EP1170994A1 - Transgene tiere, die speichelproteine exprimieren - Google Patents

Transgene tiere, die speichelproteine exprimieren

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Publication number
EP1170994A1
EP1170994A1 EP00920304A EP00920304A EP1170994A1 EP 1170994 A1 EP1170994 A1 EP 1170994A1 EP 00920304 A EP00920304 A EP 00920304A EP 00920304 A EP00920304 A EP 00920304A EP 1170994 A1 EP1170994 A1 EP 1170994A1
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European Patent Office
Prior art keywords
protein
animal
phytase
seq
transgene
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English (en)
French (fr)
Inventor
Cecil W. Forsberg
Serguei Golovan
John P. Phillips
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University of Guelph
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University of Guelph
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    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
    • A01K67/00—Rearing or breeding animals, not otherwise provided for; New or modified breeds of animals
    • A01K67/027—New or modified breeds of vertebrates
    • A01K67/0275—Genetically modified vertebrates, e.g. transgenic
    • 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
    • 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)
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
    • A01K2217/00—Genetically modified animals
    • A01K2217/05—Animals comprising random inserted nucleic acids (transgenic)
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
    • A01K2227/00—Animals characterised by species
    • A01K2227/10—Mammal
    • A01K2227/105—Murine
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
    • A01K2227/00—Animals characterised by species
    • A01K2227/10—Mammal
    • A01K2227/108—Swine
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
    • A01K2267/00—Animals characterised by purpose
    • A01K2267/01—Animal expressing industrially exogenous proteins
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
    • A01K2267/00—Animals characterised by purpose
    • A01K2267/02—Animal zootechnically ameliorated
    • 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

Definitions

  • the present invention relates to transgenic animals and, more specifically, to animals genetically modified to express a desired protein.
  • Phosphorus is an essential element for the growth of all organisms.
  • phosphorus deficiency has been described as the most prevalent mineral deficiency throughout the world and feed must often be supplemented with inorganic phosphorus in order to obtain desired growth performance of monogastric animals (e.g. pigs, poultry etc.).
  • Phytic acid, or phytate (m o-inositol 1, 2,3,4, 5,6-hexakis dihydrogen phosphate) is a major storage form of phosphorus in cereals and legumes, representing 18% to 88% of the total phosphorus content (Reddy et al. 1982).
  • the enzyme phytase belongs to the group of phosphoric rnonoester hydrolases: it catalyzes the hydrolysis of phytate (mvo-inositol hexakis phosphate) to inorganic rnonophosphate and lower phosphoric esters of m o-inositol or, in some cases, free mvo-inositol.
  • Phytases are classified either as 3-phytases or 6-phytases based on the first phosphate group attacked by the enzyme. 3 -phytase is typical for microorganisms and 6- phytase for plants (Cosgrove, 1980).
  • Phytase is either absent or present at a very low levels in monogastric animals (Bitar and Reinhold 1972; Iqbal et al. 1994). Consequently, dietary phytate is not digested or absorbed from the small intestine and instead is concentrated in fecal material, thereby contributing to phosphorus pollution in areas of intensive livestock production. Runoff from animal farms leads to contamination of rivers and streams. Such runoff has resulted in rapid drops in the oxygen concentration in rivers and streams due to excessive algal growth in water, which, in turn, has led to an increase in the mortality rate of fish and existing flora and fauna. This is becoming a global problem as pig and poultry production is increased (Miner 1999;Mallin 2000).
  • phytic acid is viewed as an anti-nutritional factor because it interacts with essential dietary minerals and proteins limiting the nutritional values of cereals and legumes in man and animals (Harland and Morris 1995).
  • various attempts have been made to enable animals to utilize available phytate in feed. Such attempts have included production of low phytate plants (Abelson 1999), addition of phytase to the animal feed (Simons et al. 1990) (Stahl et al. 1999) or transformation of the fodder plants to produce the required phytase (Pen et al. 1993, Verwoerd et al. 1995).
  • a phytase produced by Escherichia coli has been reported to exhibit the highest activity of those reported (Wodzinski and Ullah 1996).
  • This phytase from E. coli was initially cloned as an acid phosphatase gene that was designated APPA (Dassa et al. 1990).
  • Greiner et al. ( 1991 ; 1993) purified phytase from E. coli and reported that some of the kinetic properties of the acid phosphatase activity of the native phytase of E. coli were similar to those of the RR -encoded acid phosphatase.
  • the authors did not clone the phytase gene to prove that it was identical to APPA gene.
  • the invention provides a transgenic non-human animal that carries in the genome of its somatic and/or germ cells a nucleic acid sequence including a heterologous transgene construct, the construct including a trangene encoding a protein, the transgene being operably linked to a first regulatory sequence for salivary gland specific expression of the protein.
  • the invention provides a transgenic non-human animal that carries in the genome of its somatic and/or germ cells a nucleic acid sequence including a heterologous transgene construct, the construct including a trangene encoding phytase or a homologue thereof.
  • the invention provides a method of expressing a protein, the method comprising the steps of: a) introducing a transgene construct into a non-human animal embryo such that a non- human transgenic animal that develops from the embryo has a genome that comprises the transgene construct, wherein the transgene construct comprises: i) a transgene encoding the protein, and ii) at least one regulatory sequence for gastrointestinal tract specific expression of the protein, b) transferring the embryo to a foster female; and, c) developing the embryo into the transgenic animal wherein the transgene is produced in the gastrointestinal tract of the animal.
  • the invention provides a transgenic animal adapted for expressing a protein according to the above method.
  • the invention also provides for the progeny of such animal.
  • the invention provides a process for producing a protein comprising the steps of: a) obtaining saliva containing the protein from a non-human transgenic animal, the animal containing within its genome a transgene construct, wherein the transgene construct comprises: i) a transgene encoding the protein, and ii) at least one regulatory sequence for salivary gland specific expression of the protein, and extracting the protein from the saliva.
  • the invention provides a method for expressing a phytase or a homologue thereof in a non-human animal, the method comprising: a) constructing a nucleic acid sequence including a transgene construct comprising: i) a transgene encoding the phytase or a homologue thereof, and ii) at least one regulatory sequence for gastrointestinal tract specific expression of the protein, and b) transfecting the animal with the nucleic acid sequence; whereby the animal carries within the genome of its somatic and/or germ cells the transgene construct and wherein the animal expresses the phytase or a homologue thereof in its gastrointestinal tract.
  • the invention provides a nucleic acid molecule comprising a nucleic acid sequence including a gene encoding a protein, the gene being operably linked to at least one regulatory sequence for gastrointestinal tract specific expression of the protein.
  • the invention provides an antibody specific to the protein expressed by the above nucleic acid sequence and a test kit for immunologically detecting such protein. The invention also provides for hybridomas secreting such antibodies.
  • the invention provides cells that are transfected with the above nucleic acid sequence.
  • the invention provides a method for producing a protein molecule comprising a glycosylated protein secreted in the saliva that exhibits a novel physiological activity.
  • a novel physiological activity is phytase.
  • Figure 1 is a schematic diagram representing a method for producing the gene construct of the present invention containing the inducible pro line-rich protein (PRP) promoter/enhancer. More specifically, Figure 1 is a schematic diagram illustrating the steps in the construction of the transgenes R15/APPA+intron and R15/APPA used for the generation of transgenic mice.
  • PRP inducible pro line-rich protein
  • Figure 2 is a schematic diagram representing a method for producing the gene construct of the present invention containing the SV40 promoter. More specifically, Figure 2 is a schematic diagram illustrating the steps in construction of the plasmid containing the transgene SV40/APPA+intron that was introduced by transfection into mammalian cell lines.
  • Figure 3 is a schematic diagram representing a method for producing the gene _,, construct of the present invention containing the constitutive parotid secretory protein (PSP) promoter/enhancer.
  • PSP constitutive parotid secretory protein
  • Figure 3 is a schematic diagram illustrating the steps in construction of the transgenes Lama2/APPA that codes for the native AppA phytase and the Lama2/PSP/APPA that codes for the AppA phytase with the PSP signal peptide sequence.
  • Figure 4 is a schematic diagram of the Lama2-APPA plasmid containing the APPA transgene.
  • Figure 5 illustrates the nucleic acid sequence of the Lama2/APPA plasmid containing the E. coli APPA gene (S ⁇ Q ID NO: 1).
