NZ289197A - Alpha lactalbumin gene constructs and transgenic cattle - Google Patents
Alpha lactalbumin gene constructs and transgenic cattleInfo
- Publication number
- NZ289197A NZ289197A NZ289197A NZ28919795A NZ289197A NZ 289197 A NZ289197 A NZ 289197A NZ 289197 A NZ289197 A NZ 289197A NZ 28919795 A NZ28919795 A NZ 28919795A NZ 289197 A NZ289197 A NZ 289197A
- Authority
- NZ
- New Zealand
- Prior art keywords
- lactalbumin
- human
- milk
- seq
- sequence
- Prior art date
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- 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
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- 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
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- 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
-
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Description
New Zealand No. 289197 International No. PCT/GB95/01651
TO BE ENTERED AFTER ACCEPTANCE AND PUBLICATION
Priority dates:
13.07.1994; 15.12.1994;31.01.19u95;25.02.19
95;
Complete Specification Filed: 12.07.1995 Classification:^) C12N15/12; A01K67/027; A23C9/00 Publication date: 24 September 1998 Journal No.: 1432
NEW ZEALAND PATENTS ACT 1953
COMPLETE SPECIFICATION
Title of Invention:
Alpha-lactalbumin gene constructs
Name, address and nationality of applicant(s) as in international application form:
PPL THERAPEUTICS (SCOTLAND) LIMITED, Roslin, Edinburgh EH25 9PP, Great Britain
Q
0 b: t ij i
WO 96/02640 PCT/GB9S/01651
1 "Alpha-Lactalbumin Gene Constructs"
2
3 The present invention is concerned with recombinant
4 gene constructs for expressing the protein a-
lactalbumin, or functional equivalents or parts
6 thereof.
7
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. However, 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.
16
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 a-lactalbumin as the major whey protein. 2 0 Whilst a-lactalbumin is present in other milk types,
21 the concentration is relatively low and instead the
22 major whey protein is /3-lactoglobulin. The level of a-
23 lactalbumin varies from species to species, with human
24 milk containing about 2.5 mg/ml, cow milk 0.5-1.0 mg/ml
and mouse milk 0.1-0.8 mg/ml.
SUBSTITUTE SHEET (RULE 26)
ACTIVE AS AT ACCEPTANCE
. WO 96/02640 PCT/GB95/01651
2 f;. *7
1 The gene sequences encoding for the bovine-'a-
2 lactalbumin and for the human a-lactalbumin proteins
3 have been elucidated and the sequence information
4 published by Vilotte et al, in Biochemie jjj).: 609-620
(1987) and by Hall et al, in Biochem J 242; 735-742
6 (1987), respectively.
7
8 The present invention seeks to utilise genetic
9 engineering techniques to provide a recombinent gene
construct capable of producing an a-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-human animal, especially bovine,
cells,
16
17 In general terms, the present invention, provides.a
18 recombinant expression system adapted to express a-
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 a-
23 lactalbumin 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 2 8 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
ACTIVE AS AT ACCEPTANCE
WO 96/02640 PCT/GB95/01651
•
1 (non-coding portion) interrupting the protein encoding
2 region. Alternatively it is also possible for the
3 sequence of the protein-encoding region itself to
4 comprise a regulatory ability.
6 The term "functional equivalent" 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.
14
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, Harbor Laboratory, Cold
Spring Harbor, New York 1982 and "Principle of Genetic
21 Engineering", Old and Primrose, 5th edition, 1994 f both
22 incorporated herein by reference) exogenous genetic 2 3 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
2 8 micro-organisms, for example bacteria such as E. coli. 2 9 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.
33
34 Genetic alterations to mammalian species by micro-
injection of genes into the pro-nuclei of single-cell 35 embryos has been described by Brinster et al, in Cell
WO 96/02640 PCT/GB95/01651
u
1 2_7 : 223-231, 1981. Here the foreign genetic material
2 is introduced into the fertilised egg of an animal
3 which then proceeds to develop into an embryo in the
4 normal manner having been transplanted into a foster
mother. Truly transgenic animals contain copies of the
6 exogenous DNA in each cell.
7
8 Where the injected genetic material is 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 to
control the location at which the transgene integrates
16 into the host chromosome for such animals.
17
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 DNA into an embryo at
24 a later developmental stage.
26 One of the most promising application of transgenesis
27 in lfvestock 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 milk used for
human or animal consumption. For example, the
36 expression of human milk proteins in cow's milk could
.WO 96/02640 PCT/GB95/01651
" 5
1 improve its nutritional value in infant formula
2 applications (Strijker et al, in Harnessing
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.
9
The first step in developing 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-Sj-casein
21 (Strijker et., 1992, supra), beta-lactoglobulin (Wright
22 et al., Bio/Technology 5.: 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).
26
27 Newly-made gene constructs are normally tested in
28 transgenic mice before adopting them for use in cattle.
29 Milk 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.
6
PCT/GB9S/01651
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. Zygotes may
4 be obtained in vivo from the oviducts (Roschlau et al.,
Arch Tierz. Berlin JU:3-8 (1988); Roschlau et al., in
6 J. Reprod. Fertil. (suppl 38), Cell Biology of
7 Mammalian Egg Manipulation, ed Greve et al (1989); Hill
8 et al. , Theriogenology 37.: 222 (1992); Bowen et al. Biol
9 Reprod. j>0:664-448 (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 zygotes must
13 be centrifuged at 15,000 x g for several minutes to 3 4 displace opaque lipid in order to visualise the
pronuclei with phase contrast, Nomarski or Hoffman
16 interference contrast optics. 2-4 pi 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 process. Ideally, the transgenes
22 integrate at the zygote stage prior to DNA replication
23 to ensure that every 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 animals
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 50%).
36 After microinjection, embryos are either transferred
7
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 by 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 (Ebert and Schindler, 1993, supra) pigs (Rexroad
18 and Purcel, Proc 11th Intl. Congr. Anim. Reprod. A.I.
19 Dublin j>:29-35 (1988)) and cattle (Krimpenfort et al.,
1991, supra; Hill et al., 1992, supra; Bowen et al.,
21 1994, supra).
22
23 Reference is also made to WO-A-88/01648 (of Immunex
24 Corporation), to WO-A-88/00239 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 in 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.
33
34 Reference is further made to Stacey et al in molecular
and Cellular Biology 14(2); 1009-1016 (February 1994)
36 which describes a knock-out experiment in which the
WO 96/02640 fTCT/GB?5/01651
Q £:. ( '• • ' : /
S 1 ^^ v tV .v
1 mouse a-lactalbumin gene is replaced with a human a-
2 lactalbumin gene. This paper (incorporated herein by
3 reference) does not however report expression of the a-
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.
9
In general"-terms, the present invention also provides a-
11 transgenic mammalian animal, said animal having cells
12 incorporating a recombinant expression system adapted
13 to express a-lactalbumin (preferably human a-
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
19 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.
23
24 Additionally, in general terms the present invention comprises.a vector
containing such a recombinant expression system and
26 host cells transformed with such a recombinant
27 expression system (optionally in the form of a vector).
28
Accordingly, in one aspect the present invention provides a recombinant DNA construct comprising, in the 5' to 3' direction and operatively linked:
(a) the 5' flanking sequence of SEQ ID NO 21;
(b) a DNA sequence encoding
(1) a signal sequence and
(2) a-lactalbumin or a functional equivalent thereof; and
(c) the 3' flanking sequence of anyone of SEQ ID NO'S 16 to 20;
the DNA construct being adapted to express the a-lactalbumin or fi&ictionai equivalent thereof in a non-human transgenic animal.
In another aspect, the present invention provides a recombinant DNA construct comprising, in the 5' to 3' direction and operatively linked:
(a) at least about 1.8 kb of 5'-flanking sequence from the human a-lactalbumin gene including the a-lactalbumin promoter;
(b) a DNA sequence encoding
(1) a signal sequence; and
(2) a-lactalbumin or a functional equivalent thereof;
(c) at least about 3 kb of 3'-flanking sequence from the human a-lactalbumin gene the DNA construct being adapted to express the a-lactalbumin or functional equivalent thereof in a non-human transgenic animal.
In still a further aspect, the present invention provides a vector containing a recombinant DNA construct as defined above.
In further aspects the invention provides a host cell containing a vector as defined above, and a transgenic animal having a recombinant DNA construct as defined above integrated into its genome.
