AP411A - DNA sequences used in the production of recombinant human BSSL/CEL in transgenic non-human mammals, and the produced BSSSL/CEL used in infant formulas. - Google Patents

DNA sequences used in the production of recombinant human BSSL/CEL in transgenic non-human mammals, and the produced BSSSL/CEL used in infant formulas. Download PDF

Info

Publication number
AP411A
AP411A APAP/P/1993/000538A AP9300538A AP411A AP 411 A AP411 A AP 411A AP 9300538 A AP9300538 A AP 9300538A AP 411 A AP411 A AP 411A
Authority
AP
ARIPO
Prior art keywords
cel
human
bssl
mammal
gene
Prior art date
Application number
APAP/P/1993/000538A
Other versions
AP9300538A0 (en
Inventor
Karl Gunnar Bjursell
Peter Nils Ivar Carlsson
Curt Sven Magnus Enerback
Stig Lennart Hansson
Ulf Fredrik Pontus Lidberg
Jeanette Annika Nilsson
Jan Birger Fredrik Tornell
Original Assignee
Ab Astra
Priority date (The priority date 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 date listed.)
Filing date
Publication date
Priority claimed from SE9201809A external-priority patent/SE9201809D0/en
Priority claimed from SE9201826A external-priority patent/SE9201826D0/en
Priority claimed from SE9202088A external-priority patent/SE9202088D0/en
Priority claimed from SE9300902A external-priority patent/SE9300902D0/en
Application filed by Ab Astra filed Critical Ab Astra
Publication of AP9300538A0 publication Critical patent/AP9300538A0/en
Application granted granted Critical
Publication of AP411A publication Critical patent/AP411A/en

Links

Classifications

    • 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; CARE OF BIRDS, FISHES, INSECTS; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
    • A01K67/00—Rearing or breeding animals, not otherwise provided for; New breeds of animals
    • A01K67/027—New breeds of vertebrates
    • A01K67/0275—Genetically modified vertebrates, e.g. transgenic
    • A01K67/0278—Humanized animals, e.g. knockin
    • 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
    • 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
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
    • C12N9/16—Hydrolases (3) acting on ester bonds (3.1)
    • C12N9/18—Carboxylic ester hydrolases (3.1.1)
    • C12N9/20—Triglyceride splitting, e.g. by means of lipase
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01K—ANIMAL HUSBANDRY; CARE OF BIRDS, FISHES, INSECTS; 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; CARE OF BIRDS, FISHES, INSECTS; 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; CARE OF BIRDS, FISHES, INSECTS; 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; CARE OF BIRDS, FISHES, INSECTS; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
    • A01K2227/00—Animals characterised by species
    • A01K2227/10—Mammal
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01K—ANIMAL HUSBANDRY; CARE OF BIRDS, FISHES, INSECTS; 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; CARE OF BIRDS, FISHES, INSECTS; 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
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • 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
    • C12N2830/00—Vector systems having a special element relevant for transcription
    • C12N2830/008—Vector systems having a special element relevant for transcription cell type or tissue specific enhancer/promoter combination