  • Figure 6 illustrates the PCR results for transformed mice. More specifically, figure 6 is a picture of an agarose gel illustrating APPA PCR products from genomic tail DNA of third generation offspring from the transgenic female founder mouse 3-1 generated using the Xhol and Notl fragment of the Lama2/APPA construct.
  • a second generation phytase gene positive male was crossed with each of two phytase positive transgenic females 9f and 1 If (Table 3). From litter 18m x 9f offspring 3, 4, 5 & 6 are PCR positive and from litter 18m x 1 If offspring 2 and 3 are PCR positive.
  • Std is the oligonucleotide standard and the numbers on the left are the bp sizes of the standard.
  • Lane C is a negative control reaction mixture that lacks a D ⁇ A template and appA is a positive control containing an amplified segment of the phytase gene.
  • the primers used were APP A-UP2 and APP A-KP ⁇ .
  • Figure 7 illustrates the PCR results for transformed founder pigs. More specifically, Figure 7 is a picture of an agarose gel illustrating phytase gene PCR products and ⁇ -globin PCR products from genomic tail D ⁇ A of five founder piglets from litter 167. Std is a 1 kb ladder. Lane 2 using the phytase primer set is positive for the phytase gene, and all of the samples were positive for the ⁇ -globin gene. Lane C is a negative control not containing template D ⁇ A. The phytase transgene primer set included APPA-UP2 and APP A-KP ⁇ gave an expected fragment size of 750 bp.
  • Figure 8 illustrates the PCR results for transgene rearrangement tests. More specifically, Figure 8 is a picture of an agarose gel showing the PCR products of four separate primer sets used to amplify different segments of the transgene introduced into pig 167-02. The Std contained a kilobase D ⁇ A ladder.
  • the primers used included lane 1, APPA- UP2 and APPA-KP ⁇ (750 bp); lane 2, APPA -MATURE and APP A-KP ⁇ (1235 bp); lane 3 APPA MATURE and APPA-DOW ⁇ 2 (608 bp); lane 4, PIG-BGF and PIG-BGR (207 bp).
  • lane 5 a negative control without DNA template added;
  • lane 6 the appA gene & primers APPA-UP2 and APPA-KPN.
  • the numbers on the left indicate the sizes of the bands in the standard. No PCR products were detected in the absence of either DNA template or primers.
  • Figure 9 illustrates weight and salivary phytase activity of the transgenic boar 167-02 and average weight of the pen-mates at intervals during growth. Symbols: Weight of 167-02, •; Average weight ⁇ SD of four penmates, A; phytase activity of 167-02, ⁇ ; Phytase specific activity, D. Arrows indicate sampling for fecal phosphorus concentration.
  • Figure 10 illustrates weight and salivary phytase activity of the transgenic boar 282-
  • Figure 11 illustrates weight and salivary phytase activity of the transgenic boar 282- 04 and average weight of the pen-mates at intervals during growth. Symbols: Weight of 282- 04, ⁇ ; Average weight ⁇ SD of five penmates, A; phytase activity of 282-04, ⁇ ; Phytase specific activity, D. Arrows indicate sampling for fecal phosphorus concentration.
  • Figure 12 illustrates weight and salivary phytase activity of the transgenic boar 405- 02 and average weight of the pen-mates at intervals during growth. Symbols: Weight of 405- 02, ⁇ ; Average weight ⁇ SD of four penmates, A; phytase activity of 405-02, ⁇ ; Phytase specific activity, D. Arrows indicate sampling for fecal phosphorus concentration.
  • Figure 13 illustrates weight and salivary phytase activity of the transgenic boar 421- 06 and average weight of the pen-mates at intervals during growth. Symbols: Weight of 421- 06, ⁇ ; Average weight ⁇ SD of four penmates, A; phytase activity of 421-06, ⁇ ; Phytase specific activity, D. Arrows indicate sampling for fecal phosphorus concentration.
  • Figure 14 illustrates the PCR results of first generation pigs. More specifically, Figure 14 is a picture of an agarose gel showing the PCR analysis of eight liter 154 piglets.
  • the phytase transgenic boar 167-02 was used to breed a non-transgenic female. Std, 100 bp ladder, numbers on left are the sizes of the fragments in each band in bp; lane 167-02, DNA from boar 167-02 1, DNA from 167-02; lane C, is a lane without added DNA; lanes 1-8, are amplified DNA inserts from each of the offspring piglets of the litter.
  • Phytase primers were Lama-UP and APPA-DOWN4.
  • ⁇ -globin primers were PIG-BGF and PIG-BGR.
  • Figure 15 illustrates a sodium dodecylsulfate gel stained with silver demonstrating the sizes of the E. coli produced APPA phytase and the APPA phytase produced by the pig and a demonstration that the pig phytase is glycosylated. More specifically, Figure 15 is a picture of a sodium dodecylsulfate polyacrylamide gel electrophoresis (SDS-PAG ⁇ ) profile of the purified AppA phytase produced in E. coli and the purified pig salivary phytase stained directly with silver (A) and a transfer from a similar SDS-PAG ⁇ gel transferred to nitrocellulose and stained for glyoproteins (B). Creatinase is not glycosylated while transferring is glycosylated. The numbers on the left are the masses in of the molecular mass standards (Std) expressed in kDa.
  • Std molecular mass standards
  • Figure 15B is a picture of Western blot of the untreated pig AppA phytase and the same phytase after treatment with a combination of three deglycosylating enzymes.
  • Lane 1 Purified AppA phytase produced in E. coli (untreated); lane 2, purified pig phytase (untreated); lane 3, purified pig phytase treated with the combination of deglycosylating enzymes including N-glycosidase F, O-glycosidase and neuraminidase.
  • Figure 16 illustrates a Western blot of the pig phytase and the E. coli produced APPA phytase using monoclonal antibodies directed to the APPA phytase documenting that they have homologous epitopes. More specifically, Figure 6 is a Western blot of the AppA phytase from pig saliva after various purification steps and of purified phytase produced inE. coli. A monoclonal antibody prepared against the E. coli phytase was used as the primary antibody for detection.
  • Lane 1 saliva from non-transgenic pig 164-04; lane 2, saliva from transgenic pig 167-02; Lane 3, saliva fraction not bound to D ⁇ A ⁇ -Sepharose; lane 4, salivary phytase bound to D ⁇ A ⁇ -Sepharose and released with an NaCl gradient; lane 5, salivary phytase further purified by Chromato focusing with a pH gradient of 4 to 7; lane 6, phytase purified from E. coli.
  • the numbers on the left are the masses of molecular mass standards (not shown) expressed in kDa.
  • Figure 17 illustrates an SDS-Page of the E.
  • Figure 6 is a SDS-PAG ⁇ profile of the purified E. coli produced AppA phytase and the AppA phytases produced by pigs and mice stained with silver (A) and a Western blot of an identical set of protein samples (B).
  • a polyclonal antibody prepared against the E. coli phytase was used as the primary antibody for detection.
  • Lane 1 Purified AppA phytase produced in E. coli; lane 2, Saliva from a non-transgenic pig 164-01; lane 3, Saliva from a AppA producing transgenic pig 167- 02; lane 4, Purified phytase from pig 167-02; lane 5, Saliva from a non-transgenic mouse; lane 6, Saliva from a transgenic mouse containing R15/APPA transgene induced with isoproterenol; lane 7, Saliva from a transgenic mouse containing the Lama/ APPA transgene; Std, Molecular mass markers. The numbers on the left are the masses of molecular mass standards (not shown) expressed in kDa.
  • Figure 18 illustrates the nucleic acid sequence of the known segment of the R15/APPA + intron plasmid including the vector sequences of pBLCAT3 (SEQ ID NO:2).
  • Figure 19 illustrates the nucleic acid sequence of the known segment of the R15/APPA + intron transgene construct used for the generation of transgenic mice (SEQ LD NO:3).
  • Figure 20 illustrates the nucleic acid sequence of the known segment of the R15/APPA plasmid including the vector sequences of pBLCAT3 (SEQ ID NO:4).
  • Figure 21 illustrates the nucleic acid sequence of the known segment of the R15/APPA transgene construct used for the generation of transgenic mice (SEQ ID NO:5).
