CA2193513A1 - Alpha-lactalbumin gene constructs - Google Patents

Abstract

The present invention provides recombinant gene constructs for expressing the protein .alpha.-lactalbumin, especially human .alpha.-lactalbumin, in bovine cells. Novel genetic constructs, vectors and transformed cells are provided as well as transgenic animals genetically engineered for enhanced expression of .alpha.-lactalbumin. Human .alpha.-lactalbumin has been shown to be superior for human infant nutrition and the present invention enables a cheap and effective form for production of the major whey protein in human milk.

Description

W096/02640 .~ 5l~165l 21 ~35 1 3 1 "Alpha-LactAlh~lm;n Gene Constructs"

3 The present invention is concerned with recombinant 4 gene constructs for expressing the protein ~-lactalbumin, or functional equivalents or parts 6 thereof.

8 Human milk has been shown to be superior over other 9 milk types, notably cow, sheep, camel and goat milk, for human infant nutrition. ~owever, many mothers find 11 breast feeding difficult or inconvenient. Moreover, in 12 countries where infant food supplements are in great 13 demand, it would be highly desirable to be able to 14 supply a milk product with the nutritional benefits of human milk.

17 One of the major differences of human milk over milk 18 from other mammals (for example cows or sheep) is the 19 presence of ~-lac~Alh~lm;n as the major whey protein.
Whilst ~-lactalbumin is present in other milk types, 21 the concentration is relatively low and instead the 22 major whey protein is ~-lactoglobulin. The level of ~-23 lac~lh~lmin varies from species to species, with human 24 milk containing about 2.5 mg/ml, cow milk O.S-1.0 mg~ml and mouse milk 0.1-0.8 mg/ml.

SUBSTITUTE SHEET ~RVLE 26) WO'~/02640 PCT/GB9~/01651 2 ( q 3,5 j 3 1 The gene sequences encoding for the bovine ~-2 lactA1h~;n and for the human ~-lactalbumin proteins 3 have been elucidated and the sequence information 4 published by ~ilotte et al, in Biochemie 69: 609-620 (1987) and by Hall et al, in Biochem J 242: 735-742 6 ~1987), respectively.
8 The present invention seeks to utilise genetic 9 engineering technique5 to provide a recombinant gene construct capable of producing an ~-lactalbumin 11 concentration of greater than 1.0 mg/ml, for example 12 1.2 mg/ml or above, in milk when expressed in mammalian 13 cells. Generally said construct is adapted to be 14 expressed in non-humarl animal, especially bovine, cells.

17 In one aspect, the present invention provides a 18 recombinant expression system adapted to express ~-19 lactalbumin, or a functional equivalent or part thereof in cells of a non-human, preferably bovine, animal.
21 Preferably, the recombinant expression system of the 22 present invention is adapted to express the human ~-23 lact~lh~7~in protein, or a functional equivalent or part 24 thereof.
26 The term "expression system" is used herein to refer to 27 a gerretic sequence which includes a protein-encoding 28 region and is operably linked to all of the genetic 29 signals necessary to achieve expression of the protein encoding region. Optionally, the expression system may 31 also include a regulatory element, such as a promoter 32 or enhancer, to increaSe transcription and/or 33 translation of the protein-encoding region, or to 34 provide control over expression. The regulatory element may be located upstream or downstream of the 36 protein-encoding region, or may be located at an intron WO9~102C40 1 ~ I / ~D~. C 1651 ~ 3 ~ ~ 9 3 5 1 3 1 (non-coding portion) interrupting the protein encoding 2 region. Alternatively it i5 also possible for the 3 sequence of the protein-encoding region itself to 4 comprise a regulatory ability.

6 The term "functional equi~alent~ refers to any ~ 7 derivative which is functionally substantially similar 8 to the reference sequence or protein. In particular 9 the term '~functional equivalent'~ includes derivatives in which nucleotide base(s) and/or amino acid(s) have 11 been added, deleted or replaced without a significantly 12 adverse effect on biological function, especially 13 biological function in milk production.

Genetic engineering has been recognised as a powerful 16 technique not only in research but also for commercial 17 purposes. Thus, by using genetic engineering 18 techniques (see Maniatis et al Molecular Cloning, a 19 Laboratory Manual Cold Spring, ~arbor Laboratory, Cold Spring ~arbor, New York 1982 and "Principle of Genetic 21 Engineering", Old and Primrose, 5th edition, 1994, both 22 incorporated herein by reference) exogenous genetic 23 material can be transferred to a host cell and the 24 protein or polypeptide encoded by the exogenous genetic material may be replicated by and/or expressed within 26 the host. For the purposes of simplicity genetic 27 engineering is normally carried out with prokaryotic 23 micro-organisms, for example bacteria such as E. coli, 29 as host. However, use has also been made of eukaryotic organisms, in particular yeasts or algae, and in 31 certain applications eukaryotic cell cultures may also 32 be used.

34 Genetic alterations to mammalian species by micro-injection of genes into the pro-nuclei of single-cell 36 embryos has been described by Brinster et al, in Cell W096~02640 ~ 7~ 16~1 ~ . 4 ~i 7JJIJ
1 27: 223-231, 1981. Here the foreign genetlc material 2 i5 introduced into the fertilised egg of an animal 3 whish then proceeds to develop into an embryo in the 4 normal manner having been transplanted into a foster S mother. Truly transgenic animals contain copies of the 6 exogenous DNA in each cell.
8 Where the injected genetic material i5 successfully 9 incorporated into the host chromosome the animal is termed "transgenic" and the transgene is inherited in 11 the normal Mendelian manner. However, only a low 12 proportion of gene transfer operations are successful, 13 especially for large domestic animals such as pigs, 14 sheep and cattle. To date it has not been possible tu control the location at which the transgene integrates 16 into the host chromosome for such animals.

18 For the purpose of the present invention it may, in 19 certain circumstances, be sufficient simply to produce a ~mosaic'' donor animal. In this situation the 21 transgene is incorporated into the chromosome copies of 22 only certain body organs. Mosaic animals are generally 23 produced by introducing exogenous ~NA into an embryo at 24 a later developmental stage.
26 One of the most promising application of transgenesis 27 in livestock aims to utilise the mammary gland as a 28 ~-bioreactor" to produce recombinant proteins of 29 pharmaceutical or nutritional interest in milk. The mammary gland is an attractive organ in which to 31 express heterologous proteins due to its capacity to 32 produce large quantities of protein in an exocrine 33 manner. Recombinant DNA techniques may be used to 34 alter the protein composition of cow's mil}: used for human or animal consumption. Por example, the 36 expression of human milk proteins in cow's milk could W096~02640 ~ ~ r~ 5lC1~51 ~ i ~ s ~ 5 ~3 1 improve its nutritional value in infant formula 2 applications ~Strijker et al, in ~larnessing 3 Biotechnology for the 21st Century, ed Ladisch and 4 Boser, American Chemical Society, Pages 38-21 (1992~).
Both applications would benefit from ability to 6 increase production capacity inexpensively by 7 multiplying producer animals with conventional and 8 advanced breeding techniques The first step in developlng a transgene to be 11 expressed in the mammary gland is to clone the gene for 12 the protein of interest. To direct expression into 13 milk, the promoter gene for a major milk protein 14 expressed in milk is employed. Milk protein genes are tightly regulated and are not expressed in tissues 16 other than the mammary gland, a characteristic that 17 minimises the possibility of negative effects on the 18 animal from inappropriate expression in other tissues.
19 Among the regulatory genes used to express heterologous proteins in the mammary gland are alpha-SI-casein 21 (Strijker et., 1992, supra), beta-lactoglobulin (~right 22 et al., sio~Technology 9:831-834 (1991)) whey acidic 23 protein (Ebert and Schindler, Transgenic Farm animals:
24 Progress Report (1993)) and beta-casein (Ebert and Schindler, 1993, supra).

27 Newly-made gene constructs are normally tested in 28 transgenic mice before adopting them for use in cattle.
29 ~ilk obtained from the transgenic mice is assayed for quantity of recombinant protein. If enough milk is 31 available, the protein may be isolated to examine its 32 structural characteristics and bioactivity. The 33 selection of a particular construct for use in cattle 34 will depend primarily on consideration of both expression level and authenticity of the resultant 36 recombinant protein.

W09610~640 .,~ 3 5 1 3 r~ 65l 1 Transgenesis in cattle is normally initiated by 2 microinjecting several hundred copies of gene construct 3 into one of the two pronuclei in a zygote. ~ygotes may 4 be obtained in vivo from the oviducts (Roschlau et al~, Arch Tierz. ~erlin 31:3-8 (1988); Roschlau et al., in 6 J. Reprod. Fertil. (suppl 38), Cell Biology of 7 Mammalian Egg ~anipulation, ed Greve et al (1989~; Hill 8 et al., Theriogenology 37:222 (1992); ~3Owen et al. ~iol 9 Reprod. ~Q:664-448 (1994)~ or by in vitro fertilisation of in vitro matured oocytes (Krimpenforth et al., 11 Biotechnology 9:844-847 (1991); Hill et al., 1992, 12 supra; Bowen et al., 1993 supra). Bovine zyyotes must 13 be centrifuged at 15,000 x g for several minutes to 14 displace opaque lipid in order to visualise the pronuclei with phase contrast, Nomars~i or Hoffrnan 16 interference contrast optics. 2-4 pl of buffer 17 containing several hundred copies of DNA construct are 18 injected into a pronucleus. Transgenes are thought to 19 integrate into random breaks in chromosomal DNA that result from mechanical disruption during the 21 microinjection proce5s. Ideally, the transgenes 22 integrate at the zygote stage prior to DNA replication 23 to ensure that eYery cell in the adult contains the 24 transgene. In general, several "copies" of the transgene, linked together linearly, integrate in a 26 single site on a single chromosome. The site of 27 integration is random. Integration probably occurs 28 after the first round of DNA replication, and perhaps 29 as late as the 2- or 4- cell stage (Wall and Seidel, 1992), resulting in animals that are mosaic with 31 respect to the transgene. Indeed, up to 30~ of animal~
32 in which transgenes are detected in somatic tissues do 33 not transmit the transgenes to their offspring (or 34 transmit to less than the expected so~).
36 After microinjection, embryos are either transferred WO ~J6/0264~ 2 ~ 9 3 5 1 3 r~ C 16sl 1 directly into the oviducts of recipients or cultured 2 for a few days and transferred to the uterus of 3 recipient cattle. Confirmation of transgene 4 integration is obtained hy Southern blot analysis of tissues sampled from the calf after birth. Transgene 6 expression is measured by assaying for the gene product ~ 7 in appropriate tissues, or in this case milk. Embryo 8 survival after microinjection, transgene integration 9 frequency, frequency of expression and expression level, and frequency of germline transmission vary 11 according to quantity and quality of DNA construct 12 injected, strain of mice used (Brinster et al., Proc.
13 Natl. Acad Sci. USA 82:4438-4442 (1985)) and skill and 14 technique of the operator performing microinjection.
This basic approach has been routinely applied to 16 produce transgenic sheep (Wright et al., 1991, supra), 17 goats (~bert and Schindler, 1993, supra) pigs (Rexroad 18 and Purcel, Proc 11th Intl. Congr. Anim. Reprod. A.I.
19 Dublin 5:29-35 (1988)) and cattle (Krimpenfort et al., 1991, supra; Hill et al., 1992, supra; Bowen et al., 21 1994, supra).

23 Reference is also made to WO-A-88/01648 (of Immunex 24 Corporation), to WO-A-83~00239 and to WO-A-90~05188 (both of Pharmaceutical Proteins Limited) for 26 describing suitable techniques and methodologies for 27 production of recombinant gene constructs, production 28 of transgenic animals incorporating such constructs and 29 expression of the protein encoded ir the mammary gland of the lactating adult female mammal. The disclosures 31 of these references and the references recited above 32 are incorporated herein by reference.

