EP0765390A1 - Alpha-lactalbumin genkonstrukte - Google Patents

Alpha-lactalbumin genkonstrukte

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Publication number
EP0765390A1
EP0765390A1 EP95924467A EP95924467A EP0765390A1 EP 0765390 A1 EP0765390 A1 EP 0765390A1 EP 95924467 A EP95924467 A EP 95924467A EP 95924467 A EP95924467 A EP 95924467A EP 0765390 A1 EP0765390 A1 EP 0765390A1
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EP
European Patent Office
Prior art keywords
lactalbumin
milk
human
construct
lac
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Withdrawn
Application number
EP95924467A
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English (en)
French (fr)
Inventor
Julian Cooper
Angelike Schnieke
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PPL Therapeutics Scotland Ltd
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PPL Therapeutics Scotland Ltd
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Filing date
Publication date
Priority claimed from PCT/GB1994/001514 external-priority patent/WO1995002692A1/en
Priority claimed from GBGB9425326.7A external-priority patent/GB9425326D0/en
Priority claimed from GBGB9503822.0A external-priority patent/GB9503822D0/en
Application filed by PPL Therapeutics Scotland Ltd filed Critical PPL Therapeutics Scotland Ltd
Publication of EP0765390A1 publication Critical patent/EP0765390A1/de
Withdrawn legal-status Critical Current

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/85—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells
    • C12N15/8509—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells for producing genetically modified animals, e.g. transgenic
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
    • A01K67/00—Rearing or breeding animals, not otherwise provided for; New or modified breeds of animals
    • A01K67/027—New or modified breeds of vertebrates
    • A01K67/0275—Genetically modified vertebrates, e.g. transgenic
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
    • A01K67/00—Rearing or breeding animals, not otherwise provided for; New or modified breeds of animals
    • A01K67/027—New or modified breeds of vertebrates
    • A01K67/0275—Genetically modified vertebrates, e.g. transgenic
    • A01K67/0276—Knock-out vertebrates
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
    • A01K67/00—Rearing or breeding animals, not otherwise provided for; New or modified breeds of animals
    • A01K67/027—New or modified breeds of vertebrates
    • A01K67/0275—Genetically modified vertebrates, e.g. transgenic
    • A01K67/0278—Knock-in vertebrates, e.g. humanised vertebrates
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23C—DAIRY PRODUCTS, e.g. MILK, BUTTER OR CHEESE; MILK OR CHEESE SUBSTITUTES; MAKING THEREOF
    • A23C9/00—Milk preparations; Milk powder or milk powder preparations
    • A23C9/20—Dietetic milk products not covered by groups A23C9/12 - A23C9/18
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/76—Albumins
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
    • A01K2207/00—Modified animals
    • A01K2207/15—Humanized animals
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
    • A01K2217/00—Genetically modified animals
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
    • A01K2217/00—Genetically modified animals
    • A01K2217/05—Animals comprising random inserted nucleic acids (transgenic)
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
    • A01K2217/00—Genetically modified animals
    • A01K2217/07—Animals genetically altered by homologous recombination
    • A01K2217/075—Animals genetically altered by homologous recombination inducing loss of function, i.e. knock out
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
    • A01K2227/00—Animals characterised by species
    • A01K2227/10—Mammal
    • A01K2227/101—Bovine
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
    • A01K2227/00—Animals characterised by species
    • A01K2227/10—Mammal
    • A01K2227/105—Murine
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
    • A01K2267/00—Animals characterised by purpose
    • A01K2267/01—Animal expressing industrially exogenous proteins

Definitions

  • Alpha-Lactalbumin Gene Constructs The present invention is connerned with recombinant gene constructs for expressing the protein ⁇ - lactalbumin, or functional equivalents or parts
  • Human milk has been shown to be superior over other milk types, notably cow, sheep, camel and goat milk, for human infant nutrition. However, many mothers find breast feeding difficult or inconvenient. Moreover, in countries where infant food supplements are in great demand, it would be highly desirable to be able to supply a milk product with the nutritional benefits of human milk.