  • Figure 22 illustrates the nucleic acid sequence of the SV40/APPA + intron plasmid
  • Figure 23 illustrates the nucleic acid sequence of the Lama2/APPA transgene construct used for the generation of transgenic mice and transgenic pigs (SEQ LD NO: 7).
  • Promoter a DNA sequence generally described as the 5' region of a gene and located proximal to the start codon. The transcription of an adjacent gene is initiated at the promoter region. If a promoter is an inducible promoter then the rate of transcription increases in response to an inducing agent. A constitutive promoter is one that initiates transcription of an adjacent gene without additional regulation.
  • operably linked a nucleic acid sequence is “operably linked” when placed into a functional relationship with another nucleic acid sequence.
  • a promoter or enhancer is “operably linked” to a coding sequence if the promoter causes the transcription of the sequence.
  • operably linked means that the linked nucleic acid sequences are contiguous and, where it is necessary to join two protein coding regions, contiguous and in one reading frame.
  • Physically - any protein that liberates phosphate from myo-inositolhexakis-phosphate or other inositol phosphates.
  • Gene - a DNA sequence that contains a template for an R A polymerase ana contains information needed for expressing a polypeptide or protein.
  • Polynucleotide Molecule a polydeoxyribonucleic (DNA) acid molecule or a polyribonucleic acid (RNA) molecule.
  • “Expression” the process by which a polypeptide is produced from a structural gene.
  • Codoning vehicle is a plasmid or phage DNA or other DNA sequence which is capable of carrying genetic information into a host cell.
  • a cloning vehicle is often characterized by one or more endonuclease recognition sites at which such DNA sequences may be cut in a determinable fashion without loss of an essential biological function of the vehicle.
  • a cloning vehicle is a DNA sequence into which a desired DNA may be spliced in order to bring about its cloning into the host cell.
  • Vector is a term also used to refer to a cloning vehicle.
  • “Plasmid” - is a cloning vehicle generally comprising a circular DNA molecule that is maintained and replicates autonomously in at least one host cell.
  • “Expression vehicle” - a vehicle or vector similar to a cloning vehicle but which supports expression of a gene that has been cloned into it, after transformation of a host. The cloned gene is usually placed under the control of (i.e. is operably linked to) certain control sequences such as promoter sequences.
  • “Host” - a cell that is utilized as the recipient and carrier of recombinant material. "Homologous” - refers to a nucleic acid molecule that originates from the same genus or species as the host.
  • Heterologous refers to a nucleic acid molecule that originates from a different genus or species than that of the host.
  • glycoprotein refers to a peptide molecule that has undergone glycosylation.
  • glycosylation refers to the addition of carbohydrate groups to a amino acid residues of a peptide molecule.
  • transgenic animals have been developed for many purposes (Pinkert et al. 1990) (Wall et al. 1997).
  • One premise, therefore, for the present invention is that by providing a transgenic animal capable of expressing phytase, the problems discussed above would be obviated.
  • the options for heterologous phytase expression in animals include (i) salivary gland secretion of a phytase, (ii) pancreatic secretion of the enzyme into the small intestine along with the digestive enzymes, or (iii) secretion from the intestinal epithelial cells much like that of indigenous alkaline phosphatase and glycosidases (Low, 1989).
  • coli phytase would appear to be best suited for hydrolytic activity in the monogastric stomach because the enzyme has a pH optimum in the range of 2.5 to 4.5 and it is resistant to pepsin, the predominant protease active in the stomach.
  • the phytase has a periplasmic location in E. coli and has an N-terminal signal peptide sequence (Golovan et al., 1999) that seemed optimally adapted for secretion from the parotid gland.
  • Phytase could be expressed in either the pancreas for secretion into the small intestine or it could be expressed in the intestinal epithelial tissue and secreted into the intestinal milieu.
  • the salivary gland system of the pig consists of three pairs of glands, the parotid gland, which secretes through a duct on each cheek, and mandibular and submaxillary glands that have joint ducts that secrete beneath the front on the tongue.
  • Saliva secreted in the pig via these ducts is discontinuous and is produced during consumption of solid foods, and can equal the weight of food consumed when water is limited during feed consumption (Corring, 1980; Arkhipovets, 1956).
  • the quantity of saliva produced by a 45 kg pig can vary from near zero when the pig receives a mainly liquid diet to 500 g when a dry diet is consumed without access to water.
  • the salivary glands of the pig secrete amylase (Rozhkov and Galimov, 1990) and a variety of other salivary proteins and mucopolysaccharides.
  • proline-rich proteins are produced when either mice or rats consume tannins or are injected with isoproterenol. It would be advantageous to develop an animal that is transformed to express phytase, preferably in the salivary gland. In such case, the phytate naturally occurring in the animal feed can be utilized by the animal without any additives being used. This will decrease the cost of animal production, and furthermore, will avoid polluting the environment with phosphorus. Therefore, the present invention aims to overcome the deficiencies of the prior art relating to increasing phytate utilization and, particularly, to provide transgenic animals which express phytase.
  • the present invention provides an animal capable of inducible or constitutive salivary expression of a heterologous protein.
  • the mouse was chosen as the animal model and the gene constructs used for transformation were created using the rat proline-rich protein (PRP) promoter/enhancer (inducible promoter) and the mouse parotid secretory protein (PSP) promoter/enhancer (constitutive promoter).
  • PRP proline-rich protein
  • PSP mouse parotid secretory protein
  • phytase was used for expression in saliva.
  • mice transgenic for the PSP construct were produced under contract at the University of Alabama. Following the testing of the mice described above, transgenic pigs were developed by introduction into the genome a phytase transgene consisting of a constitutive promoter driving the synthesis of a highly active phytase. The pigs so generated were found to excrete less phosphorus in their feces than non-transgenic pigs.
  • Saliva is a clear colorless fluid secreted by major salivary glands (parotid, submandibular, sublingual and minor salivary) that lubricates and cleans the oral structure, as well as initiates the process of digestion.
  • the parotid glands are two of six major glands associated with the production of saliva.
  • the parotid gland is composed mainly of two cell types: acinar and interglobular duct cells.
  • the acinar cells which represent 75 to 85°/o ' of the tissue, are the sites of secretory protein synthesis (Frandson and Spurgeon 1992).
  • AZA-1 2% of poly A RNA
  • PGP parotid secretory protein
  • salivary secretion in pigs has not received the attention given to that of mice and humans. It was suggested that salivary secretion is discontinuous (less secreted between periods of meal consumption). Up to 500 g of saliva may be secreted by a 45 kg pig upon consumption of 500 g of dry feed (Coning 1980). Wide variations were detected in both the flow rate and electrolytes in saliva between animals and even between samples taken from the same animal on separate days (Tryon and Bibby 1966). Very little is known about the composition of pig's saliva or salivary enzymes. Salivary amylase was detected, although the quantity was 250 000 times less than that of pancreatic amylase, and 100 times less than in human saliva (Low 1989). There are no constructs known which would allow salivary gland- specific expression of transgene in pigs.
  • a plasmid is constructed by linking a promoter/enhancer for a saliva protein with the APPA gene, which codes for the bifunctional phytase, acid phosphatase.
  • the APPA gene used in this construction was cloned from E. coli ATCC 33965 into pBR322. This is described above (Golovan et al., 2000). Proteins, unusually high in proline, the so-called proline-rich proteins (PRPs), comprise about 70% of the total proteins in human saliva (Bennick 1982). Unlike the constitutive expression of the PRPs in humans, the salivary glands of mice, rats and hamster normally either do not express PRPs or express them in low levels.
  • PRP gene expression can be dramatically induced by diets high in tannins or by injection with the ⁇ -agonist isoproterenol (Carlson 1993). After 6 to 10 days of daily isoproterenol injection the PRPs comprised about 70% of the total soluble protein in parotid gland extracts. PRP cDNA and PRP genes have been cloned and characterized from rats (Clements et al.
  • Transgenic mice were used to locate the cis-acting DNA elements that are essential for salivary-specific and inducible expression of the rat proline-rich protein gene, R15. It was found that a parotid control region (-6 to -1.7 kb) upstream of the R15 promoter is capable of directing parotid-specific and isoproterenol-inducible expression of a heterologous promoter construct (Tu et al. 1993). The distal -10 to -6 kb region was shown to function as an enhancer, which can increase levels of expression more than 30-fold.
