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WO1992011358A1 - Expression amelioree dans des organismes transgeniques au moyen d'une seconde sequence transferee - Google Patents

Expression amelioree dans des organismes transgeniques au moyen d'une seconde sequence transferee Download PDF

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Publication number
WO1992011358A1
WO1992011358A1 PCT/GB1991/002318 GB9102318W WO9211358A1 WO 1992011358 A1 WO1992011358 A1 WO 1992011358A1 GB 9102318 W GB9102318 W GB 9102318W WO 9211358 A1 WO9211358 A1 WO 9211358A1
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Prior art keywords
sequence
dna
expression
transgene
expressed
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PCT/GB1991/002318
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English (en)
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Anthony John Clark
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The Agricultural And Food Research Council
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Priority to JP4502156A priority Critical patent/JPH06508261A/ja
Priority to AU91391/91A priority patent/AU661290B2/en
Publication of WO1992011358A1 publication Critical patent/WO1992011358A1/fr

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    • 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
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    • A01K67/00—Rearing or breeding animals, not otherwise provided for; New or modified breeds of animals
    • A01K67/027—New or modified breeds of vertebrates
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    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
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    • C07K14/811—Serine protease (E.C. 3.4.21) inhibitors
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    • C12N9/64—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25) derived from animal tissue
    • C12N9/6421—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25) derived from animal tissue from mammals
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    • C12Y304/21069—Protein C activated (3.4.21.69)
    • A—HUMAN NECESSITIES
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Definitions

  • This invention relates to the production of transgenic organisms, DNA preparations useful in such a method and transgenic animals and plants produced thereby.
  • transgenic animals for a number of purposes have been developed and work on their further development continues. Among their uses, transgenic animals offer a powerful approach for the production of recombinant proteins.
  • the expression of genes encoding a protein of interest can be targeted to the mammary gland of transgenic farm animals such as sheep, goats or cattle, and the protein product can be harvested from their milk.
  • transgenic animals for protein production in milk necessitates foreign genes being introduced into the germ line in such a way that their expression can be directed to die mammary gland. Targeting of expression to other organs or tissues is important in transgenic animals prepared for other purposes. Introduction of foreign genes into an animal's germ line has been demonstrated in WO-A-8800239, which describes the production of transgenic sheep carrying genes designed to express human Factor IX and human c ⁇ -antitrypsin in milk. In order to direct expression of these transgenes to the mammary gland, the approach taken was to take regulatory sequences from milk protein genes and fuse them to the protein coding sequences of the product of interest.
  • transgenic mice In practice, this has involved the use of transgenic mice as a model system to assess the performance of the various hybrid gene constructs; the much shorter generation time of mice enables experiments to be undertaken within a realistic time-scale.
  • a genetic construct to be useful it must express in the chosen model system at a reasonable frequency and at suitable levels.
  • genomic milk protein genes which can be regarded as natural (that is to say the same configuration as DNA sequences found in the genome of animals from which the genes were isolated; such isolated gene may be termed genomic clones and will usually contain introns) have been shown to express relatively efficiently in transgenic mice. Reference is made in particular to Example 7 of
  • WO-A-8800239 which relates to the expression of the gene coding ovine ⁇ - lactoglobulin in transgenic mice.
  • Other examples include the systems of Vilotte et al (Eur. J. Biochem. 186 43-48 (1989)) and Bayna et al Nuc. Acids. .Res. 18 2977-2985 (1990)). These genomic clones thus appear to contain all the essential regulatory sequences required for directing expression efficiently to the mammary gland.
  • hybrid genes comprising milk protein gene regulatory elements fused to DNA sequences encoding proteins of interest have also been assessed in transgenic mice; in WO-A-8800239 the proteins of interest were human Factor DC and human ⁇ j -antitrypsin. In many cases such as this, however, the level of expression of the hybrid genes is well below that which would be optimal for commercial purposes.
  • a common feature of such relatively poorly expressing constructs is that they were constructed using contiguous cDNA sequences and therefore lacked their natural introns. This problem was addressed and overcome in WO-A-9005188, which disclosed the use of a construct comprising a genomic sequence encoding an exogenous protein and containing some, but not all, of its natural introns. Such a construct performed relatively efficiently.
  • the DNA sequences for many proteins of interest are available only as cDNA sequences; indeed, it may be much easier to prepare a cDNA sequence from a corresponding mRNA sequence transcribed in a given tissue in abundance in a target tissue than to isolate a genomic DNA.
  • some genes are very large, even if the proteins that they encode are not correspondingly large; too great a size makes the inclusion of most or all of the introns difficult as a practical matter.
  • a process for the preparation of a transgenic animal or plant capable of expressing a first DNA sequence comprising co-introducing into a cell or group of cells from which an animal or plant may be derived the first DNA sequence and a second DNA sequence, wherein the second DNA sequence is, when so introduced without the first sequence, capable of being expressed as, or regulating the expression of, a transgene with greater specificity and/or a greater frequency of expression and/or at a higher level than that at which the first sequence, without the second sequence, is capable of being expressed as a transgene, and allowing a transgenic animal or plant to develop from the cell(s).
  • the most commonly used method of introducing DNA into an animal cell for the purpose of transgenesis is injection (or microinjection, as it is sometimes termed). This is the method of choice for the production of trangenic animals by means of the present invention.
  • injection or microinjection, as it is sometimes termed.
  • This is the method of choice for the production of trangenic animals by means of the present invention.
  • a few hundred linear molecules of DNA is directly microinjected into a pro-nucleus (often the male pro-nucleus) of a fertilised one cell egg; microinjected fertilised egg may then subsequently be transferred into the oviducts of pseudo-pregnant foster mothers and allowed to develop.
  • the invention is however not limited to this method of introduction; any suitable method can be used.
  • Egg or embryo cells may be used, as may embryonic stem (ES) cells.
  • Transgenic plants are usually currently prepared by different procedures.
  • DNA is transformed into plant cells using a disarmed Ti-plasmid vector and carried by ⁇ grobacterium by procedures known in the art, for example as described in EP-A-0116718 and EP-A-0270822.
  • the foreign DNA could be introduced directly into plant cells using an electrical discharge apparatus.
  • Agrobacterium is ineffective, for example where the recipient plant is monocotyledenous.