Abstract

The present invention relates to a DNA molecule containing

Description

The present invention relates to a DNA molecule containing intron sequences and encoding a human protein which is, depending on the site of action, called Bile Salt-Stimulated Lipase (BSSL) or Carboxyl Ester Lipase (CEL). The DNA molecule is advantageously used in the production of recombinant human BSSL/CEL, preferably by means of production in transgenic nonhuman mammals. The recombinant human BSSL/CEL can be used as a constituent of infant formulas used for feeding infants as a substitute for human milk, or in the manufacture of medicaments against e.g. fat malabsorption, cystic fibrosis and chronic pancreatitis .
(56) Documents cited: WO 9115234 bad original $
PRIORITY DATA CONTINUED
2. SE 9201826-6 12.06.92
3. SE 9202088-2 03.07.92
4. SE 9300902-5 19.03.93
INVENTORS CONTINUED
2. PETER NILS IVAR CARLSSON Kommendorsgatan 30A
S-414 59 Goteborg SWEDEN
3. CURT SVEN MAGNUS ENERBACK Slatthultsliden 13A S-431 69 Mondal SWEDEN
4. STIG LENNART HANSSON Bjorkvagen 50 S-902 40 Umea SWEDEN
5. ULF FREDRIK PONTUS LIDBERG Teknologgatan 4
S-411 32 Goteborg SWEDEN
6. JEANETTE ANNIKA NILSSON Seagatan 16
S-413 14 Goteborg SWEDEN
7. JAN BIRGER FREDRIK TORNELL Klaveskarsgatan 56 S-421 59 Bastra Frolunda SWEDEN
BAD ORIGINAL 4
AP Ο Ο Ο 4 1 1
HX 1147 —1—
TECHNICAL FIELD
The present invention relates tc- a DNA molecule containing intron sequences and encoding a human protein which is, depending on the site of action, called Bile Salt-Stimulated Lipase (BSSL) or Carboxyl Ester Lipase (CEL). The DNA molecule is advantageously used in the production of recombinant human BSSL/CEL, preferably by means of production in transgenic nonhuman mammals. The recombinant human BSSL/CEL can be used as a constituent of infant formulas used for feeding infants as a substitute for human milk, or in the manufacture of medicaments against e.g. fat malabsorption, cystic fibrosis and chronic pancreatitis .
BACKGROUND OF THE INVENTION
Hydrolysis of dietary lipids
Dietary lipids are an important source of energy. The energy-rich triacylglycerols constitute more than 95% of these lipids. Some of the lipids, e.g. certain fatty acids and the fat-soluble vitamins, are essential dietary constituents. Before gastro-intestinal absorption the triacylglycerols as well as the minor components, i.e. esterified fat-soluble vitamins and cholesterol, and diacylphosphatidylglycerols, require hydrolysis of the ester bonds to give rise to less hydrophobic, absorbable products. These reactions are catalyzed by a specific group of enzymes called lipases .
In the human adult the essential lipases involved are considered to be Gastric Lipase, Pancreatic ColipaseDependent Lipase (hydrolysis of tri- and bad original $
AP Ο Ο Ο 4 1 1
HX 1147 —2— diacylglycerols), Pancreatic Phospholipase A2 (hydrolysis of diacylphosphatidylglycerols) and Carboxylic Ester Lipase (CEL) (hydrolysis of cholesteryl- and fat soluble vitamin esters). In the breast-fed newborn, Bile Salt-Stimulated Lipas- (BSSL) plays an essential part in the hydrolysis of several of the above mentioned lipids. Together with bile salts the products of lipid digestion form mixed micelles from which absorption occurs.
Bile Salt-Stimulated Lipase
The human lactating mammary gland synthesizes and secretes with the milk a Bile Salt-Stimulated Lipase (BSSL) (Blackberg et al., 1987) that, after specific activation by primary bile salts, contributes to the breast-fed infant's endogenous capacity of intestinal fat digestion. This enzyme, which accounts for approximately 1% of total milk protein (Blackberg & Hernell, 1981), is not degraded during passage with the milk through the stomach, and in duodenal contents it is protected by bile salts from inactivation by pancreatic proteases such as trypsin and chymotrypsin. It is, however, inactivated when the milk is pasteurized, e.g. heated to 62.5°C, 30 min (Bjorksten et al., 1980) .
Model experiments in vitro suggest that the end products of triacylglycerol digestion are different in the presence of BSSL (Bernback et al., 1990; Hernell &
Blackberg, 1982). Due to lower intraluminal bile salt concentrations during the neonatal period this may be beneficial to product absorption.
Carboxylic Ester Lipase
The Carboxylic Ester Lipase (CEL) of human pancreatic juice (Lombardo et al., 1978) seems functionally to be identical, or at least very similar, to BSSL (Blackberg et al, 1981) . They also share common epitopes, have
BAD ORIGINAL ft
AP Ο Ο Ο 4 1 1
HX 1147 —3— identical N-terminal amino acid sequences (Abouakil et al., 1988) and are inhibited by inhibitors of serine esterases, e.g. eserine and diisopropylfluorophopsphate. In recent studies from several laboratories the cDNA structures from both the milk lipase and the pancreas lipase have been characterized (Baba et al., 1991; Hui et al., 1991; Nilsson et al., 1990; Reue et al., 1991) and the conclusion is that the milk enzyme and the pancreas enzyme are products of the same gene (in this application referred to as the CEL gene, EC 3.1.1.1). The cDNA sequence and deduced amino acid sequence of the CEL gene are described in WO 91/15234 (Oklahoma Medical Research Foundation) and in WO 91/18923 (Aktiebolaget Astra).
CEL is thus assumed to be identical to BSSL, and the polypeptide encoded by the CEL gene is in the present context called BSSL/CEL.
Lipid malabsorption
Common causes of lipid malabsorption, and hence malnutrition, are reduced intraluminal levels of Pancreatic Colipase-Dependent Lipase and/or bile salts. Typical examples of such lipase deficiency are patients suffering from cystic fibrosis, a common genetic disorder resulting in a life-long deficiency in 80% of the patients, and chronic pancreatitis, often due to chronic alcoholism.
The present treatment of patients suffering from a deficiency of pancreatic lipase is the oral administration of very large doses of a crude preparation of porcine pancreatic enzymes. However, Colipase-Dependent Pancreatic Lipase is inactivated by the low pH prevalent in the stomach. This effect cannot be completely overcome by the use of large doses of enzyme. Thus the large doses administered are
BAD ORIGINAL
AP Ο Ο ϋ 4 1 1
HX 1147 —4 — inadequate for most patients, and moreover the preparations are impure and unpalatable.
Certain tablets have been formulated which pass through 5 the acid regions of the stomach and discharge the enzyme only in the relatively alkaline environment of the jejunum. However, many patients suffering from pancreatic disorders have an abnormally acid jejunum and in those cases the tablets may fail to discharge the enzyme.
Moreover, since the preparations presently on the market are of a non-human source there is a risk of immunoreactions that may cause harmful effects to the patients or result in reduced therapy efficiency. A further drawback with the present preparations is that their content of other lipolytic activities than Colipase-Dependent Lipase are not stated. In fact, most of them contain very low levels of BSSL/CEL-activity.
This may be one reason why many patients, suffering from cystic fibrosis in spite of supplementation therapy, suffer from deficiencies of fat soluble vitamins and essential fatty acids.
Thus, there is a great need for products with ( properties and structure derived from human lipases and with a broad substrate specificity, which products may be orally administered to patients suffering from deficiency of one or several of the pancreatic lipolytic enzymes. Products that can be derived from the use of the present invention fulfil this need by themselves, or in combination with preparations containing other lipases.
Infant formulas
It is well known that human milk-feeding is considered superior to formula-feeding for infants. Not only does human milk provide a well-balanced supply of nutrients, bad original J)
AP Ο Ο 0 A 1 1
HX 1147
-5but it is also easily digested by the infant. Thus, several biologically active components which are known tc have physiological functions in the infant are either a constituent of human milk or produced during the digestion thereof, including components involved in the defense against infection and components facilitating the uptake of nutrients from human milk.
In spite of the great efforts which have been invested 10 in preparing infant formulas, it has not been possible to produce a formula which to any substantial extent has the advantageous properties of human milk. Thus, infant formulas, often prepared on the basis of cow milk, is generally incompletely digested by the infant and is lacking substances known to have effect on the physiological functions of the infant. In order to obtain an infant formula with a nutritional value similar to human milk, a number of additives including protein fragments, vitamins, minerals etc., which are normally formed or taken up during the infant's digestion of human milk, are included in the formula with the consequent risk of posing an increased strain on and possible long-term damage of important organs such as liver and kidney. Another disadvantage associated with the use of cow milk-based formulas is the increased risk for inducing allergy in the infant against bovine proteins.
As an alternative to cow milk-based infant formulas, human milk obtainable from so-called milk banks has been used. However, feeding newborn infants with human milk from milk banks has in the recent years to an increasing extent been avoided, because of the fear for the presence of infective agents such as HIV and CMV in human milk. In order to destroy the infective agents in human milk it has become necessary to pasteurize the milk before use. However, by pasteurization the nutritional value and the biological effects of the
BAD ORIGINAL
AP 0 0 0 4 1 1
-6— milk components are decreased, for example is BSSL inactivated, as mentioned above.
Addition of lipases to infant formulas
The pancreatic and liver functions are not fully developed at birth, most notably in infants born before term. Fat malabsorption, for physiological reasons, is a common finding and thought to result from low intraluminal Pancreatic Colipase-Dependent Lipase and bile salt concentrations. However, because of BSSL, such malabsorption is much less frequent in breast-fed infants than in infants fed pasteurized human milk or infant formulas (Bernback et al., 1990).
To avoid the above disadvantages associated with pasteurized milk and bovine milk-based infant formulas, it would thus be desirable to prepare an infant formula with a composition closer to that of human milk, i.e. a formula comprising human milk proteins.
BSSL/CEL has several unique properties that makes it ideally suited for supplementation of infant formulas:
• It has been designed by nature for oral administration. Thus, it resists passage through the stomach and is activated in contents of the small intestine.
• Its specific activation mechanism should prevent hazardous lipolysis of food or tissue lipids during storage and passage to its site of action.
• Due to its broad substrate specificity it has the potential to, on its own, mediate complete digestion of most dietary lipids, including the fat soluble vitamin esters.
• BSSL/CEL may be superior to Pancreatic ColipaseDependent Lipase to hydrolyze ester bonds containing long-chain polyunsaturated fatty acids.
• In the presence of Gastric Lipase and in the absence of, or at low levels of Colipase-Dependent
BAD ORIGINAL &
AP Ο Ο Ο 4 1 1
HX 1147
-710
Lipase, BSSL/CEL can ascertain a complete triacylglycerol digestion in vitro even if the bile salt levels are low such as in newborn infants. In the presence of BSSL/CEL the end products of triacylglycerol digestion become free fatty acids and free glycerol rather than free fatty acids and monoacylglycerol generated by the other two lipases (Bernback et al. , 1990). This may favour product absorption particularly when the intraluminal bile salt levels are low.
The utilization of BSSL/CEL for supplementation of infant formulas requires however access to large quantities of the product. Although human milk proteins may be purified directly from human milk, this is not a realistic and sufficiently economical way to obtain the large quantities needed for large scale formula production, and other methods must consequently be developed before an infant formula comprising human milk proteins may be prepared. The present invention provides such methods for preparation of BSSL/CEL in large quantities.
Production of proteins in milk of transgenic animals
The isolation of genes encoding pharmacologically active proteins has permitted cheaper production of such proteins in heterologous systems. An appealing expression system for milk proteins is the transgenic animal (For a review see Hennighausen et al., 19 90) .
Dietary compositions comprising bile salt-activated lipase derived from e.g. transgenic animal technology, is described in EP 317,355 (Oklahoma Medical Research Foundation).
In the transgenic animal, the protein coding sequence can be introduced as cDNA or as a genomic sequence. Since introns may be necessary for regulated gene expression in transgenic animals (Brinster et al.,
ORIGINAL
0 0 4 1 1
HX 1147 —8—
1988; Whitelaw et al., 1991) it is in many cases preferable to use the genomic form rather than the cDNA form of the structural gene. WO 90/05188 (Pharmaceutical Proteins Limited) describes the use in 5 transgenic animals of protein-coding DNA comprising at least one, but not all, of the introns naturally occurring in a gene coding for the protein.
PURPOSE OF THE INVENTION
It is an object of the present invention to provide a means for producing recombinant human BSSL/CEL, in a high yield and at a realistic price, for use in infant formulas in order to avoid the disadvantages with pasteurized milk and formulas based on bovine proteins.
BRIEF DESCRIPTION OF THE INVENTION
The purpose of the invention has been achieved by cloning and sequencing the human CEL gene. In order to improve the yield of BSSL/CEL, the obtained DNA molecule containing intron sequences, instead of the known cDNA sequence, of the human CEL gene has been used for production of human BSSL/CEL in a transgenic non-human mammal.
Accordingly, in one aspect the present invention relates to a DNA molecule shown in the Sequence Listing as SEQ ID NO: 1, or an analogue of the said DNA molecule which hybridizes with the DNA molecule shown in the Sequence Listing as SEQ ID NO: 1, or a specific part thereof, under stringent hybridization conditions.
The procedure used for isolating the human BSSL/CEL DNA molecule is outlined in the Examples below.
bad original
AP 0 0 0 4 1 1
HX 1147
-9The stringent hybridization conditions referred to above are to be understood in their conventional meaning, i.e. that hybridization is carried out according to an ordinary laboratory manual such as
Sambrook et al. (1989).
In another aspect the present invention provides a mammalian expression system comprising a DNA sequence encoding human BSSL/CEL inserted into a gene encoding a milk protein of a non-human mammal so as to form a hybrid gene which is expressible in the mammary gland of an adult female of a mammal harbouring said hybrid gene so that human BSSL/CEL is produced when the hybrid gene is expressed.
In yet a further aspect, the present invention relates to a method of producing a transgenic non-human mammal capable of expressing human BSSL/CEL, comprising injecting a mammalian expression system as defined above into a fertilized egg or a cell of an embryo of a mammal so as to incorporate the expression system into the germline of the mammal and developing the resulting injected fertilized egg or embryo into an adult female mammal.
DETAILED DESCRIPTION OF THE INVENTION
The DNA molecule shown in the Sequence Listing as SEQ
ID NO: 1, which has an overall length of 11531 bp, has the following features:
bad original ft
AP Ο Ο Ο 4 1 1
HX 1147
Feature -10- from base to b<
5'-Flanking region 1 1640
TATA box 1611 1617
Exon 1 1641 1727
5 Translation start 1653 1653
Exon 2 4071 4221
Exon 3 4307 4429
Exon 4 4707 4904
Exon 5 6193 6323
10 Exon 6 6501 6608
Exon 7 6751 6868
Exon 8 8335 8521
Exon 9 8719 8922
Exon 10 10124 10321
15 Exon 11 10650 11490
3'-Flanking region 11491 11531
In the present context, the term gene is used to indicate a DNA sequence which is involved in producing a polypeptide chain and which includes regions preceding and following the coding region (5'-upstream and 3'-downstream sequences) as well as intervening sequences, the so-called introns, which are placed between individual coding segments (so-called exons) or in the 5'-upstream or 3'-downstream region. The 5'upstream region comprises a regulatory sequence which controls the expression of the gene, typically a promoter. The 3'-downstream region comprises sequences which are involved in termination of transcription of the gene and optionally sequences responsible for polyadenylation of the transcript and the 3' untranslated region.
The DNA molecules of the invention explained herein may comprise natural as well as synthetic DNA sequences, the natural sequence typically being derived directly from genomic DNA, normally of mammalian origin, e.g. as described below. A synthetic sequence may be prepared
BAD ORIGINAL ft
AP Ο Ο Ο 4 1 1
HX 1147
-11by conventional methods for synthetically preparing DNA molecules. The DNA sequence may further be of mixed genomic and synthetic origin.
In a further aspect, the present invention relates to a replicable expression vector which carries and is capable of mediating the expression of a DNA sequence encoding human BSSL/CEL.
In the present context, the term “replicable means that the vector is able to replicate in a given type of host cell into which it has been introduced. Immediately upstream of the human BSSL/CEL DNA sequence there may be provided a sequence coding for a signal peptide, the presence of which ensures secretion of the human