  • the -6 to -1.7 kb region also seems to function as a locus control region (LCR), because it conferred copy number-dependent and chromosomal position-independent expression of a reporter gene in 15 out of 15 independent transgenic mice (Tu, Lazowski, Ehlenfeldt, Wu, Lin, Kousvelari, and Ann 1993).
  • LCR locus control region
  • the 4.3 kbp CAT PCR fragment had the initiation site of the CAT gene substituted with the optimal eukaryotic initiation sequence (Kozak 1987).
  • the fragment was purified by agarose gel electrophoresis, re-ligated to itself and used to transform E. coli ( Figure 1, step 2).
  • the CAT P C R plasmid was digested with Nco I and filled-in using T4 DNA polymerase to generate a blunt end.
  • the CAT PCR fragment was digested with Eco47III and purified by agarose gel electrophoresis ( Figure 1, step 3). Three rare codons in the APPA gene were modified during the sub-cloning steps leading to the construction of the transgene.
  • the Ala 3 coding sequence was changed from GCG to GCC
  • the Pro 2 g sequence was changed from CCG to CCC
  • the Ala 429 sequence was changed from GCG to GCT. This modification was made in order to increase the possibility of transcription of the gene in eukaryotic cells.
  • the APPA gene was amplified by PCR using the previously cloned APPA gene from the pBR322/ RR plasmid with the synthetic primers APPA-DRA and APPA-SMA.
  • the 1.3 kbp APPA PCR fragment generated by PCR was digested with Dra I and Sma I and gel-purified ( Figure 1, step 4).
  • APPApc R nd CAT PCR fragments were blunt end ligated to produce CAT/APPA+intron vector (Figure 1, step 5), which was introduced into a DH5 ⁇ strain of E. coli.
  • the insert orientation was checked by restriction digest with Sal I and ⁇ coR I.
  • the transgene in CAT/APPA+intron was checked by sequencing both strands.
  • To remove the SV40 small t intron the 2.3 kbp RR /intron/polyA fragment was excised from a plasmid by Xho I and ⁇ coR I digestion ( Figure 1, step 6a), gel purified and digested by Dra I ( Figure 1, step 6b).
  • the 1.5 kbp (APPA) and 0.2 kbp (polyA) fragments were gel-purified and linked together in three way ligation with CAT PCR digested with Xho I and ⁇ coR I ( Figure 1, step 6c).
  • the resulting plasmids CAT/ APPA and CAT/APPA+intron were digested with Xho I, gel- purified and re-ligated with R15-PRP promoter digested with Xho I ( Figure 1, step 7). Because of the low efficiency of ligation the whole ligation mixture was used to transform E.coli, total plasmid DNA was prepared and run on the agarose gel.
  • Plasmids which were larger than the original CAT/ APPA (5.6 kbp) were eluted and re-transformed in E.coli. Plasmids with the R15-PRP insert (15 kbp) were identified by electrophoresing DNA from a single colony on an agarose gel. The correct orientation was identified by PCR with R15- UP 1 and APPA-DOWN2 synthetic primers.
  • the plasmids Rl 5/APP A and Rl 5/APPA+intron were both digested with Hind III and Kpn I; transgenes were gel-purified and further purified using a Qiagen column ( Figure 1, step 8).
  • Figure 18 illustrates the nucleic acid sequence for the plasmid containing the known segment of the Rl 5/APP A + intron sequence including the vector sequences of pBLCAT3.
  • the sequence of this plasmid is designated as S ⁇ Q ID NO:2.
  • Figure 19 illustrates the nucleic acid sequence for the transgene construct containing the known segment of the Rl 5/APP A + intron sequence used for the generation of transgenic mice.
  • the sequence of this transgene is designated as S ⁇ Q ID NO:3.
  • Figure 20 illustrates the nucleic acid sequence for the plasmid containing the known segment of the Rl 5/APP A sequence including the vector sequences of pBLCAT3.
  • the sequence for this plasmid is designated as S ⁇ Q ID NO:4.
  • FIG. 21 illustrates the nucleic acid sequence for the transgene construct containing the known segment of the R15/APPA sequence used for the generation of transgenic mice.
  • the sequence of this transgene is designated as S ⁇ Q ID NO:5.
  • the SV40 promoter/enhancer was amplified by PCR from the pSV- ⁇ -galactosidase plasmid (Promega) using the synthetic primers SV-HIND and SV-XHO.
  • the SV40 promoter/enhancer fragment was digested with Xho I and Hind III, gel purified, and ligated into CAT/ APPA digested with Xho I and Hind III ( Figure 2).
  • Figure 22 illustrates nucleic acid sequence for the SV40/APPA + intron.
  • the sequence for this plasmid is designated as SEQ LD NO:6.
  • CHO, COS 7 and HELA cell lines were screened for transient expression of the APPA phytase using the SV40 promoter/enhancer. All cell lines were maintained on DMEM/F12 (Sigma) cell medium with 10 % (wt/vol) heat-inactivated fetal bovine serum at 37°C in 5% CO 2 and 95% air. Cells were grown to 70 % confluence before transfection. Two hours before transfection the medium was exchanged with fresh medium.
  • Cells were transformed with 5 ⁇ g of DNA per 60 mm culture plate (1:1 SV 40/ APPA and SV40/ ⁇ -galactosidase) using the DNA-Calcium-Phosphate method of transfection (Gorman et al 1983). After 6 hours of incubation the medium was removed and cells were subjected to glycerol shock for 3 min (Ausbel et al. 1992). Cells were washed with phosphate -buffered saline (PBS) and incubated in fresh medium under standard growth conditions.
  • PBS phosphate -buffered saline
  • Phytase activity was detected in all transfected cell lines, with COS7 cells expressing a total of 0.35 U of phytase in cell-free culture fluid (4 ml) and 0.0034 U in the cell fraction (1.1 ml) obtained from the same plate.
  • the phytase activity produced by COS7 cells was 7 times higher than that of CHO and 35 times more than the HELA cell line. More than 99% of activity was located in cell-free culture fluid, which suggests that the expressed enzyme was exported out of the cell using the bacterial signal sequence.
  • mice Rl 5/APP A+intron by Dr. CA. Pinkert at the NICHD Transgenic Mouse Development Facility (NTMDF), University of Alabama at Birmingham, Alabama.
  • NTMDF NICHD Transgenic Mouse Development Facility
  • the procedures followed in generating the mice have been standardized by the NTMDF and further information concerning this can be obtained at: http://transgenics.bhs.uab.edu/pagel.htm. the content of which is incorporated herein by reference.
  • This procedure involved the microinjection technique for transfecting mice with the desired nucleic acid sequence. To summarize, the sequences are microinjected into mouse zygotes and the surviving eggs are implanted into pseudopregnant recipient mice. The recipient mice then give birth to the resulting founder transgenic mice. It will be appreciated that various other methods of generating transgenic mice may be used in the present invention.
  • the Rl 5/APP A transgene in mice was detected by PCR using the primers CAT-UP 1 and APPA-DOWN2 that gives rise to a 700 bp fragment using the standard PCR conditions, except that the hybridization step was set at 51°C for 40 seconds and the polymerization step was at 72°C for one minute.
  • the hybridization step was set at 51°C for 40 seconds and the polymerization step was at 72°C for one minute.
  • Rl 5/APP A+intron 5 PCR positive founder mice were obtained, 3 were males and 2 were females, and one of them was found to be mosaic.
  • mice were lightly anesthetized with a ketamine/xylazine mixture (ip injection of 50 mg ketamine and 5 mg xylazine per kg body weight diluted in water) and saliva flow was induced by injection with pilocarpine/isoproterenol (ip injection of 0.5 mg pilocarpine and 2 mg isoproterenol per kg body weight dissolved in saline) (Hu et al. 1992). Between 100-250 ⁇ l of saliva was collected from each mouse over a 30 min period beginning 5 min after the pilocarpine/isoproterenol injection.
  • the saliva was collected from each mouse by holding it in one hand and withdrawing saliva from the corner of the mouth with a 20 ⁇ l pipetter. Collected saliva was transferred to a cold Eppendorf microcentrifuge tube containing 2 ⁇ l of 0.5 M EDTA (pH 8.0) and 4 ⁇ l of 10 mg ml protease inhibitor Pefabloc (Boehringer Mannheim) dissolved in water. The tubes with saliva were kept on ice until assays were conducted. Phytase activity in the saliva was assayed as described for the S V40/APPA expressed in cell culture.