  • One of the major differences of human milk over milk from other mammals is the presence of ⁇ -lactalbumin as the major whey protein. Whilst ⁇ -lactalbumin is present in other milk types, the concentration is relatively low and instead the major whey protein is ⁇ -lactoglobulin.
  • the level of ⁇ - lactalbumin varies from species to species, with human milk containing about 2.5 mg/ml, cow milk 0.5-1.0 mg/ml and mouse milk 0.1-0.8 mg/ml.
  • the gene sequences encoding for the bovine ⁇ - lactalbumin and for the human ⁇ -lactalbumin proteins have been elucidated and the sequence information published by Vilotte et al, in Biochemie 69: 609-620 (1987) and by Hall et al, in Biochem J 242: 735-742 (1987), respectively.
  • the present invention seeks to utilise genetic
  • the present invention provides a
  • the recombinant expression system adapted to express ⁇ - lactalbumin, or a functional equivalent or part thereof in cells of a non-human, preferably bovine, animal.
  • the recombinant expression system of the present invention is adapted to express the human ⁇ - lactalbumin protein, or a functional equivalent or part thereof.
  • expression system is used herein to refer to a genetic sequence which includes a protein-encoding region and is operably linked to all of the genetic signals necessary to achieve expression of the protein encoding region.
  • the expression system may also include a regulatory element, such as a promoter or enhancer, to increase transcription and/or
  • the regulatory element may be located upstream or downstream of the protein-encoding region, or may be located at an intron (non-coding portion) interrupting the protein encoding region.
  • the sequence of the protein-encoding region itself to comprise a regulatory ability.
  • derivatives which is functionally substantially similar to the reference sequence or protein.
  • functional equivalent includes derivatives in which nucleotide base(s) and/or amino acid(s) have been added, deleted or replaced without a significantly adverse effect on biological function, especially biological function in milk production. Genetic engineering has been recognised as a powerful technique not only in research but also for commercial purposes. Thus, by using genetic engineering
  • exogenous genetic material can be transferred to a host cell and the protein or polypeptide encoded by the exogenous genetic material may be replicated by and/or expressed within the host.
  • exogenous genetic material can be transferred to a host cell and the protein or polypeptide encoded by the exogenous genetic material may be replicated by and/or expressed within the host.
  • prokaryotic micro-organisms for example bacteria such as E. coli, as host.
  • eukaryotic organisms in particular yeasts or algae, and in certain applications eukaryotic cell cultures may also be used.
  • transgene is incorporated into the chromosome copies of only certain body organs.
  • Mosaic animals are generally produced by introducing exogenous DNA into an embryo at a later developmental stage.
  • transgenesis in lfvestock aims to utilise the mammary gland as a "bioreactor" to produce recombinant proteins of
  • the mammary gland is an attractive organ in which to express heterologous proteins due to its capacity to produce large quantities of protein in an exocrine manner.
  • Recombinant DNA techniques may be used to alter the protein composition of cow's milk used for human or animal consumption. For example, the
  • the gene expressed in the mammary gland is to clone the gene for the protein of interest.
  • the promoter gene for a major milk protein expressed in milk is employed.
  • Milk protein genes are tightly regulated and are not expressed in tissues other than the mammary gland, a characteristic that minimises the possibility of negative effects on the animal from inappropriate expression in other tissues.
  • the regulatory genes used to express heterologous proteins in the mammary gland are alpha-Si-casein
  • Zygotes may be obtained in vivo from the oviducts (Roschlau et al., Arch Tierz. Berlin 31:3-8 (1988); Roschlau et al., in J. Reprod. Fertil. (suppl 38), Cell Biology of
  • Bovine zygotes must be centrifuged at 15,000 x g for several minutes to displace opaque lipid in order to visualise the
  • the transgenes integrate at the zygote stage prior to DNA replication to ensure that every cell in the adult contains the transgene. In general, several "copies" of the
  • transgene linked together linearly, integrate in a single site on a single chromosome.
