Transgenic non-human mammals expressing heterologous glycosyltransferase DNA sequences produce oligosaccharides and glycoproteins in their milk
First Claim
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.
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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.
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Citations
18 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.
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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.
- 1-3galactose β
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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:
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(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. - View Dependent Claims (6, 7, 8)
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9. A method of producing milk in a non-human, transgenic mammal, said method comprising the steps of:
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(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. - View Dependent Claims (10, 11, 12)
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13. A method for producing oligosaccharides and glycoproteins in the milk of a non-human transgenic mammal, the method comprising the steps of:
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(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. - View Dependent Claims (14, 15, 16, 17, 18)
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Specification