  • the SV40 intron in the Rl 5/APP A+intron construct seems to cause a lower level of expression, and in three lines (Alf, A20f and BOm) the level of phytase was barely detectable.
  • the level of phytase expression in A2m line (Rl 5/APP A+intron) was 6.2 times lower than that of the BOm-intron line (Rl 5/APP A).
  • Preliminary experiments showed that when the enzyme was analyzed by PAGE its size was increased from 42 kDa to 60 kDa. It is likely modified by glycosylation, but stable and active.
  • the murine parotid secretory protein is the most abundantly expressed protein in the parotid gland of mice (Madsen and Hjorth 1985). After an hour of pulse labeling, the mouse parotid gland incorporates 65 to 85% of 14 C-leucine into this single protein (Owerbach and Hjorth 1980). It was estimated that PSP mRNA accumulates up to 50,000 molecules per cell and that from 3 to 5 molecules of PSP are produced for every molecule of amylase (Madsen and Hjorth 1985). Despite the predominance of the PSP in saliva its function is not well characterized.
  • the single-copy gene coding for PSP has been cloned and characterized. It has two alleles PSP a (Shaw and Schibler 1986) and PSP b (Owerbach and Hjorth 1980).
  • ThePSP b allele is also expressed in the sublingual gland, but at 1/10 of the level found in the parotid gland. It was shown that 4.6 kbp of 5' flanking sequence of PSP b is sufficient for salivary gland specific expression.
  • the level of sublingual expression approached 100% of the PSP mRNA level, whereas the parotid expression did not exceed 1% (Mikkelsen et al. 1992), which demonstrates that regulatory sequences for sublingual and parotid expression are not identical.
  • the level of expression was also dependent on the site of integration.
  • the same construct was used for expression of the C-terminal chain of the human blood coagulation factor Vlll, FVlll.
  • a high level of FVlll mRNA was detected in the sublingual gland and a low level in the parotid gland.
  • the transgenic lines also secreted the FVlll light chain into saliva at the level of about 10 units per salivation (about 0. 05 ml of saliva) (Mikkelsen et al.,1992).
  • Lama 2 is a portion of the PSP gene and comprises an 18 kbp construct that is expressed in transgenic mice at up to 56% of the endogenous PSP gene.
  • Lama 2 Because a large part of Lama 2 had not been sequenced, the construct was first disassembled and subcloned into pBluescript KS(+) and after incorporation of the APPA gene, the Lama 2 was reassembled back (Figure 3).
  • the APPA bacterial signal sequence was recognized as an efficient leader peptide and the cleavage site was correctly predicted.
  • PSORT also predicted that there is a high probability that phytase would be exported correctly outside of the cell.
  • Some bacterial signal sequences might function efficiently in mammalian cells (Williamson et al. 1994) (Hall et al. 1990).
  • Our experiments using cell culture demonstrated that the APPA signal was correctly processed with export of phytase outside of the cell.
  • This leader peptide was also efficiently recognized by PSORT with the correct cleavage site (Nakai and Kanehisa 1992).
  • PSORT with the correct cleavage site
  • the APPA gene for both constructs was amplified by PCR using as the template our previous transgenic construct Rl 5/APP A that possessed the optimal Kozak sequence and the modified codons for residues Ala3, Pro428 and Ala429 as described earlier.
  • two synthetic primers were used which introduced a Clal site near the ATG codon (APPA-CLA) and a Kpnl site near the TAA stop codon (APPA-KPN).
  • the APPA PCR 1 product was digested with Clal and Kpnl.
  • the Clal site was also introduced into Lama 2 using pKS/Lama 2 as template for PCR.
  • LAMA-UP primer was located upstream of Apal site and the LAMA-CLA primer introduced the Clal site near ATG codon ( Figure 3, step 3a).
  • Lama P c R l product was digested with Clal and Apal ( Figure 3, step 4a).
  • pKS/Lama (Apal-Kpnl), Lama P R l (Apal- Clal) and APPA PCR I (Clal-Kpnl) were combined together in a three-way ligation reaction (Figure 3, step 5a).
  • the recovered pKS/Lama/APPA plasmid was digested with RsrII, Smal and inserted back into Lama2 ( Figure 3, step 6a).
  • the synthetic APPA -KPN primer was used with the synthetic APPA -MATURE primer, which produced phytase without a signal sequence.
  • the APP A CR 2 product was blunt-ended using T4 DNA polymerase and digested with Kpnl .
  • the PSP signal sequence was produced using the LAMA-UP and LAMA -SIGNAL primer ( Figure 3, step 3b).
  • the Lama P c R 2 was blunt-ended using T4 DNA polymerase and digested with Apal ( Figure 3, step 4b).
  • Lama2/APPAsignal/APPA for the generation of transgenic mice, because we have results from our previous transgenic constructs R15/APPA and Rl 5/APP A+intron which demonstrated that phytase with optimized Kozak sequence and the APPA signal peptide was synthesized at a high level in salivary glands after induction and was efficiently exported into the salivary duct.
  • the Lama2/APP A vector was digested with Xhol and Notl, and the transgene was gel-purified and further purified using a Qiagen column ( Figure 3, step 7a).
  • Lama2-APPA plasmid A large segment of the Lama2 construct (Laursen and Hjorth 1997) used for construction of the Lama2-APPA transgene had not been reported in GenBank prior to our research. To ensure that we could more clearly describe the transgene construct, and furthermore to avoid the introduction of deleterious DNA sequences from the mouse into the pig in the process of generating transgenic pigs, we sequenced the Lama2-APPA plasmid on both strands.
  • Figure 4 illustrates schematically the structure of the Lama2-APPA plasmid.
  • Figure 5 illustrates the nucleic acid sequence (SEQ ID NO: 1) of such plasmid.
  • the full transgene sequence was reconstructed from overlapping DNA sequences using the Contig Assembly Program (CAP) (http://hercules.tigem.it/ASSEMBLY/assemble.html) developed by Huang ( 1 96; 1999) and then inspected manually for sequencing errors.
  • the transgene sequence was checked for the presence of interspersed repetitive elements using the computer program RepeatMasker (Smith and Green, RepeatMasker at http://ftp.genome.washington.edu/cgi-bin RepeatMasker). It was found that 26 % of the transgene sequence was composed of repetitive elements (Table 2). However, such repetitive elements are widely present in all mammalian genomes. For example, up to 50% of the human genome is derived from repetitive elements (Smit 1996; Kazazian 1998).
  • Figure 23 illustrates the nucleic acid sequence (SEQ ID NO:7) of the Lama2/APPA transgene construct.
  • the Lama2 high level expression cassette (Laursen and Hjorth 1997) contains the enhancer region and the promoter of the Psp gene in the parotid gland. High expression was shown to be dependent on regulatory elements between -11.5 kb and -6.5 kb and/or between +8.3 kb and +10.9 kb.
  • Svendsen et al. ( 1998a) showed that a 1.5 kb sequence between -3.1 kb and -4.6 kb had properties of a parotid and sublingual specific enhancer and was designated as the PSP proximal enhancer.
  • transgenes containing the PSP promoter and 5' flanking region located between -3.6 kb and —4.3 kb contained sequence information necessary to direct salivary gland specific expression.
  • mice a pair of founder mice, incorporating the phytase gene and a constitutive promoter, were prepared under contract by the University of Alabama. As will be discussed, these founders were used to produce offspring, which were then analyzed for the presence of the phytase gene by PCR and animals containing the gene were then tested constitutive salivary phytase production.
  • Two transgenic founder mice (a black male and a white female, 3-1) containing the phytase transgene were received from the NICHD Transgenic Mouse Development Facility at the University of Alabama. The black male was negative for salivary phytase, but the female, 3-1, exhibited a salivary phytase activity of 30 U/ml.
  • Progeny produced by crossing the black male with 4 CD-I females produced 9 out of 25 females and 13 out of 26 males that were PCR positive. All progeny were negative for salivary phytase.
  • the female founder, 3- 1 was out-crossed with a CD-I male to produce 3 litters for a total of 35 offspring. Of the progeny from these matings one phytase positive Gl male was obtained.
  • the Gl male was outcrossed with 6 CD-I females of the 6 litters 20/34 males were PCR positive and salivary phytase positive and 21/28 females were PCR positive and salivary phytase positive (Table 3).