BSSL/CEL expressed by host cells harbouring the vector. The signal sequence may be the one naturally associated with the human BSSL/CEL DNA sequence or of another origin.
The vector may be any vector which may conveniently be subjected to recombinant DNA procedures, and the choice of vector will often depend on the host cell into which it is to be introduced. Thus, the vector may be an autonomously replicating vector, i.e. a vector which ( exists as an extrachromosomal entity, the replication of which is independent of chromosomal replication; examples of such a vector are a plasmid, phage, cosmid, mini-chromosome or virus. Alternatively, the vector may be one which, when introduced in a host cell, is integrated in the host cell genome and replicated together with the chromosome (s) into which it has been integrated. Examples of suitable vectors are a bacterial expression vector and a yeast expression vector. The vector of the invention may carry any of the DNA molecules of the invention as defined above.
BAD ORIGINAL
AP Ο 0 0 4 1 1
HX 1147
-12The present invention further relates to a cell harbouring a replicable expression vector as defined above. In principle, this cell may be of any type of cell, i.e. a prokaryotic cell, a unicellular eukaryotic organism or a cell derived from a multicellular organism, e.g. a mammal. The mammalian cells are especially suitable for the purpose and are further discussed below.
In another important aspect, the invention relates to a method of producing recombinant human BSSL/CEL, in which a DNA sequence encoding human BSSL/CEL is inserted in a vector which is able to replicate in a specific host cell, the resulting recombinant vector is introduced into a host cell which is grown in or on an appropriate culture medium under appropriate conditions for expression of human BSSL/CEL and the human BSSL/CEL is recovered.
The medium used to grow the cells may be any conventional medium suitable for the purpose. A suitable vector may be any of the vectors described above, and an appropriate host cell may be any of the cell types listed above. The methods employed to construct the vector and effect introduction thereof into the host cell may be any methods known for such purposes within the field of recombinant DNA. The recombinant human BSSL/CEL expressed by the cells may be secreted, i.e. exported through the cell membrane, dependent on the type of cell and the composition of the vector.
If the human BSSL/CEL is produced intracellularly by the recombinant host, that is, is not secreted by the cell, it may be recovered by standard procedures comprising cell disrupture by mechanical means, e.g.
sonication or homogenization, or by enzymatic or chemical means followed by purification.
BAD ORIGINAL &
AP Ο Ο Ο 4 1 1
HX 1147
-13—
In order to be secreted, the DNA sequence encoding human BSSL/CEL should be preceded by a sequence coding for a signal peptide, the presence of which ensures secretion of human BSSL/CEL from the cells so that at least a significant proportion cf the human BSSL/CEL expressed is secreted into the culture medium and recovered.
The presently preferred method of producing recombinant 10 human BSSL/CEL of the invention is by use of transgenic non-human mammals capable of excreting the human BSSL/CEL into their milk. The use of transgenic nonhuman mammals has the advantage that large yields of recombinant human BSSL/CEL are obtainable at reasonable costs and, especially when the non-human mammal is a cow, that the recombinant human BSSL/CEL is produced in milk which is the normal constituent of, e.g., infant formulae so that no extensive purification is needed when the recombinant human BSSL/CEL is to be used as a nutrient supplement in milk-based products.
Furthermore, production in a higher organism such as a non-human mammal normally leads to the correct processing of the mammalian protein, e.g. with respect to post-translational processing as discussed above and proper folding. Also large quantities of substantially f pure human BSSL/CEL may be obtained.
Accordingly, in a further important aspect, the present invention relates to a mammalian expression system comprising a DNA sequence encoding human BSSL/CEL inserted into a gene encoding a milk protein of a nonhuman mammal so as to form a hybrid gene which is expressible in the mammary gland of an adult female of a mammal harbouring said hybrid gene.
The DNA sequence encoding human BSSL/CEL is preferably a DNA sequence as shown in the Sequence Listing as SEQ
BAD ORIGINAL 0
AP Ο Ο Ο 4 1 1
HX 1147
-14ID NO: 1 or a genomic human BSSL/CEL gene or an analogue thereof .
The mammary gland as a tissue of expression and genes 5 encoding milk proteins are generally considered to be particularly suitable for use in the production of heterologous proteins in transgenic non-human mammals as milk proteins are naturally produced at high expression levels in the mammary gland. Also, milk is readily collected and available in large quantities. In the present connection the use of milk protein genes in the production of recombinant human BSSL/CEL has the further advantage that it is produced under conditions similar to the its natural production conditions in terms of regulation of expression and production location (the mammary gland).
In the present context the term hybrid gene denotes a DNA sequence comprising on the one hand a DNA sequence encoding human BSSL/CEL as defined above and on the other hand a DNA sequence of the milk protein gene which is capable of mediating the expression of the hybrid gene product. The term “gene encoding a milk protein denotes an entire gene as well as a subsequence thereof capable of mediating and targeting the expression of the hybrid gene to the tissue of interest, i.e. the mammary gland. Normally, said subsequence is one which at least harbours one or more of a promoter region, a transcriptional start site, 3' and 5' non-coding regions and structural sequences. The DNA sequence encoding human BSSL/CEL is preferably substantially free from prokaryotic sequences, such as vector sequences, which may be associated with the DNA sequence after, e.g., cloning thereof.
The hybrid gene is preferably formed by inserting in vitro the DNA sequence encoding human BSSL/CEL into the milk protein gene by use of techniques known in the bad original £
AP Ο Ο Ο 4 1 1
HX 1147 —15— art. Alternatively, the DNA sequence encoding human BSSL/CEL can be inserted in vivo by homologous recombinant ion .
Normally, the DNA sequence encoding human BSSL/CEL will be inserted in one of the first exons of the milk protein gene of choice or an effective subsequence thereof comprising the first exons and preferably a substantial part of the 5' flanking sequence which is believed to be of regulatory importance.
The hybrid gene preferably comprises a sequence encoding a signal peptide so as to enable the hybrid gene product to be secreted correctly into the mammary gland. The signal peptide will typically be the one normally found in the milk protein gene in question or one associated with the DNA sequence encoding human BSSL/CEL. However, also other signal sequences capable of mediating the secretion of the hybrid gene product to the mammary gland are relevant. Of course, the various elements of the hybrid gene should be fused in such a manner as to allow for correct expression and processing of the gene product. Thus, normally the DNA sequence encoding the signal peptide of choice should be precisely fused to the N-terminal part of the DNA sequence encoding human BSSL/CEL. In the hybrid gene, the DNA sequence encoding human BSSL/CEL will normally comprise its stop codon, but not its own message cleavance and polyadenylation site. Downstream of the
DNA sequence encoding human BSSL/CEL, the mRNA processing sequences of the milk protein gene will normally be retained.
A number of factors are contemplated to be responsible for the actual expression level of a particular hybrid gene. The capability of the promoter as well of other regulatory sequences as mentioned above, the integration site of the expression system in the genome
BAD ORIGINAL ft
HX 1147
APO 0 0 4 1 1
-16of the mammal, the integration site of the DNA sequence encoding human BSSL/CEL in the milk protein encoding gene, elements conferring post-transcriptional regulation and other similar factors may be of vital importance for the expression level obtained. On the basis of the knowledge of the various factors influencing the expression level of the hybrid gene, the person skilled in the art would know how to design an expression system useful for the present purpose.
A variety of different milk proteins are secreted by the mammary gland. Two main groups of milk proteins exist, namely the caseins and the whey proteins. The composition of milk from different species varies qualitatively as well as quantitatively with respect to these proteins. Most non-human mammals produces 3 different types of casein, namely a-casein, β-casein and κ-casein. The most common bovine whey proteins are α-lactalbumin and β-lactalbumin. The composition of milk of various origins are further disclosed in Clark et al. (1987) .
The milk protein gene to be used may be derived from the same species as the one in which the expression system is to be inserted, or it may be derived from another species. In this connection it has been shown that the regulatory elements that target gene expression to the mammary gland are functional across species boundaries, which may be due to a possible common ancestor (Hennighausen et al., 1990).
Examples of suitable genes encoding a milk protein or effective subsequences thereof to be used in the construction of an expression system of the invention are normally found among whey proteins of various mammalian origins, e.g. a whey acidic protein (WAP) gene, preferably of murine origin, and a βlactoglobulin gene, preferably of ovine origin. Also
BAD ORIGINAL £
AP Ο Ο Ο 4 1 1
HX 1147 —17— casein genes of various origins may be found to be suitable for the transgenic production of human BSSL/CEL, e.g. bovine aSl-casein and rabbit β-casein. The presently preferred gene is a murine WAP gene as this has been found to be capable of providing a high level of expression of a number of foreign human proteins in milk of different transgenic animals (Hennighausen et al, 1990) .
Another sequence preferably associated with the expression system of the invention is a so-called expression stabilizing sequence capable of mediating high-level expression. Strong indications exist that such stabilizing sequences are found in the vicinity of and upstreams of milk protein genes.
The DNA sequence encoding human BSSL/CEL to be inserted in the expression system of the invention may be of genomic or synthetic origin or any combination thereof.
Some expression systems have been found to require the presence of introns and other regulatory regions in order to obtain a satisfactory expression (Hennighausen et al., 1990) . In some cases it may be advantageous to introduce genomic structures, rather than cDNA elements, as polypeptide encoding element in vector constructs (Brinster et al.). The intron and exon structure may result in higher steady state mRNA levels that obtained when cDNA based vectors are used.
In a further aspect, the present invention relates to a hybrid gene comprising a DNA sequence encoding human BSSL/CEL inserted into a gene encoding a milk protein of a non-human mammal, the DNA sequence being inserted in the milk protein gene in such a manner that it is expressible in the mammary gland of an adult female of a mammal harbouring the hybrid gene. The hybrid gene and its constituents have been discussed in detail above. The hybrid gene constitutes an important
BAD ORIGINAL
AP Ο 00 4 1 1
HX 1147
-18intermediate in the construction of an expression system of the invention as disclosed above.
In another aspect, the present invention relates to a 5 non-human mammalian cell harbouring an expression system as defined above. The mammalian cell is preferably an embryo cell or a pro-nucleus. The expression system is suitably inserted in the mammalian cell using a method as explained in the following and specifically illustrated in the Example below.
In a further important aspect, the present invention relates to a method of producing a transgenic non-human mammal capable of expressing human BSSL/CEL, comprising injecting an expression system of the invention as defined above into a fertilized egg or a cell of an embryo of a mammal so as to incorporate the expression system into the germline of the mammal and developing the resulting injected fertilized egg or embryo into an adult female mammal.
The incorporation of the expression system into the germline of the mammal may be performed using any suitable technique, e.g. as described in Manipulating the Mouse Embryo; A Laboratory Manual, Cold Spring ( Harbor Laboratory Press, 1986. For instance, a few hundred molecules of the expression system may be directly injected into a fertilized egg, e.g. a fertilized one cell egg or a pro-nucleus thereof, or an embryo of the mammal of choice and the microinjected eggs may then subsequently be transferred into the oviducts of pseudopregnant foster mothers and allowed to develop. Normally, not all of the injected eggs will develop into adult females expressing human
BSSL/CEL. Thus, about half of the mammals will from a statistically point of view be males from which, however, females can be bred in the following generations .
bad ORIGINAL π
AP Ο Ο Ο 4 1 1
HX 1147
-19Once integrated in the germ line, the DNA sequence encoding human BSSL/CEL may be expressed at high levels to produce a correctly processed and functional human BSSL/CEL in stable lines of the mammal in question.
Of further interest is a method of producing a transgenic non-human mammal capable of expressing human BSSL/CEL and substantially incapable of expressing BSSL/CEL from the mammal itself, comprising (a) destroying the mammalian BSSL/CEL expressing capability of the mammal so that substantially no mammalian BSSL/CEL is expressed and inserting an expression system of the invention as defined above or a DNA sequence encoding human BSSL/CEL into the germline of the mammal in such a manner that human BSSL/CEL is expressed in the mammal; and/or (b) replacing the mammalian BSSL/CEL gene or part thereof with an expression system of the invention as defined above or a DNA sequence encoding human BSSL/CEL.
The mammalian BSSL/CEL expressing capability is conveniently destroyed by introduction of mutations in the DNA sequence responsible for the expression of the BSSL/CEL. Such mutations may comprise mutations which make the DNA sequence out of frame, or introduction of a stop codon or a deletion of one or more nucleotides of the DNA sequence.
The mammalian BSSL/CEL gene or a part thereof may be replaced with an expression system as defined above or a DNA sequence encoding human BSSL/CEL by use of the well known principles of homologous recombination.
In a further aspect, the present invention relates to a transgenic non-human mammal prepared by a method as described above.
BAD ORIGINAL
AP 0 0 0 4 1 1
HX 1,47
-2 0While the transgenic non-human mammal of the invention in its broadest aspect is not restricted to any particular type of mammal, the mammal will normally be selected from the group consisting of mice, rats, rabbits, sheep, pigs, goats and cattle. For large scale production of human BSSL/CEL the larger animals such as sheep, goats, pigs and especially cattle are normally preferred due to their high milk production. However, also mice, rabbits and rats may be interesting due to the fact that the manipulation of these animals is more simple and results in transgenic animals more quickly than when, e.g. cattle, are concerned.
Also progeny of a transgenic mammal as defined above, capable of producing human BSSL/CEL is within the scope of the present invention.
In a further aspect the present invention includes milk from a non-human mammal comprising recombinant human
BSSL/CEL.
In a still further aspect, the present invention relates to an infant formula comprising recombinant human BSSL/CEL, in particular a polypeptide of the invention as defined above. The infant formula may be ( prepared by adding the recombinant human BSSL/CEL or polypeptide in a purified or partly purified form to the normal constituents of the infant formula. However, normally it is preferred that the infant formula is prepared from milk of the invention as defined above, especially when it is of bovine origin. The infant formula may be prepared using conventional procedures and contain any necessary additives such as minerals, vitamins etc.
BAD ORIGINAL A
HX 1147
AP Ο Ο Ο 411
-21EXAMPLES
EXAMPLE 1: GENOMIC ORGANIZATION, SEQUENCE ANALYSIS AND CHROMOSOMAL LOCALIZATION OF THE CEL GENE
Standard molecular biology techniques were used (Maniatis et al., 1982 ; Ausubel et al., 1987; Sarrbrook et al., 1989) if nothing else is mentioned.
Isolation of Genomic Recombinants
Two different human genomic phage libraries, XDASH (Clcnentech Laboratories Inc., Palo Alto, Ca, USA, and XEMBL-3 SP6/T7 (Stratagene, La Jolla, CA, USA), were screened by plaque hybridization using various subcloned cDNA restriction fragments (Nilsson et al., 1990) as probes, labeled with [a-22P]dCTP by the oligolabeling technique (Feinberg et al., 1983).
Mapping, Subcloning and Sequencing of Genomic Clones
Positive clones were digested with various restriction enzymes, electrophoresed on 1% agarose gels and then vacuumtransfered (Pharmacia LKB BTG, Uppsala, Sweden) to a nylon membrane. The membrane was hybridized with various cDNA probes. Restriction fragments, hybridizing with the probes, were isolated using the ( isotachophoreses method (Ofverstedt et al., 1984).