34 Reference is further made to Stacey et al in molecular and Cellular Biology 14(21: 1009-1016 (~ebruary 1994) 36 which describes a knock-out experiment in which the W09hlO2640 ~ 93~ 3 ~~1.~.S 1651 1 mouse ~-lactalbumin qene i5 replaced with a human ~-2 lactalbumin gene. This paper (incorporated herein by 3 reference) does not however report expression of the ~-4 lactalbumin protein.
6 In one embodiment the present invention provides an 7 expression system which has been produced by techniques 8 other than by knock-out of the gene naturally present.

In a further aspect, the present invention provides a ll transgenic mammalian animal, said animal having cells 12 incorporating a recombinant expression system adapted 13 to express ~-lactalbumin ~preferably human ~-14 lactalbumin) or a functional equivalent or part thereof. Generally the recombinant expression system 16 will be integrated into the genome of the transgenic 17 animal and will thus be heritable so that the offspring 18 of such a transgenic animal may themselves contain the l9 transgene and thus also be covered by the present invention. Suitable transgenic animals include (but 21 are not limited to~ sheep, pigs, cattle and goats.
22 Cattle are especially preferred.

24 Additionally, the present invention comprises a vector containing such a recombinant expression system and 26 host cells transformed with such a recombinant 27 exprèssion system (optionally in the form of a vector).

29 In a yet further aspect the present invention provides ~-lactalbumin produced by expression of a recombinant 31 expression system of the present invention, desirably 32 sucil ~-lactalbumin produced in a transgenic ma11lmal.
33 The ~-lactalbumin gene is naturally activated in the 34 mammary glands of the lactating female mammal. Thus the protein expressed by the recombinant expression 36 system of the present invention would be produced at W096l02640 ~ i 9 ~ 5 1 3 ~ s.[ 1651 1 such a time and would be excreted as a milk component.
2 It may also be possible for the protein of interest to 3 be produced by inducing lactation through hormonal or 4 other treatment. Processed milk products comprising such ~-lactalbumin are also covered by the present 6 invention.
8 In one preferred embodiment, the recombinant expression 9 system comprises a construct designated pHAl, pHA2, pBB~A, pOBHA, pBAHA, pBo~a-A or pBova-B. The 11 constructs pHAl, pHA2, p~BHA, pOBHA and pBAHA express 12 human ~-lactalbumin and are thus preferred, 13 particularly pHA2. The construct pHA2 was deposited on 14 15 February 1955 at NCI~B under Accession No NCIMB
40709.

17 Likewise transgenic mammals comprising the specific 18 constructs listed above are preferred.

It has further been found t~-~t, in addition to 21 increased concentrations of ~-lactalbumin per unit 22 volume of milk achieved by he present invention, where 23 a human ~-laotalbumin gene is present the volume of 24 milk produced increases also. This finding is totally unexpected and for this reason constructs containing 26 the human ~-lactalbumin gene (or functional equivalents 27 or parts thereof) and transgenic animals (especially 28 cattle) are preferred Pmho~ir Ls of the invention.

Whilst we do not wish to be bound by theoretical 31 considerations, it is further believed that the 32 promoter region of the human ~-lactalbumin gene is only 33 partially responsible for the enhanced natural 34 expression of ~-lactalbum n by humans. It is believed that enhanced expression may be obtained by including 36 within the recombinant expression system of the present 37 invention the 3' sequence flanking the protein-encoding 38 region and/or the 5' sequence flan~ing the protein-SUI~STI~UT' SHEET II~ULE 26~
.. .. .. . , _, .. _, . , . , _ _ W0961~2640 ~ 9 ~ 5 ~ 3 P~ . ~L C. _ 1651 1 encoding regiQn itself.

3 The flanking sequences 3~ and 5' to the protein-4 encoding region of the human ~-lac gene have been sequenced for the first time. Partial sequences 6 (nucleotides 1-264, 1331-2131, 2259-2496, 251g-2680 and 7 3481-3952) of the 3' flanking region are presented in 8 SEQ ID Nos. 16 to 20 whilst the full sequence of the 5-9 flanking region is presented in SEQ ID No. 21. In experiments it has been observed that inclusion of 11 either or both of these sequences give a surprisingly 12 marked increase in expression levels of the a-13 lactalbumin protein. This increase in expression may 14 be observed when the protein-encoding region is non-human ~-lactalbumin as well as human a-lactalbumin.

17 Both the sequences of SEQ ID Nos. 16-20 and 21 are now 18 believed to contribute towards the higher levels of 19 expression of a-lactalbumin in human milk, and therefore comprise a further aspect of the present 21 invention.

23 In a further aspect, the present invention provides a 24 polynucleotide having a sequence substantially as set out in any one of SEQ ID Nos. 16-20 or SEQ ID No. 21 or 26 a portion or functional equivalent thereof.

28 The polynucleotides may be in any form (for example DNA
29 or RNA, double or single stranded), but generally double stranded DNA is the most convenient. Likewise 31 the polynucleotides according to the present invention 32 may be present as part of a recombinant genetic 33 construct, which itself may be included in a vector 34 (for example an expression vector) or may be incorporated into a chromosome of a transgenic animal.
36 Any vectors or transgenic animals comprising a W096l02640 ~ / } ~1 935 ~ 3 P ., ~C1~51 1 polynucleotide as described above form a further aspect 2 of the present invention.

4 Viewed from a yet further aspect the present invention - 5 provides a recombinant expression system ~preferably 6 adapted to express ~-lactalbumin ~preferably human ~-7 lactalbumin) or a portion or functional equivalent 8 thereof), said recombinant expression system comprising 9 a polynucleotide selected from the polynucleotide located between the EcoRI and XhoI restriction sites of 11 the wild-type ~-lactalbumin gene and the polynucleotide 12 located between the BamHI and EcoRI restriction sites 13 of the wild-type human ~-lactalbumin gene, or a portion 14 or functional equivalents thereof 16 In one preferred embodiment, the recombinant expression 17 sequence of the present invention comprises both 18 polynucleotides as defined above, portions and 19 functional equivalents thereof.
21 The invention also encompasses vectors containing the 22 recombinant expression systems defined above and cells 23 transformed with such vectors. Further, the present 24 invention comprises transgenic animals wherein the transgene contains the recombinant expression system.

27 Figu~e 1 as discussed in Example 1 and shows the 28 sequence of bovine ~-lactalbumin PCR primers.

Figure 2 is discussed in Example 1 and 4 shows the 31 posltion of bovine ~-lactalbumin PCR primers and 32 products.

34 Figure 3 is discussed in Example 2 and shows a restriction map of two overlapping genomic A clones for 36 the human ~-lactalbumin gene (pHA-2 and pHA-l).

WO96~)26~ ,,,.~ q 35 1 3 ~ 5.'C16~

1 Fiyure 4 is discussed in Example 3 and shows a 2 restriction map of three overlapping genomic A clones 3 for the bovine beta-lactaglobulin gene.

Figure 5 is discussed in Example 4 and shows SDS-PAGE
6 analysis of skimmed milk from bovine a-lactalbumin 7 transgenic mice run against non transgenic mouse milk.

9 Figure 6 is discussed in Example S and shows human a-lactalbumin transgene constructs.

12 Figure 7 is discussed in Example 5 and shows SDS-PAGE
13 analysis of skimmed milk from human a-lactalbumin 14 transgenic mice run against non transgenic mouse milk.
16 Figure 8 is discussed in Example 5 and shows a Western 17 analysis of the milk from human a-lactalbumin 18 transgenic mice run against human a-lactalbumin 19 standard.
21 Pigure 9 shows the PCR cloning strategy for transgene 22 constructs P~l to PKU4 as discussed in Example 6.

2g Pigure 10 gives the se~uences of P~U primers 1 to 10 as 2S discussed in Example 6.

27 Figure 11 shows the structure of null and humanised a-28 lactalbumin alleles.
2~
Figure 12 is a Northern analysis of total RNA from a-31 lactalbumin-deficient lactating mammmary glands.

33 Figure 13 illustrates a Western analysis of a-34 lactalbumin from targeted mouse strains.
36 Figure 14 is a histological analysis of wild type and W096/0264~ 3 ~. 1 9 ~ 5 ~ 3 r~ ,l651 1 ~-lac~ lactating mammary glands.

3 Figure 15A shows an RNase protection assay used to 4 distinguish human replacement and mouse ~-lactalbumin - 5 mRNA and Figure 15~ shows an RNase protection assay of 6 mouse and human replacement ~-lactalbumin mRNA, 8 Figure 16 gi~es the quantification of ~-lactalbumin by 9 hydrophobic interaction chromatography.
11 SEQ ID Nos. 16 - 20 give parts of the sequence from the 12 BamHI site to the vector restriction sites (including 13 EcoRI sites) 3' of the protein-encoding region of the 14 endogenous human ~-lactalbumin gene, as set out below:
16 SEQ ID No. 16 : Nucleotides 1 to 264 (inclusive) 17 SEQ I3 No. 17 : Nucleotides 1331 to 2131 (inclusive) 18 SEQ ID No. 18 : Nucleotides 2259 to 24g6 (inclusive~
19 SEQ IC No. 19 : Nucleotides 2519 to 2680 (inclusive) SEQ ID No. 20 : Nucleotides 3481 to 3952 (inclusive) 22 SEQ ID No. Z1 gives the sequence Erom the EcoRI
23 restriction site to the XhoI restriction site which are 24 5' to the protein-encoding region of the endogenous human a-lactalbumin gene.

27 In more detail, in Figure 11 the upper portion shows 28 the wild type murine ~-lactalbumin locus. The positio 29 and direction of the transcribed region is indicated by the arrow. The translational stop site and RNA
31 polyadenylation sites are also indicated. The middle 32 portion shows the structure of the null allele. The 33 striped bar indictes the HPRT selectable cassette. The 34 lower portion shows the structure of the human replacement allele. The checkered bar shows the human 36 ~-lact.albumin fragment. The transcription initiation, wo g6/n2640 ~ 3 5 ~ 3 PCrlGB9S~ GSI
14 ~
1 translat.ional 8tOp and polyadenylation sites are shown.
2 Restriction enzyme sites shown are: HindIII (H); BamllI
3 (B); XbaI (Xl.

In Figure 12, the two autoradiographs shown are .repeat 6 hybridLsations of the 5ame membrane filter using a 7 human a-lactalbumin probe followed by a rat ~-casein 8 probe. The probes used are indicated under each 9 autoradiograph. The source of RNA in each lane is indicated above the lane mar~.ers.

12 In Figure 13 Lane A contains purified human o-13 lactalbumin. Lanes B-F show samples of mil~ from 14 targeted mice, genotypes are indicated abo~e the lane markers. Lanes ~ and H are a shorter exposure of Lanes 16 C and D.

18 The light micrographs shown in Figure 14 are 19 haemtoxylin~eosin stained sections of mammary tissue (original magnification lOOx). The genotypes of each 21 gland are indicated.

23 In Fiqure 15A, the 3- junction between mouse and human 24 DNA in the a-lach allele lies between the translational stop site and the polyadenylation signal. Human ~-26 lactalbumin mRNA contains 120bp of mouse sequences in 27 the 3' untranslated end. ~uman replacement and mouse 28 ~-lactalbumin mRNA were detected by hybridisation with 2~ a mouse RNA probe and distinguished by the size of RNA
3C fragments protected from ribonuclease digestion. Human 31 sequences are indicated by the chequered bar and mouse 32 sequences by the shaded bar. Restriction enzyme sites 33 shown are: HindIII (H); BaI (~3); XbaI (X).

3S In ~igure 15B the autoradiograph shown is of a 5~
36 polyacrylamide urea thin layer gel. ~he source of RNA

W09Cl02610 ~ ~ t~ L i 935 t 3 PCT/GB95101651 ~ 15 1 is indicated above the lane markers. Lane A shows a 2 wild-type RNA hybridised t.o the mouse RNA proe 3 undigested with ribonuclease. ~anes D to J show RNA
4 samples from ~-lac~ lach heterozygotes, the numbers indicate inùividual mice and are the source of the 6 quantitative estimates given in Figure 15. The 7 predicted size of protected fragments are inùicated.