  • the salivary phytase activity of different offspring from the same first generation (Gl) male ranged from 1.3 to 71.2 U/ml. There was no significant difference in the phytase activities between male or female mice.
  • PCR assays for identification of the transgenic mice were carried out with an initial heating step at 95°C for 3 min, 40 cycles using 95°C for 30 sec, 54°C for 30 sec and 72°C for 1 min) using the following primers: APPA-UP2 and APPA-KPN ( Figure 6).
  • the phytase assays were conducted as described above for the R15-PRP/APPA phytase expressed in cell culture.
  • Transgenic pigs were produced using Buffalo and Buffalo/Landrace cross gilts as the embryo donors and Yorkshire sows as the recipients.
  • the experimental procedure used was similar to that described by Wall et al. ( 1985). The detailed procedure is described below.
  • the Lama2/APPA construct with the APPA signal peptide was used as the transgene for microinjection.
  • Selected Yorkshire or Buffalo/Landrace cross gilts between 70 to 80 kg were superovulated by intramuscular injecti ⁇ rr of 2000 IU of pregnant mare's serum gonadotropin (PMSG, Ayerst Veterinary Laboratories), followed by 700 IU human chorionic gonadotropin (HCG, Ayerst Veterinary Laboratories) 60 to 72 hours later, administered in the same manner.
  • the gilts were artificially inseminated three times with a 16 hour interval between inseminations using semen from a high breeding index Yorkshire boar. Twenty-four hours after the last insemination, the gilts were slaughtered and the reproductive tract recovered.
  • Estrus was synchronized in experienced recipient sows as described for donor sows. Since synchronization and not superovulation was the goal, hormone levels were reduced to 500 IU for PSMG and 500 IU for HCG. PMSG was given the day the sow's litter was weaned, followed in 72 hours by HCG and surgery for embryo transfer was performed 54 hours thereafter.
  • Embryo collection Reproductive tracts were collected at the abattoir, inserted into bags, sealed and the bags immersed in water at 39°C for transport to the laboratory. Recovery of the embryos and microinjection with the transgene was conducted in a laboratory maintained at 32 to 33°C.
  • the oviducts were dissected from the tracts and flushed, using a syringe and a feeding tube, with 15 ml of pre-warmed HBECM-3 medium (Dobrinsky et al. 1996).
  • the media was collected in a 100 mm Petri dish and placed in an incubator at 38.5°C with an atmosphere of 5% (vol/vol) of CO 2 , 5% (vol/vol) O 2 and the balance N 2 .
  • embryos were individually collected from the flushed media using a polished transfer pipette. Embryos were rinsed twice in 3 ml volumes of pre-incubated BECM-3 and placed in 100 ⁇ l of pre-incubated BECM-3 under 3 ml of filter sterilized mineral oil until injected.
  • Embryos from one gilt were collected and placed in one ml of pre-warmed HBECM-3 in a 1.5 ml centrifuge tube and centrifuged for 6 min at 14,000 x g (Wall et al. 1985). The embryos were then collected and placed in an injection dish with 40 ⁇ l of pre-warmed HBECM-3 covered with 2.5 ml of filter sterilized mineral oil. The pronucleus in each embryo was injected (Gordon et al. 1980) with three picolitres of Lama2/APPA DNA in solution at a concentration of 5 ng of DNA per ⁇ l in 10 mM Tris, pH 7.5, 0.1 mM EDTA.
  • the embryos were placedin dishes containing 100 ⁇ l of pre-incubated BECM-3 under 3 ml of filter sterilized mineral oil. After all embryos were injected, which took no more than 4 hours since collection of reproductive tracts, the embryos were transferred to 1.8 ml cryotube (Nunc) containing 1 ml of pre-warmed HBECM-3 and transported in an incubator at 38.5°C to the swine surgery.
  • Recipient sows were anesthetized by intravenous injection of 500 mg Brietol and anesthesia maintained by inhalation of 3% halothane with 4 litres per min of nitrous oxide and 4 litres per min oxygen.
  • the oviducts were exposed through a laparotomy, just off the dorsal midline, and a catheter, containing 20 to 35 injected embryos and 3 to 6 untreated embryos, was passed into the infundibulum and down the oviduct to the isthmus and emptied. The oviduct was returned to the abdominal cavity and the incision closed.
  • the diets were provided as pelleted formulations during the weanling, grower and finishing phases are shown in Tables 4 and 5.
  • the vitamin and mineral mixes included in the diets are shown in Tables 6 and 7.
  • Tail segments from newborn piglets were collected and slices of each placed in 600 ⁇ l of 50 mM NaOH and heating at for 95°C for 15 minutes. The suspension was neutralized with 50 ⁇ l of 1 M Tris (pH 8.0) and insoluble materials removed by centrifugation for 5 min in a microcentrifuge. A 2 ⁇ l sample of each was used for PCR with primers APPA-UP2 and APPA-KPN.
  • the primers produce a 750 bp fragment if the transgene is present.
  • PIG-BGF and PIG-BGR primers were used to detect the porcine ⁇ -globin gene from the same DNA preparation (Heneine and Switzer 1996).
  • the PCR reaction was performed using the same conditions as described for detection of the phytase transgene.
  • As a negative control genomic DNA from a non-transgenic pig was used in the PCR reaction, for a positive control this DNA was spiked with a known amount of transgene (1 gene copy/per genome).
  • the method for extraction of DNA from blood was based on a method described by Higuchi ( 1989) with some modifications.
  • a 100 ⁇ l volume of whole blood was mixed with 200 ⁇ l of lysis buffer (10 mM Tris-HCl, 0.32 M sucrose, 5 mM MgCl 2 , 1% (vol/vol) Triton X-100, pH 7.5.), mixed briefly and incubated on ice for 5 min.
  • the sample was then centrifuged at 14,000 x G for 3 min, and the supernate discarded.
  • the sediment was suspended in lysis buffer, mixed, incubated and centrifuged. This procedure was repeated 2 more times, or until no hemoglobin remained.
  • the sediment was dissociated in 100 ⁇ l of 50 mM NaOH, mixed and heated at 100°C for 10 min. The contents were cooled, 10 ⁇ l of 1 M Tris-HCl (pH 8.5) added and mixed briefly. The sample was then centrifuged at 14,000 x g for 2 min and 2 ⁇ l of the supemate used for analysis by PCR.
  • the PCR reaction mixture with a total volume of 40 ⁇ l consisted of; 23.8 ⁇ l of distilled water, 4 ⁇ l of 10 X Gibco BRL PCR buffer, 1.2 ⁇ l of 50 mM MgCl 2 , 0.8 ⁇ l of 10 mM dNTPs, 40 pmol of each of the forward and reverse primers in 8 ⁇ l, 2 ⁇ l of template DNA and 0.2 ⁇ l of Taq DNA polymerase (Gibco BRL, 5 U/ ⁇ l).
  • the amplification procedure was performed with an initial heating step at 95°C for 3 min followed by 40 cycles of 95°C for 30 sec, 54°C for 30 sec and 72°C for 60 sec.
  • the transgenic pigs were detected with primers for the APPA gene (APPA-KPN with APPA-UP2), and as a control PIG-BGF with PIG-BGR primers were used for detection of the porcine ⁇ -globin gene.
  • Weanling pigs were sampled for salivary phytase by wiping under the tongue with a cotton tipped applicator, breaking the stick offand centrifuging the applicator tip in a 0.4 ml microcentrifuge tube, with a hole in the bottom, contained within a 1.5 ml microcentrifuge tube.
  • Grower and finishing pigs were sampled using 1.5 inch long #2 dental cotton absorbent rolls (Ash Temple Sundries Ltd, Don Mills, ON) attached to dental floss. These were centrifuged in 1.5 ml microcentrifuge tubes with holes in the bottom while contained in larger tubes. The saliva was collected from the larger tube and stored at -20°C until analyzed. Saliva was collected and pigs were weighed at weekly intervals.
  • Saliva samples were either assayed directly or after dilution in 0.1 M acetate buffer pH 4.5.
  • Phytase was assayed in 200 ⁇ l of 0.1 M sodium acetate buffer (pH 4.5) using sodium phytate (4 mM) as a substrate at 37°C. After 10 min of incubation the reaction was stopped by addition of 133 ⁇ l ammonium molybdate/ammonium vanadate/nitric acid mixture and the concentration of liberated inorganic phosphate determined at 405 nm (Engelen, van der Heeft, Randsdorp, and Smit 1994). This and all other assays were performed in triplicate.