Smaller fragments, <800 bp, were directly inserted into M13mpl8, M13mpl9, M13BM20 or M13BM21 vectors and sequenced, using E. coli TGI as host bacteria, whereas larger fragments were subcloned into pTZ18R or pTZ19R vectors, using E. coli DH5a as host bacteria, and further digested. (The plasmids pS309, pS310 and pS451 used in Example 2 below were produced accordingly.)
Some of the isolated fragments were also used as probes in hybridizations. All of the nucleotide sequence was determined by the dideoxy chain termination method (Sanger et al., 1977) using Klenow enzyme and either the M13 universal sequencing primer of specific
BAD ORIGINAL
AP Ο Ο Ο 4 1 1
HX 1147
-22oligonucleotides. Sequence information was retrieved from autoradiograms by the use of the software MS-EdSeq as described by Sjoberg et al. (1989). The sequences were analyzed using the programs obtained from the
UWGCG software package (Devereux et al., 1984) .
Primer Extension
Total RNA was isolated from human pancreas, lactating mammary gland and adipose tissue by the guanidinium isothiocyanate-CsCl procedure (Chirgwin et al. , 1979).
Primer extension was performed according to (Ausubel et al., 1987) using total RNA and an antisense 26-mer oligonucleotide (5'-AGGTGAGGCCCAACACAACCAGTTGC-3'), nt position 33-58. Hybridization of the primer with 20 gg of the total RNA was performed in 30 μΐ of 0.9 M NaCl, 0.15 M Hepes pH 7.5 and 0.3 M EDTA at 30°C overnight. After the extension reaction with reverse transcriptase, the extension products were analyzed by electrophoresis through a 6% denaturing polyacrylamide gel.
Somatic Cell Hybrids
DNA from 16 human-rodent somatic cell hybrid lines, obtained from NIGMS Human Genetic Mutant Cell
Repository (Coriell Institute for Medical Research,
Camden, NJ) were used for the chromosomal assignment of the CEL gene. Human-mouse somatic cell hybrids GM09925 through GM09940 were derived from fusions of fetal human male fibroblasts (IMR-91), with the thymidine kinase deficient mouse cell line B-82 (Taggart et al. , 1985; Mohandas et al., 1986). Hybrids GM10324 and GM02860 with the HPRT and APRT deficient mouse cell line A9 (Callen et al., 1986), while hybrid GM10611 resulted from a microcell fusion of the retroviral vector SP-1 infected human lymphoblast cell line
GM07890 with the Chinese hamster ovary line UV-135 (Warburton et al., 1990). Hybrid GM10095 was derived from the fusion of lymphocytes from a female with a
BAD ORIGINAL &
AP Ο Ο Ο 4 1 1
HX 1147 —23— balanced 46,X,t(X;9)(ql3;34) karyotype with the Chinese hamster cell line CHW1102 (Mohandas et al., 1979). The human chromosome content of the hybrid lines, which was determined by cytogenetic analysis as well as by
Southern blot analysis and in situ hybridization analysis, are shown in Table 1. High molecular weight DNAs isolated from mouse, Chinese hamster and human parental cell line and the 16 hybrid cell lines were digested with FcoRI, fractionated in 0.8% agarose gels, and transferred to nylon filters. A [a-22P]dCTP-labeled CEL cDNA probe (a full-length cDNA) was prepared by oligolabeling (Feinberg and Vogelstein, 1983) and hybridized to the filters. The filters were washed for 60 min each at 65°C in 6xSSC/0.5%SDS and in
2xSSC/0.5%SDS.
Polymerase Chain Reaction
Total human genomic DNA isolated from leukocytes, DNA from somatic cell hybrids and from some of the positive genomic recombinants and total RNA from human lactating mammary gland and human pancreas were amplified for exon 10 and exon 11. Two gg of DNA were used. The primers used are listed in Table 2. Thirty cycles of PCR were performed in 100 gl volume [10 mM Tris-HCl, pH
8.3, 50 mM KC1, 1.5 mM MgCl2, 200 gM of each dNTP, 100 gg/ml gelatin, 100 pmol of each primer, 1.5 U Taq DNA polymerase (Perkin-Elmer Cetus, Norwalk, CT, USA)] and the annealing temperature 55°C for all the primer pairs. The RNA sequence was amplified by the use of combined complementary DNA (cDNA) and PCR methodologies. cDNA was synthesized from 10gg total RNA in 40 Ml of a solution containing 50 mM Tris-HCl, pH 8.3, 50 mM KC1, 10 mM MgCl2, 10 gg/ml BSA, 1 mM of each dNTP, 500 ng of oligo(dt)i2_iq» 40 U ribonuclease inhibitor, and 200 U reverse transcriptase (MoMuLV), (BRL, Bethesda Research Laborataries, N.Y., USA) for 30 min at 42°C. The cDNA was precipitated and resuspended in 25 gl H2O; 2 gl of this was amplified, as described
BAD ORIGINAL
HX 1147
AP Ο Ο Ο 411
-24above. The amplified fragments were analyzed on a 2% agarose gel. Some of the fragments were further subcloned and sequenced.
Gene Structure of the Human CEL Gene
In each genomic library, 10^ recombinants were screened and the screenings yielded several positive clones, which were all isolated and mapped. Two clones, designated XBSSLl and XBSSL5A, were further analyzed.
Restriction enzyme digestions with several enzymes,
Southern blotting followed by hybridization with cDNA probes, indicated that the XBSSLSA clone covers the whole CEL gene and that the XBSSLl clone covers the 5'half and about 10 kb of 5'-flanking region (Fig. 1).
Together these two clones cover about 25 kb of human genome.
After subcloning and restriction enzyme digestion, suitable fragments for sequencing were obtained and the entire sequence of the CEL gene could be determined, including 1640 bp of the 5'-flanking region and 41 bp of the 3'-flanking region. These data revealed that the human CEL gene (SEQ ID NO: 1) span a region of 9850 bp, containing 11 exons interrupted by 10 introns (Fig. 1).
This means that the exons and especially the introns are relatively small. In fact, exons 1-10 range in sizes from 87-204 bp respectively while exon 11 is 841 bp long. The introns range in sizes from 85-2343 bp respectively. As can be noted in Table 3, all exon/intron boundaries obey the AG/GT rule and conform well to the consensus sequence suggested by Mount et al. (1982). When the coding part of the CEL gene was compared with the cDNA (Nilsson et al·., 1990), only one difference in nucleotide sequence was found; the second nt in exon 1, a C, which in the cDNA sequence is a T. Since this position is located 10 nt upstream the translation start codon ATG, this difference does not influence the amino acid sequence.
BAD ORIGINAL
AP 0 0 0 4 1 1
HX 1147
-25Seven members of the Alu class of repetitive DNA elements are present in the sequenced region, labeled Alul-Alu7(5'-3')(Fig. 1), one in the 5'-flanking region and the six others within the CEL gene.
Transcription Initiation Sites and 5'-Flanking region
To map the human CEL gene transcription initiation site(s), primer extension analysis was performed using total RNA from human pancreas, lactating mammary gland and adipose tissue. The results indicated a major transcription start site located 12 bp, and a minor start site located 8 bases, upstream of the initiator methionine. The transcription initiation sites are the same in both pancreas and lactating mammary gland whereas no signal could be detected in adipose tissue (Fig. 2). The sequenced region includes 1640 nt of 5'flanking DNA. Based on sequence similarities a TATAbox-like sequence, CATAAAT was found 30 nt upstream the transcription initiation site (Fig. 4). Neither a CAAT20 box structure nor GC boxes were evident in this region.
The 5'-flanking sequence was computer screened, in both strands, for nucleotide sequences known as transcription factor binding sequences in other mammary gland- and pancreatic-specific genes. Several putative recognition sequences were found, see Fig. 4.
Chromosomal Localization of the CEL Gene
In human control DNA the CEL cDNA probe detected four
EcoRI fragments of approximately 13 kb, 10 kb, 2.2 kb and 2.0 kb, while in the mouse and hamster control DNAs single fragments of about 25 kb and 8.6 kb, respectively, were detected. The presence of human CEL gene sequences in the hybrid clones correlated only with the presence of human chromosome 9 (Table 1). Only one of the 16 hybrids analyzed were positive for the human CEL gene; this hybrid contained chromosome 9 as the only human chromosome. No discordancies for
BAU ORIGINAL ft
AP Ο Ο Ο 4 1 1
HX 1147 —26— localization to this chromosome were found, whereas there were at least two discordancies for localization to any other chromosome (Table 1). To further sublocalize the CEL gene we utilized a human-Chinese 5 hamster hybrid (GM 10095) retaining a der(9) translocation chromosome (9pter —> 9q34:Xql3 —> Xqter) as the only human DNA. By Southern blot we failed to detect any CEL gene sequences in this hybrid, indicating that the CEL gene resides within the 9q3410 qter region.
EXAMPLE 2: CONSTRUCTION OF EXPRESSION VECTORS
To construct an expression vector for production of recombinant human CEL in milk from transgenic animals the following strategy was employed (Fig. 5).
Three pTZ based plasmids (Pharmacia, Uppsala, Sweden) containing different parts of the human CEL gene, pS309, pS310 and pS311 were obtained using the methods described above. The plasmid pS309 contains a Sphl fragment covering the the CEL gene from the 5' untranscribed region to part of the fourth intron. The plasmid pS310 contains a Sacl fragment covering the CEL gene sequence from part of the first intron to a part of the sixth intron. Third, the plasmid pS311 contains a BamHI fragment covering a variant of the CEL gene from a major part of the fifth intron and the rest of the intron/exon structure. In this plasmid, the repetitive sequence of exon 11 that normally encodes the 16 repeats was mutated to encode a truncated variant having 9 repeats.
Another plasmid, pS283, containing a part of the human CEL cDNA cloned into the plasmid pUC19 at the Bindlll and Sacl sites was used for fusion of the genomic sequences. pS283 was also used to get a convenient
BAD ORIGINAL
HX 1147
AP Ο Ο Ο 4 1 1
-27restriction enzyme site, Kpnl, located in the 5' untranslated leader sequence of CEL. Plasmid pS283 was then digested with Ncol and Sacl and a fragment of about 2.7 kb was isolated. Plasmid pS309 was digested with Ncol and BspEI and a fragment of about 2.3 kb containing the 5'-part of the CEL gene was isolated. Plasmid pS310 was digested with BspEI and Sacl and a fragment of about 2.7 kb containing a part of the middle region of the CEL gene was isolated. These three fragments were ligated and transformed into competent
E. coli, strain TG2 , and transformants were isolated by ampicillin selection. Plasmids were prepared from a number of transformants, and one plasmid called pS312 (Fig. 6), containing the desired construct was used for further experiments.
To obtain a modification of pS311, in which the BamHI site located downstream of the stop codon was converted to a Sail site to facilitate further cloning, the following method was used. pS311 was linearized by partial BamHI digestion. The linearized fragment was isolated and a synthetic DNA linker that converts BamHI to a Sail site (5'-GATCGTCGAC-3'), thereby destroying the BamHI site, was inserted. Since there were two potential positions for integration of the synthetic ( linker the resulting plasmids were analyzed by restriction enzyme cleavage. A plasmid with the linker inserted at the desired position downstream of exon 11 was isolated and designated pS313.
To obtain the expression vector construct that harbours CEL genomic sequences and encodes the truncated CEL variant, the plasmid pS314 which was designed to mediate stage and tissue specific expression in the mammmary gland cells under lactation periods was used. Plasmid pS314 contains a genomic fragment from the murine whey acidic protein (WAP) gene (Campbell et al. 1984) cloned as a Notl fragment. The genomic fragment bAU ORIGINAL ft
AP Ο Ο Ο 41 1
HX 1147
-2 8has approximately 4.5 kb upstream regulatory sequences (URS), the entire transcribed exon/intron region and about 3 kb cf sequence downstream of the last exon. A unique Kpnl site is located in the first exon 24 bp upstream of the natural WAP translation initiation codon. Another unique restriction enzyme site is the Sail site located in exon 3. In pS314, this Sail site was destroyed by digestion, fill in using Klenow and religation. Instead, a new Sail site was introduced directly downstream of the Kpnl site in exon 1. This was performed by Kpnl digestion and introduction of annealed synthetic oligomers SYM 2401 5'-CGTCGACGTAC3', and SYM 2402 5'-GTCGACGGTAC-3', at this position (Fig. 8) The human CEL genomic sequence was inserted between these sites, Kpnl and Sail, by the following strategy. First, pS314 was digested with Kpnl and Sail and a fragment representing the cleaved plasmid was electrophoretically isolated. Second, pS312 was digested with Kpnl and BamHI and a approximately 4.7 kb fragment representing the 5'part of the human CEL gene was isolated. Third, pS313 was digested with BamHI and Sail and the 3'-part of the human CEL gene was isolated. These three fragments were ligated, transformed into competent E. coli bacteria and transformants were isolated after ampicillin selection. Plasmids were prepared from several transformants and carefully analyzed by restriction enzyme mapping and sequence analysis. One plasmid representing the desired expression vector was defined and designated pS317.
In order to construct a genomic CEL expression vector encoding full-length CEL pS317 was modified as follows (Fig. 5). First, a pTZ18R plasmid (Pharmacia) containing a 5.2 kb BamHI fragment of the human CEL gene extending from the fifth intron to downstream of the eleventh exon, pS451, was digested with Bindlll and Sacl. This digestion generated a fragment of about 1.7 kb that extends from the Bindlll site located in intron
BAD ORIGINAL
HX 1147
AP Ο 00 41 1
-2 99 to the Sacl site located in exon 11. Second, the plasmid pS313 was digested with Sacl and Sail, and a 71 bp fragment containing the 3 'part of exon 11 and the generated Sail site was isolated. Third, the rest of the WAP/CEL recombinant gene and the plasmid sequences was isolated as a Sail/Hindlll fragment of about 20 kb from pS317. These three fragments were ligated and transformed into bacteria. Plasmids were prepared from several transformants. The plasmids were digested with various restriction enzymes and subjected to sequence analysis. One plasmid containing the desired recombinant gene was identified. This final expression vector was designated pS452 (Fig.7).
To remove the prokaryotic plasmid sequences, pS452 was digested with Notl. The recombinant vector element consisting of murine WAP sequence flanking the human CEL genomic fragment was then isolated by agarose electrophoresis. The isolated fragment was further purified using electroelution, before it was injected into mouse embryos .
The recombinant WAP/CEL gene for expression in mammary gland of transgenic animals is shown in Figure 8.
DEPOSITS
The following plasmids have been deposited in accordance with the Budapest Treaty at DSM (Deutsche Sammlung von Mikroorganismen und Zellkulturen):
Plasmid Deposit No. Date of deposit
pS309 DSM 7101 12 June 1992
pS310 DSM 7102
pS451 DSM 7498 26 February 1993
pS452 DSM 7499
6AD ORIGINAL ft
HX 1147
AP ο υu4 ί ί —3 0—
EXAMPLE 3: GENERATION OF TRANSGENIC ANIMALS
A Noel fragment was isolated from the plasmid pS452 according to Example 2. This DNA fragment contained the murine WAP promoter linked to a genomic sequence encoding human BSSL/CEL. The isolated fragment, at a concentration of 3 ng/μΐ, was injected into the pronucleus of 350 C57Bl/6JxCBA/2J-f2 embryos obtained from donor mice primed with 5 IU pregnant mare's serum gonadotropin for superovulation. The C57Bl/6JxCBA/2J-f animals were obtained from BomholtgArd Breeding and Research Centre LID, Ry, Denmark. After collection of the embryos from the oviduct, they were separated from the cumulus cells by treatment with hyaluronidase in the medium M2 (Hogan et al. , 1986) . After washing the embryos were transferred to the medium M16 (Hogan et al., 1986) and kept in an incubator with 5% CO2atmosphere. The injections were performed in a microdrop of M2 under light paraffin oil using
Narishigi hydraulic micromanipulators and a Nikon inverted microscope equipped with Nomarski optics.
After injection, healthy looking embryos were implanted into pseudopregnant C57Bl/6JxCBA/2J-frecipients given 0.37 ml of 2.5% Avertin intraperitoneally. Mice that had integrated the transgene were identified with PCR analysis of DNA from tail biopsy specimens obtained three weeks after birth of the animals. Positive results were confirmed with Southern blot analysis.
EXAMPLE 4: EXPRESSION OF BSSL/CEL IN TRANSGENIC MICE
Transgenic mice were identified by analysis of DNA which has been prepared from excised tail samples. The tissue samples were incubated with proteinase K and phenol/chloroform extracted. The isolated DNA was used in polymerase chain reactions with primers which amplify specific fragments if the heterologous bad ORIGINAL
AP Ο Ο Ο 4 1 1
HX 1147
-31introduced DNA representing the expression vector fragment is present. The animals were also analyzed by DNA hybridization experiments to confirm PCR data and to test for possible rearrangements, structure of the integrated vector elements and to obtain information about the copy number of integrated vector elements.
In one set of experiments, 18 mice were analyzed with the two methods and the results demonstrated that 1 mouse was carrying the heterologous DNA vector element derived from pS452. The result from the PCR analysis and the hybridization experiments were identical (Fig.
9).
The mouse identified to carry vector DNA element (founder animal) was then mated and the FI litter was analyzed for transgene by the same procedures.
Female lactating animals were injected with 2 IU oxytocin intraperitoneally and 10 minutes later anaesthetized with 0.40 ml of 2.5% Avertin intraperitoneally. A milk collecting device was attached to the nipple via a siliconized tubing and milk was collected into a 1.5 ml Eppendorf tube by gentle massage of the mammary gland. The amount of milk ( varied, dependent on the day of lactation, between 0.1 and 0.5 ml per mouse and collection.