  • Any other method that provides for the stable incorporation of the DNA within the nuclear DNA of any plant cell of any species would also be suitable. Such methods include those suitable for species of plant which are not currently capable of genetic transformation.
  • the first and second DNA sequences are in one embodiment of the invention co- introduced by introducing a mixture of them (for example by injection, in the case of animal methodology) into the recipient cell.
  • a mixture of them for example by injection, in the case of animal methodology
  • Co-introduction may also be achieved by covalently or otherwise linking the first and second DNA sequences: the two sequences may be linked in a single DNA molecule.
  • a first sequence may be sandwiched between two second sequences, and/or the tandem nature of the sequences (ie whether they are head to head or head to tail) can be fixed.
  • a process of the first aspect of the invention can therefore be seen to involve the co-introduction of a relatively inefficient, but desired, transgene with a relatively efficiently expressing transgene; this may lead to co-integration at the same site in the chromosome or at sites near to one another.
  • the first DNA sequence may comprise cDNA or other DNA encoding the desired protein and, if the purpose of the transgene is expression, sufficient regulatory sequences (for example including a promoter) operatively linked to the protein- encoding DNA to direct the expression, for example in a target tissue or organ.
  • the regulatory sequence may be derived from a milk protein, particularly a whey protein such as 3-lactoglobulin, ⁇ -lactalbumin or whey acidic protein.
  • the desired protein may be any protein (which term includes glycoprotein) sought to be and capable of being, produced in a transgenic. High value proteins, such as those having pharmaceutical activity, are particular candidates for use in the present invention.
  • Example include, but are not limited to, insulin, plasminogen activators, ⁇ j -antitrypsin, blood factors such as Factor VIII and IX, Protein C and erythropoietin.
  • the regulatory sequences used in association with the protein- coding DNA may be 5' and/or internal and/or 3' regulatory sequences.
  • the second DNA sequence is capable of being expressed, as a transgene, when introduced without the first sequence, with greater specificity and/or a greater frequency of expression and/or at a higher level (often a higher mean level) than that at which the first sequence alone is capable of being expressed.
  • the second DNA sequence is therefore relatively efficiently expressed, compared to the first DNA sequence.
  • a greater efficiency of expression may be achieved by a greater frequency of expressions.
  • a greater frequency of expression means a higher proportion of animals or plants or lines that now express the first sequence; this is clearly of importance both practically and economically.
  • the second sequence may be derived from or constituted by a gene, preferably complete with its associated regulatory sequences, normally expressed in the target organ.
  • the target organ for expression is the mammary gland
  • the second DNA sequence is preferably derived from a milk protein gene, again particularly a whey protein gene such as ⁇ -lactoglobulin or ⁇ -lactalbumin or a casein gene such ⁇ S j -casein.
  • the gene may be the same as that from which the regulatory sequences used in the first DNA sequence are derived.
  • the second sequence may be an artificial construct or a normal gene.
  • Transgenes may be expressed in many organs of animals by means of this invention; the mammary gland is only one example. In plants, transgene expression may be specific for a particular tissue, if desired.
  • DNA useful for the preparation of a transgenic animal or plant expressing a first DNA sequence comprising, on the same or separate molecules, the first sequence and a second DNA sequence, wherein the second DNA sequence is, when introduced as a transgene without the first sequence, capable of being expressed or regulating expression with greater specificity and/or with a greater frequency of expression and/or at a higher level than that at which the first sequence, when introduced as a transgene without the second sequence, is capable of being expressed.
  • the first and second DNA sequences may be embodied on the same DNA molecule, in which case the sequences may be regarded as covalently linked, with or without a linker sequence.
  • the first and second DNA sequences may be provided on different DNA molecules.
  • the DNA may take the form of a mixture of such molecules or a kit of separate preparations of the molecules.
  • the invention is not limited to the presence of a single first sequence and a single second sequence.
  • a plurality of relatively inefficiently expressing first sequences may be potentiated by one or more relatively efficiently expressing second sequence, and a plurality of second sequences may potentiate one or more second sequences. It is preferred that the number of relatively efficiently expressing second sequences be present in excess, compared to the relatively inefficiently expressing first sequence. For example from 2 to 5 or 10 copies of the second sequence may be present per copy of the first sequence.
  • a transgenic animal or plant capable of expressing a first sequence as a transgene, the said animal or plant having the first sequence and a second sequence, wherein the second sequence is such that, when introduced as a transgene without the first sequence, it is capable of being expressed or regulating expression with greater specificity and/or with a greater frequency of expression and/or at a higher level than that at which the first sequence, when introduced as a transgene without the second sequence, is capable of being expressed.
  • suitable species include those which are (a) practicable to work with and (b), if the object is to use the invention for production of a desired molecule, are capable of giving sufficient yields for practical purposes.
  • Small ammals such as rodents including mice, rats, hamsters and guinea pigs may be suitable for some applications; livestock animals such as cattle, sheep, goats and pigs may be preferred for others.
  • the invention is applicable to a wide variety of species, both monocots and dicots, depending on the intended application.
  • FIGURE 1 shows a restriction map of clone lambda SSI, which contains the gene for ovine 0-lactoglobuIin (BLG);
  • FIGURE 2 shows an SDS PAGE analysis of murine and ovine whey proteins (Comparative Example 2);
  • FIGURE 3 shows a Western blot of the gel of Figure 2 using rabbit anti-3-lactoglobulin serum
  • FIGURES 4a and 4b show the construction of plasmid pBJ16 (Comparative Example 4);
  • FIGURE 5 shows Southern blots from mice resulting from co- injection of BLG and AATD and indicates the co-segregation of the two transgenes (Example 1); Band 1 is a BLG-specific band; and Band 2 is an AATD-specific band; FIGURE 6 shows a Northern blot showing the result of hybridisation experiments indicating the tissue specific expression of an AATD transgene in mice according to the invention (Example i);
  • FIGURE 7 shows a Northern blot showing the result of hybridisation experiments which reveal the presence of two transgenes in mice according to the invention.
