Transgenic non-human mammals expressing heterologous glycosyltransferase DNA sequences produce oligosaccharides and glycoproteins in their milk

• US 6,204,431 B1
• Filed: 09/05/1997
• Issued: 03/20/2001
• Est. Priority Date: 03/09/1994
• Status: Expired due to Fees

• Current Assignee: Abbott Laboratories, Ohio University, The University of Georgia Research Foundation, Inc.
• Original Assignee: Abbott Laboratories
• Inventors: Smith, David F., Pierce, James M., Prieto, Pedro A., Kopchick, John J., Cummings, Richard D., Moremen, Kelley W.
• Primary Examiner(s): Crouch, Deborah
• Application Number:
  US08/925,834
• Time in Patent Office: 1,292 Days
• Field of Search: 800/14, 800/15, 800/16, 800/17, 800/18, 800/4, 800/7, 800/24, 800/6, 800/25, 435/69.1
• US Class Current: 800/14
• CPC Class Codes: A01K 2217/05 Animals comprising random inserted nucleic acids (transgenic)
• CPC Class Codes: A01K 2227/10 Mammal
• CPC Class Codes: A01K 2227/105 Murine
• CPC Class Codes: A01K 2227/107 Rabbit
• CPC Class Codes: A01K 2267/01 Animal expressing industrially exogenous proteins
• CPC Class Codes: A01K 67/0275 Genetically modified vertebrates, e.g. transgenic
• CPC Class Codes: A23C 9/20 Dietetic milk products not covered by groups A23C9/12 - A23C9/18
• CPC Class Codes: C12N 15/8509 for producing genetically modified animals, e.g. transgenic
• CPC Class Codes: C12N 9/1048 Glycosyltransferases (2.4)
• CPC Class Codes: C12N 9/1051 Hexosyltransferases (2.4.1)
• CPC Class Codes: C12N 9/1077 Pentosyltransferases (2.4.2)

Abstract

The subject invention relates to methods of producing non-human transgenic mammals which produce various oligosaccharides and glycoconjugates in their milk. Additionally, the subject invention relates to the mammals themselves, the milk which they produce, compositions comprising the milk, fractions of the milk, and the purified oligosaccharides, as well as glyconjugates, present in the milk.

Claims

1. A non-human, transgenic mammal, wherein the genome of said mammal comprises at least one heterologous DNA sequence encoding an enzyme, wherein said enzyme is a glycosyltransferase operatively linked to a mammary gland-specific promoter, wherein expression of said at least one DNA sequence results in the production of oligosaccharides and glycoproteins in the milk of said mammal.

2. The non-human, transgenic mammal of claim 1, wherein said mammal is selected from the group consisting of a mouse, a rat, a rabbit, a pig, a goat, a sheep and a cow.

3. The non-human, transgenic mammal of claim 1, wherein said glycosyltransferase is selected from the group consisting of a fucosyltransferase, a galactosyltransferase, an acetylase, a glucoronyltransferase, a gluconylepimerase, a sialyltransferase, a mannosyltransferase, a sulfotransferase, a S-acetylgalactosaminyltransferase and a N-acetylglucosaminyltransferase.

4. The non-human, transgenic mammal of claims, wherein said oligosaccharides are selected from the group consisting of galactose α

1-3galactose β

1-4 glucose, 2′

fucosyllactose, 3′

fucosyllactose, lacto-N-neo-tetraose, lacto-N-tetraose, lacto-N-fucopentaose, a sialylated derivative of lacto-N-fucopentaose, lacto-N-fucopentaose II, a sialylated derivative of lacto-N-fucopentaose II, lacto-N-fucopentaose III, a sialylated derivative of lacto-N-fucopentaose III, lacto-N-fucopentaose IV, a sialylated derivative of lacto-N-fucopentaose IV, lacto-N-fucopentaose V, a sialylated derivative of lacto-N-fucopentaose V, lacto-N-di-fucopentaose I, a sialylated derivative of lacto-N-di-fucopentaose, lacto-N-di-fucopentaose II, a sialylated derivative of lacto-N-di-fucopentaose II, lacto-N-hexaose, a fucosylated derivative of lacto-N-hexaose, sialyltetrasaccharide a, a fucosylated derivative of sialyltetrasaccharide a, sialyltetrasaccharide b, a fucosylated derivative of sialyltetrasaccharide b, sialyltetrasaccharide c and a fucosylated derivative of sialyltetrasaccharide c.