  • One unit (U) of enzyme activity was the amount of the enzyme releasing 1 ⁇ mol of inorganic phosphate per minute.
  • Fresh feces were collected from each pig during the grower and finisher phases. Samples were placed in aluminum trays closed with a wax paper top and immediately frozen, and kept frozen until they were lyophilized for analysis. After lyophilization the samples were transferred to room conditions overnight to reach equilibrium in moisture content. The samples were separately ground with a mortar and pestle until homogenous and sealed in plastic containers until analyzed further. Dry matter content of samples was analyzed according to AOAC (Association of Official Analytical Chemists (AOAC) 1984) by heating 1 gram samples at 110°C for 4 hours and cooling in a desiccator prior to weighing.
  • AOAC Association of Official Analytical Chemists
  • the APPA phytase was over expressed in E. coli strain BL21(D ⁇ 3) and the EDTA lysozyme extract fraction purified on DEAE-Sepharose and Sephadex-G75 as described by Jia et al. ( 1998).
  • the pig phytase was purified by chromatography on DEAE-Sepharose and the band of enzyme eluted with a sodium chloride gradient was further purified by Chromatofocusing using a pH gradient from pH 4.0 to 7.0.
  • Monoclonal antibodies specific to the E. coli APPA encoded phytase were prepared according to the procedures of Galfre and Milstein (1981). Briefly, two female Balb/c mice were immunized 7 times over a period of 59 days with a purified APPA enzyme preparation. Mouse spleens were harvested, and the cells therein fused with an NS-1 myeloma cell line (Kohler and Milstein, 1976). Fused cells were selected for their ability to grow in media containing hypoxanthine, aminopterin, and thymidine (HAT).
  • hypoxanthine, aminopterin, and thymidine HAT
  • Antibodies were prepared in two New Zealand White Rabbits by two intramuscular injections at different sites in the thigh of 50 ⁇ g of purified Escherichia coli derived APPA phytase in 0.5 ml of a 1:1 mixture of phosphate-buffered saline (PBS) and Freund's Complete Adjuvant. This was followed by repeat injections of 20 ⁇ g each of phytase in a 1 :1 mixture of PBS and Freund's Incomplete Adjuvant on days 4, 19, 25, and 39. Blood was collected via heart puncture on day 42. The serum was separated from the cell fraction and used as the source of antibodies. The basic procedures for antibody production are described in Harlow and Lane (1988).
  • Table 9 lists the transgenic pigs that were produced, their birth dates, sex and salivary phytase levels. There were 31 pigs transgenic for the phytase gene out of 203 live piglets born from embryos microinjected. These were detected by the presence of the gene in blood samples using the standard primer set, APPA-UP2 and APPA -KPN, but only 14 were detected by analysis of tail DNA preparations using the standard primer set. When the negative samples were reanalyzed using the primer set LAMA-UP 1 and APPA-down4 ( Figure 8) a further 8 tail DNA samples were found to be positive. Purification of the tail biopsy DNA probably would have led to all being PCR positive for the phytase transgene. Characteristics of the phytase transgene in transgenic pig 167-02
  • PCR to detection of transgenic pigs is exemplified by analysis of litter 167 in which one of 7 piglets tested, including one that was stillborn and one that was crushed by the sow after birth, one live piglet designated 167-02 was identified as positive for the APPA gene by generation of a PCR product (Lane 2) of approximately 750 bps from the tail chromosomal DNA (Figure 7). No rearrangements of the APPA gene were detected as documented by the positive PCR results using primers directed to the 3' region (lane 2) the whole gene (lane 3) and the 5' region (lane 4) of the APPA gene ( Figure 8).
  • Salivary phytase and weight gain during growth of transgenic and non-transgenic penmates Data on salivary phytase activity and weight gain are shown for five transgenic pigs and for weight gains of their non-transgenic penmates in Figures 9, 10, 11, 12 and 13.
  • the phytase activity in the saliva varied substantially from one sampling time to the next. This variability was attributed to a combination of environmental factors including whether the animal had just consumed food or water, and regulation of parotid and saliva secretion in relation to food and water consumption.
  • the weight gains during growth of the five transgenic pigs was within the range of the weight gains of the normal non-transgenic pigs.
  • the growth rate of the transgenic pigs was similar to that of the non-transgenic litter mates.
  • the phosphorus content of fresh fecal samples from three of the transgenic founder pigs, 167-02, 282-02, 282-04, 405-02 and 421-06 receiving weaning, grower or finisher ration is shown in Table 9.
  • the phosphorus content of the feces of the transgenic pigs ranged from 1.59 to 2.26% while that of the non-transgenic penmates ranged from 1.61 to 2.76 %.
  • the reduction in fecal phosphorus ranged from a maximum of 26% to a minimum of 8%. In most cases the differences were at the 99% level of significance.
  • the ages of the pigs at the time of fecal sampling and the corresponding phytase activities are shown in Figures 9, 10, 11, 12 & 13.
  • the rations fed contained a supplement of readily available phosphorus suitable for maximizing growth of non-transgenic pigs. Since the reduction in fecal phosphorus is measured in transgenic pigs receiving a diet high in mineral phosphorus it is very likely that the fecal phosphorus would be substantially lower if the diet lacked mineral phosphorus. Under these conditions the phosphorus released from phytate would provide a substantial proportion of the dietary phosphorus and little would reach the large intestine and be excreted in the feces.
  • the phytase enzyme was purified to homogeneity from E. coli and from saliva collected from transgenic pig 167-02. Silver stains of the purified enzymes after SDS-PAG ⁇ are shown in Figure. 15.
  • the E. coli derived enzyme has a molecular mass of approximately 45 kDa while that produced by the pig was about 55 kDa.
  • the enzymes were also electrophoresed as before, transferred to nitrocellulose and stained for glycoproteins.
  • the second part of Figure 15 shows that the pig APPA protein is glycosylated.
  • Figure 15B shows that treatment of the pig phytase with deglycosylation enzymes changes the size of the phytase from 60 kDa to 45 kDa, an observation that confirms the glycosylated nature of the recombinant phytase produced in the saliva of the pig.
  • the purified pig phytase had K m and V ma ⁇ values of 0.33 mM and 624 units per mg of protein, respectively.
  • Golovan et al. (2000) previously reported the K m and V max for the E. coli enzyme to be 0.63 mM and 2325 units per mg of protein.
  • the salivary phytase exhibits approximately 25% of the activity of the E. coli enzyme.
  • This reduction in activity may be due to glycosylation that either modifies the catalytic site of the enzyme or otherwise leads to the formation of an enzyme with lower catalytic activity.
  • the latter finding of the production of a glycosylated protein suggests a method ' of producing such proteins using transgenic animals.
  • the mature peptide lacks the glycosylation normally associated with proteins produced by higher life forms. Insulin is an example of such protein.
  • the findings of this study suggest that one means of producing the desired glycoproteins would be to generate transgenic animals such as the pig, that have been transformed, by known methods or the method described above, with a gene encoding the desired protein. When expressed by such animal, the subject protein would be produced and would undergo post-translational processing in the cell including the step of glycosylation.
  • the invention contemplates a general method of producing such glycosylated proteins.
  • the invention contemplates a method of producing glycosylated proteins through the expression in and isolation from the saliva of an animal that has been transformed with a gene encoding such protein, and wherein such gene is operably linked to a saliva protein promoter or enhancer.
  • a gene encoding such protein and wherein such gene is operably linked to a saliva protein promoter or enhancer.
  • Various methods are known in the art for the collection of glycoproteins from the parotid gland of the pig for various applications. For example, surgical techniques have been published by Denny et al. (1972) for the collection of secretions from the parotid gland and submandibular salivary ducts.
  • the DNA sequence encoding phytase may be obtained from a variety of sources such as microbial, plant or animal sources.
  • the DNA sequence is obtained from a microbial source such as bacteria.
  • Most preferred DNA sequences are obtained from Escherichia coli.
  • the cloning of a gene or a cDNA encoding a phytase protein may be achieved using various methods.