  • Band 1 is an AATD- specific transcript (— 1600 nt);
  • Band 2 is an AATB-specific transcript (- 1400 nt); and
  • Band 3 is a BLG-specific transcript
  • FIGURE 8 is a Southern blot showing the co-segregation of FIXD and BLG (Example 2); Band 1 is a BLG-specific band; Band 2 is a FIXD-specific band; and Band 3 is a non-specific junction band;
  • FIGURE 9 shows the tissue-specific expression of FIXD in BIX lines (Example 2);
  • FIGURE 10 is a Northern blot illustrating detection of FIXD and
  • Band 1 is an endogenous mouse fix transcript (-2600 nt);
  • Band 2 is a BLG-FIX transcript in FIXA51;
  • Band 3 is a FIXD transcript; and
  • Band 4 is a BLG transcript;
  • FIGURE 11 shows the construction of a S-lactoglobulin/bovine a- lactalbumin construct (Example 3). COMPARATIVE EXAMPLE 1
  • Spleen tissue was procured from a freshly slaughtered Blackface/Suffolk lamb and nuclei were isolated essentially as described by Burch and Weintraub Cell 33 65 (1983). Nuclear pellets were lysed in 0.3M NaCl, lOmM Tris.HCl, lOmM EDTA, 1% SDS pH 7.4 and 400 ⁇ g/ml Proteinase K (Sigma Co, Fancy Road, Poole, Dorset BH17 7NH) and incubated for two hours at 37°C. Repeated phenol/chloroform extractions were performed until the preparation was completely deproteinised.
  • the lambda phage EMBL3 (Frischholz et al J. Mol. Biol. 170 827 (1983)) was employed to construct the genomic library.
  • 30 ⁇ ⁇ of bacteriophage DNA were digested with 5-fold excesses of the restriction enzymes Ec ⁇ KL and BamSl (supplied by Amersham International pic, Lincoln Place, Green End, Aylesbury, Buckinghamshire, England) using the conditions recommended by the manufacturer. After digestion, spermine hydrochloride was added to a concentration of 5mM to precipitate the lambda DNA.
  • the DNA was pelleted at 10,000g for 15 minutes in a bench microfuge, washed in 70% EtOH, 300mM NaAc, lOOmM MgCl 2 , repelleted and finally resuspended in TE at a concentration of 1 mg/ml.
  • Tris.HCl, 5mM EDTA at pH 8.0 were centrifuged in a BECKMANN SW 28 rotor at 26,000 rpm for 24 hours.
  • the expression BECKMANN SW 28 is a trade mark.
  • the sucrose gradients were fractionated from the top and 1ml fractions collected. The size distribution of DNA molecules in each fraction was assessed by agarose gel electrophoresis, and fractions containing DNA molecules from 14-21 kb in size pooled. After a two-fold dilution in TE 2 volumes of EtOH were added and the DNA precipitated overnight at -20°C. The DNA was subsequently resuspended in TE to a concentration of 300 ⁇ g/ml.
  • phage buffer is lOmM Tris.HCl, lOmM MgCl 2 , 0.01 % gelatin, pH 7.4.
  • E. coli ED 8654 (Borg et al Mol. Gen. Genetics 146 199-207 (1976)) and plated out on 9cm diameter Petri dishes to obtain confluent lysis of the bacterial lawn. Confluent plates were obtained from which the top plating agar was scraped off into 10ml of phage buffer and incubated overnight with a few drops of chloroform. The bacterial debris was pelleted by centrifugation at 5000 rpm for five minutes and the phage stocks stored at 4°C. The stocks were titrated on E. coli ED 8654 to determine the pfu/ml figure.
  • RNAse A and 10 ⁇ g/ml DNAse I were added to the supernatant which was then incubated at 37°C for one hour. After this incubation NaCl was added to 40g/litre and polyethylene glycol (PEG) to 10%. The preparation was cooled to 4°C and left for at least two hours to precipitate the phage. The phage pellet was pelleted at 10,000 rpm for 15 minutes and resuspended in 16.0ml of phage buffer. 8.0ml of this suspension was layered upon a step gradient comprising
  • the purified phage particles were dialysed into 0.1M NaCl, lOmM Tris.HCl, ImM EDTA pH 8.0 and then deproteinised by successive extractions with phenol and chloroform. NaCl was added to a final concentration of 0.3M and then the phage DNA precipitated by the addition of 2 volumes of EtOH. The DNA was pelleted by centrifugation at 10,000 rpm for 20 minutes, washed with 70% EtOH, 30% TE, dried and then resuspended in TE to a final concentration of 200-400 ⁇ g/ml.
  • J-lactoglobulin clones The identity of the J-lactoglobulin clones and the precise position of the 5' and 3' ends of the gene were directly confirmed by DNA sequencing. Using suitable restriction sites, fragments were subcloned into plasmid vectors and into M13 vectors. Sequencing was carried out using the dideoxy method of Sanger ⁇ t __• (PNAS 74 5463 (1977)).
  • the mouse milk was diluted 1:5 in distilled water, centrifuged briefly in a bench centrifuge to defat and the caseins precipitated by addition of IN HC1 to a final pH of 4.6. After centrifugation in a bench centrifuge the whey proteins were removed, precipitated with 5% trichloracetic acid and analysed by polyacrylamide gel electrophoresis according to Laemmli (Nature 277, 680-684 (1970).
  • Figure 2 shows an SDS PAGE analysis of murine and ovine whey proteins.
  • Lane 1 marker proteins
  • This clone presumably contains all the necessary sequences to ensure high levels of expression in the mammary gland of a transgenic mouse and can thus be expected to function as efficiently, if not more so, in the homologous species ie in a transgenic sheep. Consequently, fusion genes derived from this clone can also be expected to express (efficiently) in the ovine mammary gland.
  • Sall-Xbal fragment from lambda SS-1 (Fig.7) was used in place of the 16.2 kb Sa l fragment (see Simons et al (Nature 328 530-532 (1987)).
  • the 10.5 kb fragment derived from lambda SS-1 was cloned into plasmid pPolyl (see WO-A-8800239 and Lathe et al (Gene 57 193-201 (1987)) at the .Xbal and Sail sites on that plasmid.
  • the resulting plasmid was termed pSSltgXS. High levels of expression were again obtained.
  • Figures 4a and 4b summarise the procedure.
  • the approach utilises sequences derived from a lambda clone, lambdaSS-1, which contains the gene for ovine jS-lactoglobulin, and whose isolation and characterisation is outlined in WO-A-8800239 (Pharmaceutical Proteins Ltd) and by AH & Clark (1988) Journal of Molecular Biology 199, 415- 426.