In a yet further aspect the present invention provides a-lactalbumin produced by expression of a recombinant expression system of the present invention, desirably such cx-lactalbumin produced in a transgenic mammal. The a-lactalbumin gene is naturally activated in the mammary glands of the lactating female mammal. Thus the protein expressed by the recombinant expression system of the present invention would be produced at
WO 96/02640 PCT/GB95/01651
9
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 a-lactalbumin are also covered by the present
6 invention.
7
8 In one preferred embodiment, the recombinant expression
9 system comprises a construct designated pHAl, pHA2,
pBBHA, pOBHA, pBAHA, pBova-A or pBova-B. The
11 constructs pHAl, pHA2, pBBHA, pOBHA and pBAHA express
12 human a-lactalbumin and are thus preferred,
13 particularly pHA2. The construct pHA2 was deposited on
14 15 February 1995 at NCIMB under Accession No NCIMB
40709.
16
17 Likewise transgenic mammals comprising the specific
18 constructs listed above are preferred.
19
It has further been found tr.-at, in addition to
21 increased concentrations of j-lactalbumin per unit
22 volume of milk achieved by -he present invention, where
23 a human a-lactalbumin 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 a-lactalbumin gene (or functional equivalents
27 or parts thereof) and transgenic animals (especially
28 cattle) are preferred embodiments of the invention.
29
3 0 Whilst we do not wish to be bound by theoretical
31 considerations, it is further believed that the
32 promoter region of the human a-lactalbumin gene is only 3 3 partially responsible for the enhanced natural
34 expression of a-lactalbumin by humans. It is believed
that enhanced expression may be obtained by including
3 6 within the recombinant expression system of the present
37 invention the 3' sequence flanking the protein-encoding
38 region and/or the 5' sequence flanking the protein-
SUBSTITUTE SHEET (RULE 26}
WO 96/02640 PCT/GB95/016S1
1 encoding region itself.
2
3 The flanking sequences 3' and 5' to the protein-
4 encoding region of the human a-lac gene have been
sequenced for the first time. Partial sequences
6 (nucleotides 1-264, 1331-2131, 2259-2496, 2519-2680 and
7 34 81-395?) 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 nonr
human a-lactalbumin as well as human a-lactalbumin.
16
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
2 0 therefore comprise a further aspect of the present
21 invention.
22
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.
27
2 8 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
3 3 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
WO 96/02640 PCT/GB95/01651
11
1 polynucleotide as described above form a further aspect
2 of the present invention.
3
4 Viewed from a yet further aspect the present invention
provides a recombinant expression system (preferably
6 adapted to express a-lactalbumin (preferably human a-
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 Xhol restriction sites of
11 the wild-type a-lactalbumin gene and the polynucleotide
12 located between the BamHI and EcoRI restriction sites
13 of the wild-type human a-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 2 4 invention comprises transgenic animals wherein the
transgene contains the recombinant expression system.
26
27 Figure 1 as discussed in Example 1 and shows the 2 8 sequence of bovine a-lactalbumin PCR primers.
29
Figure 2 is discussed in Example 1 and 4 shows the
31 position of bovine a-lactalbumin PCR primers and
32 products.
33
34 Figure 3 is discussed in Example 2 and shows a
restriction map of two overlapping genomic A clones for
36 the human a-lactalbumin gene (pHA-2 and pHA-1).
WO 96/02640 PCT/GB95/01651
12
1 Figure 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.
4
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.
8
9 Figure 6 is discussed in Example 5 and shows human a-
lactalbumin transgene constructs.
11
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 Figure 9 shows the PCR cloning strategy for transgene
22 constructs PKU1 to PKU4 as discussed in Example 6.
23
24 Figure 10 gives the sequences of PKU primers 1 to 10 as
discussed in Example 6.
26
27 Figure 11 shows the structure of null and humanised a-
28 lactalbumin alleles.
29
Figure 12 is a Northern analysis of total RNA from a-
31 lactalbumin-deficient lactating mammmary glands.
32
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
WO 96/02640 PCT/GB95/01651
13
1 a-lac" lactating mammary glands.
2
3 Figure 15A shows an RNase protection assay used to
4 distinguish human replacement and mouse a-lactalbumin
mRNA and Figure 15B shows an RNase protection assay of
6 mouse and human replacement a-lactalbumin mRNA.
7
8 Figure 16 gives the quantification of a-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 a-lactalbumin gene, as set out below:
16 SEQ ID No. 16 : Nucleotides 1 to 264 (inclusive)
Nucleotides 1331 to 2131 (inclusive) Nucleotides 2259 to 2496 (inclusive) Nucleotides 2519 to 2680 (inclusive) Nucleotides 3481 to 3952 (inclusive)
21
22 SEQ ID No. 21 gives the sequence from the EcoRI
23 restriction site to the Xhol restriction site which are
24 5' to the protein-encoding region of the endogenous
human a-lactalbumin gene.
26
27 In mo're detail, in Figure 11 the upper portion shows
28 the wild type murine a-lactalbumin locus. The position
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 a-lactalbumin fragment. The transcription initiation,
17 SEQ ID No. 17
18 SEQ ID No. 18
19 SEQ ID No. 19
SEQ ID No. 20
WO 96/02640 PCT/GB95/01651
14
1 translational stop and polyadenylation sites are shown.
2 Restriction enzyme sites shown are: Hindlll (H); BamHI
3 (B); Xbal (X).
4
In Figure 12, the two autoradiographs shown are repeat
6 hybridisations of the same 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 markers.
11
12 In Figure 13 Lane A contains purified human a-
13 lactalbumin. Lanes B-F show samples of milk from
14 targeted mice, genotypes are indicated above the lane
markers. Lanes G and H are a shorter exposure of Lanes
16 C and D.
17
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.
22
23 In Figure 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 a-
26 lactaibumin mRNA contains 120bp of mouse sequences in
27 the I' untranslated end. Human replacement and mouse
2 8 a-lactalbumin mRNA were detected by hybridisation with
29 a mouse RNA probe and distinguished by the size of RNA
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: Hindlll (H); Bal (B); Xbal (X).
34
In Figure 15B the autoradiograph shown is of a 5%
36 polyacrylamide urea thin layer gel. The source of RNA
WO 96/02640 PCT/GB95/01651
1 is indicated above the lane markers. Lane A shows a
2 wild-type RNA hybridised to the mouse RNA proe
3 undigested with ribonuclease. Lanes D to J show RNA
4 samples from a-lacm/a-lach heterozygotes, the numbers
indicate individual mice and are the source of the
6 quantitative estimates given in Figure 15. The
7 predicted size of protected fragments are indicated.
8
9 The upper portion of Figure 16 shows phenyl-Sepharose
elution profiles of three milk samples. 1, a-lach/a-lach
11 homozygote (mouse #22); 2, a-lac"7a-lach heterozygote
12 (mouse #76); 3, a-lacm/a-lacm wild type. The lower
13 portion shows a standard curve of known quantities of
14 human a-lactalbumin plotted against integrated peak
area.
16
17 The present invention will now be further described
18 with reference to the following, non-limiting,
19 examples.
21 Example 1 - Cloninc; of Bovine a-lactalbumin gene
22
23 There are three known variants of bovine a-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: Glu in A is 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 a-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
Ba-7 SEQ ID No 2
36 Ba-8 SEQ ID No 3
WO 96/02640 PCT/GB95/01651
16
1 Ba-9 SEQ ID No 4
2 The source of DNA in all the PCR reactions was blood
3 from a Holstein-Friesian cow.
4
The length of the amplified promoter region using
6 primer Ba-9 in combination with primer Ba-8 is 0.72kb.
7 This BamHI/EcoRI fragment was cloned into Bluescript
8 (pBA-P0.7).
9
The length of the amplified promoter region using
11 primer Ba-7 in combination with primer Ba-8 is 2.Q5kb.
12 This BamHI/EcoRI fragment was cloned into Bluescript
13 (pBA-P2).
14
The entire bovine a-lactalbumin gene including 0.72kb
16 of 5' and 0. 3kb of 3' flanking region we.3 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, giving rise to construct pBova-A (see Figure 2).
22
23 Ligation of the BamHI/EcoRV fragment from clone pBA-P2
24 to the EcoRV/BamHI fragment of pBOVA-a gave rise to
construct pBOVA-b (see Figure 2).