  • One method is by purification of the phytase protein, subsequent determination of the N- terminal and several internal amino acid sequences and screening of a. genomic or cDNA library of the organism producing the phytase using oligonucleotide probes based on the amino acid sequences. If at least a partial sequence of the gene is known, this information may be used to clone the corresponding cDNA using, for instance, the polymerase chain reaction (PCR) (PCR Technology: Principles and Applications for DNA Amplification, (1989) H. A.
  • PCR polymerase chain reaction
  • the desired protein may also be produced as a fusion protein containing another protein.
  • the desired recombinant protein of this invention may be produced as part of a larger recombinant protein in order to stabilize the desired protein.
  • Useful modifications within this context include, but are not limited to, those that alter post- translational modifications, size or active site, or that fuse the protein or portions thereof to another protein. Such modifications can be introduced into the protein by techniques well known in this art, such as by synthesizing modified genes by ligation of overlapping oligonucleotides or introducing mutations into the cloned genes by, for example, oligonucleotide-mediated mutagenesis.
  • the cloned phytase gene may be used as starting materials for the construction of improved phytases.
  • Improved phytases are phytases, altered by mutagenesis techniques (e.g. site-directed mutagenesis, or directed evolution), which have properties that differ from those of wild-type phytases (Kuchner and Arnold 1997). For example, the temperature or pH optimum, specific activity, temperature or protease resistance may be altered so as to be better suited for a particular application.
  • a choice of expression in cellular compartments can be used in the present invention, depending on the biophysical and biochemical properties of the phytase. Such properties include, but are not limited to pH sensitivity, sensitivity to proteases, and sensitivity to the ionic strength of the preferred compartment.
  • the DNA sequence encoding the enzyme of interest should be modified in such a way that the enzyme can exert its action at the desired location in the cell.
  • the expression construct of the present invention utilizes a bacterial signal sequence. Although signal sequences that are homologous (native) to the animal host species are preferred, heterologous signal sequences, i.e.
  • cis- acting regulatory regions useful in the invention include the promoter that drives expression of the phytase gene. Highly preferred are promoters that are specifically active in salivary gland cells.
  • mouse parotid secretory protein (PSP) promoter highly preferred are mouse parotid secretory protein (PSP) promoter, rat proline-rich protein (PRP) promoter, human salivary amylase promoter, mouse mammary tumor virus promoter (Samuelson 1996).
  • PSP mouse parotid secretory protein
  • PRP rat proline-rich protein
  • human salivary amylase promoter mouse mammary tumor virus promoter
  • the expression system or construct of this invention also includes a 3' untranslated region downstream of the DNA sequence encoding the desired recombinant protein, or the salivary protein gene used for regulation.
  • This region apparently stabilizes the RNA transcript of the expression system and thus increases the yield of the desired protein.
  • 3' untranslated regions useful in this regard are sequences that provide a polyA signal. Such sequences may be derived, e.g., from the SV 40 small t antigen late polyadenylation si nal, synthetic polyadenylation signal or other 3' untranslated sequences well known in this art (Carswell and Alwine 1989;Levitt et al. 1989).
  • the 3' untranslated region is derived from a salivary-specific protein.
  • the stabilizing effect of this region's polyA transcript is important in stabilizing the mRNA of the expression sequence.
  • LCRs locus control regions
  • MAR matrix attachment regions
  • SARs scaffold attachment regions
  • phytase Also important in increasing the efficiency of expression of phytase is a strong translation initiation site (Kozak 1987). Likewise, sequences that regulate the post- translational modification of phytase may be useful in the invention.
  • the term "animal” as used herein denotes all animals except humans. It also includes an individual animal in all stages of development, including embryonic and fetal stages.
  • a “transgenic” animal is any animal containing cells that bear genetic information received, directly or indirectly, by deliberate genetic manipulation at the subcellular level, such as by microinjection or infection with a recombinant virus.
  • Transgenic in the present context does not encompass classical crossbreeding or in vitro fertilization, but rather denotes animals in which one or more cells receive a recombinant DNA molecule. Although it is highly preferred that this molecule be integrated within the animal's chromosomes, the invention also encompasses the use of extrachromosomally replicating DNA sequences, such as might be engineered into yeast artificial chromosomes.
  • the information to be introduced into the animal may be foreign to the species of the animal to which the recipient belongs (i.e., "heterologous"), or the information may be foreign only to the particular individual recipient, or genetic information already possessed by the recipient.
  • the introduced gene may be expressed in a manner different than the native gene.
  • transgenic animals of this invention are other than human. Farm animals (pigs, goats, sheep, cows, horses, rabbits and the like), rodents (such as mice and rats), domestic pets (eg. cats and dogs), fish and poultry (eg. chickens) are included in the scope of this invention. It is highly preferred that a transgenic animal of the present invention be produced by introducing into single cell embryos appropriate polynucleotides that encode phytase, or fragments or modified products thereof, in a manner such that these polynucleotides are stably integrated into the DNA of germ line cells of the mature animal, and are inherited in normal m ⁇ ndelian fashion.
  • totipotent or pluripotent stem cells can be transformed by microinjection, calcium phosphate mediated precipitation, liposome fusion, retroviral infection or other means, the transformed cells are then introduced into the embryo, and the embryo then develops into a transgenic animal.
  • developing embryos are infected with a retrovirus containing the desired DNA, and transgenic animals produced from the infected embryo.
  • the appropriate DNAs are co-injected into the pronucleus or cytoplasm of embryos, preferably at the single cell stage, and the embryos allowed to develop into mature transgenic animals.
  • the present invention provides for other proteins to be expressed in the salivary gland of the pig.
  • proteins may be secreted into saliva to improve digestion and decrease pollution potential (for example, endoglucanases), or specifically targeted for secretion into blood and have effects on the growth and health of the animal (such as growth hormone).
  • Phytase activity may be measured via a number of assays, the choice of which is not critical to the present invention.
  • the phytase enzyme activity of the transgenic animal tissue may be tested with an ELISA-assay, Western blotting or direct enzyme assays using calorimetric techniques or gel assay system.
  • mice were between 21 and 30 days of age.
  • RECTIFIED SHEET (RULE 9V Table 4. Composition and nutrient levels of Phase II starter diet and low phytate starter diets fed to weanling pigs between 5-10 kg.
  • Vitamin A 10 million IU Vitamin D 1 million IU Vitamin E 40 thousand IU Menadione 2.5 g Panto thenic acid 15 g Riboflavin 5 g Folic acid 2 g Niacin 25 g Thiamin 1.5 g Pyridoxine 1.5 g Vitamin Bj 2 25 mg Biotin 200 mg Choline 500 g
  • Dicalcium phosphate contained 18.5% calcium and 20.5% of phosphate and normally is added at a level of 1.2% to the pig grower diet, 1.0% to the finisher diet and 1.5% to the nursing sow diet.
  • Table 8 Statistics on embryo recovery and the introduction of embryos containing the transgene into recipient sows.
  • Sows were used for up to three farrowings of potentially transgenic pigs. Sows were inseminated with Buffalo semen from a high breeding value boars.
  • the number preceeding the dash represents the litter number and the number following the dash is the pig number within the litter.
  • Saliva was sampled and assayed for phytase 2 to 4 days after birth of the piglets.
  • Zygotes used for microinjection were collected from superovulated Yorkshire or England - Landrace cross gilts.

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MXPA06014649A (es) 2004-06-21 2007-03-12 Novozymes As Proteasas.
BRPI0517539A (pt) 2004-10-04 2008-10-14 Novozymes As polipeptìdeo isolado, polinucleotìdeo isolado, construto de ácido nucleico, vetor de expressão recombinante, célula hospedeira recombinante, métodos para produzir o polipeptìdeo, e para melhorar o valor nutricional de uma ração animal, célula de planta, parte de planta ou planta transgênica, animal não-humano, transgênico, ou produtos, ou elementos do mesmo, uso de pelo menos um polipeptìdeo, aditivo de ração animal, e, composição de ração animal
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PL2342323T3 (pl) 2008-09-26 2013-11-29 Novozymes As Warianty fitazy z hafnia
CN106011159A (zh) 2009-05-21 2016-10-12 巴斯夫酶有限责任公司 肌醇六磷酸酶、编码它们的核酸及制备和使用它们的方法
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CN103695451B (zh) * 2013-08-07 2016-04-06 华南农业大学 唾液腺组织特异性表达外源蛋白的转基因载体和转基因猪及其构建方法
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