  • This construct contains the cDNA for human ⁇ -antitrypsin flanked by BLG sequences.
  • the 5' flanking sequences include the Sail to PvwII-0 BLG sequences.
  • the fusion point between the BLG and AAT sequences is in the 5'-untranslated region of the BLG first exon.
  • the 3' flanking sequences comprise exons 6 and 7 of BLG and the 3' flanking sequences of the BLG gene as far as the Xbal site.
  • This construct contains no introns and was designed to examine whether the 5' and 3' BLG sequences described above are sufficient to direct efficient mammary specific expression of cDNAs encoding human plasma proteins as exemplified by that for
  • the gel purified SphI - Xbal restriction fragment of about 6.6 kb from pSSltgXS was ligated using T4 DNA ligase to gel purified pPolyl (Lathe, Vilotte & Clark, 1987, Gene 57, 193-201) (also described in WO- A-8800239) digested with Sphl and Xbal.
  • the vector pPolyl is freely available from Professor R. Lathe, LGME-CNRS and U184 INSERM, 11 rue Humann, 67085, France.] After transformation of competent E. coli strain DHRQ! (Gibco-BRL) the correct clone was identified by restriction enzyme analysis.
  • the gel purified PvwII restriction fragment containing the origin of replication from pSSltgSpX was self-ligated using T4 DNA ligase in the presence of ImM hexamine cobalt chloride, 25mM KCI [to encourage self-ligation (Rusche & Howard-Flanders (1985) Nucleic Acids Research 13, 1997-2008)].
  • T4 DNA ligase in the presence of ImM hexamine cobalt chloride, 25mM KCI [to encourage self-ligation (Rusche & Howard-Flanders (1985) Nucleic Acids Research 13, 1997-2008)].
  • DHR ⁇ Gibco-BRL
  • oligonucleotides were then ligated using T4 DNA ligase to equimolar amounts of a gel purified 457 bp Styl - Smal fragment from ⁇ -Lg 931 (Gaye et al, op cit) and gel purified pUC19 (Pharmacia-LKB Biotechnology, Pharmacia House, Midsummer Boulevard, Central Milton Keynes, Bucks, MK9
  • the gel purified HincII - Smal restriction fragment from pUQSlacA was ligated using T4 DNA ligase to gel purified pBJ5 linearised by partial digestion with Smal. After transformation of competent E. coli strain DH5 ⁇ (Gibco-BRL) the correct clone was identified by restriction enzyme analysis.
  • the gel purified Pw II restriction fragment containing the origin of replication from pBJ7 was self-ligated using T4 DNA ligase in the presence of ImM hexamine cobalt chloride, 25mM KCI (to encourage self-ligation [Rusche & Howard-
  • the plasmid p ⁇ lppg containing a full length cDNA encoding an M variant of 0 ⁇ - antitrypsin was procured from Professor Riccardo Cortese, European Molecular Biology Laboratory, Meyerhofstrasse 1, D-6900 Heidelberg, Federal Republic of Germany (Ciliberto, Dente & Cortese (1985) Cell 41, 531-540).
  • the strategy used in the construct BLG-AAT or pSSltgXSTARG, now known as A ATA, described in WO-A-8800239 required that the polyadenylation signal sequence at the 3' end of the ⁇ j -antitrypsin cDNA be removed.
  • the polyadenylation signal was removed in the following manner. Plasmid p ⁇ lppg DNA was digested with PstI and the digestion products were separated by electrophoresis in a preparative 1% agarose gel containing 0.5 ⁇ g/ml ethidium bromide (Sigma). The relevant fragment of about 1400 bp was located by illumination with a UV lamp (Ultra-Violet Products, Inc, San Gabriel, California, USA). A piece of dialysis membrane was inserted in front of the band and the
  • DNA fragment subsequently electrophoresed onto the membrane.
  • the DNA was eluted from the dialysis membrane and isolated by use of an 'ElutipD' [Scleicher and Schull, Postfach 4, D-3354, Dassel, W. Germany], employing the procedure recommended by the manufacturer.
  • the gel purified 1400 bp PstI fragment was digested with the TaqI, electrophoresed on a preparative 1% agarose gel as described above.
  • the TaqI - PstI fragment of approximately 300 bp comprising the 3' end of the ⁇ j -antitrypsin cDNA including the polyadenylation signal sequence was eluted and purified using an Elutip as described above, as was the TaqI - TaqI fragment of 1093 bp containing the 5' portion of the cDNA.
  • the plasmid vector pUC8 (Pharmacia-LKB Biotechnology, Pharmacia House,
  • Plasmid DNA was isolated from ampicillin resistant colonies. The correct recombinants were identified by the release of a fragment of approximately 300 bp on double digestion with Accl and PstI. The plasmid generated was called pUC8.3'AT.3.
  • Plasmid pUC8.3'AT.3 was subjected to partial digestion with BstNI and the fragments) corresponding to linearised pUC8.3'AT.3 isolated from an agarose gel. There are seven BstNI sites in pUC.3'AT.3, five in the vector and two in the region corresponding to the 3 '-untranslated sequences of o ⁇ -antitrypsin.
  • the BstNI linearised and gel purified DNA was digested with PstI which cuts in the pUC8 polylinker where it joins the 3' end of the cDNA insert.
  • the PstI digested DNA was end repaired with T4 DNA polymerase in the presence of excess dNTPs and self-ligated with T4 DNA ligase.
  • the BstNI - PstI fragment containing the polyadenylation signal sequence is lost by this procedure.
  • the ligated material was used to transform E. coli strain DH-1 to ampicillin resistance. Plasmid DNA was isolated from ampicillin resistant colonies.
  • the correct clone was identified by restriction analysis and comparison with pUC8.3'AT.3. The correct clone was characterised by retention of single sites for Bam ⁇ I and Hindlll, loss of a PstI site, and a reduction in the size of the small Pvu ⁇ fragment.
  • the correct clone was termed pB5.
  • Plasmid pB5 DNA was digested witii Accl, phenol/chloroform extracted and DNA recovered by ethanol precipitation.
  • Accl cleaved pB5 DNA was treated with calf intestine alkaline phosphatase (BCL). The reaction was stopped by adding EDTA to 10 millimolar and heating at 65°C for 10 minutes. The DNA was recovered after two phenol chloroform and one chloroform extractions by precipitation with ethanol.