5. A method for producing a non-human, transgenic mammal whose somatic and germ cells contain at least one transgene, wherein expression of said at least one transgene results in the production of oligosaccharides and glycoproteins in the milk of said mammal, the method comprising the steps of:

(a) preparing at least one transgene, said at least one 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 an enzyme;

(b) introducing said at least one transgene into a non-human, mammalian embryo, and transferring the resulting embryo into a recipient female;

(c) identifying at least one female offspring, where expression of said at least one transgene results in the production of at least one enzyme which then catalyzes the production of oligosaccharides and glycoproteins in the milk of said mammal, wherein said at least one enzyme is a glycosyltransferase.

6. The method of claim 5, wherein said non-human, transgenic mammal is selected from the group consisting of a mouse, a rat, a rabbit, a pig, a goat, a sheep and a cow.

7. The method of claim 5, wherein said glycosyltransferase is selected from the group consisting of a fucosyltransferase, a galactosyltransferase, an acetylase, a glucoronyltransferase, a gluconylepimerase, a sialyltransferase, a mannosyltransferase, a sulfotransferase, a β

-acetylgalactosaminyltransferase and a N-acetylglucosaminyltransferase.

8. The method of claim 5, wherein said oligosaccharides are selected from the group consisting of galactose α

1-3 galactose β

1-4 glucose, 2′

fucosyllactose, 3′

fucosyllactose, lacto-N-neo-tetraose, lacto-N-tetraose, lacto-N-fucopentaose, a sialylated derivative of lacto-N-fucopentaose, lacto-N-fucopentaose II, a sialylated derivative of lacto-N-fucopentaose II, lacto-N-fucopentaose III, a sialylated derivative of lacto-N-fucopentaose III, lacto-N-fucopentaose IV, a sialylated derivative of lacto-N-fucopentaose IV, lacto-N-fucopentaose V, a sialylated derivative of lacto-N-fucopentaose V, lacto-N-di-fucopentaose I, a sialylated derivative of lacto-N-di-fucopentaose, lacto-N-di-fucopentaose II, a sialylated derivative of lacto-N-di-fucopentaose II, lacto-N-hexaose, a fucosylated derivative of lacto-N-hexaose, sialyltetrasaccharide a, a fucosylated derivative of sialyltetrasaccharide a, sialyltetrasaccharide b, a fucosylated derivation of sialyltetrasaccharide b, sialyltetrasaccharide c and a fucosylated derivative of sialyltetrasaccharide c.

9. A method of producing milk in a non-human, transgenic mammal, said method comprising the steps of:

(a) preparing at least one transgene, said at least one transgene comprising is operable association

     1) a promoter functional in mammary secretory cells,

     2) a DNA sequence encoding an enzyme, wherein said enzyme is a glycosyltransferase and wherein expression of said at least one transgene results in production of oligosaccharides and glycoproteins in said milk of said mammal;

(b) inserting said at least one transgene into said non-human, transgenic mammal;

(c) milking said non-human, transgenic mammal in order to obtain said milk, wherein said milk comprises said oligosaccharides and glycoproteins.

10. The method of claim 9, wherein said non-human, transgenic mammal is selected from the group consisting of a mouse, a rat, a rabbit, a pig, a goat, a sheep and a cow.

11. The method of claim 9, wherein said glycosyltransferase is selected from the group consisting of a fucosyltransferase, a galactosyltransferase, an acetylase, a glucoronyltransferase, a gluconylepimerase, a sialyltransferase, a mannosyltransferase, a sulfotransferase, a β

-acetylgalactosaminyltransferase and a N-acetylglucosaminyltransferase.