26
27 Since TAQ polymerase lacks proof-reading activity, it
28 was essential to ensure that the amplified bovine a-
2 9 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:
34
(i) Exon I at +759 C to A. 5' non-coding region;
36 (ii) Exon I at +792 CTA to CTG. Both code for Leucine
17
1 (iii) Exon II at +1231 GCG to ACG. Alanine to
2 Threonine
3 This is 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 - Cloning of Human a-lactalbumin gene
12
13 The DNA sequences of human a-lactalbumin has been
14 published (Hall et al, Biochem. J., 242 : 735-742
(1987)). Using the human 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 subcloned into the
19 pUC18 vector and used as probes to screen a commercial 2 0 (Stratagene) A genomic library. Two recombinant
21 bacteriophages, 4a and 5b.1, which contained the a-
22 lactalbumin gene, were isolated by established methods
2 3 (Sambrook et al, Molecular Cloning 2nd ed. , Cold Spring
24 Harbor Laboratory (1989)). Restriction mapping
demonstrated that both clones contained the complete
2 6 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.
31
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.
34
Example 3 - Cloning of bovine beta lactoalobulin gene
36 (bBLG^
WO 96/02640 PCT/GB95/01651
18
1 The DNA sequence of bovine BLG (bBLG) has been
2 published (Jamieson et al; Gene, 61; 85-90, (1987);
3 Wagner, unpublished, EMBL Data Library: BTBLACEX
4 (1991)). 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 bovine
8 (Stratagene) A genomic library. Three genomic A clones
9 were isolated and characterised by restriction enzyme
analysis (see Fig. 4). Two of the clones (BB13, BB17)
11 contain the complete bBLG coding region plus various
12 amounts of flanking 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 pUClB, as well
18 as EcoR I fragments from clone BB25 (the latter was
19 cloned into pBluescript (Figure 4).
21 Example 4 - Assembly and expression of bovine ct-
22 lactalbumin constructs
23
24 Transoene 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,
2 8 cloned as a 3kb BamHI fragment into Bluescript vector.
29
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 BamHI.
36
19
1 Bovine a-lactalbumin expression in transgenic mice
2
3 The two constructs pBova-A and pBova-B (Figure 2) were
4 injected into mouse embryos and gave rise to transgenic
animals. Milk analysis by SDS-PAGE gel stained with
6 Coomasie blue (referred to as "Coomassie gels") and
7 comparison to standard amounts of a-lactalbumin showed
8 expression levels of bovine a-lactalbumin varied from
9 non detectable for pBova-A and up to =0.5-lmg/ml for 10 pBova-B (see Figure 5 and Table 1).
WO 96/02640 PCT/GB95/01651
1
Table 1
2
Bovine a-lactalbumin expression in transgenic mice
3
Mouse
Coomassie
4
244.12 BOVA-a
-
2 4 4.14 BOVA-a
-
6
"7
244.15 BOVA-a
-
/ 8
245.23 BOVA-b
-
9
245.8 BOVA-b
-
24 5.4 BOVA-b
-
11
245.7 BOVA-b
+
12
245.21 BOVA-b
-
13
245.13 BOVA-b
+
14
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
-
24 9.25.7 BOVA-b
-
21
24 9.30.3 BOVA-b
- = < 0.5mg/ml
22
24 9.30.4 BOVA-b
+ = =0.5-lmg/ml
23
24 9.33.2 BOVA-b
+/++ ++ = =l-2mg/ml
24
249.33.3 BOVA-b
+/++
26
Table 1 shows the relative levels of bovine a-
27
lactalbumin in transgenic mouse milk as estimated by
28
comparison to protein standards on Coomassie gels.
WO 96/02640 PCT/GB95/01651
21
1 Example 5 - Assembly and expression of human a-
2 lactalbumin constructs
3
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 0.5-1.Omg/ml, and mouse milk 0.1-0.8mg/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 (>10mg/ml) of heterologous genes in mouse
19 milk.
21 Transgene constructs (Figure 6^
22
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/Sall fragment
26 into pucl8.
27
2 8 pHA-2 consists of the human a-lactalbumin coding
29 region, =3.7kb of 5' flank and =13kb of 3' flank
derived from A clone 4a cloned as a =19kb Sail fragment
31 into pucl8.
32
33 pOBHA (ovine beta-lactaglobulin, human a-lactalbumin)
34 was constructed from 4 DNA fragments:
1. a 4.2kb Sail/EcoRV fragment containing the ovine
36 beta-lactoglobulin promoter (see WO-A-9Q/05188 );
WO 96/02640 PCT/GB95/01651
22
1 2. a 74bp synthetic oligonucleotide corresponding to
2 a 8bp Bell linker and bases 15-77 of the human a-
3 lactalbumin sequence used as a blunt/Bgll
4 fragment;
3. a 6.2kb Bgll/PstI human a-lactalbumin fragment
6 derived from A clone 4a comprising a region
7 between a Bgll site at base 77 and a Xhol site in
8 the 3' flank;
9 4. pSL1180 (Pharmacia) cut with PstI and Sail.
11 pBBHA (bovine beta-lactoglobulin, human a-lactalbumin)
12 was constructed from 4 DNA fragments:
13 1. a 3.0kb EcoRI fragment containing the bovine beta-
14 lactoglobulin promoter derived from clone BB25-3
and used as a EcoRI/EcoRV fragment;
16 2. a 7 4bp synthetic oligonucleotide corresponding to
17 a 8bp Bell linker and bases 15-77 of the human a-
18 lactalbumin sequence used as a blunt/Bgll
19 fragment;
3. a 6.2kb Bgll/PstI human a-lactalbumin fragment
21 derived from A clone 4a comprising a region
22 between a Bgll site at base 77 and a Xhol site in 2 3 the 3' flank;
24 4. Bluescript vector cut with EcoRI and PstI.
26 pBAHA (bovine a-lactalbumin, human a-lactalbumin) was
27 constructed from 4 DNA fragments:
2 8 1. a 0.72kb BamHI to StuI fragment containing the
29 bovine a-lactalbumin promoter derived from clone
pBA-PO.7;
31 2. a 62bp synthetic oligonucleotide corresponding to
32 bases 15-7 7 of the human a-lactalbumin sequence
33 used as a blunt/Bgll fragment;
34 3. a 6.2kb Bgll/PstI human a-lactalbumin fragment
derived from A clone 4a comprising a region
36 between a Bgll site at base 77 and a Xhol site in
WO 96/02640 PCT/GB95/01651
►
1 the 3' flank;
2 4. Bluescript vector cut with BamHI and PstI.
3
4 Human a-lactalbumin expression in transgenic 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-1 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, Vol. 297, 1.2. 13-18 (1992)).
23
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 isoelectric focusing, Western blots visualised with a 2 8 commercial anti-human a-lactalbumin antibody (Sigma)
2 9 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).
1
2
3
4
6
7
8
9
11
12
13
14
16
17
18
19
21
22
23
24
26
27
28
29
31
32
33
34
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
wo 96/02640
Table 2
Human a-lactalbumin expression in transgenic mice
Mouse
Coomassie
Western
205.19 pHAl
-
—
204.10 pHAl
++
++
204.7 pHAl
+++
+++
230.15.3 pHAl
+++
n.d.
230.15.5 pHAl
+++
n.d.
230.15.6 pHAl
+++
n.d.
230.21.5 pHAl
+++
n.d.
2 30.21.1 pHAl
++
n.d.
211.18 pHA2
+
++
211.1? pHA2
-
-
211.16 pHA2
+++
+++
212.11 pHA2
+
n.d.
213.5 pHA2
++++
++++
212.13 pHA2
++
n.d.
212.19 pHA2
-
n.d.
213.4 pHA2
+++
n.d.
212.7 pHA2
—
n.d.
232.10 BBHA
233.1 BBHA
-
231.4 BBHA
++
++
232.9 BBHA
+
+
231.9 BBHA
-
n.d.
232.5 BBHA
+
+
231.3 BBHA
+
+
232.6 BBHA
-
n.d.
2 37.6 BBHA
-
n.d.
235.15 OBHA
_
n.d.
235.19 OBHA
++
++
236.6 OBHA
++
++
2 34.1 OBHA
+
+
234.4 OBHA
++
++
234.14 OBHA
+
+
239.14 BAHA
+++
+++
239.4 BAHA
-
n.d.
240.7 BAHA
-
n.d.
239.3 BAHA
-
n.d.
239.6 BAHA
-f+
++
239.12 BAHA
-
n.d.
243.1 BAHA
++
n.d.
242.9 BAHA
+++
n.d.
241.16 BAHA
+
n.d.
234.14 BAHA
-
n.d.
243.13 BAHA
+
n.d.