9 The upper portion of Figure 16 shows phenyl-Sepharose elution profiles oE three milk samples. 1, a-lacb/~-lach 11 homozygote fmouse #22); 2, ~-lac~/~-lach heterozygote 12 (mouse #76); 3, ~-lac'/~-lacm wild type. The lower 13 portion shows a standard curve of known quantities of 14 human ~-lactalbumin plotted aaainst integrated peak area.

17 The present invention will now be further described 18 with reference to the following, non-limiting, 19 examples.
21 Examole 1 - Clonina of Bovine ~-lactalb:lmin aene 23 There are three known variants of bovine ~-lactalbumin, 24 of which the B form is the most common. The A variant from Bos (Bos) nomadicus f.d. indicus differs from the 26 B variant at residue 10: ClU in A i.s substituted for 27 Arg in B. The sequence difference for the C variant 28 from Bos (Bibos) javanicus has not been established 29 (McKenzie ~ White, Advances in Protein Chemistry 41, 173-315 (1991). The bovine ~-lactalbumin gene 31 (encoding the B form~ was cloned from genomic DNA using 32 the PCR primers indicated in Figure 1. The primers 33 have been given the following sequence ID Nos:
34 Ba-2 SEQ ID No 1 ~ 35 Ba-7 SEQ ID No 2 36 Ba-8 SEQ ID No 3 ~o ~6fn264o ~ r ~ i 9 3 5 1 3 r~ 6sl 1 sa-9 SEQ ID No 4 2 The source of DNA in all the PCR reactions was blood 3 from a Holstein-Friesian cow.

The length of the amplified promoter region using 6 primer Ba-9 in combination with primer Ba-8 is a . 72kb.
7 This BamHI/EcoRI fragment was cloned into Bluescript 8 ~pBA-P0.71.

The length of the ampli~ied promoter region using 11 primer Ba-7 in combination with primer Ba-8 is 2.05kb.
12 This BamHI~EcoRI fragment was cloned into Bluescript 13 (pBA-P2).

The entire bovine a-lactalbumin gene including 0.72kb 16 of 5' and 0.3kb of 3' flanking region was amplified 17 using primer Ba-9 in combination with primer Ba-2.
18 These primers include BamHI restriction enzyme 19 recognition sites, which allowed direct subcloning of the amplified 3kb fragment into the BamHI site of 21 pUC18, yiving rise to construct pBova-A (see Figure 2).

23 Ligation of the BamHI/EcoRv fragment from clone pBA-P2 24 to the EcoRV~amHI fragment of pBOVA-a gave rise to construct pBOYA-b (see Figure 2).

27 Since TAO polymerase lacks proof-reading activity, it 28 was essential to ensure that the amplified bovlne ~-29 lactalbumin DNA was identical to the published bovine a-lactalbumin gene. Sequence analysis was carried out 31 across all the exons and the two promoter fragments.
32 Comparison of the bovine a-lactalbumin exons with those 33 published by Vilotte showed 3 changes:

(i) Exon I at ~759 C to A. 5~ non-coding region;
36 (ii) Exon I at +792 CTA to C~G. Both code Eor Leucine W096/02640 ' :-.'.p; 17 ~) 935 1 3 p~ .SCl651 1 (iii) Exon II at +1231 GCG to ACG. Alanine to 2 Threonine 3 This i5 indicative of the more common "B" form of the 4 protein.

6 Although misreading of sequence during the PCR
7 amplification cannot be ruled out, the above mismatches 8 were probably due to the difference in the source of 9 bovine DNA.

11 ExamPle 2 - Clonina of Human a-lactalbumin qene 13 The DNA sequences of human a-lactalbumin has been 14 published (Hall et al, Biochem. J., 242 : 735-742 (1987)). Using the iluman sequence, PCR primers were 16 designed to clone two small fragments from human 17 genomic DNA, one at the 5' end of the gene and the 18 other at the 3' end. These were suhcloned into the 19 pUC18 vector and used as probes to screen a commercial (Stratagene) A genomic library. Two recombinant 21 bacteriophages, 4a and 5b.1, which contained the a-22 lactalbumin gene, were isolated by established methods 23 (Sambrook et al, ~olecular Cloning 2nd ed., Cold Spring 24 Harbor Laboratory (1989)). Restriction mapping demonstrated that both clones contained the complete 26 coding sequence for the human a-lac gene but differed 27 in the amount of 5' and 3' sequences present (Figure 28 3). Sequence analysis of exons from clone 5b.1 and 29 exons and 5' flanking region of clone 4a showed that these were identical to the published sequence.

32 Portions of the 3' sequence are given in SEQ ID Nos. 16 33 to 20 and the 5' sequence is given as SEQ ID No. 21.

EY~ le 3 - Cloninq of bovine beta lactoalobulin gene 36 rbBLG) W096102640 ~ 935~ 3 rc~ . S.~1651 1 ~'he DN~ sequence of bovine BLG (bBLG! has been 2 published (Jamieson et al; Gene, 61; 85-90, (1987);
3 Wagrler, unpublished, EMBL Data Library: BTBLACE~
4 ~1591)). Using the bovine sequence, PCR primers were designed to clone a fragment from the 5' portion of the 6 bovine BLG gene. This was subcloned into the pUC18 7 vector and used as probes to screen a commercial bovir.e 8 (Stratagene) A genomic library- Three genomic A clones 9 were isolated and characterised by restriction enzyme analysis (see ~ig. 4). Two of the clone5 (BB13, BB17) 11 contain the complete bBLG coding region plus various 12 amounts of flar-king regions, while clone BB25 lacks the 13 coding region and consists entirely of 5' flanking 14 region. Sequence analysis showed that the end of this clone lies 12 bp upstream of the ATG translation start 16 site. Sal I fragments containing the entire insert of 17 clone BB13 and BB17 were subcloned into pUC18, as well 18 as EcoR I fragments from clone BB25 (the latter was 19 cloned into pBluescript (Figure 4).
21 ExamDle 4 - Assemblv ~n~ eXDression of bovine a-22 lactalbumin constructs 24 TransGene constructs (FiG. 2) 26 pBova-A consists of the bovine a-lactalbumin coding 27 region, -0.72kb of 5' flank and 0.3kb of 3' flank, 28 cloned as a 3kb BamHI fragment into Bluescript vector.
pBova-B consists of 3 fragments:
31 1. The 1.47kb BamHI to EcoRV fragment from clone pBA-32 P2.
33 2. The 2.78kb EcoRV to BamHI fragment from clone 34 pBova-A.
3. The cloning vector Bluescript digested Bam~

WOg~/026~0 ~ ; 19 l~~ 65 1 Bovine ~-lactalbumin exrression in transaenic mice 3 The two constructs pBova-A and pBova-B (Figure 2) were 4 injected into mouse embryos and gave rise to transgenic S animals. Milk analysis by SDS-PAGE gel stained with 6 Coomasie blue (referred to as "Coomassie gels~) and 7 comparison to standard amounts of ~-lactalbumin showed 8 expression levels of bovine ~-lactalbumin varied from non detectable for pBova-A and up to ~O.S-lmg~ml for pBova-B (see Figure S and Table 1).

WO 96/0264(~ . 20 ~ f 9 3 5 1 3 PCT/GB9!;/01651 1Table 1 2Bovine a-lactalbumin expression in transgenic mice 3 Mouse Coomassie 4 244.12 BOVA-a 244.14 BOVA-a 6 244.15 BOVA-a 8 245.23 BOVA-b 9 245.8 BOVA-b 245.4 BOVA-b 11 245.7 BOVA-b +
12 245.21 BOVA-b 13 245.13 BOVA-b +
lg 249.13 BOVA-b 246.15 BOVA-b 16 249.18 BOVA-b ++
17 249.23.1 BOVA-b ++
18 249.23.5 BOVA-b ++
19 249.25.3 BOVA-b 249.25.7 BOVA-b 21 249.30.3 BOVA-b - - = < 0.5mg~ml 22 249.30.4 BOVA-b - + = =0.5-lmg/ml 23 249.33.2 BOVA-b +/++ ++ = -1-2mg/ml 24 249.33.3 BOVA-b +/++
26 Table 1 shows the relative levels of bovine a-27 lactâlbumin in transgenic mouse milk as estimated by 28 comparison to protein standards on Coomassie gels.

~ WOg6102640 ,~ 21 2 ~ 935 ~ 3 r~ . tl651 1 Example 5 - Assembl~ and exoression of human a-2 lactalbumin constructs 4 a-lactalbumin is the major whey protein in humans, beta-lactoglobulin the major whey protein in sheep and 6 cow. The level of a-lactalbumin expression varies from 7 species to species, human milk contains about 2.5mg/ml, 8 cow milk 0.5-l.Omg/ml, and mouse milk O.l-O.Bmg/ml. To 9 define sequences which allow maximum expression of the human a-lactalbumin gene several different constructs 11 were designed. These contain a) different amounts of 12 5' and 3' flanking regions derived from the human a-13 lactalbumin locus, b) 5' flanking regions derived from 14 the bovine a-lactalbumin locus, or c) 5' flanking regions derived from the bovine or ovine beta-16 lactaglobulin gene. The ovine beta-lactoglobulin gene 17 promoter has been successfully used to allow high 18 expression (>lOmg/ml) of heterologous genes in mouse 19 milk.
21 Transoene constructs (Ficure 6 23 pHA-1 consists of the human a-lactalbumin coding 24 region, =1.8kb of 5' flank and 3kb of 3~ flank derived from A clone 5b.1 cloned as a 7kb EcoRI/SalI fragment 26 into puc18.

28 pHA-2 consists of the human a-lactalbumin coding 29 region, =3.7kb of 5' flank and ~13kb of 3~ flank deri-ed from A clone 4a cloned as a ~19kb SalI fragment 31 into puc18.

33 pOBHA (ovine beta-lactaglobulin, human a-lactalbumin3 34 was constructed from 4 DNA fragments:
1. a 4.2kb SalI~EcoRV fragment containing the ovine 36 beta-lactoglobulin promoter (see WO-A-90~05188);

W096~02fi40 ~ 9 3 ~ 1 3 rc~lGBg5Jol~sl ~ ' 22 I 2. a 74bp synthet;c oli~onucleotide corresponding to 2 a 8bp BclI linker and bases 15-77 of the human a-3 lactalbumin sequence used as a bluntiBglI
4 fragment;
3. a 6.2kb BglI~PstI human a-lactalbumin fragment 6 derived from A clone 4a comprising a region 7 between a BglI site at base 77 and a XhoI site in 8 the 3' flank;
9 4. pSL1180 (Pharmacia) cut with PstI and SalI.
11 pBBHA (bovine beta-lactoglobulin, human a-lactalbumin) 12 was constructod from 4 DNA fragments:
13 1. a 3.Okb EcoRI fragment containing the bovine beta-14 lactoglobulin promoter derived from clone BB25-3 and used as a EcoRI/EcoRV fragment;
16 2. a 74bp synthetic oligonucleotide corresponding to 17 a 8bp BclI linker and bases 15-77 of the human a-18 lactalbumin sequence used as a blunt/BglI
19 fragment;
3. a 6.2kb BglI/PstI human a-lactalbumin fragment 21 derived from A clone 4a comprising a region 22 between a Bgll site at base 77 and a XhoI site in 23 the 3' flank;
24 4. Bluescript vector cut with EcoRI and PstI.
26 pBAHA (bovine a-lac~lh~min~ human a-lactalbumin) was 27 constructed from 4 DNA fragments:
28 1. a 0.72kb samHI to StuI fragment containing the 29 bovine ~-lactalbumin promoter derived from clone pBA-PO.7;
31 2. a 62bp synthetic oligonucleotide corresponding to 32 bases 15-77 of the human ~-lactalbumin sequence 33 used as a blunt/BglI fragment;
34 3. a 6.2kb BglIJPstI human a-lactalbumin fragment derived from A clone 4a comprising a region 36 between a BglI site at base 77 and a XhoI site in :
W0~6/02640 j ~ F~ 5.~i65t 1the 3' flank;
2 4. Bluescript vector cut with BamHI and PstI.