  • the site of integration is random. Integration probably occurs after the first round of DNA replication, and perhaps as late as the 2- or 4- cell stage (Wall and Seidel, 1992), resulting in animals that are mosaic with respect to the transgene. Indeed, up to 30% of animals in which transgenes are detected in somatic tissues do not transmit the transgenes to their offspring (or transmit to less than the expected 50%). After microinjection, embryos are either transferred directly into the oviducts of recipients or cultured for a few days and transferred to the uterus of recipient cattle. Confirmation of transgene
  • transgene integration is obtained by Southern blot analysis of tissues sampled from the calf after birth. Transgene expression is measured by assaying for the gene product in appropriate tissues, or in this case milk. Embryo survival after microinjection, transgene integration frequency, frequency of expression and expression level, and frequency of germline transmission vary according to quantity and quality of DNA construct injected, strain of mice used (Brinster et al., Proc. Natl. Acad Sci. USA 82:4438-4442 (1985)) and skill and technique of the operator performing microinjection.
  • the present invention provides a transgenic mammalian animal, said animal having cells incorporating a recombinant expression system adapted to express ⁇ -lactalbumin (preferably human ⁇ - lactalbumin) or a functional equivalent or part
  • transgenic animals include (but are not limited to) sheep, pigs, cattle and goats.
  • the present invention comprises a vector containing such a recombinant expression system and host cells transformed with such a recombinant
  • the present invention provides ⁇ -lactalbumin produced by expression of a recombinant expression system of the present invention, desirably such ⁇ -lactalbumin produced in a transgenic mammal.
  • the ⁇ -lactalbumin gene is naturally activated in the mammary glands of the lactating female mammal.
  • the protein expressed by the recombinant expression system of the present invention would be produced at such a time and would be excreted as a milk component. It may also be possible for the protein of interest to be produced by inducing lactation through hormonal or other treatment. Processed milk products comprising such ⁇ -lactalbumin are also covered by the present invention.
  • the recombinant expression system comprises a construct designated pHAl, pHA2, pBBHA, pOBHA, pBAHA, pBova-A or pBova-B.
  • pHA1, pHA2, pBBHA, pOBHA and pBAHA express human ⁇ -lactalbumin and are thus preferred
  • pHA2 was deposited on 15 February 1995 at NCIMB under Accession No NCIMB 40709. Likewise transgenic mammals comprising the specific constructs listed above are preferred. It has further been found that, in addition to
  • promoter region of the human ⁇ -lactalbumin gene is only partially responsible for the enhanced natural
  • flanking sequences 3' and 5' to the protein- encoding region of the human ⁇ -lac gene have been sequenced for the first time. Partial sequences
  • the present invention provides a polynucleotide having a sequence substantially as set out in any one of SEQ ID Nos. 16-20 or SEQ ID No. 21 or a portion or functional equivalent thereof.
  • the polynucleotides may be in any form (for example DNA or RNA, double or single stranded), but generally double stranded DNA is the most convenient.
  • the polynucleotides according to the present invention may be present as part of a recombinant genetic
  • construct which itself may be included in a vector (for example an expression vector) or may be
  • the present invention provides a recombinant expression system (preferably adapted to express ⁇ -lactalbumin (preferably human ⁇ - lactalbumin) or a portion or functional equivalent thereof), said recombinant expression system comprising a polynucleotide selected from the polynucleotide located between the EcoRI and Xhol restriction sites of the wild-type ⁇ -lactalbumin gene and the polynucleotide located between the BamHI and EcoRI restriction sites of the wild-type human ⁇ -lactalbumin gene, or a portion or functional equivalents thereof.
  • the recombinant expression sequence of the present invention comprises both polynucleotides as defined above, portions and
  • the invention also encompasses vectors containing the recombinant expression systems defined above and cells transformed with such vectors. Further, the present invention comprises transgenic animals wherein the transgene contains the recombinant expression system.
  • Figure 1 as discussed in Example 1 and shows the sequence of bovine ⁇ -lactalbumin PCR primers.
  • Figure 2 is discussed in Example 1 and 4 shows the position of bovine ⁇ -lactalbumin PCR primers and products.
  • Figure 3 is discussed in Example 2 and shows a
  • FIG. 5 is discussed in Example 4 and shows SDS-PAGE analysis of skimmed milk from bovine ⁇ -lactalbumin transgenic mice run against non transgenic mouse milk.
  • Figure 6 is discussed in Example 5 and shows human ⁇ - lactalbumin transgene constructs.