Analyze for the presence of recombinant human BSSL/CEL was done by SDS-PAGE, transfer to nitrocellulose membranes and incubation with polyclonal antibodies generated against native human BSSL/CEL. The obtained results demonstrated expression of recombinant human BSSL/CEL in milk from transgenic mice. Figure 10 demonstrates presence of recombinant human BSSL/CEL in milk from transgenic mice: the band at about 116.5.
Stable lines of transgenic animals are generated.
BAD ORIGINAL
AP 0 0 0 4 1 1
-32In a similar manner, other transgenic animals such as cows or sheep capable of expressing human BSSL/CEL may be prepared.
REFERENCES
Abouakil, N., Rogalska, E., Bonicel, J. & Lombardo, D. (1988): Biochim. Biophys. Acta 961, 299-308.
Ausubel, F.M., Brent, R.E., Moore, D.D., Smiyh, J.A., Seidman, J.G. and Struhl, K.: Current Protocols.in Molecular Biology. (Wiley Interscience, New York 1987)
Baba, T., Downs, D., Jackson, K.W., Tang, J. and Wang,
C.S. (1991): Biochemistry 30, 500-510.
Beato, M. (1989): Cell 56, 335-344.
Bernback, S., Blackberg, L. & Hernell, 0. (1990): J.
Clin. Invest. 221-226.
BjOrksten,B., Burman, L.G., B,, Gothefors, L. & Hernell 201, 267-272.
Blackberg, FEBS Lett. L., Angquist, 217, 37-41. K.
Blackberg, L. & Hernell, 0.
116, 221-225.
Blackberg, L. Lombardo, D. ,
Olivecrona, T. (1981): FEBS
deChateau, P., Fredrikzon, 0. (1980): Br. Med. J.
., & Hernell, 0. (1987) :
(1981): Eur. J. Biochem
Hernell, 0., Guy, 0. & Lett. 136, 284-288.
Boulet, A.M., Erwin, C.R. and Rutter, W.J. (1986): Proc. Natl. Acad. Sci. U.S.A. 83, 3599-3603.
Brinster, R.L., Allen, J.M., Behringer, R.R., Gelinas, R.E. & Palmiter, R.D. (1988) : Proc. Natl. Acad. Sci. U.S.A. 85, 836-840.
Callen, D.F. (1986): Ann. Genet. 29, 235-239.
Campbell, S.M., Rosen, J.M., Hennighausen, L.G., Strech-Jurk, U. and Sippel, A.E. (1984): Nucleic Acid Res. 12, 8685-8697.
Chirgwin, J.M., Przybyla, A.E., MacDonald, R.J. and Rutter, W.J. (1979): Biochemistry 18, 5294-5299.
Clark, A.J., Simons, P., Wilmut, I. and Lahte, R.
(1987): TIBTECH 5, 20-24.
BAD ORIGINAL ft —3 3—
AH 0 0 ϋ 4 1 1
Devereux, J., Haeberli, P. and Smithies. (1984):
Nucleic Acids Res. 12, 387-395.
Feinberg, A. and Vogelstem, B. (1983) : Anal. 3iochem. 132, 6-13.
Hennighausen, L., Ruiz, L. & Wall, R. (1990): Current Opinion in Biotechnology 1, 74-78.
Hernell, O. & Blackberg, L. (19:2): Pediatr. Res. 16, 882-885.
Hogan, B., Constantini, F. and Lacy, E. (1986): Manipulating the mouse embryo. A Laboratory Manual. Cold Spring Harbor Laboratory Press.
Hui, D. and Kissel, J.A. (1990): Febs Lett. 276, 131134 .
Lombardo, D., Guy, O. & Figarella, C. (1978): Biochim. Biophys. Acta 527, 142-149.
Maniatis, T., Fritsch, E.F. & Sambrook, J.: Molecular Cloning. A Laboratory Manual. (Cold Spring Harbor, NY, 1982)
Mohandas, T., Sparkes, R.S., Sparkes, M.C., Shulkin, J.D., Toomey, K.E. and Funderburk, S.J. (1979): Am. J. Hum. Genet. 31, 586-600.
Mohandas, T., Heinzmann, C., Sparkes, R.S. Wasmuth, J., Edwards, P. and Lusis, A.J. (1986): Somatic Cell. Mol. Genet. 12, 89-94.
Mount, S.M. (1982): Nucleic Acids Res. 10, 459-472.
Nilsson, J., Blackberg, L., Carlsson, P., Enerback, S., Hernell, 0. and Bjursell, G. (1990): Eur. J. Biochem. 192, 543-550.
Qasba, Μ., and Safaya, S.K. (1984): Nature 308, 377380 .
Reue, K., Ronk, Μ., Ameis, D. 267-276.
Zambaux, J., Wong, H., Lee, Shively, J.E., Sternby, B., and Schotz, M.C. (1991): J.
G., Leete, Borgstrom, Lipid. Res
T.H. , Β. ,
32,
Sambrook, J., Fritsch, E.F. and Maniatis, T.E.: Molecular Cloning. A Laboratory Manual. (Cold Spring Harbor, NY, 1989)
Sanger, F., Nicklen, S. and Coulson, A.R. (1977): Proc. Natl. Acad. Sci. U.S.A. 74, 5463-5467.
Sjoberg, S., Carlsson, P., EnerbSck, S. and Bjursell,
G. (1989): Comput. Appl. Biol. Sci. 5,41-46.
BAD ORIGINAL £
AP 0 0 0 4 1 1
HX 1147
-34Taggart. R.T., Mohandas, Τ., Shows, T.B. and Bell, G.I. (1985): Proc. Natl. Acad. Sci. U.S.A. 82, 6240-6244.
Warburton, D., Gersen, S., Yu, M.T., Jackson, C.,
Handelin, B. and Housman, D. (1990): Genomics 6, 358366 .
Whitelaw et al. (1991): Transgenic Research 1, 3-13.
Yu-Lee, L., Richter-Mann, L., Couch, C., Stewart, F.,
Mackinlay, G. and Rosen, J. (1986): Nucleic. Acid. Res. 14, 1883-1902.
Ofverstedt, L.G., Hammarstrom, K., Balgobin, Ν.,
Hjerten, S., Petterson, U. and Chattopadhyaya, J. (1984): Biochim. Biophys. Acta 782, 120-126.
bad ORIGINAL &
HX 1147
AP 0 0 0 4 1 1
-3 5X
CM
CM
CMl
Table 1. Correlation of CEL sequences with human chromosomes in 16 human-rodent somatic cell hybrids.
CQ CZ3 w
Σ O
C/5 o 2 o oi s u z <
s s fc m ¢/3
J ►J w
U fcso ω
o <
H
Z tu u
Qi ω
e.
o
CM
O'
OO
CM
W
O ¢/3
O si S
O Pi 06 A >
SB
o © © OO © 04 ro © © © © CM © © © © © CMlif
x mi if
o © C4 © © © © © © © © © © O' © © rr O'
sO r- © © © O' o 00 oo 04 O' oo © © © © © © © © ©lif
- © © 04 00 CM O' © © 00 © O o © C- © © © © © ml if
00 Os 00 ro 04 04 © 04 CO © © rr CM © © oo © o © o © ©lif
'-O O' oo 04 © CM © oc © © rr rt © rt © © © © © IZ-,| ~-f
•Ό O' ro O' © 00 O' © © O' © OO © W) © © © © © © © © © OCtif
© © cil©
00 — © © © oo © ro © © CM © © © © θ
o 00 © 00 O' 00 © © © ©
•*r © © © © © © © © © © © © OO O' © © CMl if
00 r-- © & C4 04 © 04 © © 00 © CM © © rr CM © o © © © ml if
sC oo o O' © WO 04 oo © OO O' 04 04 O' 00 OO © (— © oo © o © © © ©|©
O' O' © © WO © 00 © O' CM © © © © © © © «Cllif
00 o © © 04 04 © CM © W-\ O' 00 04 C4 O CM rr CM © © © © © © ©j©
τΓ © CO ro © OO CM © O' O' © -T m © © (*) © © © · © © ©lif
o 04 oO © © CM © © OO © rr © © © © © © mi if
X
© © © © © © © © © © © © © © O' © rr O'
00 Γ O' © © oo wn O' oo © © © r- rr CM © © © © © ©lif
C4 00 © O' 04 © 04 © 04 © TT © © t— rf <C1 © oo rr oo © © © ©
© ro © ro TT © © rr © O' © 04 © rr CM © oo oo © © © © © o|©
© r-- © © 04 © oo TT wn ro 00 00 04 © © © © © o © © © ml if
Γ' O' O' © wo OO © © © © 00 00 cn oo oo © © © © © ml if
© wo O' © 04 © © oo © 04 wo © rT Ό CM © rr © © © © oolif
04 co 00 © <T ro © © © © © © © © © © © © w-l| if
O' © © © © © © W) © © © © © © © © © © rrl if
< <
<nr— Ο'©0ΜΟ'1'Τ<ϩ× OOOrrr--—'Xl CM CM CM CO <O CO Γ*Ί <*Ί <O CO CO -31 CM Ό — O'
O' O' O' O' O' O' O' O' O' O' O' CO Ό © O
O' O' O' O' O' O' O' O' O' O' O' O' O O O O ooooooooooo ©· —« — — — s s s s s 2 22SS2S222S qoqooooqoooqooqo o
a rt •o
Ul
S o as ·— &
a In general, a human chromosome has to be present in more than 20 to 22% of the cells lo he detected by Southern blot analyst b Contains 9pter->q34 and Xq 13->q,er.
BAD ORIGINAL ft
HX 1147 —3 6—
Q.
g
Ό
Φ
U c
Φ σ
ο w
<o c
o •H
4-) •rd cn
O a
4J c
APO 0 0 4 1 1
g o
o cH •Ή
U-I c c
o 0
tn X X
l-i Φ Φ
Φ
g Ol c
• c4 G •H
G •H
a X c
C 0
x 3 •H
U Ο u
• rd G • Ή
5 G
3 Φ
o tn a
φ
tn G
c G
o o φ
X X X
Cd Φ E-
00 o in 00 CN kO LO 00 on co IN in rn <3 cn t σ\ ο rH σ> I CN Γ- ΟΊ I
t m 1 <r> 1 O 1 kO 1 cn 1 (Ν
<Ti CN rH co CN
’ϊ kO CN O ο Γ
00 co r*· σ\ σ\ σι
Primers Used for DNA Amplification
Ο
Φ r-H υ
G o
Oi
η ι 0 co ι 0 ΓΟ 1 0 0 ΓΟ I
γο ΓΟ 0 Ε- < Ε-
ι 1 Ε- Ε- 0 0
Ο 0 0 0 0 0
Ε Ε-1 0 Ε-< < <
0 < Ε- < 0 0
0 0 Η 0 0 Ε-1
< Ε- 0 0 0 0
Ε Ε 0 0 Ε
0 0 0 0 Ε-1
0 0 0 0 < 0
ο 0 Ε- Η 0 0
ο 0 0 0 0
< <
Ε- Ε- 0 0 0
0 0 0 0 0
0 0 0 *ΐ Η
|< 0 0 0 0 0
0 0 < 0 <
< < F 0 I 0 I I Ο
1 -
LO ιη ιη m ιη ιη
·· ·« ·· ·· ·♦ ··
<Ή (Ν ιη <ο θ'
CU Οι CX. 04 CL CU
cn φ X 0
φ 4J 4J
ιη (0 φ tn
X G Φ
>4-1 (0 a tn ti
Ο g X
G ο ο ο
Φ 3 C ο •U) G ν> 1£> χΗ X
Φ X <
Φ Ό «3 2
X G α
4_) Ο - υ
tn G rd g
(0 Η .. 0
C Ο 2 G <4-1
Φ > G Ο Q -
•Η X Η Ο
σι Φ χΗ
W .ο Ο Cd G
-Η tn 0 0
G X
G •Η χ c φ
Ο Μ-Ι *-> g -
•G 4-1 Φ •Η 3 5 -t >4-1
•G X Ο Ο
ιη 4-1 G Ο
Ο 0 G
a >4-1 •Η φ Ο
0 u X Η
φ -Η 4_> 4->
η 4-> ιη <α
-Η G Ο G ο
4J (0 a ή Ή
Ο U <4-1
φ Μ Φ G •Η
X Φ γ—4
ο Φ Ή X
3 X 4J 6 g
G 4J 0 3 (0
Φ g Φ G ι—1 Μ
X Ο Ο Φ ο
Ε-< G 3 X Cb
<0 >4-1 G 4J X
BAD ORIGINAL
ΗΧ 1147
-37ω ►4
CQ
AK U ύ υ η 1 1
m tn r- 00 P- 03 tO «-Η 00
’J 00 p* 00 r- Ί* to σ\ o 03
m (N 03 ’O' i-H 03 m
OJ r2 »-2
l—tt—«·—!>>>—I»—II—«X W H Μ > l-l l-l H
Μ > Μ >
υ Ρ σ Ε- φ φ Ρ ο φ Φ
υ Ρ ο < <.) < Ρ 4 Φ φ
J2 ο Φ φ Η Η φ < ο φ φ
4J ο Φ φ Φ ο Ε-1 φ ο Ε- φ
a, ο Φ φ Η Φ φ < Ο
Φ ο < Η 2 Φ Ο φ ο < ο
C ϋ ο Φ Ο e- Φ Ρ φ φ Φ φ
0 Ό Η ο Η ο Φ C <
Ή ο < φ
XJ Φ
0 ϋ ο © Οϊ Οι σι σι σι σι σι σι
c Ή <0 41 <5 <0 « «0 Ό <0 <0 β
3 ι-2 U υ υ υ XJ υ U υ υ υ
Τ’* a σ» σι 4J U Ο υ υ υ υ σι
ω υ XJ υ 0 υ υ υ υ U 4J
SZ JJ υ υ υ U Ό υ U υ
0 m ϋ 0 «β υ υ υ υ υ υ 4->
kl υ <α υ ο XJ Ο υ jj υ υ
4J υ υ υ 4J σι 4J U jj υ ια
c 4-> υ υ υ σι υ jj 43 4-> υ
ϋ σ CJ U σι XJ U σι Ο 4U
1 JJ 4J υ U JJ jj 4J Λ3 4J υ
c σι υ 4J Ο 0 σι υ U υ 4J
0 4J υ υ 4J 4J Ο σι υ jj 4J
X 0> 4J οι υ 3J XJ σ <0 σ CJ
Φ • *
Φ
C φ
ο j
ω υ
φ
Λ
XJ
Ο
C ο
•rl xj ιθ
Ν
Ή
Ε (0 σι ο
c ο
Μ
XJ £
H £
Ο
C ο
X
U
•a « υ XI © σ XI υ σ 43
φ σι σι © © υ β © © σ ©
ο Μ « σι XI υ XI « ιθ 4) 43 43
C 0 <0 © υ © υ « © «3 © 43
φ C XI XI XI XI XI XI χι XI χ) XI
□ 0 σι © © © © © © © © σ
σ’ •σ
φ φ φ φ
ω V < < Ρ
υ 2 φ φ φ υ 2 Η φ 2 Φ
♦Η
r-2 Ρ < < Ρ rf Φ φ φ «<
a φ 2 2 φ Ρ 2 < φ φ Φ
0) φ φ φ φ φ 2 Φ 4 Φ <
tn φ φ Φ Ρ ε· Φ Ρ φ ε rf
φ Ρ Ρ φ φ Ρ ε Λ
φ Ρ 2 φ Ε- φ φ φ 2
η •σ •r-l υ
ο
C
-γΗ <0 m ο οι ιη to
Ό ό σι m oo Ό m m to to to
C 03
ιη ιη to 03 to 03 r2 ’ί 03 oO tn
, 03 ο* 42 00 03 to o to ro cn «i
η 4“2 4-2 03 03 n ro ’J' r*
0 a 1 «—4 to to Γ- cn Γ cn Λ tn ro σ»
οι Γ* r2 00 03 to o to ro σ»
4-2 4-2 03 03 ro ro
Ο 4J C C φ — [^ΗΠΦΗΦΛΓ'^ΟΟΗ
ΦυΊΓίΟί^ΟΗΦΟΟ^1 i-2r24-2c24-24-24-2Olr2C0
TJ c p- 4-2 at TJ< ro 00 OO 4-2 Ol 4-2 o
•H 0 00 00 00 to 00 to 03 GO oo 00 in
XJ •H in r* 04 to σ» Ol 00 Ol to 00
0 XJ Ol Ol ro *3« tn to o- 00 at
Φ •H 1 1 1 1
rH 0) 4-2 4-2 r* ro r2 c2 in at Tj· o
u 0 ro to to in to 4-2 at r* 00 4-2
3 a rf to o in 00 4-2 to o O
C Ol Ol ro <· in to r- <30 at
(Ν (*) IP Φ Γ* Φ Ο Ο 4-2 φ
CO o
ai m XJ XJ
<a CD CO
43 tl (0 CD CO
1X4 a 10
O ε o Λ
tl CD £ u 0 xJ o KO ϊ—1
£ tl CD Ό Ό
CD 42 Ό tl (0
XJ ω 0 £ i—l
(0 l-l 6
£ ID £ 2
> O Q
•rl Cn X CD M CX
ω XJ Cd
•rH m tl cn .
£ •rl 42 £
O <X4 XJ c
•rl •H £
XJ CD 5 r—1
•rl 42 o
CO o xJ £ O υ
O. 1X4 •rl CD
0 XJ 42
CD •rl XJ
Ό XJ CO
•rl tl O £
XJ o (β XJ Ol •rl
CD CO CD tl
Γ—| Ό CD
O CD •rl Λ
3 J2 XJ ε
£ XJ O CD £ £
CD ε r-l CD
43 O O
E-> tl 3 42
43 1X4 £ XJ
BAD ORIGINAL
HX 1147
-38FIGURE LEGENDS
Figure 1
The CEL gene locus. Localization and restriction enzyme 5 map of the two partly overlapping clones, XESSLl and
XBSSL5A are shown. The exon-intron organization and used restriction enzyme site are shown below. Exons are represented by boxes numbered 1-11. Asp=Asp700,
B=BamHI, E=EcoRI, S=SacI, Sa = SalI, Sp=Sphl and X=Xbal.
Positions and orientation of Alu repetitive elements are shown by bold arrows, a-h represent different subcloned fragments.
Figure 2
Primer extension analysis of RNA from human lactating mammary gland, pancreas and adipose tissue. An endradiolabeled 26-mer oligonucleotide, which is complementary to nt positions 33 to 58 of the CEL gene, was used to prime reverse transcription of the RNA.
Lane A is a molecular size marker (a sequencing ladder), lane B pancreatic RNA, lane C adipose tissue RNA and lane D lactating mammary gland RNA.
Figure 3
Dotplot analysis of the human CEL and rat CEL gene 5'flanking regions. The homology regions are labeled A-H and the sequences representing these parts are written, upper is human and lower is rat.
Figure 4
Analysis of 5'-flanking sequence of the human CEL gene. The putative recognition sequences are either highlighted underline or underline representing the complementary strand. Bold letters show the locations of the homologies to the rCEL (regions A-H). The TATAbox is underlined with dots.
BAD ORIGINAL fi
AP Ο Ο Ο 4 1 1
HX 1147
-39There are two sequences that both show a 80% similarity to the consensus sequence of the glucocorticoid receptor binding site, GGTACANNNTGTTCT, (Beato, Μ. , 1989), the first one on the complementary strand at nt position -231 (IA) and the second one at nt position
-811 (IB). Moreover, at nt position -861 (2) there is a sequence that shows 87% similarity to the consensus sequence of the estrogen receptor binding site,
AGGTCANNNTGACCT, (Beato, Μ. , 1989).
Lubon and Henninghausen (1987) have analyzed the promoter and 5'-flanking sequences of the whey acidic protein (WAP) gene and established the binding sites for nuclear proteins of lactating mammary gland cells.
One of them, an 11 bp conserved sequence, AAGAAGGAAGT, is present in a number of milkprotein genes studied e.g. the rat α-lactalbumin gene (Qasba et al., 1984) and the rat α-casein gene (Yu-Lee et al., 1986). In the CEL gene's 5'-flanking region, on the complementary strand at nt position -1299 (3) there is a sequence that shows 82% similarity to this conserved sequence.
In a study of the β-casein gene's regulation, a tissue specific mammary gland factor (MGF) was found in nuclear extracts from pregnant or lactating mice and its recognition sequence was identified (ANTTCTTGGNA). In the human CEL gene's 5'-flanking region there are two sequences, one on the complementary strand at nt position -368 (4A) and the other at nt position -1095 (4B), they both show 82% similarity to the consensus sequence of the MGF binding site. Beside these two putative MGF binding sites in the 5'-flanking region there is a sequence on the complementary strand at nt 275 in intron I, AGTTCTTGGCA, which shows 100% identity to the consensus sequence of the MGF binding site.
Furthermore, there are four sequences which all show 65% similarity to the consensus sequence of rat
BAD ORIGINAL ft
HX 1147
AP 0 00 41 1
-4 0pancreas-specific enhancer element,
GTCACCTGTGCTTTTCCCTG, (Boulet et al. , 1986), one at nt position -359 (5A), the second at nt position -718 (5B), the third at nt position -1140 (50 and the last at nt position -1277 (5D).
Figure 5
Method for production of the plasmid pS452. For further details, see Example 2.
Figure 6
Schematic structure cf the plasmid pS312.
Figure 7
Schematic structure of the plasmid pS452.
Figure 8
Physical map representing the physical introduction of human BSSL/CEL genomic structure in the first exon of the WAP gene as described in Example 2.
Figure 9
A. Schematic representation of the localization of PCR-primers used for identification of transgenic animals. The 5'-primer is positioned within the WAP sequence starting at the position -148 bp upstream of the fusion between the WAP and BSSL/CEL. The 3'-primer is localized in the first BSSL/CEL intron ending 398 bp downstream of the fusion point.
B. The sequences of the PCR primers used.
C. Agarose gel showing a typical analysis of the PCR analysis of the potential founder animals. M: molecular weight markers. Lane 1: control PCR-product generated from the plasmid pS452. Lanes 2-13: PCR reactions done with DNA preparations from potential founder animals.
BADORIGINAL $
ΑΡ Ο Ο Ο 4 1 1
Ηλ 1147 —41—
Fiqure 10
Immunoblot analysis of milk from a mouse line transgenic for the recombinant murine WAP/human CEL gene of pS452. The proteins were separated on SDS-PAGE, transferred to Immobilon membranes (Millipore) and visualized with polyclonal rabbit antibodies generated using highly purified human native CEL, followed by alkaline phosphatase labelled swine anti-rabbit IgG (Dakopatts). Lane 1, Low molecular weight markers, 106,
80, 49.5, 32.5, 27.5, and 18.5 kDa, respectively. Lane
2, High molecular weight markers, 205, 116.5, 80 and
49.5 kDa, respectively. Lane 3, 25 ng purified nonrecombinant CEL from human milk. Lane 4, 2 μΐ milk sample from a CEL transgenic mouse diluted 1:10. Lanes
5 and 6, 2 μΐ milk samples from two different non-CEL transgenic mice, diluted 1:10, as control samples.
BAD ORIGINAL ft
AP Ο Ο Ο 4 1 1
ΗΧ 1147 —42—
SEQUENCE LISTING i! GENERAL INFORMATION:
(l) APPLICANT:
(A) NAME: A3 ASTRA (Bi STREET: Kvarnbergagatan li (C) CITY: Sodertalje (E) COUNTRY: Sweden (F) POSTAL CODE (ZIP): S-151 35 (G) TELEPHONE: +46-8-553 26000 (H) TELEFAX: +46-8-553 28820 (I) TELEX: 19237 astra s (ii) TITLE OF INVENTION: New DNA Sequences (iii) NUMBER OF SEQUENCES : 1 (iv) COMPUTER READABLE FORM:
(A) MEDIUM TYPE: Floppy disk (B) COMPUTER: IBM PC compatible (C) OPERATING SYSTEM: PC-DOS/MS-DOS (D) SOFTWARE: Patentln Release #1.0, Version #1.25 (EPO) (vi) PRIOR APPLICATION DATA:
(A) APPLICATION NUMBER: SE 9201809-2 (B) FILING DATE: ll-JUN-1992 (vi) PRIOR APPLICATION DATA:
(A) APPLICATION NUMBER: SE 9201326-6 (B) FILING DATE: 12-JUN-1992 (vi) PRIOR APPLICATION DATA:
(A) APPLICATION NUMBER: SE 9202088-2 (B) FILING DATE: 03-JUL-1992 (vi) PRIOR APPLICATION DATA:
(A) APPLICATION NUMBER: SE 9300902-5 (B) FILING DATE: 19-MAR-1993 (2) INFORMATION FOR SEQ ID NO: 1:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 11531 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: double (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (vi) ORIGINAL SOURCE:
(A) ORGANISM: Homo sapiens (F) TISSUE TYPE: Mammary gland (ix) FEATURE:
(A) NAME/KEY : CDS (B) LOCATION: join (1653..1727, 4071..4221, 4307..4429, 4707
..4904, 6193..6323, 6501..6608, 6751..6868, 8335 ..8521, 8719..8922, 10124..10321, 10650..11394) (ix) FEATURE:
(A) NAME/KEY: mat_peptide (B) LOCATION: join(1722..1727, 4071..4221, 4307..4429, 4707
..4904, 6193..6323, 6501..6608, 6751..6868, 8335 ..8521, 8719..8922, 10124..10321, 10650..11391)
BAD ORIGINAL ft
AP ο Ο Ο 4 1 1
HX 1147
-43(D) OTHER INFORMATION: /EC_number= 3.1.1.1 /product= Bile Salt-Stimulated Lipase (ix) FEATURE:
(A) NAME/KEY: 5 ' UTR (B) LOCATION: 1..1640 (ix) FEATURE:
(A) NAME/KEY: (B) LOCATION: TATA_signal
1611. . 1617
(ix) FEATURE : (A) NAME/KEY: (B) LOCATION: exon 1641. .1727
(ix) FEATURE: (A) NAME/KEY: (B) LOCATION: exon 4071. .4221
( ix) FEATURE : (A) NAME/KEY: (B) LOCATION: exon 4307 . .4429
(ix) FEATURE : (A) NAME/KEY: (B) LOCATION: exon 47 07. .4904
(ix) FEATURE: (A) NAME/KEY: (B) LOCATION: exon 6193 . .6323
( ix) FEATURE: (A) NAME/KEY: (B) LOCATION: exon 6501. .6608
(ix) FEATURE : (A) NAME/KEY: (B) LOCATION: exon 6751. .6868
(ix) FEATURE: (A) NAME/KEY: (B) LOCATION: exon 8335. .8521
(ix) FEATURE : (A) NAME/KEY: (B) LOCATION: exon 8719. .8922
(ix) FEATURE : (A) NAME/KEY: (B) LOCATION: exon 10124 . . 10321
(ix) FEATURE: (A) NAME/KEY: (B) LOCATION: exon 10650 . . 11490
(ix) FEATURE: (A) NAME/KEY: (B) LOCATION: 3 'UTR 11491 ..11531
(xi) SEQUENCE DESCRIPTION : SEQ ID NO: 1
GGATCCCTCG AACCCAGGAG TTCAAGACTG CAGTGAGCTA TGATTGTGCC ACTGCACTCT 60