4 ~luman a-lactalbumin ex~ression in transcenic mice 6 5 constructs were injected into mouse embryos and gave 7 rise to transgenic animals- All constructs expressed 8 human a-lactalbumin in the milk of mice. pHA-1 and 9 pHA-2, which contain the human a-lactalbumin gene and various amounts of flanking regions expressed between 1 11 to ~18mg/ml (213.5 pHA-2) in the majority of animals.
12 pOBHA and pBBHA containing the human a-lactalbumin gene 13 driven by either the ovine or bovine BLG promoter had 14 slightly lower levels of expression. pBAHA containing the human a-lactalbumin gene driven by the 0.72kb 16 bovine a-lactalbumin promoter had expression levels 17 similar to pHA-l or pHA-2 but a lower percentage of 18 transgenic animals expressed detectable levels of 19 protein. This finding is surprising as the same bovine promoter sequence driving the bovine a-lactalbumin gene 21 gave very poor results (see Example 4 and Vilotte et 22 al; FEBS, ~ol. 297, 1.2. 13-18 ~1992)).

24 Table 2 gives a summary of the relative amount of the transgenic protein. Skimmed milk from these animals 26 was analysed by SDS-PAGE stained with Coomasie blue, 27 isoeIectric focusing, Western blots visualised with a 28 commercial anti-human a-lactalbumin antibody (Sigma) 29 and chromatofocusing. The results from these analyses showed that the transgenic protein was of the correct 31 size, pI and antigenicity when compared to a human a-32 lactalbumin standard (Sigma).

W096/02640 ,~ i 935 1 3 PCT/GB9~01GSI

1 Table 2 3Human a-lactalbumin expression in transgenic mice Mouse Coomassie Western 6 205.19 pHAl 7 204.10 pHAl ++ ++
8 204.7 pHAl +++ +++
9 230.1S.3 pHA1 +++ n.d.
230.15.5 pHAl +++ n.d.
11 230.15.6 pHA1 +++ n.d.
12 230.21.5 pHA1 +++ n.d.
13 230.21.1 pHA1 ++ n.d.

211.18 pHA2 + ++
16 211.17 pHA2 - -17 211.lfi pHA2 +++ +++
18 212.11 pHA2 + n.d.
19 213.5 pHA2 ++++ ++++
212.13 pHA2 ++ n.d.
21 212.19 pHA2 - n.d.
22 213.4 pHA2 +++ n.d.
23 212.7 pHA2 - n.d.

232.10 B5HA
26 233.1 BBHA
27 231.4 BBHA ++ ++
28 232.9 BBHA + +
29 231.9 BBHA - n.d.
232.5 BBHA + +
31 231.3 BBHA + +
32 232.6 BBHA - n.d.
33 237.6 BBHA - n.d.

235.15 OBHA - n.d.
36 235.19 OBHA ++ ++
37 236.6 OBHA ++ ++
38 234.1 OBHA + +
39 234.4 OBHA ++ ++
234.~4 OBHA + +

42 239.~4 BAHA +++ +++
43 239.4 BAHA - n.d.
44 240.7 BAHA - n.d.
239.3 BAHA - n.d.
46 239.6 BAHA +~- ++
47 239.12 BAHA - n.d.
48 243.1 BAHA ++ n.d.
49 242.9 BAHA +++ n.d.
241.16 BAHA + n.d.
51 234.14 BAHA - n.d.
52 243.13 BAHA + n.d.
53 243.10 BAHA - n.d.
54 243.4 BAHA - n.d.

W0~6/02~0 ~193~13 r~ 75,~l65l ~ Z5 2 Table 2 shows the relative levels of human a-3 lactalbumlrl in transgenic mouse milk as estimsted by 4 comparison to proteil- standards on Coomassie gels and Western Blots.

7 - = < 0.5 mg/ml 8 + = ~0.5-lmg/ml 9 ++ = ~1-2 mg~ml ++~ = ~2-3 mg/ml 11 ++++ = ~5 mg/ml 12 n.d. = not determined 14 The results from several mice are shown in Figs. 7 and 8. Fig. 7 shows an SDS-PAGE analysis of skimmed 16 transgenic mouse milk run against a non-transgenic 17 control mouse milk. Fig. 8 shows a Western blot of 18 human a-lactalbumin transgenic milks run against a lg human a-lactalbumin standard.
21 Ex~r~le 6 - Exr~ression of ~utacenised Bovino a-22 Lact.album;n under the control of the Human a-23 Lactalbumin Promoter in vivo Expression of the human a-lactalbumin transgene is 26 considerably higher than that of the native bovine a-27 lactalbumin transgene, reflecting the difference in 28 expression levels of the endogenous bovine and human 29 genes. As this might be caused by differences in the 5- control regior, the 5' region of the bovine a-31 lactalbumin transcriptional start sit.e was substituted 32 with sequences from the human a-lactalbumin gene.

34 Two constructs were made, namely PKU-5 and PKU-l~, which incorporate the amino acid substitutions shown in 36 Table 3.

~ 1 935 1 ~
~'096102640 ~ JI~5I
J ~ 26 1 The follo~ing SEQ ID Nos. have beer. assiqned to the PCR
2 primers used.

4 PKU-1 SRQ ID No. 5 PKU-2 SEQ ID No. 6 6 PKU-2L SEQ ID No. 7 7 PKU-3 SEQ ID No. 8 8 PKU-4 SEQ ID No. 9 9 PKU-5 SEQ ID No. 10 PKU-6 SEQ ID No. 11 11 PRU-7 SEQ ID No. 12 12 PKU-8 SEQ ID No. 13 13 PKU-9 SEQ ID No. 14 14 PKU-10 SEQ ID No. 15 18 In a first cloning step three frayments were subcl.oned 19 into the EcoRI/Eam~I site of pUC18:
21 tl) the Eco~I to PvuI fragment derived by PCR
22 amplification using PKU-primer 7 in combination 23 wit.h 8 (see Figure 10);

2S (2~ the PvuI to ~saBI frayment derived by PCR
26 amplification using PKU-primer 9 in combination 27 'with 10 (see Fiyure 10); and 29 (3) The ~saBI to HindIII fragment derived from pBA.
31 The final construct included 6 DNA fra~ments:

33 (1) the 3.7kb SalI to KpnI fragment contai.ning the 34 human a-lactalbumin promoter derived from ~ clone 4a (Figure 3);

W096/02640 ' '~ r.~ 1f'1 27 ,~l j 935 ~ 3 1 (2~ the 152bp synthetic oligorlucleotide containing 2 human ~-lactalbumin sequences from the KpnI site 3 to the AUG and bovine a-lactalbumin sequences from 4 the AUG to the ~apI site;
6 ~3) the 1.25kb HpaI to ~lindIII fragment from the first 7 subcloning step;

9 ~4) the 0.95 kb HindIII to BglII fragment derived from pBA;

12 (5) the 3.7kb BamHI to X~oI fragment from the 3' flank 13 of the human ~-lactalbumin gene derived from A
14 clone 4a (Figure 3) used as a BamHI fragment; and 16 (6) a Bluescript KS- vector cut with SalI and BamHI.

18 PKU-lH was constructed in the same way as PKU-5 with 19 the exception of fragment (3), which was derived from PKU-1.

22 PKU-] was constructed from six DNA fragments (see 23 Figure 9):

(1) a 2.04kb SstI to HpaI fragment derived from 26 pBOVA-6;

28 (2) a 0.46kb HpaI to PvuI fragment derived from PCR
29 product A (PKU-primer pair 1 and 2; see Figure 10);

32 (3) a 0.60kb PvuI to BsaB-r fragment derived from PCR
33 product B (primer pair 3 and 4; see Figure 10);

(4) a 0.22kb BsaBI to HindIII fragment derived from 36 pBoVA-6;

W096f0264(1 ~j . 2 1 ~ 3 5 1 3 r~

~ A 0.95kb HindIII to BglII frayment derived from 2 pBO~A-6;

4 (6~ the vector pSL1180 digested wlth SstI and BglII.
6 Table 3 8 Amino Acid Substitutions present in Transgene 9 Constructs 11 Substi.tutions Human promoter Plasmid 12 Human 3' flank 13 pos~n 9 30 53 80 14 Tyr, Tyr, Tyr, Tyr + pPKU-lll Ser, Tyr, Leu, Leu + pPKU-5 17 Expression in transgenic mice 19 The two constructs PK~-lH and P~U-5 have been injected 20 into mouse embryos. So far transgenic animals were 21 derived ~or the PKU-5 construct. These animals are set 22 up for breeding to allow milk analysis.

24 E~Amnle 7 - Effect on Lactation bv disruption of ~-Lactalbu~in deficiency and insertion of human a-26 Lactalbumin cene re~lacement in mice 28 Materials and Methods 29 Mouse lines 31 Mice carrying the null ~-lactalbumin allele and the 32 humanised ~-lactalbumin replacement allele were derived 33 by breeding chimeras produced from the targeted 34 embryonic stem cell clones M2 and Fh respectively against Balb/c mates, as described previously 36 (Fitzgerald et al J. Biol. Chem 245:2103-2108). During W096/02640 .~ 29 2 93 r~ .l651 1 the breeding of these strains, ~-lactalbumin genotypes 2 were detenDined by Southern analysis o~ genomic DNA
3 prepared from tail biopsies.

RNA analysis 7 Total RNA was prepared by the method of Auffray and 8 Rougeon (Eur. J. 8iochem 107:303-14) from ~h~l in~l 9 mammary glands of female mice 506 days postpartum.
Northern analysis was carried out according to standard 11 procedures ~Sambrook et al, Molecular cloning). Probes 12 used for hybridisation were: a 3.5kb Bam~l fragment 13 containing the complete mouse ~-lactalbumin gene; and a 14 l.lkb rat ~-casein cDNA (Blackburn et al Nucl. Acids Res 10:2295-2307).

17 RNAse protection analysis 19 32P-CTP radiolabelled antisense RNA was transcribed by T7 RNA polymerase (Promega) from a 455 bp ~indIII-BalI
21 mouse ~-lactalbumin fragment ~Figure 15A) cloned in 22 Bluescript ~S. The conditions for the transcription 23 reaction, solution hybridisation and RNAse digestion 24 were as rec~ ied by Promega. Protected fragments were separated by polyacrylamide gel electrophoresis 26 and visualised by autoradiography.

28 Nilk composition and yield analysis ,29 Milk samples were collected between days 3-7 of 31 lactation under Hypnorm (~oche)~Hypnovel (Janssen) 32 anaesthesia. 150mU of oxyt.ocin ~Intervet) was 33 administered by intraperitoneal injection and milk 34 expelled by gentle massage. Milk fat content was measured as described by Fleet and Linzell (J. Physiol 36 175:15). Defatted milk waa assayed for protein W096102640 .~ l651 ~ 30 ~ ~ 9 ~ 5 7 3 1 (Bradford Analyt. Biochem 72:24a-54) and lactose was 2 measured enzymatically by sequential incubation with l~-3 galactosidase, (Boehringer) glucose oxidase and 4 peroxidase (Sigma) by a method adapted Erom that of Bergmeyer and Bernt (Methods in Enzyme Analysis 3:1205-6 1212).

8 Milk yield was estimated using a titrated water 3 technique described by Knight et al., (Comp. Biochem.
Physiol ~ 127-133) in mice suckling young over a 43 11 hour period between days 3 and 6 of lactation.

13 Milk ~-lactAl' in analysis and quantification Milk samples were analysed on 16% of SDS-PAGE gels 16 (Novex) and western blotted onto lmmobilon P membrane.
17 a-lactalbumin was detected by absorption with rabbLt 18 anti-human ~-lactalbumin antiserum (Dako), followed by 19 goat anti-rabbit LgG peroxidase antibody conjugate and visualised with an enhanced chemiluminescence system 21 (Amersham).