  • Figure 7 is discussed in Example 5 and shows SDS-PAGE analysis of skimmed milk from human ⁇ -lactalbumin transgenic mice run against non transgenic mouse milk.
  • Figure 8 is discussed in Example 5 and shows a Western analysis of the milk from human ⁇ -lactalbumin
  • transgenic mice run against human ⁇ -lactalbumin
  • Figure 9 shows the PCR cloning strategy for transgene constructs PKU1 to PKU4 as discussed in Example 6.
  • Figure 10 gives the sequences of PKU primers 1 to 10 as discussed in Example 6.
  • Figure 11 shows the structure of null and humanised ⁇ - lactalbumin alleles.
  • Figure 12 is a Northern analysis of total RNA from ⁇ - lactalbumin-deficient lactating mammmary glands.
  • Figure 13 illustrates a Western analysis of ⁇ - lactalbumin from targeted mouse strains.
  • Figure 14 is a histological analysis of wild type and ⁇ -lac- lactating mammary glands.
  • Figure 15A shows an RNase protection assay used to distinguish human replacement and mouse ⁇ -lactalbumin mRNA
  • Figure 15B shows an RNase protection assay of mouse and human replacement ⁇ -lactalbumin mRNA
  • Figure 16 gives the quantification of ⁇ -lactalbumin by hydrophobic interaction chromatography.
  • SEQ ID Nos. 16 - 20 give parts of the sequence from the BamHI site to the vector restriction sites (including EcoRI sites) 3' of the protein-encoding region of the endogenous human ⁇ -lactalbumin gene, as set out below:
  • SEQ ID No. 16 Nucleotides 1 to 264 (inclusive)
  • SEQ ID No. 17 Nucleotides 1331 to 2131 (inclusive)
  • SEQ ID No. 18 Nucleotides 2259 to 2496 (inclusive)
  • SEQ ID No. 19 Nucleotides 2519 to 2680 (inclusive)
  • SEQ ID No. 20 Nucleotides 3481 to 3952 (inclusive)
  • SEQ ID No. 21 gives the sequence from the EcoRI
  • polyadenylation sites are also indicated.
  • the middle portion shows the structure of the null allele.
  • the striped bar indictes the HPRT selectable cassette.
  • the lower portion shows the structure of the human
  • the checkered bar shows the human ⁇ -lactalbumin fragment.
  • the transcription initiation, translational stop and polyadenylation sites are shown. Restriction enzyme sites shown are: Hindlll (H); BamHI (B); Xbal (X).
  • H Hindlll
  • B BamHI
  • Xbal X
  • the two autoradiographs shown are repeat hybridisations of the same membrane filter using a human ⁇ -lactalbumin probe followed by a rat ⁇ -casein probe. The probes used are indicated under each autoradiograph. The source of RNA in each lane is indicated above the lane markers.
  • Lane A contains purified human ⁇ - lactalbumin.
  • Lanes B-F show samples of milk from targeted mice, genotypes are indicated above the lane markers.
  • Lanes G and H are a shorter exposure of Lanes C and D.
  • the light micrographs shown in Figure 14 are
  • Lanes D to J show RNA samples from ⁇ -lac m / ⁇ -lac h heterozygotes, the numbers indicate individual mice and are the source of the quantitative estimates given in Figure 15.
  • Figure 16 shows phenyl-Sepharose elution profiles of three milk samples. 1, ⁇ -lac h / ⁇ -lac h homozygote (mouse #22); 2, ⁇ -lac m / ⁇ -lac h heterozygote (mouse #76); 3, ⁇ -lac m / ⁇ -lac m wild type.
  • the lower portion shows a standard curve of known quantities of human ⁇ -lactalbumin plotted against integrated peak area.
  • Example 1 Cloning of Bovine ⁇ -lactalbumin gene
  • Bos Bos
  • the A variant from Bos (Bos) nomadicus f.d. indicus differs from the B variant at residue 10: Glu in A is substituted for Arg in B.
  • the sequence difference for the C variant from Bos (Bibos) javanicus has not been established (McKenzie & White, Advances in Protein Chemistry 41, 173-315 (1991).
