U.S. patent application number 10/358037 was filed with the patent office on 2003-11-13 for transgenically produced non-secreted proteins.
Invention is credited to Chen, Li-How, DiTullio, Paul, Meade, Harry.
Application Number | 20030213003 10/358037 |
Document ID | / |
Family ID | 21903106 |
Filed Date | 2003-11-13 |
United States Patent
Application |
20030213003 |
Kind Code |
A1 |
Meade, Harry ; et
al. |
November 13, 2003 |
Transgenically produced non-secreted proteins
Abstract
The invention provides a method of making and secreting a
non-secreted protein. The method includes expressing the protein
from a nucleic acid construct which includes: (a) a mammary
epithelial specific promoter; (b) a milk protein specific signal
sequence which can direct the secretion of a protein; (c)
optionally, a sequence which encodes a sufficient portion of the
amino terminal coding region of a secreted protein to allow
secretion in the milk of a transgenic mammal, of the non-secreted
protein; and (d) a sequence which encodes a non-secreted protein,
wherein elements (a), (b), optionally (c), and (d) are preferably
operatively linked in the order recited. Both glutamic acid
decarboxylase (GAD) and myelin basic protein (MBP), which are
cytoplasmic proteins, have been produced by the methods of the
present invention. The invention also provides methods for treating
diabetes and multiple sclerosis using proteins produced by the
methods of the present invention.
Inventors: |
Meade, Harry; (Newton,
MA) ; Chen, Li-How; (Acton, MA) ; DiTullio,
Paul; (Northboro, MA) |
Correspondence
Address: |
GTC BIOTHERAPEUTICS, INC.
175 CROSSING BOULEVARD, SUITE 410
FRAMINGHAM
MA
01702
US
|
Family ID: |
21903106 |
Appl. No.: |
10/358037 |
Filed: |
February 4, 2003 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10358037 |
Feb 4, 2003 |
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09028551 |
Feb 24, 1998 |
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6528699 |
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60038998 |
Feb 25, 1997 |
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Current U.S.
Class: |
800/7 |
Current CPC
Class: |
C07K 14/4713 20130101;
A61K 38/00 20130101; A01K 67/0275 20130101; A01K 2227/102 20130101;
A01K 2227/105 20130101; A01K 2217/05 20130101; C12N 2830/008
20130101; A01K 2217/00 20130101; C12N 9/88 20130101; C12N 15/8509
20130101; A61P 37/06 20180101; A61P 29/00 20180101; A01K 67/0278
20130101; A01K 2267/01 20130101; A01K 2207/15 20130101; A61P 3/10
20180101; C07K 2319/036 20130101 |
Class at
Publication: |
800/7 |
International
Class: |
C12P 021/00 |
Claims
1. A method of making and secreting a non-secreted protein
comprising expressing the protein from a nucleic acid construct
which comprises: (a) a mammary epithelial specific promoter; (b) a
milk protein specific signal sequence which can direct the
secretion of a non-secreted protein; (c) a sequence which encodes a
sufficient portion of the amino terminal coding region of a
secreted protein to allow secretion in the milk of a transgenic
mammal of a non-secreted protein; and (d) a sequence which encodes
a non-secreted protein, wherein elements (a), (b),(c), and (d) are
preferably operatively linked in the order recited, thereby
producing the non-secreted protein in the milk of the transgenic
mammal.
2. The method of claim 1, wherein the mammary epithelial specific
promoter is the .beta.-casein promoter sequence.
3. The method of claim 1, wherein the milk protein specific signal
sequence is the .beta.-casein signal sequence.
4. The method of claim 1, wherein the non-secreted protein-coding
sequence is of a human origin.
5. The method of claim 1, wherein the non-secreted protein-coding
sequence codes for a truncated, nuclear, or a cytoplasmic
polypeptide.
6. The method of claim 1, wherein the non-secreted protein-coding
sequence codes for glutamic acid decarboxylase or myelin basic
protein.
7. The method of claim 1, wherein the non-secreted protein is a
mutant protein which lacks a biological activity of the wild type
protein.
8. The method of claim 1, wherein the transgenic mammal is selected
from the group consisting of sheep, mice, pigs, cows and goats.
9. An isolated nucleic acid construct which comprises: (a) a
mammary epithelial specific promoter; (b) a milk protein specific
signal sequence which can direct the secretion of a protein; (c) a
sequence which encodes a sufficient portion of the amino terminal
coding region of a secreted protein to allow secretion in the milk
of a transgenic mammal of a non-secreted protein; and (d) a
sequence which encodes a non-secreted protein, wherein elements
(a), (b), (c), and (d) are preferably operatively linked in the
order recited, thereby producing the non-secreted protein in the
milk of the transgenic mammal.