23 ~-lactalbumin in milk samples was quantified by a 24 modification of the method of Lindahl et al., (Analyt.
Biochem 140:394-402) for calcium dependant purification 26 of ~-lactalbumin by phenyl-Sepharose chronatography.
27 Milk samples were diluted 1:10 with 27% w/v ammonium 28 sulphate solution, incubated at room temperature for 10 29 minutes and centrifuged. Supernatant was mixed with an equal volume of lOOmM Tris~Cl, pH 7.5, 70mM EDT~ and 31 loaded onto a column (200~1 packed volume) of phenyl-32 Sepharose (Pharmacia) pre-equilibrated with 50mM
33 Tris/Cl, pH 7.5, lmM E~TA. The column was washed with 34 the same buffer and ~-lactalbumin eluted with 50mM
Tris~Cl, pH 7.5, lmM CaCl~. The optical absorbance at 36 280nm of the column was monitored and integrated peak W096/02640 r~ ;. 1651 ~i935~3 1 areas corresponding to the a-lactalbumin fraction 2 computed. A standard curve was constructed using known 3 quantities of purified human a-lactalbumin from 0 to 4 2.46 mg~ml (Figure 16).
6 .-iiistology 8 Pups were removed for two hours from lactating mothers 9 on the sixth day postpartum, mothers sacrificed and thoracic mammary glands were dissected, preserved in 11 neutral buffered formalin, paraffin embedded and 12 stained with haematoxylin/eosin by standard methods.

14 Results 16 Nouse a-lactalbumin gene deletion 18 A line of mice in which a 2.7kb fragment covering the 19 complete mouse a-lactalbumin coding region and a 0.57kb of promoter has been deleted and replaced with a 2.7kb 21 fragment containing a hypoxanthine phosphoribosyl-22 transferase (HPRT) selectable marker gene was 23 established as described in Stacey et al, 1994 suDra 24 (see Figure 11). Animals carrying this allele are designated a-lac-. The wild type mouse a-lactalbumin 26 allele is designated ~-lac~.

28 Northern analysis of RNA from mammary glands taken on 29 the fifth day of lactation showed that a-lactalbumin mRNA was absent in a-lac-/a-lac- homosygotes (see 31 Figure 12) confirming that the targeted a-lactalbumin 32 gene has been removed and that no other source of ~-33 lactalbumin mRNA exists. Hybridisation of the same RNA
34 samples with a ~-casein RNA in all samples (see Figure 12).

~ 3Z ~ 5 1 3 . ~ ~ 1651 1 ~-Lactalbumin deflciency has no apparent effect in mice 2 other than during lactation. ~-lac-/a-lac- homozygotes 3 and ~-lacm/~-lac-heterozygotes of both sexes are normal 4 in appearance, behaviour and fertility. However, ~-S lac-/~-lac- homozygous females cannot rear litters 6 successfully. Their pups fail to thrive and die within 7 the first 5-10 days of life. Off5pring of homozygous 8 ~-lac-/~-lac- females do survive normally when g transferred to wild type foster mothers. Conversely, offspring from wild type mothers transferred to 11 homozygous a-lac-/a-lac- mothers are not sustained.
12 Table 4 shows that pups raised by ~-lac-/a-lac- mothers 13 are approximately half the weight of those raised by ~-14 lac=/Q-lac~ wild type mice. Estimates of milk yield are consistent with this, ~-lacm/~-lac- heterozygotes 16 produce similar quantities of milk as wild type, but 17 the yield of a-lac-/~-lac- hl ~yyuLes was severely 18 reduced (Table 4).

' Table 4 I Milk composition, pup weight, mammary tissue weight and milk yield in targeted mouse lines Genotype ~-lac=/~-lac' ~-lac=/~-lac- ~-lac-/~-lac- ~-lac=~~-lact' ~-lact'/o-lac~
Fat (~ 28.23 + 1.65(7) 29.6 + 1.3(6) 45.25 + 2.15(6) 25.25 + 1.36(7) 21.2 + 0.23(4)*
v/v ) Protein 87.52 ~ 5.82(7) 95.81 + 9.5(5) 164.63 + 13.92(8)~i 94.51 + 5.97(7) 77.07 + 1.05(4) (mg/ml) - -Lactose 62.44 + 9.27(7) 42.7 + 4.2(6) 0.7 + 0.34(3) 42.40 +1.93(7)56.85 + 3.8(g) ;~-(mM) Single 2.82 + 0.25(8) 3.14 + 0.1(7) 1.52 ~ 0.12(10)~ 2.9 + 0.15(8) 3.4 + 0-75(4) pup weight (g) ~lamnialy 0.34 ~ 0.06(7) 0.4 + 0.1(7) 0.35 ~ 0.05(8)0.31 + 0.04(6) 0.51 + 0.09(4) tissue weight w per pup (g) ,~.
Milk 7.51 +- 0.44(4) 6.7 + 0.38(6) 1.37 + 0.48(4) n.t. 9.94 + 0.65(5)*
yield (g/day) Statistical analysis by unpaired t-test, * p<0.05; **p<0.01; ***p<0.001;
Values are means + S~.
Figures in brackets indicate the num~er of mothers analysed.
n.t., not tested WO~61U264~ 34 ~o~ 93~1 3 P~JI~ III6SI

l Milk was obtained from each genotype hy manual milking 2 and the composition of key components analysed. ~lilk 3 from a-lac=/~-lac-heterozygote5 was indistinguishabLe in 4 appearance from wild type milk and showed fat and protein contents similar to wild type (Table 4). ~hile 6 lactose concentration appeared to be slightly reduced 7 in a-lac~/a-lac- heterozygotes, statistical analysis 8 showed that the difference was not significant. In 9 contrast, milk from a-lac-~a-lac- homozygotes w~s viscous, difficult to express from the teats and was ll markedly different in composition to wild type. Fat 12 content was -60% greater than wild type, protein content 13 was -88~ greater, and lactose was effectively absent.
14 The apparent 0.7mM lactose detected in a-lac-/a-lac-females represents milk glucose content, since the 16 lactose assay used involved the enzymatic conversion of 17 lactose to glucose. Direct assay of glucose in wild 18 type milk indicated a concentration of 1.8mM.

Western analysis of milk protein failed to detect a-21 lactalbumin in milk from a-lac-/a-lac- homozygotes (see 22 Figure 13, Lane F). This was confirmed by phenyl-23 Sepharose chromatography, a technique used to 24 specifically identify a-lactalbumin which has been adapt.ed to obtain quantitative estimates of milk a-26 lactalbumin content (Table 5; see also Figure 16).
27 ~hen~applied to milk from ~-lac-/a-lac-homozygotes no 28 a-lactalbumin was detected. In contrast, a-lactalbumin 29 concentration in ~-lac=/a-lac- heterozygote milk was estimated as 0.043mg/ml, appro~imately half that of 31 wild type (Table 5).

~ W096~026~ 35 ~ t 935 ~ 3 r~ ,~C~l6~l 1 Table 5 2 Milk ~-lactalbumin con~en~.

4 Source a-lactalbumin (mc/ml) Human 2.9 + 0.1(2) 6 a-lac=/a-lac~ mice 0.09 + 0.005(6 7 a-lac-/a-lac-mice 0 (3) 8 a-lacm/a-lac-mice 0.043 + 0.004(5) 9 a-la~=/Q-lach mice 0.65 + 0.07(4) a-lacb/a-lach mice 1.38 + 0.12(5) 12 a-Lactalbumin content of milk samples were estimated by 13 phenyl-Sepharose chromatography.

Values are means t SE.

17 Figures in brackets indicate the number of mothers 18 analysed.

a-Lactalbumin deficiency has no apparent effect on 21 mammary gland development. Table 4 shows that total 22 mammary tissue weights of wild type, heterozygous a-23 lac=/a-lac- and homozygous a-lac-/a-lac- lactating 24 mothers were not significantly different. ~ight microscopic analysis of mammary glands (Figure 14) 26 revealed that heterozygous and homozygous a-lac-/a-lac-27 glands were hi5tologically normal. However, the 28 alveoli and ducts of homozygous glands were distended 29 and clogged with material rich in lipid droplets.
31 Replacement of mouse a-lactalbumin by human ~-32 lac~l' in 34 We have generated mice carrying the human a-lactalbumin gene at the mouse a-lactalbumin locus. The 2.7kb mouse 36 a-lactalbumin fragment deleted at the a-lac- null 37 allele was replaced by a 2.97kb fragment containing the 38 complete human a-lactalbumin coding region and 5' WOg61(1~640 ~ r . ' 36 ~7 ~ 9351 3 ~ '.ilt6~1 1 flanking se~uences. The human fragment stretches from 2 0.77kb upst:ream of the human transcription initiation 3 site to an Eco~I site 136bp 3' of the human 4 translational stop site. Junctions with mouse sequences were made at a Bam~I site 0.57~.b upstream of 6 the mouse transcription initiation site and at an Xbal 7 site 147bp 3' of the mouse translational stop site (see 8 Stacey et al, 1994, ~Y2E~; see also Figure 11). Here 9 we describe our analysis of animals carrying this allele, designated ~-lach.

12 Deletion of the murine ~-lactalbumln gene established 13 that ~-lactalbumin deficiency blocks lactose synthesis 14 and severely disrupts milk production. We have used the ~-lacb allele to test the ability of human ~-16 lactalbumin to restore milk production in the absence 17 of mouse ~-lactalbumin. ~-lac~/~-lach heterozygous and 18 ~-lach/~-lach homozygous mice were normal in appearance, 19 fertility and behaviour.
21 In contrast to ~-lac-J~-lac- mice, ~-lac~ lach 22 homozygous mothers produce apparently normal. milk and 23 rear oftspring successfully. Table 4 shows that pups 24 raised by ~-lac~-lach heterozygous and ~-lach/a-lach hr Gy~ous females are similar in weight to those of 26 wild type mothers. This is supported by our 27 observation that these animals raised successive 28 litters of pups entirely normally. These data 29 constitute clear evidence that the human gene can functionally replace the mouse gene. Analysis of milk 31 composition (Table 4) shows that lactose concentration 3Z is similar in all genotypes. Although both protein and 33 fat concentrations seem reduced in ~-lach/~-lach 34 homozygous animals, only the fat reduction was judged statist.ically siqnificant by unpaired t-test. These 36 animals show an lncrease in milk volume over wild type 37 (Table 4).

~ wOg6/0264n J~ 37 2 1 ~351 3 r~ 3~l651 1 Relative quantification of human and mouse a-2 lactalbumin RNA.

4 ~luman milk contains considerably more ~-lactalbumin (2.5mg/ml) than murine milk (O.lmg/ml). We wished to 6 determine whether the human a-lactalbumin fragment 7 retained a high level of expression when placed at the 8 mouse locus, or assumed a lower level more 9 characteristic of the rrlouse gene. ~-lacm/a-lach heterozygous mice provided an ideal means of addressing 11 this question, as the expression of the human gene 12 could be directly compared with its mouse counterpart 13 in the same animal.

Figure 15A shows the strategy used to compare levels of 16 mouse and human ~-lactalbumin mRNA. Because the 17 junction between human and mouse a-lactalbumin 18 sequences lies upstream of the polyadenylation site, ~-19 lach mRNA contains a "tag~' of 120 bases of untranslated mouse sequences at the 3' end. A uniformly 21 radiolabelled mouse RNA probe was u.sed in a 22 ribonuclease protection assay to detect and distinguish 23 human and mouse a-lactalbumin mRNA in the same RNA
24 sample. The relative abundance of each mRNA was calculated from the amount of label in fragments 26 protected by human and mouse mRNAs.