  • T4 DNA ligase was used to ligate the 1093 bp TaqI - TaqI fragment described above to pB5, Accl cleaved and phosphatased DNA and the ligation products were used to transform E.
  • Plasmid pUC8 ⁇ lAT.73 was digested with Accl and HindUI and the resulting fragment containing the c- i -antitrypsin cDNA minus its polyadenylation signal was end-repaired using Klenow fragment of E. coli DNA polymerase in the presence of excess dNTPs. This blunt ended fragment was gel purified and ligated using T4 DNA ligase to gel purified pBJ8 linearised with PvuII. After transformation of competent E. coli strain DH5 ⁇ (Gibco-BRL) the correct clone was identified by restriction enzyme analysis.
  • Plasmid pSSltgSpS was digested with BglR and end-repaired using the Klenow fragment of E. coli DNA polymerase in the presence of excess dNTPs.
  • the blunt- ends were modified using HindUI synthetic linkers (New England Biolabs Inc, 32 Tozer Road, Beverly, MA 01915-5510, USA) and the resulting fragment self- ligated using T4 DNA ligase in the presence of ImM hexamine cobalt chloride,
  • the gel purified HindlH - SphI fragment from pBJl and the gel purified SphI - Xbal fragment from pBJ12 were ligated using T4 DNA ligase to gel purified pUC19 (Pharmacia-LKB Biotechnology, PharmaciaHouse, Midsummer Boulevard, Central Milton Keynes, Bucks, MK9 3HP, UK) digested with Hndm and Xbal.
  • Plasmid pBJ16 was digested with HindUI and Xbal and the resulting 8.0 kb AATD fragment was isolated from a gel using DE81 paper (Dretzen et al (1981) Analytical Biochemistry 112, 285-298). After separation from the DE81 paper the DNA was phenol/chloroform extracted, ethanol precipitated and finally resuspended in TE buffer (10 mM Tris-HCl, ImM EDTA pH 8) ready for microinjection.
  • RNA isolated from various tissues was examined for the presence of AATD transcripts and milk from the females was assayed for the presence of ⁇ j - antitrypsin by Western blotting; both of these analyses are as described in Example 2 of WO-A-9005188. The results are shown in Table 1 below: TABLE 1
  • the jS-lactoglobulin construct prepared in Comparative Examples 1 and 3, containing the 10.5 kb SaH-Xbal fragment prepared from pSSltgXS ("BLG”) and the ⁇ i-antitrypsin construct prepared in Comparative Example 3 ("AATD”) were co-injected into mouse eggs as before in an equimolar ratio in a total DNA concentration of about 3 ⁇ g/ml.
  • 20 transgenic founder mice were detected by Southern blotting and 11 of these were found to carry both AATD and BLG sequences. These mice were designated BAD (BLG and AATD). 9/11 transmitted both transgenes to the Gl progeny. Analysing a number of progeny in each line showed that in each case the two transgenes had segregated together ( Figure 5) indicating that they were integrated very close together and in all probability were co-integrated at the same site.
  • FIG. 5 shows the co-segregation of AATD and BLG.
  • DNA samples from the various BAD lines resulting from co-injection with AATD and BLG were restricted with Ec ⁇ RI, Southern blotted and probed with an EcoBI-Sphl fragment that hybridised to the 5' end of the two transgenes.
  • BLG and AATD transgenes can be distinguished by Eco ⁇ l digestion and die specific JEc ⁇ RI fragments are indicated.
  • DNA from a number of Gl animals has been analysed.
  • the similar pattern of AATD and BLG restriction fragments in Gl animals from the same line ie BAD 1; BAD99 etc
  • Note tiiat line BAD93 may have two co-integrated loci which are segregating in the Gl generation.
  • AATD transcripts of the expected size were detected in mammary RNA in 6 out of the 9 lines: 3 lines expressed AATD mRNA at low levels, 1 at medium level and 2 at high level. AATD transcripts were not detected in other tissues (Figure 6).
  • Figure 6 shows tissue-specific expression of AATD in BAD lines.
  • RNA prepared from a variety of tissues was probed witii human AAT-specific sequences.
  • the — 1600 nt AATD transcripts (arrowed D) are seen specifically in the mammary gland of BAD mice 99.3 and BAD 135.13.
  • Sa salivary gland
  • H heart
  • K kidney
  • Sp spleen
  • L liver
  • M mammary gland.
  • Human ⁇ j -antitrypsin has been measured in d e two high expressing lines and estimated at about 140 ⁇ g/ml and about 600 ⁇ g/ml (mean values of two) respectively.
  • the BLG transgene has also been analysed for expression: the 6 lines that expressed AATD mRNA also expressed BLG mRNA, whereas the three
  • FIG. 7 illustrates detection of AATD and BLG transcripts in BAD mice.
  • AAT ⁇ lAT
  • the filters were stripped and re-hybridised to a BLG-specific probe (BLG); the same samples showed strong hybridisation to the — 800 nt BLG transcript, also detected in sheep mammary gland RNA (SM); AATB 35, mammary gland RNA from transgenic line
  • AATD expression may be associated with an actively expressing BLG gene. Comparing the results of this example with those of Comparative Example 5, it appears that the co-injection (and presumably co-integration) of AATD with BLG has significantly increased d e efficiency of expression of AATD. Considering RNA data only, when AATD was injected alone 0/8 animals expressed die transgene. When co-injected (and presumable co-integrated) witii BLG, expression of AATD mRNA was detected in 6/9 animals as shown in Table 2 below.
  • ⁇ lAT determined by ELISA sensitivity >0.2 ⁇ g/ml.
  • BAD lines values represent average from 2 mice.
  • Example 4 Construction of AATD
  • tiiat the DNA sequence encoding the polypeptide of interest encodes Factor IX.
  • a Nhel- H ⁇ ndHI fragment comprising 1553 bp of the insert from p5'G3'CVl [see WO-A-
  • FIX construct described in Comparative Example 6 above was used to generate transgenic mice by die method described in Example 1 of WO-A- 9005188.
  • RNA isolated from various tissues was examined for the presence of FIX transcripts and milk from the females was assayed for the presence of factor IX by Western blotting and/or ELISA; both of these analyses are as described in
  • transgenic mice carrying FIX D expressed die transgene as determined by analysis of milk proteins or by Northern blotting of mammary gland RNA.