AGCCTGGGTG ACAGAGACCC TGTCTCAAAA AAACAAACAA ACAAAAAACC TCTGTGGACT 120
CCGGGTGATA ATGACATGTC AATGTGGATT CATCAGGTGT TAACAGCTGT ACCCCCTGGT 180
BAD ORIGINAL ft
AP Ο Ο Ο 4 1 1
ΗΧ 1147
-44-
GGGGGATGTT GATAACGGGG GAGACTGGAG TGGGGCGAGG ACATACGGGA AATCTCTGTA 240
ATCTTCCTCT AATTTTGCTG TGAACCTAAA GCTGCTCTAA AAATGTACAT AGATATAAAC 300
TGGGGCCTTC GCCCTGCCCC AGCCCTCCCC CACCTCCTTC CTCTCCCTGC 360
TGCCTCCCCT CTGCCCTCCC CTTTCCTCCT TAGCCACTGT AAATGACACT GCAGCAAAGG 420
TCTGAGGCAA ATGCCTTTGC CCTGGGGCGC CCCAGCCACC TGCAGGCCCC TTATTTCCTG 430
TGGCCGAGCT CCTCCTCCCA CCCTCCAGTC CTTTCCCCAG CCTCCCTCGC CCACTAGGCC 540
TCCTGAATTG CTGGCACCGG CTGTGGTCGA CAGACAGAGG GACAGACGTG GCTCTGCAGG 600
TCCACTCGGT CCCTGGCACC GGCCGCAGGG GTGGCAGAAC GGGAGTGTGG TTGGTGTGGG 660
AAGCACAGGC CCCAGTGTCT CCTGGGGGAC TGTTGGGTGG GAAGGCTCTG GCTGCCCTCA 720
CCCTGTTCCC ATCACTGCAG AGGGCTGTGC GGTGGCTGGA GCTGCCACTG AGTGTCTCGG 780
TGAGGGTGAC CTCACACTGG CTGAGCTTAA AGGCCCCATC TGAAGACTTT GTTCGTGGTG 840
TTCTTTCACT TCTCAGAGCC TTTCCTGGCT CCAGGATTAA TACCTGTTCA CAGAAAATAC 900
GAGTCGCCTC CTCCTCCACA ACCTCACACG ACCTTCTCCC TTCCCTCCCG CTGGCCTCTT 960
TCCCTCCCCT TCTGTCACTC TGCCTGGGCA TGCCCCAGGG CCTCGGCTGG GCCCTTTGTT 1020
TCCACAGGGA AACCTACATG GTTGGGCTAG ATGCCTCCGC ACCCCCCCAC CCACACCCCC 1080
TGAGCCTCTA GTCCTCCCTC CCAGGACACA TCAGGCTGGA TGGTGACACT TCCACACCCT 1140
TGAGTGGGAC TGCCTTGTGC TGCTCTGGGA TTCGCACCCA GCTTGGACTA CCCGCTCCAC 1200
GGGCCCCAGG AAAAGCTCGT ACAGATAAGG TCAGCCACAT GAGTGGAGGG CCTGCAGCAT 1260
GCTGCCCTTT CTGTCCCAGA AGTCACGTGC TCGGTCCCCT CTGAAGCCCC TTTGGGGACC 1320
TAGGGGACAA GCAGGGCATG GAGACATGGA GACAAAGTAT GCCCTTTTCT CTGACAGTGA 1380
CACCAAGCCC TGTGAACAAA CCAGAAGGCA GGGCACTGTG CACCCTGCCC GGCCCCACCA 1440
TCCCCCTTAC CACCCGCCAC CTTGCCACCT GCCTCTGCTC CCAGGTAAGT GGTAACCTGC 1500
ACAGGTGCAC TGTGGGTTTG GGGAAAACTG GATCTCCCTG CACCTGAGGG GGTAGAGGGG 1560
AGGGAGTGCC TGAGAGCTCA TGAACAAGCA TGTGACCTTG GATCCAGCTC CATAAATACC 1620
CGAGGCCCAG GGGGAGGGCC ACCCAGAGGC TG ATG CTC Met Leu -23 ACC ATG GGG CGC CTG Thr Met Gly Arg Leu -20 1673
CAA CTG GTT GTG TTG GGC CTC ACC TGC TGC TGG Gin Leu Val Val Leu Gly Leu Thr Cys Cys Trp -15 -10 GCA GTG GCG AGT GCC Ala Val Ala Ser Ala -5 1721
GCG AAG GTAAGAGCCC AGCAGAGGGG CAGGTCCTGC TGCTCTCTCG CTCAATCAGA Ala Lys 1 1777
TCTGGAAACT TCGGGCCAGG CTGAGAAAGA GCCCAGCACA GCCCCGCAGC AGATCCCGGG 1837
CACTCACGCT CATTTCTATG GGGACAGGTG CCAGGTAGAA CACAGGATGC CCAATTCCAT 1897
TTGAATTTCA GATAAACTGC CAAGAACTGC TGTGTAAGTA TGTCCCATGC AATATTTGAA 1957
ACAAATTTCT ATGGGCCGGG CGCAGTGGCT CACACCTGCA ATCCCACCAG TTTGGGAGGC 2017
BAD ORIGINAL
AP Ο Ο Ο 4 1 1
HX, 1147
-45-
CGAGGTGGGT GGATCACTTG AGGTCAGGAG TTGGAGACCA GCCTGGCCAA CATGGTGAAA 2077
CCCCGTCTCT ACTAAAAATA CAAATATTAA TCGGGCGTGG TGGTGGGTGC CTGTAATCCC 2137
AGCTACTCGG GAGGCTGAGG CAGGAGAACC GCTTGAAGCT GGGAGGTGGA GATTGCGGTG 2197
AGCTGAGATC ACGCTACTGC ACTCCAGCCT GGGTGACAGG GCGAGACTCT GTCTCAAAAA 2257
ATAGAAAAAG AAAAAAATGA AACATACTAA AAAACAATTC ACTGTTTACC TGAAATTCAA 2317
ATGTAACTGG GCCTCTTGAA TTTACATTTG CTAATCCTGG TGATTCCACC TACCAACCTC 2377
TCTGTTGTTC CCATTTTACA GAAGGGGAAA CGGGCCCAGG GGCAGGGAGT GTGGAGAGCA 2437
GGCAGACGGG TGGAGAGAAG CAGGCAGGCA GTTTGCCCAG CATGGCACAG CTGCTGCCTC 2497
CTATTCCTGT GCAGGAAGCT GAAAGCCGGG CTACTCCACA CCCGGGTCCG GGTCCCTCCA 2557
GAAAGAGAGC CGGCAGGCAG GAGCTCTCTC GAGGCATCCA TAAATTCTAC CCTCTCTGCC 2617
TGTGAAGGAG AAGCCACAGA AACCCCAAGC CCCACAGGAA GCCGGTGTCG GTGCCCGGCC 2677
CAGTCCCTGC CCCCAGCAGG AGTCACACAG GGGACCCCAG ATCCCAACCA CGCTGTTCTG 2737
CTGCCTGCGG TGTCTCAGGC CCTGGGGACT CCTGTCTCCA CCTCTGCTGC CTGCTCTCCA 2797
CACTCCCTGG CCCTGGGACC GGGAGGTTTG GGCAGTGGTC TTGGGCTCCT GACTCAAAGG 2857
AGAGGTCACC TTCTTCTTGG GCGAGCTCTT CTTGGGGTGC TGAGAGGCCT TCGGCAGGTC 2917
ATCACGACCC CTCCCCATTT CCCCACCCTG AGGCCCTCTG GCCAGTCTCA ATTGCACAGG 2977
GATCACGCCA CTGGCACAAG GAGACACAGA TGCCTCGCAG GGGATGCCCA CGATGCCTGC 3037
ATGTGTTGCT TCTGGTTCCT TTCCTCCAGT TCCAACCGCC GCACTCTCCC ACACCAGTGT 3097
GACAGGGGGC CCATCACCCT AGACTTCAGA GGGCTGCTGG GACCCTGGCT GGGCCTGGGG 3157
GTGTAGGGCC ACCCTGCCCT TCCCCACCTG GAACCTGGCA CAGGTGACAG CCAGCAAGCA 3217
ATGACCTGGT CCCACCATGC ACCACGGGAA GAGGGAGCTG CTGCCCAAGA TGGACAGGAG 3277
GTGGCACTGG GGCAGACAGC TGCTTCTCAA CAGGGTGACT TCAAGCCCAA AAGCTGCCCA 3337
GCCTCAGTTC CGTCAGGGAC AGAGGGTGGA TGAGCACCAA CCTCCAGGCC CCTCGTGGGG 3397
GTGGACAGCT TGGTGCACAG AGGCCATTTT CATGGCACAG GGAAGCGTGG CGGGGGTGGG 3457
AGGTGTGGTC CCTAGGGGGT TCTTTACCAG CAGGGGGCTC AGGAACTGTG GGGACTTGGG 3517
CATGGGGCCA TCGACTTTGT GCCCAGCCAG CTAGGCCCTG TGCAGGGAGA TGGGAGGAGG 3577
GAAAAGCAGG CCCCACCCCT CAGAAAGGAG GAAGGTTGGT GTGAAACATC CCGGGTACAC 3637
TGAGCATTGG GTACACTCCT CCCGGGAGCT GGACAGGCCT CCCATGTGAT GGCAAACAGG 3697
CCGACAGGAG ACACGGCTGT TGCTCGTCTT CCACATGGGG AAACTGAGGA TCGGAGTCAA 3757
AGCTGGGCGG CCATAGCCAG AACCCAAACC TCCATCCCAC CTCTTGGCCG GCTTCCCTAG 3817
TGGGAACACT GGTTGAACCA GTTTCCTCTA AGATTCTGGG AGCAGGACAC CCCCAGGGAT 3877
AAGGAGAGGA ACAGGAATCC TAAAGCCCTG AGCATTGCAG GGCAGGGGGT GCTGCCTGGG 3937
TCTCCTGTGC AGAGCTGTCC TGCTTTGAAG CTGTCTTTGC CTCTGGGCAC GCGGAGTCGG 3997
CTTGCCTTGC CCCCTCCGGA TTCAGGCCGA TGGGGCTTGA GCCCCCCTGA CCCTGCCCGT 4057
bad ORIGINAL
AP Ο Ο Ο 4 1 1
ΗΧ 1147 —46—
GTCTCCCTCG CAG CTG GGC GCC GTG TAC ACA GAA GGT GGG TTC GTG GAA 410 6
Leu Gly Ala Val Tyr Thr Glu Glv Gly Phe Val Glu
10
GGC Gly 15 GTC Va l· ΛΛ i rtb Γί AAG Lys AAG Lys CTC Leu 20 GGC Gly CTC Leu CTG Leu GGT Gly GAC Asp 25 TCT Ser GTG Val GAC Asp ATC lie TTC Phe 30 4154
AAG GGC ATC CCC TTC GCA GCT CCC ACC AAG GCC CTG GAA AAT CCT CAG 4202
Lys Gly lie Pro Phe Ala Ala Pro Thr Lys Ala Leu Glu Asn Pro Gin
35 40 45
CCA CAT CCT GGC TGG CAA G GTGGGAGTGG GTGGTGCCGG ACTGGCCCTG 42 51
Pro His Pro Gly Trp Gin
CGGCGGGGCG GGTGAGGGCG GCTGCCTTCC TCATGCCAAC TCCTGCCACC TGCAG GG 4308
Gly
ACC Thr CTG Leu 55 Λ ' Lys GCC Ala AAG Lys AAC Asn TTC Phe 60 AAG Lys AAG Lys AGA TGC Arg Cys CTG Leu 65 CAG Gin GCC Ala ACC Thr ATC He 4356
ACC CAG GAC AGC ACC TAC GGG GAT GAA GAC TGC CTG TAC CTC AAC ATT 4404
Thr Gin Asp Ser Thr Tyr Gly Asp Glu Asp Cys Leu Tyr Leu Asn lie
70 75 80 85
TGG GTG CCC CAG GGC AGG AAG CAA G GTCTGCCTCC CCTCTACTCC 4449
Trp Val Pro Gin Gly Arg Lys Gin
CCAAGGGACC CTCCCATGCA GCCACTGCCC CGGGTCTACT CCTGGCTTGA GTCTGGGGGC 4509
TGCAAAGCTG AACTTCCATG AAATCCCACA GAGGCGGGGA GGGGAGCGCC CACTGCCGTT 4569
GCCCAGCCTG GGGCAGGGCA GCGCCTTGGA GCACCTCCCT GTCTTGGCCC CAGGCACCTG 4629
CTGCACAGGG ACAGGGGACC GGCTGGAGAC AGGGCCAGGC GGGGCGTCTG GGGTCACCAG 4689
CCGCTCCCCC ATCTCAG TC TCC CGG GAC CTG CCC GTT ATG ATC TGG ATC 4733
Val Ser Arg Asp Leu Pro Val Met lie Trp He
100
TAT GGA GGC GCC Ala TTC Phe CTC ATG GGG TCC GGC Ser Gly CAT GGG GCC AAC Asn TTC Phe CTC Leu 120 4786
Tyr 105 Gly Gly Leu Met 110 Gly His 115 Gly Ala
AAC AAC TAC CTG TAT GAC GGC GAG GAG ATC GCC ACA CGC GGA AAC GTC 4834
Asn Asn Tyr Leu Tyr Asp Gly Glu Glu He Ala Thr Arg Gly Asn Val
125 130 135
ATC GTG GTC ACC TTC AAC TAC CGT GTC GGC CCC CTT GGG TTC CTC AGC 4832
lie Val Val Thr Phe Asn Tyr Arg Val Gly Pro Leu Gly Phe Leu Ser
140 145 150
ACT GGG GAC GCC AAT CTG CCA G GTGCGTGGGT GCCTTCGGCC CTGAGGTGGG 4934
Thr Gly Asp Ala Asn Leu Pro
155
GCGACCAGCA TGCTGAGCCC AGCAGGGAGA TTTTCCTCAG CACCCCTCAC CCCAAACAAC 4994
CAGTGGCGGT TCACAGAAAG ACCCGGAAGC TGGAGTAGAA TCATGAGATG CAGGAGGCCC 5054
TTGGTAGCTG TAGTAAAATA AAAGATGCTG CAGAGGCCGG GAGAGATGGC TCACGCCTGT 5114
AATCCCAGCA CTTTAGGAGG CCCACACAGG TGGGTCACTT GAGCGCAGAA GTTCAAGACC 5174
BAD ORIGINAL
AH υ υ Ο 4 1 1
HX 1147
—47—
AGCCTGAAAA TCACTGGGAG ACCCCCATCT CTACACAAAA ATTAAAAATT AGCTGGGGAC 5234
TGGGCGCGGC GGCTCACCTC TGTAATCCCA GCACGTTGGG AGCCCAAGGT GGGTAGATCA 5294
CCTGAGGTCA GGAGTTTGAG ACCAGCCTGA CTAAAATGGA GAAACCTCTT CTCTACTAAA 5354
AATACAAAAT TAGCCAGGCG 7GGTGGCGCT TGCCTGTAAT CCCAGCTACT CGGGAGGCTG 5414
AGGCAGGAGA ATCGCTTGAA C7CAGGAGGC GGAGGTTGCG GTGAGCCGAG ATCATGCCAC 5474
TGCACTCCAG CCTGGAGAAC AA3AGTAAAA CTCTGTCTCA AAAAAAAAAA AAAAAAAAAA 5534
ATAGCCAGGC GTGGTATCTC ATGCCTCTGT CCTCAGCTAC CTGGGAGGCA GAGGTGGAAG 5594
GATCGCTTGA GCCCAGGGGT TCAAAGCTGC AGTGAGCCGT GGTCGTGCCA CTGCACTCCA 5654
GCCTGGGCGA CAGAGTGAGG CCCCATCTCA AAAATAAGAG GCTGTGGGAC AGACAGACAG 5714
GCAGACAGGC TGAGGCTCAG AGAGAAACCA GGAGAGCAGA GCTGAGTGAG AGACAGAGAA 5774
CAATACCTTG AGGCAGAGAC AGCTGTGGAC ACAGAAGTGG CAGGACACAG ACAGGAGGGA 5834
CTGGGGCAGG GGCAGGAGAG GTGCATGGGC CTGACCATCC TGCCCCCGAC AAACACCACC 5894
CCCTCCAGCA CCACACCAAC CCAACCTCCT GGGGACCCAC CCCATACAGC ACCGCACCCG 5954
ACTCAGCCTC CTGGGACCCA CCCACTCCAG CAACCAACGT GACCTAGTCT CCTGGGACCC 6014
ACCCCCTCCA GCACCCTACC C3ACCCAGCT TCTTAGGGAC CCACCATTTG CCAACTGGGC 6074
TCTGCCATGG CCCCAACTCT GTTGAGGGCA TTTCCACCCC ACCTATGCTG ATCTCCCCTC 6134
CTGGAGGCCA GGCCTGGGCC ACTGGTCTCT AGCACCCCCT CCCCTGCCCT GCCCCCAG GT Gly 160 6194
AAC TAT GGC CTT CGG GAT CAG CAC ATG GCC ATT GCT TGG GTG 1 AAG AGG 6242
Asn Tyr Gly Leu Arg Asp Gin His Met Ala lie 165 170 Ala Trp Val Lys Arg 175
AAT ATC GCG GCC TTC GGG GGG GAC CCC AAC AAC ATC ACG CTC TTC GGG 6290
Asn lie Ala Ala Phe Gly Gly Asp Pro Asn Asn 180 185 He Thr Leu 190 , Phe Gly
GAG TCT GCT Glu Ser Ala 195 GGA GGT GCC AGC GTC TCT CTG CAG Gly Gly Ala Ser Val Ser Leu Gin 200 GTCTCGGGAT CCCTGTGGGG 6343
AGGGCCTGCC CCACAGGTTG AGAGGAAGCT CAAACGGGAA GGGGAGGGTG GGAGGAGGAG 6403
CGTGGAGCTG GGGCTGTGGT GCTGGGGTGT CCTTGTCCCA GCGTGGGGTG GGCAGAGTGG 6463
GGAGCGGCCT TGGTGACGGG ATTTCTGGGT CCCGTAG ACC : CTC TCC CCC TAC AAC 6518
Thr Leu Ser Pro Tyr Asn
205
AAG GGC CTC ATC CGG CGA GCC ATC AGC CAG AGC GGC GTG GCC CTG AGT 6566
Lys Gly Leu lie Arg Arg Ala He Ser Gin Ser Gly Val Ala Leu Ser
210 215 220 225
CCC TGG GTC ATC CAG AAA AAC CCA CTC TTC TGG GCC AAA AAG 6608
Pro Trp Val He Gin Lys Asn Pro Leu Phe Trp Ala Lys Lys
230 235
GTAAACGGAG GAGGGCAGGG CTGGGCGGGG TGGGGGCTGT CCACATTTCC GTTCTTTATC 6668
CTGGACCCCA TCCTTGCCTT CAAATGGTTC TGAGCCCTGA GCTCCGGCCT CACCTACCTG 6728
bad original
AP Ο Ο Ο 4 1 1
ΗΧ 1147 —48—
CTGGCCTTGG TTCTGCCCCC AG GTG GCT GAG AAG GTG GGT TGC CCT GTG GGT 67 8 0
Val Ala Glu Lys Val Gly Cys Pro Val Gly 240 245
GAT Asp 250 GCC Ala GCC Ala AGG Arg ATG Met GCC Ala 255 CAG Gin TGT Cys CTG AAG GTT ACT GAT CCC Pro CGA Arg GCC Alci 265 6328
Leu Lys Val 260 Thr Asp
CTG ACG CTG GCC TAT AAG GTG CCG CTG GCA GGC CTG GAG T < 3TGAGTAGCT 6878
Leu Thr Leu Ala Tyr Lys Val Pro Leu Ala Gly Leu Glu
270 275
GCTCGGGTTG GCCCATGGGG TCTCGAGGTG GGGGTTGAGG GGGGTACTGC CAGGGAGTAC 6938
TCCGGAGGAG AGAGGAAGGT AAGGCCCCAG GCCAGAGCTG CGGTCTTGTC CTGTCACCAA CTAGCTGGTG 6998
TCTCCCCTCG CTGTAAGGGA GAGGGGGTGC CGTTTCTTCT TTTTTTTTGA 7058
GATGGAGTCT CACTGTTGCC CAGGCTGGAG TGCAGTGTCA CGATCTCAGC TCACTGCAAC 7113
CTCCACCTCC TGGGTTCAAG TGATTCTCTG ACTCAACCTC CCATGTAGCT GGGACTACAG 7178
GCACATGCCA CCATGCCCAG ATAATTTTTC TGTGTGTTTA GTAGGGATGG AGTTTCATCG 7238
TGTTAGCTAG GATGATCTCG GTCTTGGGAC CTCATGATCT GCCCACCTCG GCCTCCCAAA 7298
GTGCTGGAAT TACAGGCGTG AGCCACTGTG CCCGGCCCCT TCTTTATTCT TATCTCCCAT 7358
GAGTTACAGA CTCCCCTTTG AGAAGCTGAT GAACATTTGG GGCCCCCTCC CCCACCTCAT 7418
GCATTCATAT GCAGTCATTT GCATATAATT TTAGGGAGAC TCATAGACCT CAGACCAAGA 7478
GCCTTTGTGC TAGATGACCG TTCATTCATT CGTTCATTCA TTCAGCAAAC ATTTACTGAA 7538
CCGTAGCACT GGGGCCCAGC CTCCAGCTCC ACTATTCTGT ACCCCGGGAA GGCCTGGGGA 7598
CCCATTCCAC AAACACCTCT GCATGTCAGC CTTACCAGCT TGCTACGCTA AGGCTGTCCC 7658
TCACTCATTC TTCTATGGCA ACATGCCATG AAGCCAAGTC ATCTGCACGT TTACCTGACA 7718
TGAGCTCAAC TGCACGGGCT GGACAAGCCC AAACAAAGCA ACCCCCACGG CCCCGCTAGA 7778
AGCAAAACCT GCTGTGCTGG GCCCAGTGAC AGCCAGGCCC CGCCTGCCTC AGCAGCCACT 7838
GGGTCCTCTA GGGGCCCGTC CAGGGGTCTG GAGTACAATG CAGACCTCCC ACCATTTTTG 7898
GCTGATGGAC TGGAACCCAG CCCTGAGAGA GGGAGCTCCT TCTCCATCAG TTCCCTCAGT 7958
GGCTTCTAAG TTTCCTCCTT CCTGCTTCAG GCCCAGCAAA GAGAGAGAGG AGAGGGAGGG 8018
GCTGCCGCTG AAGAGGACAG ATCTGGCCCT AGACAGTGAC TCTCAGCCTG GGGACGTGTG 8078
GCAGGGCCTG GAGACATCTG TGATTGTCAC AGCTGGGGAG GGGGTGCTCC TGGCACCTCG 8138
TGGGTCGAGG CCGGGGATGC TCTAAACATC CTACAGGGCA CAGGATGCCC CTGATGGTGC 8198
AGAATCAACC CTGCCCCAAG TGTCCATAGA TCAGAGAAGG GAGGACATAG CCAATTCCAG 8258
CCCTGAGAGG CAAGGGGCGG CTCAGGGGAA ACTGGGAGGT ACAAGAACCT GCTAACCTGC 8318
TGGCTCTCCC ACCCAG AC Tyr CCC ATG CTG CAC TAT GTG GGC TTC GTC CCT Pro Met Leu His Tyr Val Gly Phe Val Pro 8366
280 285
GTC ATT GAT GGA GAC TTC ATC CCC GCT GAC CCG ATC AAC CTG TAC GCC 8414
Val lie Asp Gly Asp Phe lie Pro Ala Asp Pro lie Asn Leu Tyr Ala
290 295 300 305
BAD ORIGINAL
AP Ο Ο Ο 4 1 1
ΗΧ 1147
AAC Asn GCC Ala GCC Ala GAC Asp ATC lie 310 GAC Asp TAT Tyr ATA He GCA Ala GGC Gly 315 ACC Thr AAC Asn AAC Asn ATG Met GAC Asp 320 GGC Gly 3462
CAC ATC TTC GCC AGC ATC GAC ATC CCT GCC ATC AAC J-lAG GGC AAC AnG 3510
His He Phe Ala Ser He Asp Met Pro Ala He Asn Lys Gly Asn Lys
325 330
AAA GTC ACG GA GTAAGCAGGG GGCACAGGAC TCAGGGGCGA CCCGTGCGGG 3561
Lys Val Thr Glu
340
AGGGCCGCCG GGAAAGCACT GGCGAGGGGG CCAGCCTGGA GGAGGAAGGC ATTGAGTGGA 8621
GGACTGGGAG TGAGGAAGTT AGCACCGGTC GGGGTGAGTA TGCACACACC TTCCTGTTGG 8681
CACAGGCTGA GTGTCAGTGC CTACTTGATT CCCCCAG G GAG GAC TTC TAC AAG 8734
Glu Asp Phe Tyr Lys
345
CTG GTC AGT GAG TTC ACA ATC ACC AAG GGG CTC AGA GGC GCC AAG ACG 8782
Leu Val Ser Glu 350 Phe Thr lie Thr Lys 355 Gly Leu Arg Gly Ala 360 Lys Thr
ACC TTT GAT GTC TAC ACC GAG TCC TGG GCC CAG GAC CCA TCC CAG GAG 8830
Thr Phe Asp Val Tyr Thr Glu Ser Trp Ala Gin Asp Pro Ser Gin Glu
365 370 375
AAT AAG AAG AAG ACT GTG GTG GAC TTT GAG ACC GAT GTC CTC TTC CTG 8878
Asn Lys Lys Lys Thr Val Val Asp Phe Glu Thr Asp Val Leu Phe Leu
380 385 390
GTG CCC ACC GAG ATT GCC CTA GCC CAG CAC AGA GCC AAT GCC AA 8922
Val Pro Thr Glu lie Ala Leu Ala Gin His Arg Ala Asn Ala Lys
395 400 405
GTGAGGATCT GGGCAGCGGG TGGCTCCTGG GGGCCTTCCT GGGGTGCTGC ACCTTCCAGC 8982
CGAGGCCTCG CTGTGGGTGG CTCTCAGGTG TCTGGGTTGT CTGGGAAAGT GGTGCTTGAG 9042
TCCCCACCTG TGCCTGCCTG ATCCACTTTG CTGAGGCCTG GCAAGACTTG AGGGCCTCTT 9102
TTTACCTCCC AGCCTACAGG GCTTTACAAA CCCTATGATC CTCTGCCCTG CTCAGCCCTG 9162
CACCCCATGG TCCTTCCCAC TGGAGAGTTC TTGAGCTACC TTCCATCCCC CATGCTGTGT 9222
GCACTGAGAG AACACTGGAC AATAGTTTCT ATCCACTGAC TCTTATGGGC CTCAACTTTG 9282
CCCATAATTT CAGCCCACCA CCACATTAAA AATCTTCATG TAATAATAGC CAATTATAAT 9342
AAAAAATAAG GCCAGACACA GTAGCTCATG CCTGTAATCC CAGCACATTG GGAGGTCAAG 9402
GTGGGAGGAT CACTTGAGGT CAGGAGTCTG AGACTAGTCT GGCCAACATG GCAAAACCCC 9462
ATCTCTACTA AAAATACAAA AATTATCCAG GCATGGTGGT GCATGCCTAT AATCCTAGCT 9522
ACTCAGGAGG CTGAGGTAGC AGAATTGATT GACCCAGGGA GGTGGAGGTT GCAGTGAGCC 9582
GAGATTACGC CACTGCACTC CAGCAGGGGC AACAGAGTGA GACTGTGTCT CGAATAAATA 9642
AGTAAATAAA TAATAAAAAT AAAAAATAAG TTAGGAATAC GAAAAAGATA GGAAGATAAA 9702
AGTATACCTA GAAGTCTAGG ATGAAAGCTT TGCAGCAACT AAGCAGTACA TTTAGCTGTG 9762
AGCCTCCTTT CAGTCAAGGC AAAAAGGGAA ACAGTTGAGG GCCTATACCT TGTCCAATCT 9822
AATTGAAGAA TGCACATTCA CTTGGAGAGC AAAATATTTC TTGATACTGA ATTCTAGAAG 9882
BAD original
AP 0 0 0 4 11
HX 1147 —50—
GAAGGTGCCT CACAATGTTT TGTGGAGGTG AAGTATAAAT TCAGCTGAAA TTGTGGAACC 9942
CATGAATCCA TGAATTTGGT TCTCAGCTTT CCCTTCCCTG GGTGTAAGAA GCCCCATCTC 10002
TTCATGTGAA TTCCCCAGAC ACTTCCCTGC CCACTGCCCG GGACCTCCCT CCAAGTCCGG 10062
TCTCTGGGCT GATCGGTCCC CAGTGAGCAC CCTGCCTACT TGGGTGGTCT CTCCCCTCCA 10122
G G AGT GCC AAG ACC TAC GCC TAC CTG TTT TCC CAT CCC TCT CGG ATG 10169
Ser Ala Lys Thr Tyr Ala Tyr Leu Phe Ser His Pro Ser Arg Met
410 415 420
CCC GTC TAC CCC AAA TGG GTG GGG GCC GAC CAT GCA GAT GAC ATT CAG 10217
Pro Val Tyr Pro Lys Trp Val Gly Ala Asp His Ala Asp Asp Ile Gin
425 430 435 440
TAC GTT TTC GGG AAG CCC TTC GCC ACC CCC ACG GGC TAC CGG CCC CAA 10265
Tyr Val Phe Gly Lys Pro Phe Ala Thr Pro Thr Gly Tyr Arg Pro Gin
445 450 455
GAC AGG ACA GTC TCT AAG GCC ATG ATC GCC TAC TGG ACC AAC TTT GCC 10313
Asp Arg Thr Val Ser Lys Ala Met Ile Ala Tyr Trp Thr Asn Phe Ala
460 465 470
AAA ACA GG GTAAGACGTG GGTTGAGTGC AGGGCGGAGG GCCACAGCCG 10361
Lys Thr Gly
475
AGAAGGGCCT CCCACCACGA GGCCTTGTTC CCTCATTTGC CAGTGGAGGG ACTTTGGGCA 10421
AGTCACTTAA CCTCCCCCTG CATCGGAATC CATGTGTGTT TGAGGATGAG AGTTACTGGC 10431
AGAGCCCCAA GCCCATGCAC GTGCACAGCC AGTGCCCAGT ATGCAGTGAG GGGCATGGTG 10541
CCCAGGGCCA GCTCAGAGGG CGGGGATGGC TCAGGCGTGC AGGTGGAGAG CAGGGCTTCA 10601
GCCCCCTGGG AGTCCCCAGC CCCTGCACAG CCTCTTCTCA CTCTGCAG G GAC CCC 10656
Asp Pro
AAC ATG GGC GAC TCG GCT GTG CCC ACA CAC TGG GAA CCC TAC ACT ACG 10704
Asn Met Gly Asp Ser Ala Val Pro Thr His Trp Glu Pro Tyr Thr Thr
480 485 490
GAA AAC AGC GGC TAC CTG GAG ATC ACC AAG AAG ATG GGC AGC AGC TCC 10752
Glu Asn Ser Gly Tyr Leu Glu Ile Thr Lys Lys Met Gly Ser Ser Ser
495 500 505
ATG AAG CGG AGC CTG AGA ACC AAC TTC CTG CGC TAC TGG ACC CTC ACC 10800
Met Lys Arg Ser Leu Arg Thr Asn Phe Leu Arg Tyr Trp Thr Leu Thr
510 515 520 525
TAT CTG GCG CTG CCC ACA GTG ACC GAC CAG GAG GCC ACC CCT GTG CCC 10848
Tyr Leu Ala Leu Pro Thr Val Thr Asp Gin Glu Ala Thr Pro Val Pro
530 535 540
CCC ACA GGG GAC TCC GAG GCC ACT CCC GTG CCC CCC ACG GGT GAC TCC 10896
Pro Thr Gly Asp Ser Glu Ala Thr Pro Val Pro Pro Thr Gly Asp Ser
545 550 555
GAG ACC GCC CCC GTG CCG CCC ACG GGT GAC TCC GGG GCC CCC CCC GTG 10944
Glu Thr Ala Pro Val Pro Pro Thr Gly Asp Ser Gly Ala Pro Pro Val
560 565 570
CCG CCC ACG GGT GAC TCC GGG GCC CCC CCC GTG CCG CCC ACG GGT GAC 10992
Pro Pro Thr Gly Asp Ser Gly Ala Pro Pro Val Pro Pro Thr Gly Asp
575 580 585
BAD ORIGINAL fl
AP Ο Ο Ο 411
ΗΧ 1147
TCC Ser 590 GGG Gly GCC Ala CCC Pro CCC Pro GTG Val 595 CCG Pro CCC Pro ACG GGT GAC Asp 600 TCC GGG GCC CCC Pro CCC Pro 605 11040
Thr Gly Ser Gly Ala
GTG CCG CCC ACG GGT GAC TCC GGG GCC CCC CCC GTG CCG CCC ACG GGT 1103S
7a 1 Pro Pro Thr Gly 610 Asp Ser Gly Ala Pro 615 Pro Val Pro Pro Thr 620 Gly
GAC TCC GGG GCC CCC CCC GTG CCG CCC ACG GGT GAC TCC GGC GCC CCC 11135
Asp Ser Gly Ala 625 Pro Pro Val Pro Pro 630 Thr Gly Asp Ser Gly 635 Ala Pro
CCC GTG CCG CCC ACG GGT GAC GCC GGG CCC CCC CCC GTG CCG CCC ACG 11184
Pro Val Pro 640 Pro Thr Gly Asp Ala 645 Gly Pro Pro Pro Val 650 Pro Pro Thr
GGT GAC TCC GGC GCC CCC CCC GTG CCG CCC ACG GGT GAC TCC GGG GCC 11232
Gly Asp 655 Ser Gly Ala Pro Pro 660 Val Pro Pro Thr Gly 665 Asp Ser Gly Ala
CCC CCC GTG ACC CCC ACG GGT GAC TCC GAG ACC GCC CCC GTG CCG CCC 11280
Pro 670 Pro Val Thr Pro Thr 675 Gly Asp Ser Glu Thr 680 Ala Pro Val Pro Pro 685
ACG GGT GAC TCC GGG GCC CCC CCT GTG CCC CCC ACG GGT GAC TCT GAG 11328
Thr Gly Asp Ser Gly 690 Ala Pro Pro Val Pro 695 Pro Thr Gly Asp Ser 700 Glu
GCT GCC CCT GTG CCC CCC ACA GAT GAC TCC AAG GAA GCT CAG ATG CCT 11376
Ala Ala Pro Val Pro Pro Thr Asp Asp Ser Lys Glu Ala Gin Met Pro
705 710 715
GCA GTC ATT AGG ΤΤΤ TAGCGTCCCA TGAGCCTTGG TATCAAGAGG CCACAAGAGT 11431
Ala Val lie Arg Phe
720
GGGACCCCAG GGGCTCCCCT CCCATCTTGA GCTCTTCCTG AATAAAGCCT CATACCCCTG 11491
TCGGTGTCTT TCTTTGCTCC CAAGGCTAAG CTGCAGGATC 11531
BAD ORIGINAL