12. The method of claim 9, wherein said oligosaccharides are selected from the group consisting of galactose α

1-3 galactose β

1-4 glucose, 2′

fucosyllactose, 3′

fucosyllactose, lacto-N-neo-tetraose, lacto-N-tetraose, lacto-N-fucopentaose, a sialylated derivative of lacto-N-fucopentaose, lacto-N-fucopentaose II, a sialylated derivative of lacto-N-fucopentaose II, lacto-N-fucopentaose III, a sialylated derivative of lacto-N-fucopentaose III, lacto-N-fucopentaose IV, a sialylated derivative of lacto-N-fucopentaose IV, lacto-N-fucopentaose V, a sialylated derivative of lacto-N-fucopentaose V, lacto-N-di-fucopentaose I, a sialylated derivative of lacto-N-di-fucopentaose, lacto-N-di-fucopentaose II, a sialylated derivative of lacto-N-di-fucopentaose II, lacto-N-hexaose, a fucosylated derivative of lacto-N-hexaose, sialyltetrasaccharide a, a fucosylated derivative of sialyltetrasaccharide a, sialyltetrasaccharide b, a fucosylated derivation of sialyltetrasaccharide b, sialyltetrasaccharide c and a fucosylated derivative of sialyltetrasaccharide c.

13. A method for producing oligosaccharides and glycoproteins in the milk of a non-human transgenic mammal, the method comprising the steps of:

(a) preparing at least one transgene, said at least one 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 an enzyme, wherein said enzyme is a glycosyltransferase;

(b) introducing said at least one transgene into a non-human, mammalian embryo, and transferring the resulting embryo into a recipient female;

(c) identifying at least one female offspring, where expression of said at least one transgene results in the production of at least one enzyme which then catalyzes the production of said oligosaccharides and glycoproteins in the milk of said mammal, wherein said at least one enzyme is a glycosyltransferase;

(d) milking said mammal; and

(e)isolating said oligosaccharides and glycoproteins from said milk.

14. The method of claim 13, wherein said non-human transgenic mammal is selected from the group consisting of a mouse, a rat, a rabbit, a pig, a goat, a sheep and a cow.

15. The method of claim 14, wherein said non-human transgenic mammal is a cow.

16. The method of claim 13 wherein said glycosyltransferase is selected from the group consisting of glycosyltransferase is selected from the group consisting of a fucosyltransferase, a galactosyltransferase, an acetylase, a glucoronyltransferase, a gluconylepimerase, a sialyltransferase, a mannosyltransferase, a sulfotransferase, a B-acetylgalactosaminyltransferase and a N-acetylglucosaminyl-transferase.

17. The method of claim 13, wherein said oligosaccharides are selected from the group consisting of galactose a 1-3 galactose β

1-4 glucose, 2′

fucosyllactose, 3′

fucosyllactose, lacto-N-neo-tetraose, lacto-N-tetraose, lacto-N-fucopentaose, a sialylated derivative of lacto-N-fucopentaose, lacto-N-fucopentaose II, a sialylated derivative of lacto-N-fucopentaose II, lacto-N-fucopentaose III, a sialylated derivative of lacto-N-fucopentaose III, lacto-N-fucopentaose IV, a sialylated derivative of lacto-N-fucopentaose IV, lacto-N-fucopentaose V, a sialylated derivative of lacto-N-fucopentaose V, lacto-N-di-fucopentaose I, a sialylated derivative of lacto-N-di-fucopentaose, lacto-N-di-fucopentaose II, a sialylated derivative of lacto-N-di-fucopentaose II, lacto-N-hexaose, a fucosylated derivative of lacto-N-hexaose, sialyltetrasaccharide a, a fucosylated derivative of sialyltetrasaccharide a, sialyltetrasaccharide b, a fucosylated derivation of sialyltetrasaccharide b, sialyltetrasaccharide c and a fucosylated derivative of sialyltetrasaccharide c.

18. The method of claim 13, wherein said glycoproteins are human milk proteins.