  • Figure 8 shows the co-segregation of FIX D and BLG.
  • DNA samples from the various BIX lines resulting from co-injection with FIX D and BLG were restricted witii BamHL, Southern blotted and probed witii an Ec ⁇ SI-Sphl fragment that hybridised to the 5' end of die two transgenes.
  • BLG and FIX D transgenes can be distinguished by BamHI digestion and die specific Bam ⁇ I fragments are indicated.
  • DNA from a number of Gl animals has been analysed.
  • the similar pattern of FIX D and BLG restriction fragments in Gl animals from the same line (eg BIX 34; BIX 99 etc) is indicative of co-segregation.
  • line BIX 29 may have two co-integrated loci which are segregating in the Gl generation.
  • FIXD mRNA has been analysed in 11 of the 12 lines of die double transgenic mice.
  • the FIX D transcripts of the expected size were detected in mammary RNA in all these lines: 4 lines expressed FIX mRNA at low levels, 4 at medium level and 3 at high level. (Some variation in die level of expression between individual mice of a given line was observed in some cases.) FIX D transcripts were not detected in other tissues (Figure 9).
  • Figure 9 shows tissue-specific expression of FIX D in BIX lines.
  • RNA prepared from a variety of tissues was probed witii fIX-specific sequences.
  • the — 1800 nt FIX D transcripts (arrowed) are seen specifically in the mammary gland of BIX mice 12.1, 30.2 and 33.1.
  • the -2600 nt transcript from the endogenous FIX gene (arrowed B) is present in liver samples, CM, control mouse mammary gland
  • RNA see also Example 2 of WO-A-9005188
  • CL control liver RNA.
  • Sa salivary gland
  • H heart
  • K kidney
  • Sp spleen
  • L liver
  • M mammary gland.
  • FIX A51 mammary gland RNA from a transgenic mouse carrying a second FIX transgene, FIX A that expresses a ⁇ 2400nt FIX transcript (arrowed A).
  • Human factor IX was detected in the milk from mice from 6 out of the 10 lines which have been analysed - see Table 4 below.
  • the BLG transgene has also been analysed for expression; all the lines analysed were shown to express BLG mRNA also expressed BLG mRNA.
  • Figure 10 illustrates detection of FIX D and BLG transcripts in BIX mice.
  • RNA samples Mammary gland (M) and liver (L) RNA samples, blotted onto Hybond membranes, were probed witii fTX specific sequences.
  • - 1800 nt FIX D transcripts are detectable in mammary gland RNA samples from BIX mice 34.1, 37.1, 43.3, 66.2, 10.3, 12.1, 30.4, 33.1, 22.13, 29 and 131.2.
  • the filters sere stripped and re-hybridised to a BLG-specific probe (BLG); the same samples showed hybridisation to the - 800 nt BLG transcript, FIX A 51, mammary gland RNA from transgenic mouse FIX A 51 containing -2400 nt FIX A transcript; C, control mouse samples.
  • a 0-lactoglobulin/bovine ⁇ -lactalbumin construct (BLG/alacTR.D - BAT) was prepared in the following way.
  • the 3.0kb Xhol fragment comprising regulatory sequences derived from the bovine ⁇ -lactalbumin gene fused to a segment of DNA encoding ovine trophoblastin was excised from the plasmid vector (for full details of this plasmid construct see Stinnakre et al (1991), FEBS Letters, 284 1, 19-22).
  • This fragment was inserted directiy into the plasmid pSSltgXS (see WO-A- 8800239) at the unique Sa l site ( Figure 11).
  • the resulting 13.5kb insert comprising the alacTR gene linked to the BLG gene was excised from d e plasmid vector by digestion with Xbal. This fragment was purified by gel electrophoresis and used for die direct microinjection of mouse eggs, as previously described.
  • the multiple cloning site of the vector pUC18 (Yanisch-Perron et al, (1985) Gene 33:103-119) was removed and replaced witii a synthetic, double stranded, oligon ucleotide containing th e n ew res tric ti o n si tes : PmVMluVSaWx ⁇ co ⁇ .WIXbaVP ⁇ ixiIIMluI, and flanked by 5 '-overhangs compatible witii the restriction sites JEc ⁇ RI and HindUI.
  • pUC18 DNA was cleaved with both
  • the jS-lactoglobulin gene sequences from the plasmid pSSltgXS were excised on a Sa l and Xbal fragment and recloned into the vector pUC.PM, cut with Sai and Xbal, to give plasmid pUCXS.
  • the plasmid pSSltgSE contains jS-lactoglobulin gene sequences from the SphI site at position 754 to the EcoR site at 2050, a region spanning a unique Not! site at position 1148.
  • This insert contains a single PvuII site (832) which lies in the 5'-untranslated of the /3-lactoglobulin mRNA.
  • the DNA sequences bounded by SphI and Noil were then excised and used to replace the equivalent fragment in the plasmid pUCXS, tiius effectively introducing a unique Ec ⁇ RV site into the 0-lactoglobulin gene placed in such a way as to allow the insertion of any additional DNA sequences under the control of the ⁇ - lactoglobulin gene promoter and 3' to the initiation of transcription.
  • the resulting plasmid was called pUCXS/RV.
  • pUCXS/RV A derivative of pUCXS/RV, containing only the 4.2kbp of the /3-lactoglobulin gene which lie 5' to the transcription initiation site (the promoter), was constructed by subcloning the SaH-Ec ⁇ RV fragment into pUC.PM; this plasmid is called pUCSV.
  • a fragment of the 3' flanking sequence of die 3-lactoglobulin gene were subcloned in such a way as to eliminate all introns.
  • Plasmid DNA of pUCXS/RV which was partially digested with Smal by performing an enzyme titration with lower and lower concentrations of enzyme at a fixed concentration of DNA. The Smal protein was removed by phenol-chloroform extraction and ethanol precipitation and die DNA resuspended in water. This DNA was subsequently digested to completion with the enzyme Xbal. DNA cut once at the Smal site, position 5286, and then cleaved with Xbal gave a characteristic band of size 2. lkbp. This band was purified from an agarose gel slice and ligated into Smal and Xbal cut pBSIISK- (Stratagene Ltd, Cambridge Science Park, Cambridge, UK) to give d e plasmid pBLAClOO.