  • the source of DNA in all the PCR reactions was blood from a Holstein-Friesian cow.
  • the length of the amplified promoter region using primer Ba-9 in combination with primer Ba-8 is 0.72kb.
  • This BamHI /EcoRI fragment was cloned into Bluescript (pBA-PO.7).
  • the length of the amplified promoter region using primer Ba-7 in combination with primer Ba-8 is 2.05kb.
  • This BamHI/EcoRI fragment was cloned into Bluescript (pBA-P2).
  • the entire bovine ⁇ -lactalbumin gene including 0.72kb of 5' and 0.3kb of 3' flanking region was amplified using primer Ba-9 in combination with primer Ba-2.
  • These primers include BamHI restriction enzyme
  • PCR primers were designed to clone two small fragments from human genomic DNA, one at the 5' end of the gene and the other at the 3' end. These were subcloned into the pUC18 vector and used as probes to screen a commercial (Stratagene) ⁇ genomic library. Two recombinant bacteriophages, 4a and 5b.1, which contained the ⁇ - lactalbumin gene, were isolated by established methods (Sambrook et al, Molecular Cloning 2nd ed., Cold Spring Harbor Laboratory (1989)). Restriction mapping
  • PCR primers were designed to clone a fragment from the 5' portion of the bovine BLG gene. This was subcloned into the pUC18 vector and used as probes to screen a commercial bovine (Stratagene) ⁇ genomic library. Three genomic ⁇ clones were isolated and characterised by restriction enzyme analysis (see Fig. 4). Two of the clones (BB13, BB17) contain the complete bBLG coding region plus various amounts of flanking regions, while clone BB25 lacks the coding region and consists entirely of 5' flanking region. Sequence analysis showed that the end of this clone lies 12 bp upstream of the ATG translation start site.
  • pBova-B consists of 3 fragments:
  • Table 1 shows the relative levels of bovine ⁇ - lactalbumin in transgenic mouse milk as estimated by comparison to protein standards on Coomassie gels.
  • ⁇ -lactalbumin is the major whey protein in humans, beta-lactoglobulin the major whey protein in sheep and cow.
  • the level of ⁇ -lactalbumin expression varies from species to species, human milk contains about 2.5mg/ml, cow milk 0.5-1.0mg/ml, and mouse milk 0.1-0.8mg/ml.
  • flanking regions derived from the human ⁇ - lactalbumin locus contain a) different amounts of 5' and 3' flanking regions derived from the human ⁇ - lactalbumin locus, b) 5' flanking regions derived from the bovine ⁇ -lactalbumin locus, or c) 5' flanking regions derived from the bovine or ovine beta- lactaglobulin gene.
  • the ovine beta-lactoglobulin gene promoter has been successfully used to allow high expression (>10mg/ml) of heterologous genes in mouse milk.
  • pOBHA ovine beta-lactaglobulin, human ⁇ -lactalbumin
  • ⁇ clone 4a derived from ⁇ clone 4a comprising a region between a Bgll site at base 77 and a Xhol site in the 3' flank;
  • pSL1180 Pulcoa
  • pBBHA bovine beta-lactoglobulin, human ⁇ -lactalbumin
  • ⁇ clone 4a derived from ⁇ clone 4a comprising a region between a Bgll site at base 77 and a Xhol site in the 3' flank;
  • pBAHA bovine ⁇ -lactalbumin, human ⁇ -lactalbumin
  • bovine ⁇ -lactalbumin promoter derived from clone pBA-P0.7;
  • ⁇ clone 4a derived from ⁇ clone 4a comprising a region between a Bgll site at base 77 and a Xhol site in the 3 ' flank ;
  • Figs. 7 and 8 show an SDS-PAGE analysis of skimmed transgenic mouse milk run against a non-transgenic control mouse milk.
  • Fig. 8 shows a Western blot of human ⁇ -lactalbumin transgenic milks run against a human ⁇ -lactalbumin standard.
  • Example 6 Expression of Mutagenised Bovine ⁇ - Lactalbumin under the control of the Human ⁇ - Lactalbumin Promoter in vivo Expression of the human ⁇ -lactalbumin transgene is considerably higher than that of the native bovine ⁇ - lactalbumin transgene, reflecting the difference in expression levels of the endogenous bovine and human genes.