10. The method of claim 9, wherein the mammary epithelial specific
promoter sequence is the .beta.-casein promoter sequence.
11. The method of claim 9, wherein the milk protein specific signal
sequence is the .beta.-casein signal sequence.
12. The method of claim 9, wherein the non-secreted protein-coding
sequence is of a human origin.
13. The method of claim 9, wherein the non-secreted protein-coding
sequence codes for a truncated, nuclear, or a cytoplasmic
polypeptide.
14. The method of claim 9, wherein the non-secreted protein-coding
sequence codes for glutamic acid decarboxylase or myelin basic
protein.
15. The method of claim 9, wherein the non-secreted protein is a
mutant protein which lacks a biological activity of the wild type
protein.
16. A method for providing a non-secreted protein in the milk of a
transgenic mammal, comprising obtaining milk from a transgenic
mammal having introduced into its germline a nucleic acid construct
comprising a heterologous non-secreted protein-coding sequence
operatively linked to a milk protein specific signal sequence and a
mammary epithelial specific promoter sequence that result in the
preferential expression of the protein-coding sequence in mammary
gland epithelial cells, thereby secreting the heterologous
non-secreted protein in the milk of the mammal
17. The method of claim 16, wherein the non-secreted protein is
inactive.
18. The method of claim 16, wherein the transgenic mammal is
selected from the group consisting of sheep, mice, pigs, cows and
goats.
19. The method of claim 16, wherein the mammary epithelial specific
promoter is selected from the group consisting of the beta
lactoglobulin promoter, whey acid protein promoter, .beta.-casein
promoter and the lactalbumin promoter.
20. The method of claim 16, wherein the milk protein specific
signal sequence is .beta.-casein signal sequence.
21. The method of claim 16, wherein the non-secreted protein-coding
sequence is of a human origin.
22. The method of claim 16, wherein the non-secreted protein-coding
sequence codes for a truncated, nuclear, or a cytoplasmic
polypeptide.
23. The method of claim 16, wherein the non-secreted protein-coding
sequence codes for glutamic acid decarboxylase or myelin basic
protein.
24. The method of claim 16, wherein the non-secreted protein-coding
sequence codes for an inactive form of a glutamic acid
decarboxylase.
25. The method of claim 16, wherein the non-secreted polypeptide is
purified from the milk of a transgenic mammal.
26. A method of inducing tolerance in a subject to an antigen,
comprising: providing a tolerogen expressed in a transgenic mammal
which comprises the antigen; and administering the tolerogen to the
subject in an amount sufficient to induce tolerance to the
antigen.
27. The method of claim 26, wherein the tolerogen is administered
orally to the subject in the milk of a transgenic mammal.
28. The method of claim 26, wherein the tolerogen is an inactive
protein.
29. The method of claim 26, wherein the tolerogen is a non-secreted
protein.
30. The method of claim 26, wherein the tolerogen is a protein
antigen fused to all or part of a secreted protein.
31. The method of claim 26, wherein the antigen is an antigen which
is characteristic of an autoimmune disorder selected from the group
consisting of diabetes, lupus, multiple sclerosis and rheumatoid
arthritis.
32. The method of claim 26, wherein the subject is at risk of
developing, or has, an anutoimmune disorder selected from the group
consisting of diabetes, lupus, multiple sclerosis and rheumatoid
arthritis.
33. A method of treating insulin-dependent diabetes mellitus (IDDM)
in a subject, comprising administering to the subject
therapeutically effective amount of a transgenically produced
tolerogen which comprises glutamic acid decarboxylase, or an
effective amount of a fusion protein which comprises glutamic acid
decarboxylase.
34. The method of claim 33, wherein the subject is orally
administered milk from a transgenic mammal which expresses the
transgenically produced tolerogen which comprises glutamic acid
decarboxylase, or an effective amount of a fusion protein which
comprises glutamic acid decarboxylase.
35. The method of claim 33, wherein the transgenically produced
tolarogen is in inactive form.
36. A method for treating multiple sclerosis in a subject,
comprising administering to the subject therapeutically effective
amount of a transgenically produced tolerogen which comprises
myelin basic protein, or an effective amount of a fusion protein
which comprises myelin basic protein.