243.10 BAHA
-
n.d.
243.4 BAHA
-
n.d.
1
2 Table 2 shows the relative levels of human a-
3 lactalbumin in transgenic mouse milk as estimated by
4 comparison to protein standards on Coomassie gels and
Western Blots.
6
7
8 +
9 ++
+++
11 ++++
12 n.d.
13
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
19 human a-lactalbumin standard.
21 Example 6 - Expression of Mutaaenised Bovine a-
22 Lactalbumin under the control of the Human a-
23 Lactalbumin Promoter in vivo
24
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 region, the 5' region of the bovine a-
31 lactalbumin transcriptional start site was substituted
32 with sequences from the human a-lactalbumin gene.
33
34 Two constructs were made, namely PKU-5 and PKU-1H,
which incorporate the amino acid substitutions shown in
36 Table 3.
37
<0.5 mg/ml = -0.5-lmg/ml = =1-2 mg/ml =2-3 mg/ml = >5 mg/ml = not determined
WO 96/02640 PCT/GB95/01651
26
1 The following SEQ ID Nos. have been assigned to the PCR
2 primers used.
3
4
PKU-1
SEQ
ID
No.
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.
PKU-6
SEQ
ID
No.
11
11
PKU-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.
16 PKU-5
17
18 In a first cloning step three fragments were subcloned
19 into the EcoRI/BamHI site of pUC18:
21 (1) the EcoRI to Pvul fragment derived by PCR
22 amplification using PKU-primer 7 in combination
23 with 8 (see Figure 10);
24
(2) the Pvul to BsaBI fragment derived by PCR
26 amplification using PKU-primer 9 in combination
27 "with 10 (see Figure 10); and
28
29 (3) The BsaBI to Hindlll fragment derived from pBA.
31 The final construct included 6 DNA fragments:
32
33 (1) the 3.7kb Sail to Kpnl fragment containing the
34 human a-lactalbumin promoter derived from A clone
4a (Figure 3);
36
WO 96/02640 PCT/GB95/01651
27
1 (2) the 152bp synthetic oligonucleotide containing
2 human a-lactalbumin sequences from the Kpnl site
3 to the AUG and bovine a-lactalbumin sequences from
4 the AUG to the Hapl site;
6 (3) the 1.25kb Hpal to Hindlll fragment from the first
7 subcloning step;
8
9 (4) the 0.95 kb Hindlll to BgJlI fragment derived from
pBA;
11
12 (5) the 3.7kb BamHI to Xhol fragment from the 3' flank
13 of the human a-lactalbumin gene derived from A
14 clone 4a (Figure 3) used as a BamHI fragment; and
16 (6) a Bluescript KS- vector cut with Sail and BamHI.
17
18 PKU-1H was constructed in the same way as PKU-5 with
19 the exception of fragment (3), which was derived from
PKU-1.
21
22 PKU-1 was constructed from six DNA fragments (see
23 Figure 9):
24
(1) a 2.04kb SstI to Hpal fragment derived from
26 pBOVA-6;
27
2 8 (2) a 0.4 6kb Hpal to Pvul fragment derived from PCR
29 product A (PKU-primer pair 1 and 2; see Figure
10);
31
32 (3) a 0.60kb Pvul to BsaBI fragment derived from PCR
33 product B (primer pair 3 and 4; see Figure 10);
34
(4) a 0.22kb BsaBI to Hindlll fragment derived from
36 pBOVA-6;
WO 96/02640 PCT/GB95/01651
1 (5) a 0.95kb Hindlll to Bglll fragment derived from
2 pBOVA-6;
3
4 (6) the vector pSL1180 digested with SstI and Bglll.
6 Table 3
7
8 Amino Acid Substitutions present in Transgene
9 Constructs
11 Substitutions Human promoter Plasmid
12 Human 3' flank
13 pos'n 9 30 53 80
14 Tyr, Tyr, Tyr, Tyr + pPKU-lH
Ser, Tyr, Leu, Leu + pPKU-5
16
17 Expression in transgenic mice
18
19 The two constructs PKU-IH and PKU-5 have been injected
into mouse embryos. So far transgenic animals were
21 derived for the PKU-5 construct. These animals are set
22 up for breeding to allow milk analysis.
23
24 Example 7 - Effect on Lactation bv disruption of a-
Lactalbumin deficiency and insertion of human a-
26 Lactalbumin gene replacement in mice
27
2 8 Materials and Methods
29 Mouse lines
31 Mice carrying the null a-lactalbumin allele and the
32 humanised a-lactalbumin replacement allele were derived
3 3 by breeding chimeras produced from the targeted
34 embryonic stem cell clones M2 and F6 respectively
against Balb/c mates, as described previously
36 (Fitzgerald et al J. Biol. Chem 245:2103-2108). During
29
1 the breeding of these strains, a-lactalbumin genotypes
2 were determined by Southern analysis of genomic DNA
3 prepared from tail biopsies.
4
RNA analysis
6
7 Total RNA was prepared by the method of Auffray and
8 Rougeon (Eur. J. Biochem 1^7:303-14) from abdominal
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 BamHI fragment
13 containing the complete mouse a-lactalbumin gene; and a
14 1. lkb rat /?-casein cDNA (Blackburn et al Nucl. Acids
Res 10:2295-2307).
16
17 RNAse protection analysis
18
19 32P-CTP radiolabelled antisense RNA was transcribed by
T7 RNA polymerase (Promega) from a 455 bp Hindlll-Ball
21 mouse a-lactalbumin fragment (Figure 15A) cloned in
22 Bluescript KS. The conditions for the transcription 2 3 reaction, solution hybridisation and RNAse digestion
24 were as recommended by Promega. Protected fragments
were separated by polyacrylamide gel electrophoresis
26 and visualised by autoradiography.
27
28 Milk composition and yield analysis
29
Milk samples were collected between days 3-7 of
31 lactation under Hypnorm (Roche)/Hypnovel (Janssen)
32 anaesthesia. 150mU of oxytocin (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 17 5:15). Defatted milk was assayed for protein
WO 96/02640 PCT/GB95/01651
1 (Bradford Analyt. Biochern 7.2 :248-54 ) and lactose was
2 measured enzymatically by sequential incubation with /?-
3 galactosidase, (Boehringer) glucose oxidase and
4 peroxidase (Sigma) by a method adapted from that of
Bergmeyer and Bernt (Methods in Enzyme Analysis 3.: 1205-
6 1212).
7
8 Milk yield was estimated using a titrated water
9 technique described by Knight et al. , (Comp. Biochern.
Physiol 84A:127-133) in mice suckling young over a 48
11 hour period between days 3 and 6 of lactation.
12
13 Milk ct-lactalbumin analysis and quantification
14
Milk samples were analysed on 16% of SDS-PAGE gels
16 (Novex) and western blotted onto Immobilon P membrane.
17 a-lactalbumin was detected by absorption with rabbit
18 anti-human a-lactalbumin antiserum (Dako), followed by
19 goat anti-rabbit IgG peroxidase antibody conjugate and
visualised with an enhanced chemiluminescence system
21 (Amersham).
22
23 a-lactalbumin in milk samples was quantified by a
24 modification of the method of Lindahl et al., (Analyt.
Biochern 140:394-402) for calcium dependant purification 2 6 of a-lactalbumin by phenyl-Sepharose chromatography.
27 Milk'samples were diluted 1:10 with 27% w/v ammonium
28 sulphate solution, incubated at room temperature for 10
2 9 minutes and centrifuged. Supernatant was mixed with an
equal volume of lOOmM Tris/Cl, pH 7.5, 7 0mM EDTA and
31 loaded onto a column (200^1 packed volume) of phenyl-
32 Sepharose (Pharmacia) pre-equilibrated with 50mM
3 3 Tris/Cl, pH 7.5, ImM EDTA. The column was washed with
34 the same buffer and a-lactalbumin eluted with 50mM
Tris/Cl, pH 7.5, ImM CaCl2- The optical absorbance at
36 2 80nm of the column was monitored and integrated peak
WO 96/02640 PCT/GB95/01651
31
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 Histology
7
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.
13
14 Results
16 Mouse a-lactalbumin gene deletion
17
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 supra
24 (see Figure 11). Animals carrying this allele are
designated a-lac-. The wild type mouse a-lactalbumin
26 allele is designated a-lac™.
27
2 8 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- homozygotes (see
31 Figure 12) confirming that the targeted a-lactalbumin
32 gene has been removed and that no other source of a-
33 lactalbumin mRNA exists. Hybridisation of the same RNA
34 samples with a /?-casein RNA in all samples (see Figure
12).
