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Production of recombinant polypeptides by bovine species and transgenic methods

a technology of recombinant polypeptides and bovine species, which is applied in the fields of peptides, enzymology, transferrins, etc., can solve the problems of little, if any, success in producing transgenic cows, and achieve the effects of facilitating identification of successful transgenesis, facilitating integration, and resisting digestion

Inactive Publication Date: 2005-09-08
PHARMING INTPROP BV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Benefits of technology

[0022] Further, the invention includes methods for producing transgenic non-human mammals having a desirable phenotype. The method comprises first causing the methylation of a transgene capable of conferring the desirable phenotype when incorporated into the cells of a transgenic non-human animal, e.g., by transforming an appropriate bacterium, such as E. coli MM 294, with a plasmid containing the transgene. The methylated transgene is then excised and introduced into fertilized oocytes of the non-human animal to permit integration into the genome. The oocytes are then cultured to form pre-implantation embryos thereby replicating the genome of each of the fertilized oocytes. Thereafter, at least one cell is removed from each of the pre-implantation embryos and treated to release the DNA contained therein. Each of the released DNAs are then digested with a restriction endonuclease capable of cleaving the methylated transgene but incapable of cleaving the unmethylated form of the transgene formed after integration into and replication of the genomic DNA. Those pre-implantation embryos which have integrated the transgene contain DNA which is resistant to cleavage by the restriction endonuclease in the region containing the transgene. This resistance to digestion, which can be detected by electrophoresis of the digest after PCR amplification of the DNA and hybridization with a labelled probe for the transgene, facilitates the identification of successful transgenesis.

Problems solved by technology

However, little, if any, success has been achieved in producing transgenic cows.

Method used

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  • Production of recombinant polypeptides by bovine species and transgenic methods
  • Production of recombinant polypeptides by bovine species and transgenic methods
  • Production of recombinant polypeptides by bovine species and transgenic methods

Examples

Experimental program
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Effect test

example 1

Construction of a Probe Specific for Bovine αS1 Casein Sequences

A. Isolation of Chromosomal DNA

[0118] Placental tissue was obtained from the slaughterhouse. Surrounding connective tissue was removed and pieces of about 30 grams were quickly frozen in liquid N2. Chromosomal DNA was isolated as follows: 30 grams of tissue was homogenized (on ice) with 35 ml of Buffer 1 containing 300 mM Sucrose; 60 mM KCl; 15 mM NaCl; 60 mM Tris.HCl pH 8.2; 0.5 mM spermidine; 0.15 mM spermine; 2 mM EDTA; 0.5 mM EGTA. 65 ml of icecold buffer 1 containing 1% NP40 was added and the mixture was incubated for five minutes on ice. After centrifugation for five minutes at 3000×g the pellet was rinsed with buffer 1 containing 1% NP40. After repeating the centrifugation step the pellet was resuspended in 5 ml of buffer 1. 5 ml 0.5 M EDTA was quickly added. Final volume was now 15 ml. 0.15 ml of a 10% SDS solution was added. After mixing, RNAse A and T1 were added to final concentrations of 0.4 mg / ml and 6 u...

example 2

Cloning of Human Lactoferrin Gene

A. Materials

[0130] Restriction endonucleases, T4 ligase, and T7 polynucleotide kinase were obtained from Boehringer-Mannheim, New England Biolabs, or Bethesda Research Laboratories. Radio-isotopes were purchased from Amersham. A human mammary gland cDNA library in bacteriophage λgt11 was obtained from Clontech, Inc., Palo Alto, Calif.

B. Isolation of the Human Lactoferrin Gene

[0131] The human mammary gland library was screened by standard plaque hybridization technique (Maniatis, et al. (1982) Molecular Cloning: A Laboratory Manual) with three synthetic oligomers. Two of the oligomers were 30-mers corresponding to the cDNA sequence of Rado et al., supra, at amino acid positions 436-445 and 682-691. The third was a 21-mer “best guess” probe based on human codon bias and coding for amino acid sequence of HLF between amino acid residues 18 and 24. Respectively, they were:

(Seq. ID No.: 9)(1)5′-CTTGCTGTGGCGGTGGTTAGGAGATCAGAC-3′(Seq. ID No.: 10)(2)5...

