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Production of collagen in the milk of transgenic mammals

a technology of transgenic mammals and collagen, which is applied in the field of transgenic nonhuman mammals, can solve the problems of complex process by which collagen is expressed, processed and ultimately assembled into mature collagen fibers, and the mammalian cellular expression system is not entirely satisfactory for recombinant protein production, and it is unpredictable whether this technology could be extended to the expression of multimeric proteins requiring extensive post-translational modification and assembly, such as collagen

Inactive Publication Date: 2005-08-04
COHESION TECH +2
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  • Summary
  • Abstract
  • Description
  • Claims
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AI Technical Summary

Problems solved by technology

The process by which collagen is expressed, processed and ultimately assembled into mature collagen fibers is complex.
Mammalian cellular expression systems are not entirely satisfactory for production of recombinant proteins because of the expense of propagation and maintenance of such cells.
Although the feasibility of expressing several recombinant proteins in the milk of transgenic animals has been demonstrated, it was unpredictable whether this technology could be extended to the expression of an multimeric protein requiring extensive posttranslational modification and assembly, such as collagen.
Moreover, the large size of trimeric procollagen (>420 kDa) in comparison with other milk protein might have been expected to clog the secretory apparatus.
The health and even viability of transgenic animals expressing exogenous collagen in their mammary glands was also uncertain.
Inappropriate accumulation of collagen in the mammary gland might have impaired mammary gland development and resulted in cessation of lactation.
Even low levels of secondary expression in tissues other than the mammary gland could have resulted in lethal accumulation of collagen deposits.

Method used

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  • Production of collagen in the milk of transgenic mammals
  • Production of collagen in the milk of transgenic mammals
  • Production of collagen in the milk of transgenic mammals

Examples

Experimental program
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Effect test

example 1

Vectors for Collagen Expression

[0070] a. Proα1(l) Collagen cDNA Based Expression Vector

[0071] A plasmid vector was constructed containing the bovine αS1-casein 5′-flanking region including the proximal promoter operably linked to a cDNA sequence encoding the human proα(I) collagen gene, which is in turn operably linked to a 3′-flanking sequences derived from the human genomic collagen gene. The fusion product of the collagen cDNA [XbalSalI fragment] and the casein promoter at a Clal site yields the following nucleotide sequence:

[0072] The XbaI-EcoRI fragment (4363 bp) of a collagen cDNA was subcloned into the XbaI-EcoRI site of pGEM-7B, giving rise to the pGCOLXE plasmid (FIG. 1). This collagen cDNA fragment lacks the region encoding for the last 10 amino acids of the protein. The full-length coding region of the human proα1(I) collagen was reconstituted by fusing a 5.7 kb EcoRI fragment (Schnieke et al., Proc. Natl. Acad. Sci. USA 84, 764-768 (1987)) derived from the GC103 geno...

example 2

Expression of Constructs in Mammary Cell Culture

[0087] This example shows the feasibility of expressing, assembling and secreting an α1(I) procollagen in mammary gland cells, a cell type that does not normally express this gene. The cDNA and genomic vectors described above were transfected in their circular form into the mouse mammary epithelial cell line HC11 (Ball et al., EMBO 7, 2089-2095 (1988)). The cells were maintained as monolayers in RPMI 1640 (10% FCS, 2 mM L-Glutamine, 50 μg / ml gentamicin, 5 μg / ml insulin, 10 ng / ml EGF). 30-40 μg of each construct together and a hygromycin-resistance cotransfecting plasmid were complexed with 50 μg lipofectine (Gibco) and allowed to fuse with 2-3×106 cells. After 48 hr of growth in normal medium, selection medium was applied to select for stable transfectants. Two independent transfection rounds have been performed, and resistant colonies were scored after about 2 weeks (Table 1).

TABLE 1TRANSFECTION OF COLLAGEN EXPRESSION VECTORSNUMBER...

example 3

Production of Transgenic Animals Expressing α1(I) Procollagen

[0098] (1) Transgenesis

[0099] Collagen transgene fragments were excised from the three vectors described in Example 1 by NotI digestion and purified by 0.65% agarose gel electrophoresis and electroelution. (FIG. 2, panel A). Fertilized mouse eggs (CBA / BrAxC57B1 / 6) were microinjected (with 100-200 copies of the fragment) and transferred into pseudo-pregnant females as described (Hogan et al., supra). Total genomic DNA was prepared from a short segment of mouse tail to check for integration of the injected DNA. EcoRI-digested tail DNA was analyzed by Southern blotting (Sambrook et al., supra). The probe used to check for integration of the transgene was a 300 bp NcoI-NsiI fragment, spanning the region from −680 to −250 (relative to the major transcription start site) of the bovine αS1-casein gene. The probe was labeled with 32P using random hexanucleotide primers (Sambrook et al., supra). The numbers of transgenic mice con...

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Abstract

The invention provides transgenic nonhuman mammals capable secreting exogenous procollagen or collagen into their milk. The mammals are healthy and capable of producing procollagen or collagen at high levels, usually in trimeric form. Suitable transgenes for incorporation into the mammals are also provided.

Description

CROSS-REFERENCE TO RELATED APPLICATION [0001] This application is a continuation-in-part of U.S. Ser. No. 08 / 281,493 filed Jul. 27, 1994, which is incorporated by reference in its entirety for all purposes.TECHNICAL FIELD [0002] The invention relates generally to transgenic nonhuman mammals producing procollagen or collagen in their milk. BACKGROUND [0003] Collagen is a family of fibrous proteins present in all multicellular organisms. Collagen forms insoluble fibers having a high tensile strength. Collagen is the major fibrous element of skin, bone tendon cartilage, blood vessels and teeth. It is present in nearly all organs and serves to hold cells together in discrete units. Recently, collagen has assumed a therapeutic importance in reconstructive and cosmetic surgical procedures. [0004] The process by which collagen is expressed, processed and ultimately assembled into mature collagen fibers is complex. At least 28 distinct collagen genes have been reported, whose expression pro...

Claims

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Application Information

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Patent Type & Authority Applications(United States)
IPC IPC(8): A01K67/027A23C9/00A23C9/20A23J1/20A23J3/06C07K14/78C12N5/10C12N15/09C12N15/85
CPCA01K67/0278A01K2207/15A01K2217/00A01K2217/05A01K2227/10A01K2227/101C12N15/8509A01K2267/01A23C9/20A23J1/20A23J3/06C07K14/78C07K2319/02A01K2227/105A61P19/02A61P29/00
Inventor KARATZAS, COSTASPIEPER, FRANKDE WIT, INEKEBERG, RICHARDPLATENBURG, GERARDTOMAN, PAUL
Owner COHESION TECH
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