28 A ribonuclease protection assay was performed and the 29 results are shown in Figure 15B. Lane A shows the undigested 455 base probe and Lane ~ shows that yeast 31 tRNA did not protect any fragments. Wild type mouse 32 RNA protected a fragment consistent with the predicted 33 305 base RNA from endogenous mouse ~-lactalbumin RNA
34 ~see Lane B). Homozygous ~-lact/~-lac}' gland RNA
protected a smaller band consistent with the predicted 36 120 base RNA protected by human ~-lactalbumin mRNA (see 37 Lane C). Lanes D-J show results consistent with a 38 series of hetero7ygous a-lac~-lach animals were W096/02640 ~ ; 38 2 i 9 3 5 7 3 r~~ c 6!, 1 obtained ~see Lanes D to J). Small and large protected 2 iragments in each sample indicate thc presence c~ both 3 human and mouse ~-lactalbumin mRNA. Protected 4 fraqments were excised from the gel, radioisotope content measured, adjusted for the size difference and 6 the ratio of human to mouse ~-lactalbumin mRNA
7 estimated. Table 6 shows the amount of radioisotope 8 present in the 305 base and 120 base fragments excised ~ from Lanes D to J of the gel shown in Figure 15B, and the calculated ratio of human to mouse ~-lactalbumin 11 mRNA in each ~-lac=/~-lac~ heterozygote. It is apparent 12 that, although there was variation between individual 13 animals, human a-lactalbumin mRNA was significantly 14 more abundant than mouse mRNA. Averaging the seven ~-lS lac~l~-lac~ heterozygotes gives a value of 15-fold 16 greater expression for human ~-lactalbumin mRNA.

';
WOg6l02640 ~ ~ 3g ~ 93513 r~ J~il01651 1 Table 6 3 Relat.ive quantification of human and mouse ~-4 lactalbumin mRNA in ~-iacm/~-lach mammary glands S
6 Lane Mouse# 120 base 305 base human/mouse 7 fragment' fragment' RNA ratiob 9 D 2 5957 1000 15:1 11 E 3 5770 547 26:1 13 F 4 4825 810 15:1 G 76 6018 1077 14:1 17 H 98 5206 1452 9:1 19 I 99 5481 1117 12:1 21 J 79 26858 3561 19:1 26 Lane designations indicate the source of protected 27 frag~ents and correspond to those shown in Figure 15B.

29 a. numbers are expressed in counts per minute (c.p.m.) 32 b. Ratio between c.p.m. of 120 ba.se fragment 33 multiplied by 2.54 (to adjust for size difference 34 and c.p.m. of 305 base fragment.

9 ~ 5 1 3 W0 ~2640 1 Human a-lactalbumin protein expression 3 A Western analysis of ~-lactalbumin in targeted mouse 4 lines was conducted. Human ~-lactalbumin can be distinguished irom mouse ~-lactalbumin boy its faster 6 electrophoretic mobility (see Lanes A, L). A prominent 7 lower band in a-lach/~-lach homozygotes and ~-lach/~-lacm 8 heterozygotes was observed (see Lanes C, D, G, H), and 9 corresponds to the position of the human ~-lactalbumin standard and was only observed in mice which e~press 11 human a-lactalbumin generated either by gene targeting 12 or by pronuclear microinjection ~data not shown). This 13 identity was confirmed by phenyl-Sepharose 14 chromatography (See Figure 16) and analysis of peptides lS released by cyanogen bromide cleavage (data not shown).
16 The band with slower mobility, similar to mouse a-17 lactalbumin, is also a human ~-lactalbumin gene product 18 the nature of which is unknown. This species varied in 19 intensity with ~-lacb gene dosage (see Lanes G, H) and was also present in milk from human ~-lactalbu~in 21 transgenic mice generated by pronuclear microinjection 22 (data not shown).

24 The ~-lact~lh~lmin content of milk samples was quantified by phenyl-Sepharose chromatography. Figure 26 16 shows superimposed absorbance profiles of column 27 eluates of three illustrative milk samples including 28 the ~-lac=~-lach heterozygote and ~-lac~/~-lac~
2Y h~ z~ote shown in Figure 13. The peaks corresponding to eluted ~-lactalbumin are marked. ~-Lactalbumin 31 contents were estimated by comparing the integrated 32 peak areas with the human ~-lactalbumin standard curve 33 shown. The relationship between integrated peak area 34 and ~-lactalbumin quantity was linear and highly reproducible. ~-Lactalbumin content for the samples 36 shown in Figure 16 were estimated as follows: ~-lacm/~-37 lacm wild-type O.lmg/ml; ~-lacmi~-lach heterozygote #76 38 0.45mg~ml; ~-lachi~-lach homozygote #22 8.9mg~ml. Table ~ W096~26~ ~ 41 ~ 7 935 ~ 3 r~ Jl~l65l 1 S shows the concentration of a-lactalbumin in milk 2 samples from targeted mouse lines an~ lactating women.
3 It is clear that the concentration of a-lactalbumin in 4 milk is directly related to gene dosage, eg a-laP/a-lac- heterozygotes shown an a-lactalbumin concentration 6 half that of wild type- Given that the volumes of milk 7 produced by these mice are similar (Table 4), the 8 concentration of a-lactalbumin provides a reasonable 9 indication of the quantity synthesised. The relative proportions of human and mouse a-lactalbumin c, o.lents 11 in a-lac=~a-lach heterozygote milk were estimated by 12 assuming that a-lactalbumin expression from a single 13 mouse allele was 0.043mg/ml and the rest represented 14 human a-lactalbumin. This is consistent with the amounts of a-lactalbumin expressed by a-lac~/a-lac-16 heterozygotes and wild type mice. Therefore, a-lac=/a-17 lac-heterozygotes were estimated as expressing 18 0.61mg/ml human and 0.043mg/ml mouse a-lactalbumin.
19 Thus, human a-lactalbumin is approximately 14-fold more abundant than mouse a-lactalbumin in a-lac~/a-lach 21 heterozygote milk. This is remarkably consistent with 22 the relative proportions of mRNA.

24 ~Y~le 8 Enhanced ex~ression of a heteroloaous cene.
26 These data confirm that the upstream promoter region 27 (AUG to about -3.7 kb) which is included in the pHA-2 28 construct enhances expression of a heterologous gene.
29 Table 7 shows the results of milk analysis from pHA-2 transgenic founder females. Out of 10 females, 6 31 animals expressed high levels of human a-lac. 3 32 animals failed to express detectable levels of human a-33 lac (less than 0.2 mg/ml in this assay~, all 3 also 34 failed to transmit the transgene. ~e can neither be certain whether they were low expressors or not 36 transgenic.

W096l02640 ~ 4~ 2 I q3~ l 3 P~ 1651 1 Table 8: Constructs PKU-O to BALT-B all contain the 2 Lovine ~-lac promoter ~about 2kb). Constructs PKU~
3 to PRU-16 all contain the human ~-lac promoter (3.7kb).
4 Using the human ~-lac promoter increased the expression of the transgene to almost 100%.

7 These data show that the use of the human ~-lac 8 promoter achieves a higher level of expression than the 9 use of the bovine promoter and inducefi expression in more animals than the bovine promoter.

CONSTRUCT NOUSE SEX C mg/ml TRANS. MI FREQ.
MILK
~ALYSIS
pHA2 210-17 M - - 1~21=5%
211-12 F ~1 - - 14/54 =26 211-16 F 10 5 3~4 211-17 F ~>10 ND 0/8 212-7 F 10 ND 0/5 8~77=10 212-11 F <10 1 0~4 212/13 F ~1 1-5 2~4 212-19 F >10 ND 0~3 213-4 F C10 5 0~2 2~14=14&
213-5 F 10 10 2~8 OVERALL MIF 25/166=15%
ND = Not detectable TBA = To be analysed TBO = To bred on C = Copy number TRANS. = Transmission MI FR~Q. = Integration frequency WO96101640 ~ 43 Z 1935 1 3 r~ l016SI

T~3LE 8 Constr. Transgenics Expres5ers max. expr.
PRU-0 6F/5M 3~5 500 PKU-l 18F~23M 5/18 200 PRU-2 8F/7M 3/8 goo*
PKU-3 6F/5M 2/6 100*
PKU-4 7F/3M 5/18 30U*
8ALT-A 34F/24M n.t. n.t, BALT-B lOF/18M n.t. n.t.
PKU-lH 6F/5M 4/5 100 PKU-7 4F/llM 1/1 <20 PKU-16 13F/12M n.t. n.t.

HaPKU-l n.t. n.t. n.t.
HaPKU-2 n.t. n.t. n.t.

n.t. = not tested * estimate W09610264~ ? 1 935 ~ 3 PCI'IGB9~1016$1 SEQUEtlCE !,}STII; .

~1) GENER~L INFOY~ATION:
~1) AppLIcAr3T
A) NAME: PPL THERAPEUTICS ~SCOTLAtlDj LIMITED
~Bj STREET: RO5LIN
IC~ CITY: EDINBUFGH
~E) COUNTYY D'NITED ~INGDOM
~F) POSTAL CODE. ~ZIPj: EH25 9PP
iij TITLE OF IN;ENTIO?~: Alph2-Lactalbumin Gene Cor.structs ~li.) NUMEER OE SEQUENCES: 21 ~iv) COMPUTER READABLE FORM:
~A) ~SEDIUM TYPE: Flcppy disk ~B) COMPUTER: I8.V. PC compatihle ~') OPERAT;tlG SYSTE!~: PC-DOS¦~S-DOS
~ù~ SOFTWAP.E: Pasor.~Ir Release 1 0 version 1.30 ~EPO) ~2) INFOR~T;ON FOR SEQ ID NO: 1:
111 SEQ~E?3CE cHA~cTERrsTIcs ~A) LENGTH: 27 base pairs ~E) TYPE: nucleic ac id }C! STRANDED?3ES5: sinsle ~Dj ropoLoGv: 'incar ~is) VOLECUI.FA TYPE: cDNA

(xi) 52QUENCE DESCRIPTrO?!: SEO ID NO: 1:

~2j IriFo~yATIorl FOP. SEQ ID NO: 2:
~i; SEOUENCE CHAP~CTERISTICS:
~A) LENGTH: 35 base pairs ~B) TYPE: nUcle~c acld ~C~ STRANDEDNESS: single ~D~ TOPOLOGY: lsr.Oar (ii) MOLECULE TYPE: cDtA

(xi) SEQUENCE DEscRrpTIo?! SE~ ID NO: 2:

SUBSTITUTE ~HEET ~ULE 26J

~ W 096/02640 ' '~ r~ .'O1651 4~ ~ j93~1 3 CATGGATCCT GGGTGGTC,.T TGAA~CGACT GATCC 35 12) }NFOQ~ATIo~ FOR SEQ ID NO: 3:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 43 base pairs (B) TYPE: nucleic acid (C1 STRANDEDNESS: sinyle (D) TOPOLOGY: llnear (ii) MOLECULE TYPE: cDNA

(xi) SEQUENCr DESCRIPTION: Si'.Q ID NO: 3:

(2) INFOR~ATION FOR SEQ ID NO: 4:
(i~ SEQUENCE CHAQACTER}STICS:
(A) LENGT.H: 33 Dase pa.rs (a) TYPE: nucleic acid (C) STQANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA

(xi) SEQDENCE DESCRIPTION: SEQ ID NO: 4:

(2) INFORMATIoN FOR S-Q ID NO: S:
(i) SEQUENCE CHAQACTERISTICS:
(A) LE~GTH: 46 base pai-s (B) TYP2: nucleic acid (C) STQANDEDNESS: sinyle ~D)-TOPOLOGY: linear (ii) MOLECULE Typr: cONA

(xi) SEQUENCE DESCRIPTTON: S--Q ID NO: S:
GCTGAATTCG TTAACAAAAT GTGAGGTGTP. TCGGGAGCTG AAAGAC 46 (2) INFOP~ATION FOR SEQ ID NO: 6:

SUBSTITUTE 5HEET (RULE 263 W0 9~l02640 ' ~ ~s~ ; 4~ 9~5 ~ 3 r~ ;5~ol~sl EQuEr~cE CHAPP~CTrP.I ST rcs:
~A; LENGTH 58 base pairs ~Bj TYPE nucleic acLd ~c) STRANCEDN_~S~S: singLe (D) TOPOLOGY- iinear ~Lij MOLECULE TYPE cDNA

~xi) SEQUENCE DESCRIPTION: SEQ ID NO 6:
GCGGATCCCA TCGCTTGTC.T GrcATAAcc~ CTGGTATGGT ACGCGGTACA GACCCCTG S8 (2j INFOP~L~TION FOR SEQ ID r:o: 7:
(l~ SEQUENCE cHARp~cTERIsTrcs (A~ LENGTH: 58 bAse pairs (~ TYPE nucleic aclrl ~Cj sT~p-ANDEDr1Ess slngle ~D) TOPOLOGY linear (ii) UOLECULE Typr cDriA