example 3

Construction of bovine αS1-casein CAT vectors

[0134] In order to determine whether the αS1 casein fragments obtained in Example 1 had promoter and other properties needed to express a heterologous gene, expression plasmids were constructed containing variable amounts of 5-′ and 3′-flanking regions from the αS1-casein gene. The chloramphenicol Acetyl transferase gene (CAT) was used as a heterologous gene in these vector constructs. The CAT gene is useful to detect the expression level for a heterologous gene construct since it is not normally present in mammalian cells and confers a readily detectable enzymatic activity (see Gorman, C. N., et al. (1983), Mol. Cell. Biol. 2, 1044-1051) which can be quantified in the cells or animals containing an expressible gene.

A. DNA Sequences

[0135] 681 bp of a αS1-casein promoter plus the first non-coding exon plus approximately 150 bp of the first intervening sequence (IVS) were isolated from a 5′-flanking genomic clone from Example 1 by PCR a...

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Abstract

Transgenes for producing recombinant polypeptides transgenic bovine species. A transgene for producing recombinant polypeptides in the milk of transgenic bovine species comprises at least one expression regulation sequence, a secretory DNA sequence encoding a secretory signal sequence which is functional in mammary secretory cells of the bovine species and a recombinant DNA sequence encoding the recombinant polypeptide. Also included are methods for producing transgenic bovine species. The method includes introducing the above transgene into an embryonal target cell of a bovine species, transplanting the transgenic embryonic target cell formed thereby into a recipient bovine parent and identifying at least one female offspring which is capable of producing the recombinant polypeptide in its milk. The invention also includes transgenic bovine species capable of producing recombinant polypeptides in transgenic milk as well as the milk from such transgenic bovine species and food formulations containing one or more recombinant polypeptide. Methods are also provided for producing transgenic non-human mammals having a desirable phenotype. The method comprises first methylating a transgene followed by introduction into fertilized oocytes. The oocytes are then cultured to form pre-implantation embryos. Thereafter, at least one cell is removed from each of the pre-implantation embryos and the DNA digested with a restriction endonuclease capable of cleaving the methylated transgene but incapable of cleaving the unmethylated form of the transgene. Those pre-implantation embryos which have integrated the transgene contain DNA which is resistant to cleavage by the restriction endonuclease in the region containing the transgene.

Description

[0001] This a continuation-in-part of U.S. patent application Ser. No. 08 / 077,788, filed Jun. 15, 1993, which is a continuation-in-part of U.S. patent application Ser. No. 07 / 898,956, filed Jun. 15, 1992, which is a continuation-in-part of U.S. patent application Ser. No. 07 / 619,131 filed Nov. 27, 1990, which is a continuation-in-part of U.S. patent application Ser. No. 07 / 444,745 filed Dec. 1, 1989 (now abandoned). Each of the above applications is incorporated by reference in its entirety for all purposes.FIELD OF THE INVENTION [0002] The invention relates to the production of recombinant polypeptides by transgenic bovine species and to methods for producing transgenic non-human mammals having a desired phenotype. BACKGROUND OF THE INVENTION [0003] There is a plethora of literature relating to the expression of heterologous genes in lower organisms such as unicellular bacteria, yeast and filamentous fungi, and in higher cell types such as mammalian cells. There are also numerous r...

Claims

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Application Information

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IPC IPC(8): A01K67/027A23C9/20C07K14/47C07K14/765C07K14/79C12N9/36C12N9/64C12N15/12C12N15/85
CPCA01K67/0275C12Y304/21069A01K2207/15A01K2217/00A01K2217/05A01K2217/072A01K2227/101A01K2227/105A01K2267/01A23C9/20C07K14/4732C07K14/765C07K14/79C12N9/2462C12N9/6464C12N15/85C12N15/8509C12N2800/30C12N2830/008C12N2830/15C12N2830/42C12N2830/85C12N2840/44A01K67/0278
Inventor DEBOER, HERMANSTRIJKER, REINHEYNEKER, HERBERTPLANTENBURG, GERARDLEE, SANGPIEPER, FRANKKRIMPENFORT, PAUL
Owner PHARMING INTPROP BV
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