymorphisms in characters controlling

Table 1. Summary of the main characteristics of the four lines of transgenic mice

Line 23 Line 58 Line 101 Line 158

Sex of G0 mice female female male maleGermline transmission rate of G0 mice 13/21 (61.9%) 3/27 (11.1%) 6/51 (11.8%) 1/50 (2%)Germline mosaica No Yes Yes YesTransgene copy number in G0 mice 2–3 1–2 ,1 ,1Transgene copy number in G1 mice 2–3 2–4 1 1Somatic mosaicb No Yes Yes YesIntegrity of transgene Yes One copy deleted Only copy deleted Yes

at 39 end at 39 endBLG in milk (mg mlÿ1)c 0.2–0.5 0–0.02 n.d. 0.3–0.4

aFounder mice with transmission rates ,50%bFounder mice with ,1 transgene copy/cell or with a transgene copy number lower than their G1 progenycBLG content in the milk of the two transgenic hemizygous female mice analysed from each transgenic lineBLG, â-lactoglobulin; n.d., not detected (,10 ìg mlÿ1)

Caprine â-lactoglobulin expression in transgenic mice 71

directly and indirectly milk protein production (Persuy etal., 1992).

The results reported in this study are quite differentfrom those reported previously on the high expression of

ovine BLG transgenes in mice (Simons et al., 1987).Ovine BLG transgenes with 4 kb of 59 flankingsequences are expressed, in all transgenic mice analysed,at very high levels (3–23 mg mlÿ1) and in a position-independent manner (Whitelaw et al., 1992). Similarly,using ovine BLG transgenes with approximately 3 kb of59 flanking sequences, Shani et al. (1992) obtained highlevel expression of the exogenous protein in the milk oftransgenic mice (1–8.5 mg mlÿ1), though at lower levelsthan those obtained by Simons et al. (1987).

The caprine BLG transgene used in our study wasexpressed at lower levels than those found for ovine BLGand in a position-dependent manner. However, compari-son of ovine (Harris et al., 1988) and caprine (Folch etal., 1994) BLG genes reveals a high degree of sequencesimilarity and conservation of the binding sites fortranscription factors that regulate the expression of thisgene.

Several factors could account for the different expres-sion profiles displayed by ovine and caprine transgenes:

(1) low sample size of transgenic lines generated inour study with respect to that of Simons et al. (1987) andWhitelaw et al. (1992);

Fig. 2. Characterization of transgenic mice by Southern blotanalysis. Southern blot experiments were carried out on Hin dIIIdigested genomic DNA as described in Materials and methods.Arrows indicate the size of the bands expected for the BLGtransgene: the 7.6 kb fragment corresponding to exons 1, 2 and 3and the 5.2 kb fragment corresponding to exons 4, 5, 6 and 7. (A)Southern blot analysis of founder transgenic mice. Lane M:genomic DNA from a non-transgenic mouse; lanes 1–4: genomicDNA from transgenic founder mice 23, 58, 101 and 158,respectively; lane G: goat genomic DNA; lanes a and b: genomicDNA from a non-transgenic mouse (10 ìg) mixed with caprineBLG transgene in amounts equivalent to 0.01 and 1 copies percell, respectively; lanes c–f: linearized caprine BLG transgene inamounts equivalent to 0.02, 0.4, 0.6 and 0.8 copies per cell,respectively. (B) Southern blot analysis of transgenic hemizygousG1 offspring from each founder mouse. Lane M: genomic DNAfrom non-transgenic mouse; lanes 1–4: genomic DNA fromtransgenic offspring of founder mice 23, 58, 101 and 158,respectively; lane G: goat genomic DNA; lanes a–f as in (A).

Fig. 3. Detection of caprine BLG in the milk of transgenic miceby western blot analysis. Two hemizygous transgenic offspring ofeach founder mice were used for expression analysis and westernblots were performed as described in Materials and methods. Wheyproteins were loaded in quantities equivalent to 1 ìl of milk forcontrol goat (lanes G), control non-transgenic mouse (lanes M) andtransgenic offspring of founder mice 23 and 158 (lanes 1 and 4,respectively). Quantities equivalent to 2 ìl of milk were loaded fortransgenic offspring of founder mice 58 and 101 (lanes 2 and 3,respectively). Lanes a–d: 20, 100, 500 and 1000 ng of purifiedbovine BLG, respectively. A and B correspond to the two animalsanalysed from each transgenic line.

72 Ibanez et al.

(2) transgene copy number, ranging from 1 to 4 in thisstudy and from 1 to 30 in that of Whitelaw et al. (1992);

(3) differences in the regulation of expression of BLGencoding genes between goat and sheep. BLG content inmilk has been estimated at 1.4 mg mlÿ1 in goat and at2.8 mg mlÿ1 in sheep (Hambling et al., 1992);

(4) differences in the DNA constructs used for theproduction of the transgenic animals. In this study, aBLG transgene comprising 6.1 kb and 3.7 kb of the 59and 39 flanking sequences, respectively, was used. Withovine BLG transgenes, high expression levels wereobtained with different constructs comprising from406 bp to 4.3 kb of the 59 flanking sequence and 1.9 or7.3 kb of the 39 flanking sequence (Whitelaw et al.,1992) and also with constructs comprising 3 kb and0.8 kb of the 59 and 39 flanking DNA, respectively (Shaniet al., 1992). Differences in the length of the 39 flankingregion seem to have no effects on the expression of ovinetransgenes, while, in contrast, resection of the 59proximal region to 146 bp results in a dramaticallyreduced frequency of expression (Whitelaw et al., 1992).On the other hand, extension of the 59 flanking sequenceto 5.5 or 10.8 kb also resulted in a reduced frequency ofexpression, and animals that expressed the foreign proteindid so at lower levels. Presence of potential inhibitorycis-acting elements within these further upstream se-quences of the ovine gene was suggested as one possiblecause for this effect (Shani et al., 1992). This could alsobe the case for the caprine BLG transgene used in ourstudy, which contains 6.1 kb of the 59 flanking DNA.

Nucleotide sequence differences between caprine BLGgene and its ovine counterpart reside basically in introns(95.6% similarity) and in the 59 and 39 flanking regions(95.7% and 93.3% similarity, respectively) (Folch et al.,1994). Ovine BLG transgenes are one of the few milk-protein-encoding genes that are expressed in a position-dependent manner in the mammary gland of transgenicmice, and it has been proposed by Clark et al. (1994)that this position-independent expression requires thepresence of cis-acting regulatory elements, which may belocated within as well as downstream of the gene. Thus,presence of such putative cis-acting elements in the ovinetransgene, otherwise absent in the caprine transgene,could also account for the different performances of bothconstructs in transgenic mice. The existence of regulatoryelements within ovine BLG gene has been recentlyreported by Barash et al. (1996).

In conclusion, the results reported here show that thecaprine BLG construct used contains the cis-actingregulatory elements necessary for directly the expressionof the caprine protein to the mammary gland oftransgenic mice. However, the lower levels of expressionof the caprine BLG transgene with respect to thosereported for the ovine BLG transgenes might reflect

differences in the content of regulatory elements betweenboth constructs.

Acknowledgements

This work received financial support from CICYT (Spain)(project AGF 93/0560), Ministerio de Educacion y Ciencia(Spain) (FPI grants to E.I. and J.M.F.), Generalitat deCatalunya (DGU grant to A.C.) and Institut de CulturaJuan Gil-Albert (Diputacio d’Alacant, Spain) (grant toE.I.).

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