Claims (31)

1. A DNA molecule encoding for biologically functional BSSL/CEL and containing intron sequences .
2. A DNA molecule according to claim 1 which is shown in SEQ ID NO: 1 in the Sequence Listing.
3. An analogue of the DNA molecule according to claim 2 which contains intron sequences and which hybridizes with the DNA sequence shown in SEQ ID NO: 1 in the Sequence Listing or a specific parr thereof under stringent hybridization conditions.
4. A replicable expression vector which carries and is capable of mediating the expression of a DNA molecule according to any one of claims 1-3, encoding human BSSL/CEL.
5. A vector according to claim 4 capable of encoding biologically funtional BSSL/CEL and containing regulatory elements directing expression to the mammary gland of a non-human mammal.
6. A vector according to claim 4 which is the vector pS452 (DSM 7499).
7. A cell derived from a multicellular organism and harbouring a vector according to any one of claims 4-6.
8. A process for production of human BSSL/CEL, comprising (a) inserting a DNA molecule as defined in any one of claims 1-3 in a vector which is able to replicate in a specific host cell; (b) introducing the resulting recombinant vector into a host cell; (c) growing the
BAD ORIGINAL fi
AP Ο Ο Ο 4 1 1
HX 1147 —53— resulting cell in or on a culture medium for expression of the polypeptide; and (d) recovering the polypeptide.
5
9. A mammalian expression system, comprising a hybrid gene which is expressible in the mammary gland of an adult female of a non-human mammal harbouring said hybrid gene, so that human BSSL/CEL is produced when the hybrid gene is
10 expressed, said hybrid gene being produced by inserting a DNA molecule according to any one of claims 1-3 into a gene regulating a milk protein gene of a non-human mammal.
10. A hybrid gene as defined in claim 9.
11. A mammalian cell harbouring an expression system as defined in claim 9.
12. A non-human mammalian cell according to claim 11 which is an embryo cell.
13. A process of producing a transgenic non-human mammal capable of expressing human BSSL/CEL,
25 comprising (a) introducing an expression system as defined in claim 9 into a fertilized egg or a cell of an embryo of a non-human mammal so as to incorporate the expression system into the germline of the mammal and (b) developing the
30 resulting introduced fertilized egg or embryo into an adult female non-human mammal.
14. A process of producing a transgenic non-human mammal capable of expressing human BSSL/CEL and
35 substantially incapable of expressing BSSL/CEL from the mammal itself, comprising (a) destroying the mammalian BSSL/CEL expressing capability of the mammal so that substantially no mammalian
BAD ORIGINAL ft
AP Ο Ο Ο 41 1
HX 1147
-54BSSL/CEL is expressed and inserting an expression system as defined in claim 9 into the germline of the mammal in such a manner that human BSSL/CEL is expressed in the mammal; and/or (b) replacing
5 the mammalian BSSL/CEL gene or part thereof with an expression system as defined in claim 9.
15. A transgenic non-human mammal harbouring in its genome a DNA molecule according to any one of
10 claims 1-3.
Ιό. A transgenic non-human mammal according to claim 15 in which the DNA molecule is present in the germline of the mammal.
17. A transgenic non-human mammal according to claim 15 or 16 in which the DNA molecule is present in a milk protein gene of the mammal.
20
18. A transgenic non-human mammal prepared by the process of claim 13 or 14.
19. A transgenic non-human mammal according to any one of claims 15-18 which is selected from the
25 group consisting of mice, rats, rabbits, sheep, pigs and cattle.
20. Progeny of a transgenic non-human mammal according to any one of claims 15-19.
21. Milk obtained from a transgenic non-human mammal according to any one of claims 15-20.
22. An infant formula comprising milk as defined in
35 claim 21.
23. A process for production of an infant formula by supplementing an infant food formula with a
BAD ORIGINAL &
AP Ο Ο Ο 4 1 1
HX 1147 —55— polypeptide encoded by a DNA molecule according to any one of claims 1-3.
24. Use of a DNA molecule according to any one of
5 claims 1-3 for the production of human BSSL/CEL.
25. The use according to claim 24 for production of a transgenic non-human mammal expressing human BSSL/CEL.
26. The use according to claim 24 for production of milk, containing human ESSL/CEL, derived from a transgenic non-human mammal.
15
27. The use according to claim 24 for production of an infant formula comprising milk, containing human BSSL/CEL, derived from a transgenic nonhuman mammal.
28. The use of a DNA molecule according to any oneof claims 1-3 in the manufacture of a medicament for the treatment of a pathological condition related to exocrine pancreatic insufficiency.
25
29. The use according to claim 28 in the manufacture of a medicament for the treatment of cystic fibrosis .
30. The use according to claim 28 in the manufacture
30 of a medicament for the treatment of chronic pancreatitis .
31. The use according to claim 28 in the manufacture of a medicament for the treatment of fat
35 malabsorption.
BAD ORIGINAL
AP Ο Ο Ο 4 1 1
HX 1147
32 .
5 33 .
—56—
The use according to claim 28 in the manufacture cf a medicament for the treatment of malabsorption of fat soluble vitamins.
The use according to claim 28 in the manufacture of a medicament for the treatment of fat malabsorption due to physiological reasons.
APAP/P/1993/000538A 1992-06-11 1993-06-10 DNA sequences used in the production of recombinant human BSSL/CEL in transgenic non-human mammals, and the produced BSSSL/CEL used in infant formulas. AP411A (en)