36
WO 96/02640 PCT/GB95/01651
32
1 a-Lacta) bum.in deficiency has no apparent effect in mice
2 other than during lactation. a-lac-/a-lac- homozygotes
3 and a-lacVa-lac-heterozygotes of both sexes are normal
4 in appearance, behaviour and fertility. However, a-
lac-/a-lac- homozygous females cannot rear litters
6 successfully. Their pups fail to thrive and die within
7 the first 5-10 days of life. Offspring of homozygous 8. a-lac-/a-lac- females do survive normally when
9 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 a-lac-/a-lac- mothers
13 are approximately half the weight of those raised by a-
14 lacm/a-lacm wild type mice. Estimates of milk yield are
consistent with this, a-lacVa-lac- heterozygotes
16 produce similar quantities of milk as wild type, but
17 the yield of a-lac-/a-lac- homozygotes was severely
18 reduced (Table 4).
Table 4
Milk composition, pup weight, mammary tissue weight and milk yield in targeted mouse lines
Genotype a-lacm/ot-lacm a-lacm/cx-lac-
a-lac-/cx-lac-
a.- lac"/ot— lach a-lach/ot-lach
Fat ( % v/v)
28.23 + 1.65(7)
29.6 ± 1.3(6)
45.25 ± 2.15(6)***
.25 + 1.36(7)
21.2 + 0.23(4)*
Protein (mg/ml)
87.52 + 5.82(7)
95.81 ± 9.5(5)
164.63 + 13.92(8)***
94.51 + 5.97(7)
77.07 ± 1.05(4)
Lactose (mM)
62.44 + 9.27(7)
42.7 + 4.2(6)
0.7 ± 0.34(3)***
42.40 ±1.93(7)
56.85 t 3.8(4)
Single pup weight
(g)
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)
Mammary tissue weight per pup
(g)
0.34 i 0.06(7)
0.4 + 0.1(7)
0.35 ± 0.05(8)
0.31 ± 0.04(6)
0.51 ± 0.09(4)
Milk yield
(g/day)
7.51 ± 0.44(4)
6.7 + 0.38(6)
1.37 ± 0.48(4)***
n. t.
9.94 + 0.65(5)*
Statistical analysis by unpaired t-test, * p<0.05; **p<0.01; ***p<0.001; Values are means t SE.
Figures in brackets indicate the number of mothers analysed.
n.t., not tested
WO 96/02640 , PCT/GB95/01651
3 A
1 Milk was obtained from each genotype by manual milking
2 and the composition of key components analysed. Milk
3 from a-lacVa-lac-heterozygotes was indistinguishable in
4 appearance from wild type milk and showed fat and
protein contents similar to wild type (Table 4). While
6 lactose concentration appeared to be slightly reduced
7 in a-lacVa-lac- heterozygotes, statistical analysis
8 showed that the difference was not significant. In
9 contrast, milk from a-lac-/a-lac- homozygotes was
viscous, difficult to express from the teats and was
11 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.
19
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-2 3 Sepharose chromatography, a technique used to
24 specifically identify a-lactalbumin which has been
adapted to obtain quantitative estimates of milk a-
26 lactalbumin content (Table 5; see also Figure 16).
27 When.applied to milk from a-lac-/a-lac-homozygotes no
2 8 a-lactalbumin was detected. In contrast, a-lactalbumin
2 9 concentration in a-lacm/a-lac- heterozygote milk was
estimated as 0.04 3mg/ml, approximately half that of
31 wild type (Table 5).
1 Table 5
2 Milk a-lactalbumin content.
3
4 Source
Human
6 a-lacVa-lac" mice
7 a-lac-/a-lac-mice
8 a-lac'Va-lac-mice
9 a-lac"/a-lach mice
a-lach/a-lach mice
11
12 a-Lactalbumin content of milk samples were estimated by
13 phenyl-Sepharose chromatography.
14
Values are means ± SE.
16
17 Figures in brackets indicate the number of mothers
18 analysed.
19
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 lacm/a-lac- and homozygous a-lac-/a-lac- lactating
24 mothers were not significantly different. Light
microscopic analysis of mammary glands (Figure 14)
26 revealed that heterozygous and homozygous a-lac-/a-lac-
27 glands were histologically normal. However, the
2 8 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 a-
32 lactalbumin
33
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 ceding region and 5'
g-lactalbumin (ma/ml) 2.9 + 0.1(2) 0.09 i 0.005(6)
0 (3)
0.043 + 0.004(5) 0.65 ± 0.07(4) 1.38 + 0.12(5)
WO 96/02640 36 PCT/GB95/01651
1 flanking sequences. The human fragment stretches from
2 0.77kb upstream of the human transcription initiation
3 site to an EcoRI site 136bp 3' of the human
4 translational stop site. Junctions with mouse
sequences were made at a BamHI site 0.57kb 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, supra; see also Figure 11). Here
9 we describe our analysis of animals carrying this
allele, designated a-lach.
11
12 Deletion of the murine a-lactalbumin gene established
13 that a-lactalbumin deficiency blocks lactose synthesis
14 and severely disrupts milk production. We have used
the a-lach allele to test the ability of human a-
16 lactalbumin to restore milk production in the absence
17 of mouse a-lactalbumin. a-lac°/a-lach heterozygous and
18 a-lach/a-lach homozygous mice were normal in appearance,
19 fertility and behaviour.
21 In contrast to a-lac-/a-lac- mice, a-lach/a-lach
22 homozygous mothers produce apparently normal milk and
23 rear offspring successfully. Table 4 shows that pups
24 raised by a-lactt/a-lach heterozygous and a-lach/a-lach
homozygous 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
2 9 constitute clear evidence that the human gene can
functionally replace the mouse gene. Analysis of milk
31 composition (Table 4) shows that lactose concentration
32 is similar in all genotypes. Although both protein and
33 fat concentrations seem reduced in a-lach/a-lach
34 homozygous animals, only the fat reduction was judged
statistically significant by unpaired t-test. These
36 animals show an increase in milk volume over wild type
37 (Table 4) .
38
37
PCT/GB9S/01651
1 Relative quantification of human and mouse a-
2 lactalbumin RNA.
3
4 Human milk contains considerably more a-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
S characteristic of the mouse gene. a-lacVa-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.
14
Figure 15A shows the strategy used to compare levels of
16 mouse and human a-lactalbumin mRNA. Because the
17 junction between human and mouse a-lactalbumin
18 sequences lies upstream of the polyadenylation site, a-
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 used 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.
27
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 K 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 a-lactalbumin RNA
34 (see Lane B) . Homozygous a-lach/a-lach gland RNA
protected a smaller band consistent with the predicted
36 120 base RNA protected by human a-lactalbumin mRNA (see
37 Lane C). Lanes D-J show results consistent with a
38 series of heterozygous a-lac"7a-lach animals were
1
2
3
4
6
7
8
9
11
12
13
14
38
PCT/GB9S/01651
obtained (seo Lanes D to J). Small and large protected fragments in ^ach sample indicate the presence of both human and mouse a-lactalbumin mRNA. Protected fragments were excised from the gel, radioisotope content measured, adjusted for the size difference and the ratio of human to mouse a-lactalbumin mRNA estimated. 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 a-lactalbumin mRNA in each a-lacn/'a-lach heterozygote. It is apparent that, although there was variation between individual animals, human a-lactalbumin mRNA was significantly more abundant than mouse mRNA. Averaging the seven a-lacm/a-lach heterozygotes gives a value of 15-fold greater expression for human a-lactalbumin mRNA.
39
1
2
3
4
6
7
8
9
11
12
13
14
16
17
18
19
21
22
23
24
26
27
28
29
31
32
33
34
Table 6
Relative quantification of human and mouse a-lactalbumin mRNA in a-lac°/a-lach mammary glands
Lane
Mouse#
2
3
120 base 305 base human/mouse fragment3 fragment" RNA ratiob
H
76
98
99 79
5957 5770 4825 6018 5206 5481 26858
1000 547 810 1077 1452 1117 3561
:1 26:1 15:1 14:1 9:1 12:1 19:1
Lane designations indicate the source of protected fragments and correspond to those shown in Figure 15B.
a. numbers are expressed in counts per minute (c.p.m.)
b. Ratio between c.p.m. of 120 base fragment multiplied by 2.54 (to adjust for size difference) and c.p.m. of 305 base fragment.