  • Plasmid pMAD The /S-lactoglobulin cloning vector pMAD was constructed to allow rapid insertion of cDNAs under die control of the jS-lactoglobulin gene promoter and 3 '-flanking sequences. Such constructs contain no introns.
  • the plasmid pBLAClOO was opened by digestion witii both Ec ⁇ RV and Sail, the vector fragment was gel purified. Into this was ligated the 4.2kbp promoter fragment from the plasmid pUCSV as a SaR-Ec ⁇ RW fragment.
  • This construct is termed pSTl and constitutes a /8-lactoglobulin mini-gene encoding die 4.2kbp promoter and 21kbp of 3'- flanking sequences.
  • a unique ⁇ fc ⁇ RV site is present to allow blunt-end cloning of any additional DNA sequences.
  • Mlul the entire mini-gene from pSTl was excised on a xXh ⁇ l-Notl fragment, the DNA termini made flush with Klenow polymerase, under standard conditions, and blunt-end cloned into the EcoRV site of pUC.PM to give pMAD.
  • Plasmid pCORP2 A 1450bp cDNA of the protein C gene, flanked by Kpnl sites, was obtained in the form of plasmid pWAPC2. The cDNA was excised as a Kpnl fragment, the 3' overhangs made flush by treatment with T4 DNA polymerase, the fragment gel purified and blunt-end cloned into the £lc ⁇ RV site of pMAD. Orientation was determined by restriction digest and confirmed by DNA sequencing. This construct is plasmid pCORP2 and contains the protein C cDNA under die transcriptional control of the / S-lactoglobulin gene 5' and 3' flanking sequences. There are no introns.
  • Transgenic mice were generated by direct pro-nuclear injection essentially as described by Gordon and Ruddle, "Metiiods in Enzymology", Vol 101, [(1983) Eds. Wu, Grossman and Moldave], Academic Press pp 411-432.
  • the ' ⁇ - Lactoglobulin' construct and the 'Protein C construct were co-injected at both equimolar ratio and non-equimolar ratio (1:3 with respect to Protein C) at an overall concentration of 6 ⁇ g/ml. 12 founder mice were produced, all of which had integrated botii transgenes.

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Abstract

On produit des animaux et des plantes transgéniques pouvant exprimer une protéine désirée en introduisant conjointement dans une cellule d'un ÷uf ou d'un embryon d'un animal, ou dans des cellules réceptrices appropriées d'une plante, une première séquence codant la protéine désirée et une seconde séquence d'ADN plus efficacement exprimée. L'efficacité d'expression est ainsi conférée à la première séquence, ce qui permet d'obtenir un rendement ou un ciblage amélioré, ou les deux. L'introduction simultanée peut être effectuée lorsqu'on injecte conjointement un mélange des deux séquences d'ADN dans un ÷uf fertilisé, dans le cas d'un animal. L'invention peut être utilisée pour améliorer l'efficacité d'expression de protéines choisies, telles que celles présentant une activité biologique dans les glandes mammaires d'un animal transgénique femelle.
PCT/GB1991/002318 1990-12-24 1991-12-24 Expression amelioree dans des organismes transgeniques au moyen d'une seconde sequence transferee WO1992011358A1 (fr)

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Cited By (14)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0591219A1 (fr) * 1991-01-11 1994-04-13 American Red Cross Expression de la proteine c humaine active dans les tissus mammaires d'animaux transgeniques
WO1995023868A1 (fr) * 1994-03-03 1995-09-08 Zymogenetics, Inc. Production de fibrinogene chez des animaux transgeniques
WO1995027782A1 (fr) * 1994-04-08 1995-10-19 Ppl Therapeutics (Scotland) Ltd Production de peptides utiles en tant que proteines de fusion dans du lait de mammifere transgenique
WO1998016634A1 (fr) * 1996-10-14 1998-04-23 Biotechnology And Biological Sciences Research Council Expression transgene
US5831141A (en) * 1991-01-11 1998-11-03 United States Of America As Represented By The Department Of Health And Human Services Expression of a heterologous polypeptide in mammary tissue of transgenic non-human mammals using a long whey acidic protein promoter
US5851796A (en) * 1995-06-07 1998-12-22 Yale University Autoregulatory tetracycline-regulated system for inducible gene expression in eucaryotes
US5898094A (en) * 1996-10-21 1999-04-27 University Of South Florida Transgenic mice expressing APPK670N,M671L and a mutant presenilin transgenes
WO1999058703A1 (fr) * 1998-05-08 1999-11-18 Shanghai Institute Of Medical Genetics, Shanghai Children's Hospital Procede d'obtention d'un mouton transgenique
US6046380A (en) * 1994-05-03 2000-04-04 Ppl Therapeutics (Scotland) Limited Factor IX production in transgenic non-human mammals and factor IX DNA sequences with modified splice sites
WO2000030436A1 (fr) * 1998-11-19 2000-06-02 Ppl Therapeutics (Scotland) Ltd. Stabilisation du lait provenant d'animaux transgeniques
US6262336B1 (en) 1991-01-11 2001-07-17 American Red Cross Expression of a heterologous protein C in mammary tissue of transgenic animals using a long whey acidic protein promoter
AU774247B2 (en) * 1996-11-04 2004-06-24 E.I. Du Pont De Nemours And Company Hydrofluorocarbon compositions
US7030289B2 (en) 1998-11-19 2006-04-18 Ppl Therapeutics (Scotland) Ltd Stabilization of milk from transgenic animals
US7045677B2 (en) 1999-06-23 2006-05-16 Pharming Intellectual Property Bv Fusion proteins incorporating lysozyme

Citations (9)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1988000239A1 (fr) * 1986-06-30 1988-01-14 Pharmaceutical Proteins Ltd Production de peptides
WO1990005188A1 (fr) * 1988-11-11 1990-05-17 Pharmaceutical Proteins Limited Structure genetique dont l'adn codant des proteines comprend des introns et est conçu pour faire produire des proteines a des animaux transgeniques