(~i) SEQUEN~;E DESCRI2TION SEQ ID ~lo GCGGATCCGA TCGCTTGTGT GTCATAACCA CTGC-.. TGGA GCGCGGTACA GACCCCTG 58 ~2) IrlFoRuATIoN FOR SEQ ID UO: 8:
~i) SEQUENCE CHAP~CTERISTrcs ~A) LErlGTH: 69 Dase pairs ~Ej TYPE ruclele acid ~Cj sTpAriDED~Ess single ID) TOPOLOGY line~r (li) UOLECULE TYPE: cDNA

~xi) SEQuEricE DEscRIpTroN SEQ rs NC a GCGGATCCGA TCGTACA~AA CAATGACAGC ACAG.;.;T.A~G GACTCTACC.; GP.T.~AT.~P.T 60 AAAATTTGG s9 (2~ INFO~ ATION FOR SEQ ID NO 9 ~L) SEQUENCE cHAp-AcrERIsTIcs SUBSTITUTE SHEET (RULE 26) W096~2640 ;'' ,~ 1651 4~ ~- I q~5 ~ 3 (~) LENCTk: 34 b_se pairr (3) TYPE: nucle~c ~cid (C) STRANDEDNESS: single (D) TOPOLOCY: linear ~ii) MOLECULE TYPE: cDNA

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 9:
GCTCTAGATC ATCATCCAGG TACTCTGGCA GGAG ~4 (2) INFORMATION FOR SEQ ID No 10 (i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 24 base pairs (S) TYPE: nuclelc acid IC~ STRANDEDNESS: single ID~ TOPOLOGY: l~near (ii) MOLECULE TYPE: cDNA

(xl) SEQUENCE DESCRIPTION: SEQ ID rlo: lo:
GCTGA,.GCTT C~CTTACTTC AC-C 24 (2) INFOP~ATION FOR SEQ I3 NO: 11:
(i) SEQUENCE CHARACTERISTICS:
(.~l LENGTH: 65 base pairs (31 TYPE: nuc!e:c acid (C) STRANDEDNESS: sinyle (D) TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA

(xi) SEQUENCE DZSCRIPTION: SEQ ID NO: 11:
GCGGATCCAA AGACAGCAGG TGTTCACCGT CGACGACGCC TACGT.3ACTT CTCACAGAGC 60 (2~ INFORMATION FOR SEQ ID NO: 12:
(i) SEQ"ENCE CHARACTEP.ISTICS.
(Al LENGTH: 46 base pairs SU~STITUTE SHEET (RULE 26) W 096/02640 . .' 2 i ~3~ 1 3 PCT/GB95/ot6Cl ~ ~ 48 lB ! TY?E: nucLeic ac~d ~CI STRANDEDNESS: s~ngle ~D) TOPOLOGY: linear (LiJ HOLECULE TYPE: cDNA

(xi) SEQUENCE DESCR~PTIOI: SEQ ID NO: 2:
GCTGP~.TICG TT.~.caia.AT GTGAGGTG.~G CCGGGAGC-G AAAGAC :5 (2) rNFoR~lATloN FOR SEQ ID NO: 13:
(i1 SEQUENCE CHARACTEP~ISTICS:
~A) LENGTH: 54 base ralrs (B1 TYPE: nucLeic acid ~C~ STRANDEDNESS: s:ngle (D! TOPOLOGY: linesr (ii) l~OLECUL~ TY:-_: cDNA

(XL) SEQUENCE DESCRIPTION: SEO I3 l30: 13:
GCGGATCCGP mCGCTTGTC.T GTCAT.~CC. CTGGTPTG.~.? P.CGCGGTP.CA GACC 54 (21 }NFOR.~TION FOR SEQ ID :IO: 14:
(i1 SEQUENCE CHAR~CTERISTICS:
(al LENGTH: 6g basc pairs (?) TYPE: r.ucieic acld (~I STRANDEDNESS: s!ngle ~D! TO?OLOGY: linear ( Li 1 HOLECULE TYPE: cDNA

(xi1 SEQUENCE OESCP.IPTION: SEQ ID NO: 14:
CCGGATCCGA TCGTACA~A CAATGACAGC ACAGAATAmG GACTCCTCCA GATAAATAAT 60 A~AATTTGG 69 ~2) TlJFoRl~ATIoN FOR SEQ ID NO: iS:
(i) SEQUE~JCE C~:P.RACTERISTICS:
~A! LENGTH: 34 base raLrs (B) TYPE: nucieic acid SUBSTI~UTE SHEET lRVLE 261 W 096/02640 '~ 5 ~ 3 r~,5. 1651 (Cj sTRArJDEGtJEss singLe l~) TOPOLOGY: linear (ilj MOLECULE TYPE: cDNP, (xl1 SEQUENCE DESCP~IPTION: SEQ rD rlo: lS:

(2j INFORMATrON FOR SEQ ID rlo: 16:
(:) SEQUENCE C.:APiCTERISTrCS:
(A~ L-NGT~: 264 base Fairs (E) TYPE: nucleic acid (C) STRANDEDNESS: double (Dl TOPOLOGY: llnear (il) /MOL.ECULE Typr: cnNA

(Xl) SEOUENCE DESC?.IPT}ON: SEQ ID NO: 16:
GGATCCAAAG TTGGCT.~AAC ACTGGCCGGG TGCAGTGCTT CCACCTGTAA TTCCAGCACT 60 TTGGAAGGCT GAGGTGGGCA Gr.TTGCTTGA GGTC.'!GG.i_r T-GP.G~CCAG C?TGGCTAAC 12C
AGCAAPACCC TGTCTCTACC .~ AGTACAA P.AP.T-ATCTG GGI~ ~u CAGGCGCCTG 13G

~2) IrJFoR~ATIoN FOR SEQ ID NO: 17:
(l) SEQUENCE C~ARACTERISTICS:
~A) LENGT.-': 303 base p2Lr~
(B~' TYPE: nucleic acid (C) STRP.NDEDNE~SS do~bL~
(D) TOPOLOGY: li~ear (ii) MOLECULE TYPE: cDrJA

(xi) sEQuErJcE D~5CP.LPTION SEQ Ir~ NO 17:
TCTTTTTCAA TTATTCATTT GTTACAGTGG GTTATGATAC .iAATGTTTAT AGATGCCTAC 6C

SUBSTITUTE SHEET (RULE 26) W 0~6~2640 ,; ,~ , 2 1 93~13 r~ 7."~165 5'~
TCTGTACTAG TACT.ACACAG CAC.-TT.CT GTG,TTT.- - .;T T5ACTTCAA- TGT,;CICrC: 12~.
TGAGTTCTAT W-v.~r.r~T'_CAT GTATTAAATC, AAATAAAcr~r~ hCAAAATGCC ATGTTCTTTG 180 GTACAAGCAA CACTCACCh~; AGGCATTTGG CGTCTGCATT TGCAATTCTC ACCC,AAACTC 240 L ~1 U CTACTCTCTA C.TATTTTCC CCACACTACC TT.rTCTATAT ATATTTTTCA 300 GATTGGAGTI ~UOU~I~LL~ CCCACGCTC~G AGTCCAGTGG CACCATTCTT GGCTCACGAG 360 ACCTCCACGT CTTGGGTI'AA AGCGTTTC-C CTCCCTCACC CTCCTGACTA CTCCGATTAC 420 AGGCGCC-GC CACCATGCCC GGCTP~;rTrT TGTATTTTT.r. GTAGAGATGG GCTTTCACCA 460 TGTTGCTC~C CCTGCTCTTG A~;CTCC,CCP. CCTCGGCCCT TCCCA~-GC GCTCCGATTA Sq0 CAGGTGTGAG CCACACTGCC TCGCCTGTP.C ATTTTTTAAA TTTCP.ATGTC TA.rTATGGTG 600 TCCACTGP'~T TAAGAATTCT TTTGAGAAAA TGAATCAAT.r. PATCTATACA OL~1UUII 660 TArCCAGTGA GGTATGGCTG G.;TC.'.GCTTC ATGACAT.~C.r. -GCCAG.ACT TcTcCrcCTC 72C
~ .L~ l ACAAATAAhA AT-GTATP.TG TTGAACG-CT .'.CAACTTG.riT GTTTGTTATP. 730 ~2) INFOR~rTION FOR SEQ D IIO: la:
(i) SEQUE~ICE C'rlARACTE~ISTICS:
(A) LENGT~: 233 base prlirS
~3) TYPE: r,uc lo ! C acLd (C) STPUJDEDNESS double ~D) TOPOLOGY: llnear (li) MOLECUEE T'YPE: cD~lA

(~i) SEQUENCE DESCPI?TrOrl: SEQ ID NO: lB:
TTTGCGTP.GA ACrCAGACAGT AAACTTGCTG Il_l~LI~_~ C.. G.ArCTTTT GTTGAGP.TGC 60 TGAATAGGAG GCAGCATGGC AGCTGAGCTA TCTGTTCT5C ---CTCTACC ru~rrl~i~ i2D
TCCCTTAGGC cT~r~AAATGAA GCTCTAAGCC PAGCA.~CGT C-GAAGTCAT CCAGAcTAr.~ lB0 IGGGAAGCGG GTAGGCTCCA GGGAGTGGCT CTCAGAGA:C AGACCP.TTTA CTGAGCTC 338 r2) INFOI~ATION FOP SEO ID NO: 19:
~-! SEQUENCE Cr.A'.~CTEP.ISTICS:
'A) LENGTP:: 162 ba66 pa~rs (B) TYPE: nucl~c acid SUBSTITUTE SHEET (RULE 26) W 096/02640 . ~ - ' r ~,5!01651 ~ 5~ 3 5 ~ 3 ~C1 STRAr~DED~IESS ioubl.
(rj TOPOLOCY: llnear ~il) MOLEC~LE TYPE: cDNA

(X1) SEQUENCE 3ESCPIPTION: SEQ ID NO: 19:
AATACAGACT l L1~1 L~l - - ACTCLTATCC I~CL L I~L-;~ TCCCTCCTAC lLlL.~ ~A 60 CACCTATCTT GTTGTGAAGA CAGG~TTGC ATT.~CATAAA ATCA~ TCTT TTTTATTTTT 120 TTTTGAGATG GAATCTTGCT CTGTTTCCAG CCTC.GAGTGC TG162 (2) INFORMATION FOR SEQ ID NO: 2D:
(i) SEQUEN'CE CHARACTERrSTICS:
A) LENGTH: 472 base pairs ~3 j TYPE: nuc!eic aciù
~C) STRANDEDNESS: doub ie ~D) TOPOLOGY: !:~ear (ii) MOLECULE TYPE: cDN.'.

(x~) SEQ'uENCE 3ESCRrPTrO';: S~Q I3 NO: 2G:
ATCTGGTCAG CAGTGAAGCT CAGTGTACAC ATTCP.TTCCT TCCTI'CACTG CTTGATTTGT 60 CACCAAGTGG TTATTGAGGA TATGCTG-TT GCT.9GGTACT ACTTTACTTA TTTATTTGTT 120 TATTTAGAGA TGGGGTCTCr CAATGTTGC;' CAGTCTACAG GAC.;GTGGCT P.TTCACAGGT 180 TCTCGAGTAG CTGGGACTAC AGGGP.TGTGC CACCACACAT GGCTTAGGCT CTACTTTAGC 300 TGCTACTTGA AGGATGAAC.A T.;GGAGGAGA CACTCTTATT TT.;TTTGATT Ll_Ll L 1 ~1 1 1 36G
111~L~IL. L TTGACAGAGT TTTGCTCTGT TGCCAGGCTG GAGTGCTCAC TGCAACCTCC 42D
ACCTCCAGGT CAAGCPATTC TCCTGCTCAC CCTCCGAG-A GT''5GACCAA GG 472 ~2j INFORA~ATI3N FOR SEQ ID NO: 2!:
~i) SEQUENCE CHAP..;CTERIS-ICS:
(A) LENGTH: 2}1~ base pairs ~31 TYPE: nucLe1c acLd (Cj STRANDEDNESS: double (D~ TOPOLOGY: llnear SUBSTITUTE SHEET ~RULE 26~' WO 9~il0264 , " t ~ 52 ~ I 5 3 ~ ~ 3 ~ ~ "~ O~

~ MOLEGULE TLPE: C:DI~P.