WO 96/02640 PCT/GB95/01651
1 Human a-lactalbumin protein expression
2
3 A Western analysis of a-lactalbumin in targeted mouse
4 lines was conducted. Human a-lactalbumin can be
distinguished from mouse a-lactalbumin by its faster
6 electrophoretic mobility (see Lanes A, B). A prominent
7 lower band in a-lach/a-lach homozygotes and a-lach/a-lacm
8 heterozygotes was observed (see Lanes C, D, G, H), and
9 corresponds to the position of the human a-lactalbumin
standard and was only observed in mice which express
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
released by cyanogen bromide cleavage (data not shown).
16 The band with slower mobility, similar to mouse a-
17 lactalbumin, is also a human a-lactalbumin gene product
18 the nature of which is unknown. This species varied in
19 intensity with a-lach gene dosage (see Lanes G, H) and 2 0 was also present in milk from human a-lactalbumin
21 transgenic mice generated by pronuclear microinjection
2 2 (data not shown).
23
2 4 The a-lactalbumin 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 2 8 the a-lac"/a-lach heterozygote and a-lach/a-lach
29 homozygote shown in Figure 13. The peaks corresponding
to eluted a-lactalbumin are marked. a-Lactalbumin
31 contents were estimated by comparing the integrated
32 peak areas with the human a-lactalbumin standard curve
33 shown. The relationship between integrated peak area
34 and a-lactalbumin quantity was linear and highly
reproducible. a-Lactalbumin content for the samples
36 • shown in Figure 16 were estimated as follows: a-lacVa-
37 lacm wild-type O.lmg/ml; a-lac°'/a-lach heterozygote #76
38 0.45mg/ml; a-lach/a-lach homozygote #22 0.9mg/ml. Table
WO 96/02640 .. PCT/CB95/01651
1 5 shows the concentration of a-lactalbumin in milk
2 samples from targeted mouse lines and lactating women.
3 It is clear that the concentration of a-lactalbumin in
4 milk is directly related to gene dosage, eg a-lac°/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 components
11 in a-lacm/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-lacn/a-lac-
16 heterozygotes and wild type mice. Therefore, a-lacVa-
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-lacVa-lach
21 heterozygote milk. This is remarkably consistent with
22 the relative proportions of mRNA.
23
24 Example 8 Enhanced expression of a heterologous crene.
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. We can neither be
certain whether they were low expressors or not
36 transgenic.
37
WO 96/02640 k2 PCT/GB9S/01651
1 Table 8: Constructs PKU-0 to BALT-B all contain the
2 bovine a-lac promoter (about 2kb) . Constructs PKU-IH
3 to PKU-16 all contain the human a-lac promoter (3.7kb).
4 Using the human a-lac promoter increased the expression
of the transgene to almost 100%.
6
7 These data show that the use of the human a-lac
8 promoter achieves a higher level of expression than the
9 use of the bovine promoter, and induces expression in 10 more animals than the bovine promoter.
TABLE 7
CONSTRUCT
MOUSE
SEX
C
mg/ml
MILK
ANALYSIS
TRANS.
MI FREQ.
pHA2
210-17
M
-
—
1/21=5%
211-12
F
<1
-
-
14/54 = 26%
211-16
F
3/4
211-17
F
>>10
ND
0/8
211-24, 27, 29, 31, 36, 37, 42, 46, 47, 54
M
212-7
F
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
0/8
212-36, 44, 45, 46
M
213-4
F
<10
0/2
2/14=14%
213-5
F
>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 FREQ. = Integration frequency
A3
TABLE 8
Constr.
Transgenics
Expressers max. expr.
PKU-0
6F/5M
3/5
500
PKU-1
18F/23M
/18
200
PKU-2
8F/7M
3/8
400*
PKU-3
6F/5M
2/6
100*
PKU-4
7F/3M
/18
300*
BALT-A
34F/24M
n.t.
n.t.
BALT-B
10F/18M
n.t.
n.t.
PKU-IH
6F/5M
4/5
100
PKU-5
13F/14M
13/13
800
PKU-6
3F/4M
2/2
100
PKU-7
4F/11M
1/1
<20
PKU-16
13F/12M
n.t.
n.t.
HctPKU-1
n.t.
n.t.
n.t.
HaPKU-2
n.t.
n.t.
n.t.
n.t. = not tested * estimate
hh sequence: listing
(1) GENERAL INFORMATION:
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(ii) TITLE OF INVENTION: Alpha-lactalbumin Gene Constructs (iii) NUMBER OF SEQUENCES: 21
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(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 1:
GCGGATCCAC AACTGAAGTG ACTTAGC 27
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(2) INFORMATION FOR SEQ ID NO: 6:
45
GATGGATCCT GGGTCCTCAT TGAAAGGACT GATCC (2) INFORMATION FOR SEQ ID NO: 3:
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46
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(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 6:
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AAAATTTGG 69
(2) INFORMATION FOR SEQ ZD NO: 9:
I
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SUBSTITUTE SHEET (RULE 26)
47
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(2) INFORMATION FOR SEQ ID NO: 10:
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(Xi) SEQUENCE DESCRIPTION: SEQ ID NO: 10:
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(2) INFORMATION FOR SEQ ID NO: 11:
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GCGGATCCAA AGACAGCAGG TGTTCACCGT CGACGACGCC TACGTAACTT CTCACAGAGC 60
CACTG 65
(2) INFORMATION FOR SEQ ID NO: 12:
1
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SUBSTITUTE SHEET (RULE 26)
48
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(i) SEQUENCE CHARACTERISTICS:
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GCGGATCCGA TCGCTTGTGT GTCATAACCA CTGGTATGAT ACGCGGTACA GACC
(2) INFORMATION FOR SEQ ID NO: 14:
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(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 14:
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aaaatttgg gg
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SUBSTITUTE SHEET (ROLE 26)
WO 96/02640 PCT/GB95/01651
49
(C) STRANDEDNESS: single
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GCTCTAGATC ATCATCCACC AGCTCTGGCA GGAG 34
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GGATCCAAAG TTGGCTAAAC ACTGGCCGGG TGCAGTGCTT CCACCTGTAA TTCCAGCACT 60
TTGGAAGGCT GAGGTGGGCA GATTGCTTGA GC-TCAGGAGT TTGAGACCAG CTTGGCTAAC 120
AGCAAAACCC TGTCTCTACC AAAAGTACAA AAATTATCTG GGTGTGGTGG CAGGCGCCTG 180
TAATCCCAGC TACTCGGGAG GCTGAGGCAG AAGAATTGTT TGAACCTGGG AGGCAGAGGT 2 40
TGTAGTGAGC TGAGATCGCT CATT 264 (2) INFORMATION FOR SEQ ID NO: 17:
(i) SEQUENCE CHARACTERISTICS:
(n) LENGTH: 803 base pairs
(B)" TYPE: nucleic acid
(C) STRANDEDNESS: double
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: cDNA
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 17:
TCTTTTTCAA TTATTCATTT GTTACAGTGG GTTATGATAC AAATGTTTAT AGATGCCTAC 60
SUBSTITUTE SHEET (RULE 26)
WO 96/02640 PCT/GB95/01651
50
TC7GTAC7AG TACTACAGAG CACTTT7TCT GTGTTTATAT TCAGTTCAAT TGTAGTGTGT 120
TGAGTTGTAT WWTAATCCAT GTATTAAATC AAATAAACAA ACAAAATGCC ATGTTCTTTG 180
GTACAAGCAA CACTCACCAA AGGCATTTGG GGTCTGCATT TGGAATTCTC AGGCAAACTC 240
TCTCTTGTTC CTAGTCTGTA C77A777TCC CCACACTAGC T7A7G7ATAT ATATTTTTGA 300.