WO1990006363A1 (fr) * 1988-12-08 1990-06-14 Damon Biotech, Inc. Procede d'expression d'une proteine par induction grace au gene mutant dihydrofolate reductase (dhfr)
WO1990007936A1 (fr) * 1989-01-23 1990-07-26 Chiron Corporation Therapies de recombinaison pour infections et troubles hyperproliferatifs
WO1990013645A1 (fr) * 1989-05-10 1990-11-15 Somatogen, Inc. Production d'hemoglobine et de ses analogues dans des bacteries et de la levure
EP0420055A2 (fr) * 1989-09-21 1991-04-03 W.R. Grace & Co.-Conn. Expression augmentée de gènes en réponse à des signaux de lactation
WO1991006666A1 (fr) * 1989-11-06 1991-05-16 Cell Genesys, Inc. Production de proteines par recombinaison homologue
WO1991013151A1 (fr) * 1990-02-22 1991-09-05 Biogen, Inc. Expression amelioree de polypeptides
WO1991015111A1 (fr) * 1990-03-30 1991-10-17 President And Fellows Of Harvard College Systeme genetique binaire commandant l'expression d'un gene mutant chez un animal a genes mutants

Patent Citations (9)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1988000239A1 (fr) * 1986-06-30 1988-01-14 Pharmaceutical Proteins Ltd Production de peptides
WO1990005188A1 (fr) * 1988-11-11 1990-05-17 Pharmaceutical Proteins Limited Structure genetique dont l'adn codant des proteines comprend des introns et est conçu pour faire produire des proteines a des animaux transgeniques
WO1990006363A1 (fr) * 1988-12-08 1990-06-14 Damon Biotech, Inc. Procede d'expression d'une proteine par induction grace au gene mutant dihydrofolate reductase (dhfr)
WO1990007936A1 (fr) * 1989-01-23 1990-07-26 Chiron Corporation Therapies de recombinaison pour infections et troubles hyperproliferatifs
WO1990013645A1 (fr) * 1989-05-10 1990-11-15 Somatogen, Inc. Production d'hemoglobine et de ses analogues dans des bacteries et de la levure
EP0420055A2 (fr) * 1989-09-21 1991-04-03 W.R. Grace & Co.-Conn. Expression augmentée de gènes en réponse à des signaux de lactation
WO1991006666A1 (fr) * 1989-11-06 1991-05-16 Cell Genesys, Inc. Production de proteines par recombinaison homologue
WO1991013151A1 (fr) * 1990-02-22 1991-09-05 Biogen, Inc. Expression amelioree de polypeptides
WO1991015111A1 (fr) * 1990-03-30 1991-10-17 President And Fellows Of Harvard College Systeme genetique binaire commandant l'expression d'un gene mutant chez un animal a genes mutants

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
EXPERIENTIA vol. 47, no. 9, 15 September 1991, BASEL, CH pages 905 - 912; WILMUT, J. ET AL.: 'Production of pharmaceutical proteins in milk' Abstract, lines 10-11 *

Cited By (20)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0591219A4 (en) * 1991-01-11 1997-04-23 American Nat Red Cross Expression of active human protein c in mammary tissue of transgenic animals
US5831141A (en) * 1991-01-11 1998-11-03 United States Of America As Represented By The Department Of Health And Human Services Expression of a heterologous polypeptide in mammary tissue of transgenic non-human mammals using a long whey acidic protein promoter
US6262336B1 (en) 1991-01-11 2001-07-17 American Red Cross Expression of a heterologous protein C in mammary tissue of transgenic animals using a long whey acidic protein promoter
EP0591219A1 (fr) * 1991-01-11 1994-04-13 American Red Cross Expression de la proteine c humaine active dans les tissus mammaires d'animaux transgeniques
WO1995023868A1 (fr) * 1994-03-03 1995-09-08 Zymogenetics, Inc. Production de fibrinogene chez des animaux transgeniques
US5639940A (en) * 1994-03-03 1997-06-17 Pharmaceutical Proteins Ltd. Production of fibrinogen in transgenic animals
USRE42704E1 (en) 1994-03-03 2011-09-13 Pharming Intellectual Property B.V. Production of fibrinogen in transgenic animals
CN1079433C (zh) * 1994-03-03 2002-02-20 酶遗传学公司 在转基因动物中生产纤维蛋白原
US6197946B1 (en) 1994-04-08 2001-03-06 Ppl Therapeutics (Scotland) Limited Peptide production as fusion protein in transgenic mammal milk
WO1995027782A1 (fr) * 1994-04-08 1995-10-19 Ppl Therapeutics (Scotland) Ltd Production de peptides utiles en tant que proteines de fusion dans du lait de mammifere transgenique
US6211427B1 (en) 1994-04-08 2001-04-03 Ppl Therapeutics (Scotland) Limited Peptide production as fusion protein in transgenic mammal milk
US6046380A (en) * 1994-05-03 2000-04-04 Ppl Therapeutics (Scotland) Limited Factor IX production in transgenic non-human mammals and factor IX DNA sequences with modified splice sites
US5851796A (en) * 1995-06-07 1998-12-22 Yale University Autoregulatory tetracycline-regulated system for inducible gene expression in eucaryotes
WO1998016634A1 (fr) * 1996-10-14 1998-04-23 Biotechnology And Biological Sciences Research Council Expression transgene
US5898094A (en) * 1996-10-21 1999-04-27 University Of South Florida Transgenic mice expressing APPK670N,M671L and a mutant presenilin transgenes
AU774247B2 (en) * 1996-11-04 2004-06-24 E.I. Du Pont De Nemours And Company Hydrofluorocarbon compositions
WO1999058703A1 (fr) * 1998-05-08 1999-11-18 Shanghai Institute Of Medical Genetics, Shanghai Children's Hospital Procede d'obtention d'un mouton transgenique
WO2000030436A1 (fr) * 1998-11-19 2000-06-02 Ppl Therapeutics (Scotland) Ltd. Stabilisation du lait provenant d'animaux transgeniques
US7030289B2 (en) 1998-11-19 2006-04-18 Ppl Therapeutics (Scotland) Ltd Stabilization of milk from transgenic animals
US7045677B2 (en) 1999-06-23 2006-05-16 Pharming Intellectual Property Bv Fusion proteins incorporating lysozyme

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EP0566596A1 (fr) 1993-10-27
JPH06508261A (ja) 1994-09-22
AU661290B2 (en) 1995-07-20
GB9028062D0 (en) 1991-02-13
AU9139191A (en) 1992-07-22

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