  • mice carrying the null ⁇ -lactalbumin allele and the humanised ⁇ -lactalbumin replacement allele were derived by breeding chimeras produced from the targeted
  • ⁇ -lactalbumin genotypes were determined by Southern analysis of genomic DNA prepared from tail biopsies. RNA analysis Total RNA was prepared by the method of Auffray and Rougeon (Eur. J. Biochem 107 : 303-14) from abdominal mammary glands of female mice 506 days postpartum.
  • ⁇ -lactalbumin in milk samples was quantified by a modification of the method of Lindahl et al., (Analyt. Biochem 140:394-402) for calcium dependant purification of ⁇ -lactalbumin by phenyl-Sepharose chromatography.
  • Milk ⁇ samples were diluted 1:10 with 27% w/v ammonium sulphate solution, incubated at room temperature for 10 minutes and centrifuged. Supernatant was mixed with an equal volume of 100mM Tris/Cl, pH 7.5, 70mM EDTA and loaded onto a column (200 ⁇ l packed volume) of phenyl- Sepharose (Pharmacia) pre-equilibrated with 50mM
  • mice were removed for two hours from lactating mothers on the sixth day postpartum, mothers sacrificed and thoracic mammary glands were dissected, preserved in neutral buffered formalin, paraffin embedded and stained with haematoxylin/eosin by standard methods.
  • Results Mouse ⁇ -lactalbumin gene deletion A line of mice in which a 2.7kb fragment covering the complete mouse ⁇ -lactalbumin coding region and a 0.57kb of promoter has been deleted and replaced with a 2.7kb fragment containing a hypoxanthine phosphoribosyltransferase (HPRT) selectable marker gene was
  • Milk was obtained from each genotype by manual milking and the composition of key components analysed. Milk from ⁇ -lac m / ⁇ -lac-heterozygotes was indistinguishable in appearance from wild type milk and showed fat and protein contents similar to wild type (Table 4). While lactose concentration appeared to be slightly reduced in ⁇ -lac m / ⁇ -lac- heterozygotes, statistical analysis showed that the difference was not significant. In contrast, milk from ⁇ -lac-/ ⁇ -lac- homozygotes was viscous, difficult to express from the teats and was markedly different in composition to wild type. Fat content was -60% greater than wild type, protein content was -88% greater, and lactose was effectively absent.
  • ⁇ -Lactalbumin content of milk samples were estimated by phenyl-Sepharose chromatography. Values are means ⁇ SE. Figures in brackets indicate the number of mothers analysed. ⁇ -Lactalbumin deficiency has no apparent effect on mammary gland development. Table 4 shows that total mammary tissue weights of wild type, heterozygous ⁇ - lac m / ⁇ -lac- and homozygous ⁇ -lac-/ ⁇ -lac- lactating mothers were not significantly different. Light microscopic analysis of mammary glands ( Figure 14) revealed that heterozygous and homozygous ⁇ -lac-/ ⁇ -lac- glands were histologically normal.
  • mice carrying the human ⁇ -lactalbumin gene at the mouse ⁇ -lactalbumin locus The 2.7kb mouse ⁇ -lactalbumin fragment deleted at the ⁇ -lac- null allele was replaced by a 2.97kb fragment containing the complete human ⁇ -lactalbumin coding region and 5' flanking sequences.
  • the human fragment stretches from 0.77kb upstream of the human transcription initiation site to an EcoRI site 136bp 3' of the human
  • mice were normal in appearance, fertility and behaviour. In contrast to ⁇ -lac-/ ⁇ -lac- mice, ⁇ -lac h / ⁇ -lac h homozygous mothers produce apparently normal milk and rear offspring successfully.
  • Table 4 shows that pups raised by ⁇ -lac m / ⁇ -lac h heterozygous and ⁇ -lac h / ⁇ -lac h homozygous females are similar in weight to those of wild type mothers. This is supported by our
  • FIG. 15A shows the strategy used to compare levels of mouse and human ⁇ -lactalbumin mRNA. Because the junction between human and mouse ⁇ -lactalbumin
  • ⁇ - lac h mRNA contains a "tag" of 120 bases of untranslated mouse sequences at the 3' end.