GAT7GGAGTT GCCCTTGTTG CCCAGGCTGG AGTGCAGTGG CACGATTCTT GGCTCACGAG 360
ACCTCCACGT CTTGGGTTAA AGCGTTTCTC CTGCCTCACC CTCCTGACTA CTGGGATTAC 420
AGGCGCCTGC CACCA7GCCC GGCTAA777T TGTAT7777A GTAGAGA7GG GCT77CACCA 480
TG7TGCTCAG GCTGG7CTTG AAACTCCCCA CCTCGGCCC7 TCCCAAATGC GCTGGGATTA 540
CAGG7GTGAG CCACAGTGCC TGGCCTGTAC A7T7TTTAAA T7TCAA7GTC 7AATATGGTG 600
TCCAC7GAAT TAAGAATTC7 77TGAGAAAA TGAA7CAA7A AATC7A7ACA CTGCCTCCTT 660
7ATCCAG7GA GGTATGGCTG GA7CAGCT7C A 7 G A C AT A C A 7GCCAGTAGT TCTCCTCCTC 720
CTCCTTTT7T ACAAATAAAA ATTG7ATA7G T7GAAGG7G7 ACAAC77GAT GTTTG7TATA 780
TG7ATACACT TAAATGTCAC CAC 303 (2) INFORMATION FOR SEQ ID NO: 18:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 233 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: double
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: cDNA
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 18:
TTTGGGTAGA AGCAGACAGT AAACTTGCTG TTCTCTTCCT GAGATCTTTT GTTGAGATGC 60
TGAATAGGAG GCAGCATGGC AGCTGAGCTA TCTGTTCTGC T77C7C7ACC TCTGTCTCTT 120
TCCCTTAGGC CTAAAATGAA GCTCTAAGCC AAGCAAAGG7 C7GAAGTCAT CCAGACTAAT 180
7GGGAAGCGG GTAGGCTCCA GGGAGTGGCT CTCAGAGAGC AGACCATTTA CTGAGCTC 238 (2) INFORMATION FOR SEQ ID NO: 19:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: lo2 base pairs
(B) TYPE: nucleic acid
SUBSTITUTE SHEET (RULE 26)
WO 96/02640 PCT/GB95/01651
51
(C) STRANDEDf-iESS : COuble
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE:
cDNA
(xi) SEQUENCE DESCRIPTION': SEQ ID NO: 19:
AATACAGAGT TTTGTTCTCT ACTCTTATCC TGCTTTCTCC TCCCTCCTAC TTTTCCCTGA 60
CACCTATCTT GTTGTGAAGA CAGGAATTGC ATTAGATAAA ATCAAATCTT TTTTATTTTT 120
TTTTGAGATG GAATCTTGCT CTGTTTCCAG GCTGGAGTGC TG 162
(2) INFORMATION FOR SEQ ID NO: 20:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 472 bass pairs (3) TYPE: nucleic acid
(C) STRANDEDNESS: double
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: cDNA
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 20:
ATCTGGTCAG CAGTGAAGCT CAGTGTACAC ATTCATTCCT TCCTTCACTG CTTGATTTGT 60
CACCAAGTGG TTATTGAGGA TATGCTGTTT GCTAGGTACT ACTTTACTTA TTTATTTGTT 120
TATTTAGAGA TGGGGTCTCA CAATGTTGCC CAG7CTACAG GACAGTGGCT ATTCACAGGT 180
GTGAGCACAG CACACTACAG CCTCAAACTC CTGAGTTCAA GAGATCCTCC TGCCTCAGTC 240
TCTCGAGTAG CTGGGACTAC AGGGATGTGC CACCACACAT GGCTTAGGCT CTACTTTAGC 300
TGCTACTTGA AGGATGAAGA TAGGAGGAGA CACTCTTATT TTATTTGATT TCTTTTTTTT 360
TTTTTTTTTT TTGACAGAGT TTTGCTCTGT TGCCAGGCTG GAGTGCTCAC TGCAACCTCC 420
ACCTCCAGGT CAAGCAATTC TCCTGCTCAG CCTCCGAG7A GTCGGACCAA GG 47 2 (2) INFORMATION FOR SEQ ID NO: 21:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 2119 base pairs (3) TYPE: nucleic acid
(C) STRANDEDNESS: double
(D) TOPOLOGY: linear
SUBSTITUTE SHEET {RULE 26)'
WO 96/02640 PCT/GB95/01651
52
(ii) MOIECULE TY?Z: cDNA
(xi) SEQUENCE DESCRIPTION: SI GGAATTCCCA TTCCTGTCCT GTACCCTTGC AGTGGCTCCA CTTCTGTTGT GTTTCTGAAC CATCCCTTCT GGCACACCAA GATCCTCCAC GGTTATCCAT GGTACAGAAG ACAGGATTTA CATGTAGGAG ATAACGATAT GGAAGACCTT GATAAATGTT CCCTGAAACA TAAGAAACAG TTGGGAGGGC CGAGGCCAGG CAGGCAAATT GCCAACATGC AGAAACTCCG TCTCTACTAA CGTGCCTGTA GTCCCAGCTA CTCGGGAGGC GCAGAGGTTG CAGTGAGCCA AGATCGCGCC ACTTGGTCAA AAAAAAAAAA AAAAAAAAAA GAGATGAGGA CAAAGAAGAC GAATCGGTGG TGACATGAAG CTTCATGCCA GCAAATTAAA TACTTGCTCA GGGGCACTGA CCTTATAGAG TCCAATCTTT CCACTGGCTT GGTCCTTCCC CAAGTTATTG GTCTTAGATT TATGTAATGT CGGTAGGAGT GGTTAGGGGT GGGGAATCTG TTTTTTTTTT TTTTTTTTAA AGATAGGGTC GTGCAGTGGA GTGAACATGG CTCACTGCAG TGCCTCAGCC CCTCAAGTAG CTGGGACTAC TTTTGTAGAG ATGGGATTTT ACCATGTTGC TGATCCACCA GACTCGGCCT CCCAAAATGC GCCTAGATGC TTTCATACAG GCTTTTCAAT GATCCAAGTT ATATCGGATT GTTGTAGTCT
Q ID NO: 21:
AGTGCCTCTG GGTGGAATGC CGACAAATGG 60
ATGTATCTCT TGCTATCAGA ACTTTCTGCT 120
ATTCCCTTCA CTCATGCCAC TTCATATACT 180
ACTGAGAGGA CTTTTCCCTG ACTCTGAATA 240
CAGTATGTAA GTCTTAAATA GATTGGTTGG 300
CGCAGCGGCT CCTGTCTGTA ATCTAGCACT 3 60
GCCTGAGCTC AGAAGTTTGA GACCAGCCTG 4 20
AAATACATAA ATTAACCGGG CATGGTAACA 480
TGAGGCAGGA GAA7CACTTG AGCCTGGGAG 540
ACTGCATTCC AGCCTGGCCA ACAGAGTGAG 600
AAAAGGAAGA AGAAGAAGAA ATCAGGTTTA 660
CATGAAGGAG CTAAGAGCTA CTTGTCACCA 720
GGAGCTATTC AGAACTAGTA TCCTCAACTC 780
ATTCCAGACA TAAGCTTGTT CAGCCTTAAG 840
ACTTTCTGTG GCCAACTCTG AGGTTGTCTA 900
CTCAATGCCA GTGTAGTATT TGGTTATTTA 960
ATAATAGCTC GTAGGATAGC TAGATTCTTT 1020
TCACTTTGTC TCCCAGGATG GATGGATGGA 1080
CCTCGACCTC CTGTGCTCAA G7GTTCCTCC 1140
AGGCACATGT CACCATGCCC AGCTAATTTT 1200
CCAGGCTGGT CTCGAGCTCC TGGGCTCAAG 1260
CGGGATTACA GGTGTGAGCC ACTGTGCCTG 1320
TATGCATTTT CCTTAAGTAG GAAGTCTTAA 1380
ACGTTCCCAT ATTCTATTCC TATTTCTGAG 1440
SUBSTITUTE SHEET (RULE 26)
CC7TCAC7CA i GG _ . GGACrt C7CCACAT7C A7777C7GA7 CnC.-^GnC.nC i
GGGAAGCAGG GATACTGGGA CG77TCTGTC CCAGAGCCCC TTCAGGTTCT C7G77CCC7G GACATAGA7G
7GAGC . AC-.-. —---vvj
AG7GCCAGC7 C7GA7CC7GG TGCATGTC7C 7AGG«jGGGAA TGCCTCAC77 C77A7A7TGC GC77CCCAGA ACCAACCC7A ATCA7GG • ii A/*iC - - . XC GAGGGAAACG AAAAG7AGGG TTGGCATGAC CAGTC7C7C7 TGAGGCTT7C 7GCATGAA7A TGGGGG7AGC CAAAA7GAGG CCA7CCTGGC ~AAGC.---.77C G77A7GGAG
53
C 4 AA» ,C.uw GAC7CTGGCA GCGGGAAGCT CCCCATGCCC C A A G A A A C A A TGG CC.-aG.-.O P1. 7GAA7TATGG 7CAT7C7C77 TAAATAAATG TTC7TTGTCC ACAA.-.A7G7G
GCC77G77AC 7G7GATGACA CGGTATACAA 77CTTTCTTC AGGGCTAAAC ACAATACCTG AAGGAAGCTG CC7AGATGTA AAACTGAGTG CTCTGT7CCT
- vj — GG A7 TACACCCCC7 CCTT7ATTGT CTCAAG7AAC AAACCCAAA7 C « ATGGAC7A GCAGGCTCAG GGCC7TGGTA ATGCTTCCAT C-GTGGGCA7C GC7GC7GAAA
1500 1560 1620 1680. 1740 1800 1860 1920 1980 2040 2100
SUBSTITUTE SHEET (RULE 26)
54
INDICATIONS RELATING TO A DEPOSITED MICROORGANISM
(PCTRule 13 bis)
A. The indications made below relate to the microorganism referred to in the description 9 , line 13-15
on page
B. IDENTIFICATION OF DEPOSIT
Further deposits are identified on an additional sheet | |
Name of depositary institution
NATIONAL COLLECTIONS OF INDUSTRIAL AND MARINE BACTERIA LTD
Address of depositary institution (includingpostal code and country)
23 ST MACHAR DRIVE ABERDEEN AB2 1RY SCOTLAND -UNITED KINGDOM
Date of deposit
FEBRUARY 1995
Accession Number
NCIMB 40709
C. ADDITIONAL INDICATIONS (leave blank if not applicable) This information is continued on an additional sheet | |
ESCHERICHIA COLI DH5 <*. F' (K12) CONTAINING PLASMID pHA-2
D. DESIGNATED STATES FOR WHICH INDICATIONS AREMADE (if the indications ore not for all designated States)
ALL STATES
E. SEPARATE FURNISHING OF INDICATIONS (leave blank if not applicable)
The indications listed below will be submined to the International Bureau later (specify the general nature of the indications e.g., 'Accession Number of Deposit')