(x13 SEQUE~CE DESC~I~TlOII: SEC ID NO: 21:
GGAATTCCCA LL~-L~I-L~I GTACCCTTGC AGTGCCTCTG GGTGCAATGC GGAGA'~ATGG 60 AGTGGCTCC.A Oll~L~IL~I GTTTCTGP-~C ATG?~TCTCT TGCTATC.~GA ACTTTCTGCT lZ0 CATCCCTTCT GGCACACC-3~- G~TCCTCCAC A~TCCCTTCA CTCATGCC.;C TTCATATACT 180 GGTTATCCAT GGTACAGA.~G ACAGG~TTT.3'. ACTGAGAGGA ~111 - -L IG ACI'CTGAATA 240 CATGTAGGAG hT.3'ACGATAT GG~AGACCTT CAGTATGTPA GTC?TAAAT.~ GATTGGTTGG 3GO
GATAAATGTT CCCTGAA9CA TAAGPAACAG CGCAC~CGGCT CCTGTCTGTA ATCTAGCACT 360 TTGGGAGGGC CGAGGCCAGG C.'LGGCAAATT GCCTGAGCTC AGAAGTTTGA GACCAGCCTG 420 GCCAACATGC AGAAACTCCG TCTCTACTAA AAATACATA.9 ATT.3ACCGC.G CATGGTAACA 480 CGTGCCTGTA GTCCCAGCTA CTCGGGAGGC TGP.GCCAGGA GAATCACTTG AGCCTGGGAG 545 GCAGAGGTTG CAGTGAGCCA AGATCGCGCC r.CTGCATTCC AGCCTGGGCA ACAG.'.GTGAG 60Q
ACTTGGTCAA AAAAAAAA.AA A,3AA.3'A3LAAA AAP.LGGAAGA AGAAGAAGAA P.TCAGGTTTA 660 GAG.;TGAGG.A CP9AGAAG.'C GAATCGGTGG CATGAAGG;LG CTAAGAGC?A C-TGTCACCA 720 TGACATGAAG CTTCATGCCA GC.3'..AATTAA.'. GGAGCTA?TC P.GAACTAGTA TCCTC.3U~CTC 780 TACTTGCTC.i GGGGCACTGA CCTTATAGhG ATTCCAGACA TAAGCTTGTT CAGCCTTAAG 840 TCCALTCTTT CCACTGGCTT ~L - _I L~'~ ACTT?CTGTG GCCAACTCTG AGGTTGTCTA 900 CAAGTTATTG GTCTTAGATT TATGTAATGT CTCAATGCCA GTGTAGTAT? TGCTTATTTA 960 LLLLlLI L ! TTTTTTTT.~A AGATAGGC-TC TCACTTTGTC TCCCAGGATC- GA?GGATGGA lG80 GTGCAGTGGA GTGPACATGG C?CACTGCAG CCTCGP.CCTC CTGTGCTCAA ~ ~ ~OL~ 1140 TGCCTCAGCC CCTCAAGTAG CTGGGACTA.C AGGC.AC;L.LC? CACCATGC.-C AGCTA.3rTTT lZ50 TTTTGTAG.3G ATCGC-P.TT?T ACCArGT?GC CCP.GGCTGG- CTCGAGCTCC TGGGCTCAAG 126;J
TGATCCACCA GACTCGGCCT CCCA.4AATGC CGGGATT.~CA GCTGTGAGCC ACTGTGCCTG 1320 GCCTAGATGC TTTCATACAG GCTTTTCA.AT T.'TGCAT'rrr CCTTAAGTAG GL~GTCTTAA 13a5 GATCCAAGTT ATATCGGATT GTTGTAGTCT ACGTTCCCAT ATTCTATTCC TP.TTTCTGAG 144.l SUBSTITUTE SHEET ~RULE 2ti) ~ W 096~02640 2 t 9 3 5 1 3 1~ a~OlG .

CCTTC,jGTC.i TGAGCTAC ~ TT ~;Gr~ CT.'_;~.T.'.~ C_TTC...'.C .'TGG_TGG.:T i;OJ
TGGTTGGACA P.GTGCCAGCT CTC~A~CCTGG GACTCTGGC.; TGTGATGACA TACACCCCCT 1560 CTCCACATTC TGCATGTC C TAGG'GGGAA GGGGGA~C. '_GG.ATAGAA CCTT;.iTT'? 7620 ATTTTCTGAT TGCCTCAC.T CTTA~--TTGC CCCC..TGCCC ~.L L !~L ! - CTCA'.GTAAC 1680.
CAGAGACAGT GCTTC.CC.9G; ACCA.CCCT.~ C.~.~GA.;.~C.;. .~GCGCTP.AAC A.iAGCC,'u~iT 1740 GGGAAGCAGG ATC.iTGG-TT GA-~C~C-TTC TGGCC.iGiGr rC..~ATACCTG CTATGGAcTA 1800 GAT.;CTGGG.i GL.GGG.~.iGG .~i.~AC ;GGG TG---}.T-'TGG ;';GGA.iGCTG GCAGGCTCAG 1860 CGTTTCTGT_ TTGGCATG.':C C.~GTC CTCA TCA ICTCTT C'T.'.G.'TGIA GGGCTTGGTA 19~0 CCAGAGCCCC TGAGGCTTTC -GCATG.~.'; A T.~'T.~A~TG AA~'TGAGrG ATGCTTCCAT 1980 TTCAGGTTCT TGGGGGT.~GC C.'Ar..-.~.iGC TT'.TTG C~ CTCTCT-CCT CC'TGGGCA-C '04G
~ C_,;TCCTC~ .'C.~.................... '.. :~~~~ . ~_~-.; C~GC-G.~ 0 GAC,iTAGA.C. G-!.i-GG.i~ -L119 SUBSTITUTE SHEET (RULE 26) W096/02640 ~ ~ r P~ Iru._ !rnl651 j4 2 ~ ~3 ~ ~ 3 lNDlCAI lONS }3~:LA~ING TO A DEPOSITED hnCROORGANIShl ~PCI Rule 13bis) A . Thc ind~ ons rrl~de below telu le to the ~ relerred to in ~be descripBon on p~ge 9 , iine 13-1 S
B. }DENT~iCATlON OF DEPOSIT Furtber deposits 9rc idtnrified on ~n ~ddi~ional shce~ iO
Name of deposiary institutiun NATIONAL COLLECTIONS OF INDUSTRIAL AND
MARINE BACTERIA LTD
Address of deposiurry insdtution (i~:tlulinl~partalco/tcarutcou~urr) ABER,DEEN AB2 lRY
SCOTLAND
UNITED KINGDOM
D~e of deposil ¦ Accession Nutnber 15 FEBRUARY 1995 ¦ NCIi'~B 40709 C. ADDITIONAL ~DICATIONS flaavcbtariifrclapplicablcl This inform~tion isi continued on~n ~dditionrl sheet O

~.~rH~RTrHTA COLI DH50~ F' (K12) CONTAINING PLASHID pHA-2 D. DE.SlGl~ATErl STATES FOR WEIICII INDICATIONS ARE .5~DE fiJrtusirulicuiorlrarcr~tforaUreri~atct~r~s ALL STATES

E. SEPARATE FURNlSnTNG OF INDICATIONS ItarYc bu~u4 ilnGI applieablc~
Thcindirttionsiisledbeiowwillbesubminedtotb-lnt-rnalionslBuraul~ter(.~ b~ln~uurcofrhc~ri~ucarior~e~ Aecr-rion NuntbsrrfDapcrf~ j AC OE SSION ~iO : 40709 DATE OF DEPOSII' : 15 FEBRUARY 1995 CONSTRUCT DEPOSITED : pHA-2 i'or receivin~ Of ~ce use only For Intetlurionul Bure-u use only Thi5 sbee~ WIS reaived wilh Ihe intern~lion~l IppliGttion O This shect wss rcceivcd by the Intern~tion~l Burau ort:
~11 SEPTEMBER 1995 Authori~cd of ~Iccr AutboriIcd of Bs es D. J. MAcKERNEss tMRSl ortn PCI/ROtl3~ (Julv i992)

Claims (34)

1. A recombinant gene construct adapted to express .alpha.-lactalbumin or a functional equivalent or part thereof in cells of a bovine animal.

2. The construct as claimed in Claim 1 adapted to express human .alpha.-lactalbumin or a functional equivalent or part thereof.

3. The construct as claimed in Claim 1 adapted to express bovine .alpha.-lactalbumin or a functional equivalent or part thereof.

4. A vector containing the recombinant gene construct as claimed in Claim 1.

5. A host cell containing the vector as claimed in Claim 4.

6. A transgenic animal having the construct as claimed in Claim 1 integrated into its genome.

7. The transgenic animal as claimed in Claim 6, said animal being capable of transmitting the comprising to its progeny.

8. Transgenic cattle as claimed in Claim 6.

9. A method of producing milk having an enhanced content of .alpha.-lactalbumin, said method comprising extracting milk from a lactating female transgenic animal as claimed in Claim 6.

10. Milk produced according to Claim 9.

11. .alpha.-lactalbumin extracted from milk according to Claim 10.

12. A recombinant gene construct adapted to express .alpha.-lactalbumin or a functional equivalent or part thereof, said construct having a flanking sequence selected from the group consisting of:

a. the 3' flanking sequence of any one of SEQ ID
Nos. 16 to 20;

b. the 5' flanking sequence of SEQ ID No. 21;
and c. parts of such sequences.

13. The construct as claimed in Claim 12 having as a first 3' flanking sequence a sequence selected from the group consisting of the sequence of any one of SEQ ID Nos. 16 to 20 and a substantial portion thereof, and having as a second 5' flanking sequence a sequence selected from the group consisting of the sequence of SEQ ID No. 21 or a substantial portion thereof.

14. The construct as claimed in Claim 12 adapted to express human .alpha.-lactalbumin or a functional equivalent or part thereof.

15. The construct as claimed in Claim 12 adapted to express bovine .alpha.-lactalbumin or a functional equivalent or part thereof.

16. A vector containing the recombinant gene construct as claimed in Claim 12.

17. A host cell containing the vector as claimed in Claim 16.

18. A transgenic animal having the construct as claimed in Claim 12 integrated into its genome.

19. The transgenic animal as claimed in Claim 18, said animal being capable of transmitting the construct to its progeny.

20. Transgenic cattle as claimed in Claim 18.

21. A method of producing milk having an enhanced content of .alpha.-lactalbumin, said method comprising extracting milk from a lactating female transgenic animal as claimed in Claim 18.

22. Milk produced according to Claim 21.

23. .alpha.-lactalbumin extracted from milk according to Claim 21.

24. A recombinant genetic construct selected from the group consisting of pBBHA, pOBHA, pBAHA, pBova-A, pBova-B, pHA1, pHA2 and constructs derived thereform.

25. A transgenic animal having the construct as claimed in Claim 24 integrated into its genome.

26. The transgenic animal as claimed in Claim 25, said animal being capable of transmitting the construct to its progeny.

27. Transgenic cattle as claimed in Claim 25.

28. A method of producing milk having an enhanced content of .alpha.-lactalbumin, said method comprising extracting milk from a lactating female transgenic animal as claimed in Claim 25.

29. Milk produced according to Claim 28.

30. .alpha.-lactalbumin extracted from milk according to Claim 28.

31. A polynucleotide having a sequence selected from the group consisting of:

a. the sequence of SEQ ID No. 16;

b. the sequence of SEQ ID No. 17; and c. parts of such sequences.

32. A recombinant genetic construct containing the polynucleotide of Claim 31.

33. A transgenic animal containing the construct of Claim 32.

34. Transgenic cattle as claimed in Claim 33.