  • ribonuclease protection assay to detect and distinguish human and mouse ⁇ -lactalbumin mRNA in the same RNA sample. The relative abundance of each mRNA was calculated from the amount of label in fragments protected by human and mouse mRNAs. A ribonuclease protection assay was performed and the results are shown in Figure 15B. Lane A shows the undigested 455 base probe and Lane K shows that yeast tRNA did not protect any fragments. Wild type mouse RNA protected a fragment consistent with the predicted 305 base RNA from endogenous mouse ⁇ -lactalbumin RNA (see Lane B).
  • Table 6 shows the amount of radioisotope 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 mRNA in each ⁇ -lac m / ⁇ -lac h heterozygote. It is apparent that, although there was variation between individual animals, human ⁇ -lactalbumin mRNA was significantly more abundant than mouse mRNA. Averaging the seven ⁇ - lac m / ⁇ -lac h heterozygotes gives a value of 15-fold greater expression for human ⁇ -lactalbumin mRNA.
  • Lane designations indicate the source of protected fragments and correspond to those shown in Figure 15B. a. numbers are expressed in counts per minute
  • Human ⁇ -lactalbumin protein expression A Western analysis of ⁇ -lactalbumin in targeted mouse lines was conducted. Human ⁇ -lactalbumin can be distinguished from mouse ⁇ -lactalbumin by its faster electrophoretic mobility (see Lanes A, B).
  • Figure 16 shows superimposed absorbance profiles of column eluates of three illustrative milk samples including the ⁇ -lac m / ⁇ -lac h heterozygote and ⁇ -lac h / ⁇ -lac h
  • ⁇ -Lactalbumin content for the samples shown in Figure 16 were estimated as follows: ⁇ -lac m / ⁇ - lac m wild-type 0.1mg/ml; ⁇ -lac m / ⁇ -lac h heterozygote #76 0.45mg/ml; ⁇ -lac h / ⁇ -lac h homozygote #22 0.9mg/ml.
  • Table 5 shows the concentration of ⁇ -lactalbumin in milk samples from targeted mouse lines and lactating women. It is clear that the concentration of ⁇ -lactalbumin in milk is directly related to gene dosage, eg ⁇ -lac m / ⁇ - lac- heterozygotes shown an ⁇ -lactalbumin concentration half that of wild type. Given that the volumes of milk produced by these mice are similar (Table 4), the concentration of ⁇ -lactalbumin provides a reasonable indication of the quantity synthesised.
  • Example 8 Enhanced expression of a heterologous gene. These data confirm that the upstream promoter region (AUG to about -3.7 kb) which is included in the pHA-2 construct enhances expression of a heterologous gene. Table 7 shows the results of milk analysis from pHA-2 transgenic founder females. Out of 10 females, 6 animals expressed high levels of human ⁇ -lac. 3 animals failed to express detectable levels of human ⁇ - lac (less than 0.2 mg/ml in this assay), all 3 also failed to transmit the transgene. We can neither be certain whether they were low expressors or not
  • bovine promoter achieves a higher level of expression than the use of the bovine promoter, and induces expression in more animals than the bovine promoter.
  • MI FREQ. Integration frequency

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EP95924467A 1994-07-13 1995-07-12 Alpha-lactalbumin genkonstrukte Withdrawn EP0765390A1 (de)

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US381691 1982-05-25
WOPCT/GB94/01514 1994-07-13
PCT/GB1994/001514 WO1995002692A1 (en) 1993-07-16 1994-07-13 Modified alpha-lactalbumin
GB9425326 1994-12-15
GBGB9425326.7A GB9425326D0 (en) 1994-12-15 1994-12-15 Gene constructs
US08/381,691 US5852224A (en) 1994-12-15 1995-01-31 α-lactalbumin gene constructs
GBGB9503822.0A GB9503822D0 (en) 1995-02-25 1995-02-25 "Alpha-lactalbumin gene constructs"
GB9503822 1995-02-25
PCT/GB1995/001651 WO1996002640A1 (en) 1994-07-13 1995-07-12 Alpha-lactalbumin gene constructs

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