ACCESSION NO DATE OF DEPOSIT CONSTRUCT DEPOSITED
40709
FEBRUARY 1995 pHA-2
For receiving Office use only
This sheet was received with the international application
111 SEPTEMBER 1995
Authorized officer
D. J. MACKERNESS (MRS!
For International Bureau use only
[""I Tb's sheet was received by the International Bureau on:
Authorized officer
Form PCT/RO/134 (July 1992)
55 P20170NZ
ifi-
Claims (16)
1. A recombinant DNA construct comprising, in the 5' to 3' direction and operatively linked: (a) the 5' flanking sequence of SEQ ID NO 21; (b) a DNA sequence encoding (1) a signal sequence and (2) a-lactalbumin or a functional equivalent thereof; and (c) the 3' flanking sequence of any one of SEQ ID NO'S 16 to 20; the DNA construct being adapted to express the a-lactalbumin or functional equivalent thereof in a non-human transgenic animal.
2. A recombinant DNA construct comprising, in the 5' to 3' direction and operatively linked: (a) at least about 1.8 kb of 5'-flanking sequence from the human a-lactalbumin gene including the a-lactalbumin promoter; (b) a DNA sequence encoding (1) a signal sequence; and (2) a-lactalbumin or a functional equivalent thereof; (c) at least about 3 kb of 3'-flanking sequence from the human a-lactalbumin gene - 56 the DNA construct being adapted to express the a-lactalbumin or functional equivalent thereof in a non-human transgenic animal.
A recombinant DNA construct as claimed in claim 1 or 2 comprising about 3.7 kb of 5' flanking sequence and about 13 kb of 3' flanking sequence.
A recombinant DNA construct as claimed in claim 1, 2 or 3, wherein the a-lactalbumin is human a-lactalbumin.
A recombinant DNA construct as claimed in any one of claims 1 to 4, wherein the functional equivalent of a-lactalbumin is an a-lactalbumin mutein incorporating amino acid substitutions as compared to wild type a-lactalbumin.
The recombinant DNA construct pHA-1 as described herein.
The recombinant DNA construct pHA-2 as described herein. •?
A vector containing a recombinant DNA construct as claimed in any one of claims 1 to 7.
A host cell containing a vector as claimed in claim 8.
A transgenic animal having a recombinant DNA construct as claimed in any one of claims 1 to 7 integrated into its genome.
A transgenic animal as claimed in claim 10 which is capable of transmitting the construct to its progeny. ~ t yp n 57 QP20.170NZ /y i~
12. A transgenic animal as claimed in claim 10 or 11 which is a lactating female.
13. Transgenic cattle as claimed in claim 10, 11 or 12. 5
14. A method of producing milk having an enhanced content of a-lactalbumin, the method comprising extracting milk from a lactating female transgenic animal as claimed in claim 12. 10
15. Milk produced by a method as claimed in claim 14.
16. a-lactalbumin extracted from milk as claimed in claim 15. END OF CLAIMS
Applications Claiming Priority (5)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
PCT/GB1994/001514 WO1995002692A1 (en) | 1993-07-16 | 1994-07-13 | Modified alpha-lactalbumin |
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" |
PCT/GB1995/001651 WO1996002640A1 (en) | 1994-07-13 | 1995-07-12 | Alpha-lactalbumin gene constructs |
Publications (1)
Publication Number | Publication Date |
---|---|
NZ289197A true NZ289197A (en) | 1998-09-24 |
Family
ID=56289646
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
NZ289197A NZ289197A (en) | 1994-07-13 | 1995-07-12 | Alpha lactalbumin gene constructs and transgenic cattle |
Country Status (7)
Country | Link |
---|---|
EP (1) | EP0765390A1 (en) |
JP (1) | JPH10502816A (en) |
CN (1) | CN1157635A (en) |
AU (1) | AU700224B2 (en) |
CA (1) | CA2193513A1 (en) |
NZ (1) | NZ289197A (en) |
WO (1) | WO1996002640A1 (en) |
Families Citing this family (6)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN100335632C (en) * | 2000-12-08 | 2007-09-05 | 李宁 | Seven kinds of yak milk protein gene sequence |
CN100445379C (en) * | 2005-04-21 | 2008-12-24 | 李宁 | Human alpha-lacto albumin gene transgenic cloned macro domectic animal production method |
CN101104635B (en) * | 2007-04-30 | 2010-11-03 | 北京济普霖生物技术有限公司 | Method for purifying recombination human alpha-whey albumin from transgene cow milk |
WO2010119088A2 (en) * | 2009-04-15 | 2010-10-21 | Bodo Melnik | Milk and milk-based products modified to exhibit a reduced insulinemic index and/or reduced mitogenic activity |
CN102590413B (en) * | 2012-01-18 | 2013-12-25 | 浙江省疾病预防控制中心 | Quantitative detection method for bovine alpha-lactalbumin |
CN114736287B (en) * | 2022-04-20 | 2023-07-07 | 中国农业科学院生物技术研究所 | Hypoallergenic alpha-lactalbumin, and preparation method and application thereof |
Family Cites Families (3)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
WO1993025567A1 (en) * | 1992-06-15 | 1993-12-23 | Gene Pharming Europe B.V. | Production of recombinant polypeptides by bovine species and transgenic methods |
GB9314802D0 (en) * | 1993-07-16 | 1993-08-25 | Pharmaceutical Proteins Ltd | Modified proteins |
AU1454495A (en) * | 1993-12-29 | 1995-07-17 | Gene Pharming Europe Bv | Recombinant production of modified proteins lacking certain amino acids |
-
1995
- 1995-07-12 CA CA002193513A patent/CA2193513A1/en not_active Abandoned
- 1995-07-12 WO PCT/GB1995/001651 patent/WO1996002640A1/en not_active Application Discontinuation
- 1995-07-12 CN CN95194129A patent/CN1157635A/en active Pending
- 1995-07-12 NZ NZ289197A patent/NZ289197A/en unknown
- 1995-07-12 AU AU28962/95A patent/AU700224B2/en not_active Ceased
- 1995-07-12 EP EP95924467A patent/EP0765390A1/en not_active Withdrawn
- 1995-07-12 JP JP8504802A patent/JPH10502816A/en active Pending
Also Published As
Publication number | Publication date |
---|---|
JPH10502816A (en) | 1998-03-17 |
AU700224B2 (en) | 1998-12-24 |
AU2896295A (en) | 1996-02-16 |
CN1157635A (en) | 1997-08-20 |
EP0765390A1 (en) | 1997-04-02 |
WO1996002640A1 (en) | 1996-02-01 |
CA2193513A1 (en) | 1996-02-01 |
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