Applications Claiming Priority (4)

Application Number Priority Date Filing Date Title
SE9201809A SE9201809D0 (en) 1992-06-11 1992-06-11 NEW DNA SEQUENCES
SE9201826A SE9201826D0 (en) 1992-06-12 1992-06-12 NEW DNA SEQUENCES II
SE9202088A SE9202088D0 (en) 1992-07-03 1992-07-03 NEW DNA SEQUENCES III
SE9300902A SE9300902D0 (en) 1993-03-19 1993-03-19 NEW DNA SEQUENCES IV

Publications (2)

Publication Number Publication Date
AP9300538A0 AP9300538A0 (en) 1993-07-31
AP411A true AP411A (en) 1995-09-28

Family

ID=27484751

Family Applications (1)

Application Number Title Priority Date Filing Date
APAP/P/1993/000538A AP411A (en) 1992-06-11 1993-06-10 DNA sequences used in the production of recombinant human BSSL/CEL in transgenic non-human mammals, and the produced BSSSL/CEL used in infant formulas.

Country Status (31)

Country Link
US (2) US5616483A (en)
EP (1) EP0651793B1 (en)
JP (1) JP4231105B2 (en)
KR (1) KR100357670B1 (en)
CN (1) CN1071791C (en)
AP (1) AP411A (en)
AT (1) ATE447012T1 (en)
AU (1) AU670598B2 (en)
CA (1) CA2137815C (en)
CZ (1) CZ288677B6 (en)
DE (1) DE69334298D1 (en)
DK (1) DK0651793T3 (en)
ES (1) ES2335626T3 (en)
FI (1) FI945793A (en)
HR (1) HRP930935A2 (en)
HU (1) HU220338B (en)
IL (1) IL105874A (en)
IS (1) IS4029A (en)
MA (1) MA22905A1 (en)
MX (1) MX9303439A (en)
NO (1) NO944715L (en)
NZ (1) NZ253391A (en)
PH (1) PH31680A (en)
PT (1) PT651793E (en)
RU (1) RU2128708C1 (en)
SG (1) SG52580A1 (en)
SI (1) SI9300319A (en)
SK (1) SK286993B6 (en)
TN (1) TNSN93067A1 (en)
UA (1) UA41322C2 (en)
WO (1) WO1993025669A1 (en)

Families Citing this family (40)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
MX198456B (en) * 1994-03-09 2000-09-05 Abbott Lab Transgenic animals producing oligosaccharides and glycoconjugates.
US5907080A (en) * 1995-11-30 1999-05-25 Nexia Biotechnologies, Inc. Method for development of transgenic dwarf goats
EA003293B1 (en) * 1996-12-06 2003-04-24 Авентис Фармасьютикалз Продактс Инк. Polypeptides encoded by a human lipase-like gene, compositions containing them and methods of use of compositions
US7008776B1 (en) * 1996-12-06 2006-03-07 Aventis Pharmaceuticals Inc. Compositions and methods for effecting the levels of high density lipoprotein (HDL) cholesterol and apolipoprotein AI very low density lipoprotein (VLDL) cholesterol and low density lipoprotein (LDL) cholesterol
FR2763958A1 (en) * 1997-05-29 1998-12-04 Transgene Sa COMBINATION PRODUCT COMBINING A NUCLEIC ACID WITH A SUBSTANCE DISORGANIZING THE EXTRACELLULAR MATRIX FOR GENE THERAPY
US6699672B1 (en) 1997-09-02 2004-03-02 Insight Biopharmaceuticals Ltd. Heparanase specific molecular probes and their use research and medical applications
US20020088019A1 (en) * 1997-09-02 2002-07-04 Oron Yacoby-Zeevi Methods of and pharmaceutical compositions for improving implantation of embryos
US6177545B1 (en) * 1997-09-02 2001-01-23 Insight Strategy & Marketing Ltd. Heparanase specific molecular probes and their use in research and medical applications
US20010006630A1 (en) * 1997-09-02 2001-07-05 Oron Yacoby-Zeevi Introducing a biological material into a patient
US20040213789A1 (en) * 1997-09-02 2004-10-28 Oron Yacoby-Zeevi Heparanase activity neutralizing anti-heparanase monoclonal antibody and other anti-heparanase antibodies
SE9801424D0 (en) * 1998-04-22 1998-04-22 Astra Ab Expression methods
US20030217375A1 (en) * 1998-08-31 2003-11-20 Eyal Zcharia Transgenic animals expressing heparanase and uses thereof
AU2878600A (en) * 1999-03-01 2000-09-21 Hadasit Medical Research Services & Development Company Ltd Polynucleotide encoding a polypeptide having heparanase activity and expression of same in genetically modified cells
AU4074400A (en) * 1999-04-09 2000-11-14 Human Genome Sciences, Inc. 49 human secreted proteins
WO2001089553A1 (en) * 1999-05-19 2001-11-29 Barnes Jewish Hospital Enhanced triglyceride digestion in a deficiency of bile salts
WO2002010217A2 (en) 2000-08-02 2002-02-07 The Johns Hopkins University Endothelial cell expression patterns
IT1319655B1 (en) 2000-11-15 2003-10-23 Eurand Int PANCREATIC ENZYME MICROSPHERES WITH HIGH STABILITY AND RELATIVE PREPARATION METHOD.
US20100268658A1 (en) * 2001-05-14 2010-10-21 Prolacta Bioscience Method for collecting, testing and distributing milk
US20020182243A1 (en) * 2001-05-14 2002-12-05 Medo Elena Maria Method of producing nutritional products from human milk tissue and compositions thereof
US6671189B2 (en) * 2001-11-09 2003-12-30 Minebea Co., Ltd. Power converter having primary and secondary side switches
US20030213007A1 (en) * 2002-03-27 2003-11-13 Slattery Charles Wilbur Human milk produced by human mammary tissue implanted in non-human host animals and uses thereof
US20060269552A1 (en) * 2003-06-09 2006-11-30 Oron Yacoby-Zeevi Heparanase activity neutralizing anti-heparanase monclonal antibody and other anti-heparanase antibodies
BRPI0616323A2 (en) 2005-09-20 2011-06-14 Prolacta Bioscience Inc Method for determining whether a donated mammary fluid was obtained from a specific individual
WO2007089133A1 (en) * 2006-02-03 2007-08-09 Academisch Medisch Centrum Bij De Universiteit Van Amsterdam Means and methods for influencing interactions between dc-sign and dc-sign ligands
EP2099321B1 (en) * 2006-11-29 2017-04-19 Prolacta Bioscience, Inc. Human milk compositions and methods of making and using same
EP2101597B1 (en) 2006-12-08 2012-10-17 Prolacta Bioscience, Inc. Compositions of human lipids and methods of making and using same
MY163469A (en) 2007-02-20 2017-09-15 Allergan Therapeutics LLC Stable digestive enzyme compositions
WO2009109856A2 (en) 2008-03-07 2009-09-11 Axcan Pharma Inc. Method for detecting infectious parvovirus in pharmaceutical preparations
JP2012510476A (en) 2008-12-02 2012-05-10 プロラクタ バイオサイエンス，インコーポレイテッド Human milk permeation composition and methods of making and using
PL2621476T5 (en) 2010-10-01 2022-06-06 Société des Produits Nestlé S.A. Enteric coated, low-strength pancrelipase formulations
JP5850940B2 (en) * 2010-10-21 2016-02-03 スウェディッシュ オーファン バイオビトラム パブリーク アクチエボラグ Methods for increasing the growth rate of human infants
RU2013123056A (en) 2010-10-21 2014-11-27 Сведиш Орфан Биовитрум Аб (Пабл) METHOD FOR INCREASING SUCTION OF UNSATURATED FATTY ACIDS IN BREAST CHILDREN
ES2655019T3 (en) 2011-08-03 2018-02-16 Prolacta Bioscience, Inc. Microfiltration of human milk to reduce bacterial contamination
RU2016119726A (en) 2011-08-08 2018-11-02 Апталис Фарма Лтд. The method of conducting a test for the dissolution of solid compositions containing digestive enzymes
CN103088000A (en) * 2012-03-23 2013-05-08 北京济福霖生物技术有限公司 Method for overexpression of cholate activation lipase in mammary gland of mammal
AU2013287353B2 (en) * 2012-07-12 2018-11-08 Proqr Therapeutics Ii B.V. Oligonucleotides for making a change in the sequence of a target RNA molecule present in a living cell
PT2967094T (en) 2013-03-13 2021-01-14 Prolacta Bioscience Inc High fat human milk products
AU2014306222A1 (en) 2013-08-09 2016-02-18 Aptalis Pharma Ltd. Digestive enzyme composition suitable for enteral administration
EP3157568A1 (en) 2014-06-19 2017-04-26 Aptalis Pharma Limited Methods for removing viral contaminants from pancreatic extracts
IL260327B2 (en) 2015-12-30 2023-10-01 Prolacta Bioscience Inc Human milk products useful in pre- and post-operative care

Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1991015234A1 (en) * 1990-04-04 1991-10-17 Oklahoma Medical Research Foundation Recombinant bile salt activated lipases

Family Cites Families (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US3256150A (en) * 1964-04-20 1966-06-14 Dairyland Food Lab Method for treating malabsorption syndrome
GB1509866A (en) * 1975-06-10 1978-05-04 Johnson & Johnson Enteric coated digestive enzyme compositions
US4873316A (en) * 1987-06-23 1989-10-10 Biogen, Inc. Isolation of exogenous recombinant proteins from the milk of transgenic mammals
US4944944A (en) * 1987-11-19 1990-07-31 Oklahoma Medical Research Foundation Dietary compositions and methods using bile salt-activated lipase
US5173408A (en) * 1989-11-13 1992-12-22 Lange Louis George Iii Mammalian pancreatic cholesterol esterase
WO1991015534A1 (en) * 1990-03-30 1991-10-17 Allied-Signal Inc. Electrically conductive poly(aromatic vinylenes) and poly(heteroaromatic vinylenes)
SE9001985D0 (en) * 1990-06-01 1990-06-01 Astra Ab NEW CHEMICAL PRODUCTS

Patent Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1991015234A1 (en) * 1990-04-04 1991-10-17 Oklahoma Medical Research Foundation Recombinant bile salt activated lipases

Also Published As

Publication number Publication date
HRP930935A2 (en) 1994-12-31
HUT71789A (en) 1996-02-28
DE69334298D1 (en) 2009-12-10
FI945793A0 (en) 1994-12-09
UA41322C2 (en) 2001-09-17
CN1086262A (en) 1994-05-04
AU4366793A (en) 1994-01-04
DK0651793T3 (en) 2010-03-15
MA22905A1 (en) 1993-12-31
EP0651793A1 (en) 1995-05-10
HU9403536D0 (en) 1995-02-28
CZ288677B6 (en) 2001-08-15
IL105874A0 (en) 1993-10-20
CA2137815A1 (en) 1993-12-23
CZ309194A3 (en) 1995-07-12
HU220338B (en) 2001-12-28
RU2128708C1 (en) 1999-04-10
JPH07507687A (en) 1995-08-31
JP4231105B2 (en) 2009-02-25
SI9300319A (en) 1994-03-31
MX9303439A (en) 1994-04-29
SK148694A3 (en) 1995-08-09
NO944715D0 (en) 1994-12-07
CA2137815C (en) 2010-11-09
SK286993B6 (en) 2009-09-07
PT651793E (en) 2010-01-20
FI945793A (en) 1995-02-09
TNSN93067A1 (en) 1994-03-17
KR100357670B1 (en) 2003-04-11
ATE447012T1 (en) 2009-11-15
IS4029A (en) 1993-12-12
PH31680A (en) 1999-01-18
IL105874A (en) 1998-12-27
ES2335626T3 (en) 2010-03-30
AU670598B2 (en) 1996-07-25
CN1071791C (en) 2001-09-26
US5716817A (en) 1998-02-10
AP9300538A0 (en) 1993-07-31
NO944715L (en) 1995-02-07
EP0651793B1 (en) 2009-10-28
RU94046360A (en) 1997-06-10
WO1993025669A1 (en) 1993-12-23
SG52580A1 (en) 1998-09-28
NZ253391A (en) 1996-01-26
US5616483A (en) 1997-04-01

Similar Documents

Publication Publication Date Title
AP411A (en) DNA sequences used in the production of recombinant human BSSL/CEL in transgenic non-human mammals, and the produced BSSSL/CEL used in infant formulas.
US5827683A (en) Nucleic acids encoding BSSL variants
US6525241B1 (en) Expression methods
PL175404B1 (en) Isolated dna molecule and method of obtaining human protein bssl/cel
LT4008B (en) Novel polypeptides