# US 5,700,671 A · Methods of making transgenic animals producing oligosaccharides and glycoproteins

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- Number: US 5,700,671 A
- Document ID: US5700671A
- Status: Granted
- Kind: A
- Application number: 08/434,151
- Filed: 1995-05-02
- Granted: 1997-12-23
- Earliest claimed: 1994-03-09
- Assignees: Abbott Laboratories
- Inventors: Pedro Antonio Prieto; David Fletcher Smith; Richard Dale Cummings; John Joseph Kopchick; Pradip Mukerji; Kelley Wilson Moremen; James Michael Pierce
- Web page: https://patentgrep.com/patent/US5700671A

## CPC

- C12N 9/1051: {Hexosyltransferases (2.4.1)}
- A01K 67/0278: Knock-in vertebrates, e.g. humanised vertebrates
- C12N 15/8509: {for producing genetically modified animals, e.g. transgenic}
- A01K 2207/15: Humanized animals
- A01K 2217/00: Genetically modified animals
- A01K 2217/05: Animals comprising random inserted nucleic acids (transgenic)
- A01K 2227/10: Mammal
- A01K 2227/105: Murine
- A01K 2267/01: Animal expressing industrially exogenous proteins
- C12N: MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA (microbiological testing media C12Q1/00)
- A01K: ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS

## Abstract

The invention relates to transgenic non-human mammals characterized in that the genome of said mammals contain at least one heterologous gene encoding for the production of heterologous catalytic entity selected from the group consisting of enzymes and antibodies, and wherein said catalytic entity produces a second heterologous product in the milk of said mammal. Especially useful in the practice of the invention are human glycosyltransferases and transgenic sheep, goats and cows. The heterologous product includes oligosaccharides and glycoconjugates.

## Claims

### Claim 1

A method for producing a non-human transgenic mammal whose somatic and germ cells contain a transgene, wherein expression of the transgene results in the production of oligosaccharides or glycoproteins in the milk of said mammal, the method comprising the steps of:
(a) preparing a transgene, said transgene comprising in operable association 1) at least one expression regulatory sequence functional in mammary secretory cells, 2) a DNA sequence encoding a signal sequence functional in mammary secretory cells, and 3) a DNA sequence encoding a heterologous glycosyltransferase;
(b) introducing said transgene into a non-human mammalian embryo, and transferring the resulting embryo into a recipient female; and
(c) identifying at least one female offspring where expression of said transgene results in the production of a heterologous glycosyltransferase which then catalyzes the production of oligosaccharides or glycoproteins in the milk of said mammal.

### Claim 2 (depends on 1)

The method according to claim 1 wherein said mammal is selected from the group consisting of mice, rats, rabbits, pigs, goats, sheep, and cows.

### Claim 3 (depends on 1)

The method according to claim 1 wherein said glycosyltransferase is .alpha.-1,2-fucosyltransferase.

### Claim 4 (depends on 1)

The method according to claim 1 wherein said oligosaccharides are selected from the group consisting of lactose, 2-fucosyllactose, lacto-N-tetraose, lacto-N-neotetraose, lacto-N-fucopentaose I, lacto-N-fucopentaose II, lacto-N-fucopentaose III, lacto-N-difucopentaose I, sialyllactose, 3-sialyllactose, sialyltetrasaccharide a, sialyltetrasaccharide b, sialyltetrasaccharide c, disialyltetrasaccharide and sialyl lacto-N-fucopentaose.

### Claim 5 (depends on 1)

The method according to claim 1 wherein said glycoproteins are human milk proteins.

### Claim 6 (depends on 5)

The method according to claim 5 wherein said human milk proteins are selected from the group consisting of secretory immunoglobulins, lysozyme, lactoferrin, kappa-casein, alpha-lactalbumin, beta-lactalbumin, lactoperoxidase and bile salt stimulated lipase.

### Claim 7

A method for producing a non-human transgenic mammal whose somatic and germ cells contain a transgene, wherein expression of the transgene results in the production of oligosaccharides or glycoproteins in the milk of said mammal, the method comprising the steps of:
(a) preparing a transgene which encodes a heterologous glycosyltransferase;
(b) methylating said transgene;
(c) introducing the transgene of step (b) into fertilized eggs of a non-human mammal such that the transgene is integrated into the genome of the fertilized eggs;
(d) culturing the eggs which result from step (c) to pre-implantation embryos;
(e) removing at least one cell from each of said pre-implantation embryos, and lysing at least one cell;
(f) incubating DNA molecules released in step (e) with a restriction endonuclease which cleaves said methylated transgene but does not cleave an unmethylated form of the transgene;
(g) identifying those embryos of step (e) which contain the methylated transgene integrated into their genome;
(h) implanting at least one of the embryos identified in step (g) into a recipient female; and
(i) developing to term said non-human transgenic mammal, where the expression of said transgene results in the production of oligosaccharides or glycoproteins in its milk.

### Claim 8 (depends on 7)

The method according to claim 7 wherein said oligosaccharide is 2'-fucosyllactose.