37. The method of claim 36, wherein the subject is orally
administered milk from a transgenic mammal which expresses the
transgenically produced tolerogen which comprises myelin basic
protein, or an effective amount of a fusion protein which comprises
myelin basic protein.
38. The method of claim 36, wherein the transgenically produced
tolarogen is in inactive form.
39. A therapeutic composition which comprises a therapeutically
effective amount of the transgenically produced myelin basic
protein and a pharmaceutically-acceptable carrier or diluent.
40. The method of claim 39, wherein the pharmaceutically-acceptable
carrier or diluent comprises the milk of a transgenic mammal.
41. A fusion protein which comprises: a non-secreted protein; a
milk protein specific signal sequence which directs the secretion
of the protein; and a sequence which encodes a sufficient portion
of the amino terminal coding region of a secreted protein to allow
secretion in the milk of a transgenic mammal, of the non-secreted
protein.
42. A method of inducing tolerance in a transgenic mammal to an
antigen, comprising expressing a tolerogen in the milk of the
transgenic mammal at a level sufficient to induce tolerance.
43. The method of claim 42, wherein the antigen is selected from
the group consisting of a xenoantigen, an alloantigen and an
autoantigen.
44. The method of claim 42, wherein the transgenic mammal is a
transgenic dairy mammal.
45. The method of claim 44, wherein the transgenic dairy mammal is
selected from the group consisting of goats, sheep and cows.
46. The method of claim 42, wherein the tolerogen is a non-secreted
protein.
47. The method of claim 42, wherein the antigen is an antigen which
is characteristic of an autoimmune disorder selected from the group
consisting of diabetes, lupus, multiple sclerosis, and rheumatoid
arthritis.
48. A transgenically produced non-secreted polypeptide, wherein the
polypeptide is secreted.
49. The transgenically produced non-secreted polypeptide of claim
48, wherein the transgenically produced non-secreted polypeptide is
secreted into the milk of a transgenic mammal.
50. The transgenically produced non-secreted polypeptide of claim
48, wherein the transgenically produced non-secreted polypeptide is
selected from the group consisting of a truncated, nuclear, and
cytoplasmic polypeptide.
51. The transgenically produced non-secreted polypeptide of claim
51, wherein the cytoplasmic polypeptide is glutamic acid
decarboxylase or myelin basic protein.
52. The transgenically produced non-secreted polypeptide of claim
51, wherein glutamic acid decarboxylase is expressed in an inactive
form.
53. The transgenically produced non-secreted polypeptide of claim
49, wherein the transgenic mammal is selected from the group
consisting of sheep, mice, pigs, cows and goats.
Description
[0001] This application claims the benefit of a previously filed
Provisional Application No. 60/038,998, filed Feb. 25, 1997, which
is hereby incorporated by reference.
FIELD OF THE INVENTION
[0002] This invention relates to the production and secretion of
proteins which are not ordinarily secreted.
BACKGROUND OF THE INVENTION
[0003] A growing number of recombinant proteins are being developed
for therapeutic and diagnostic applications. However, many of these
proteins may be difficult or expensive to produce in a functional
form and/or in the required quantities using conventional methods.
Conventional methods involve inserting the gene responsible for the
production of a particular protein into host cells such as
bacteria, yeast, or mammalian cells, e.g., COS cells, and then
growing the cells in culture media. The cultured cells then
synthesize the desired protein. Traditional bacteria or yeast
systems may be unable to produce many complex proteins in a
functional form. While mammalian cells can reproduce complex
proteins, they are generally difficult and expensive to grow, and
often produce only mg/L quantities of protein. In addition,
non-secreted proteins are relatively difficult to purify from
procaryotic or mammalian cells as they are not secreted into the
culture medium.
SUMMARY OF THE INVENTION
[0004] In general, the invention features, a method of making and
secreting a protein which is not normally secreted (a non-secreted
protein). The method includes expressing the protein from a nucleic
acid construct which includes:
[0005] (a) a promoter, e.g., a mammary epithelial specific
promoter, e.g., a milk protein promoter;
[0006] (b) a signal sequence which can direct the secretion of a
protein, e.g. a signal sequence from a milk specific protein;
[0007] (c) optionally, a sequence which encodes a sufficient
portion of the amino terminal coding region of a secreted protein,
e.g., a protein secreted into milk, to allow secretion, e.g., in
the milk of a transgenic mammal, of the non-secreted protein;
and
[0008] (d) a sequence which encodes a non-secreted protein,
[0009] wherein elements (a), (b), optionally (c), and (d) are
preferably operatively linked in the order recited.
[0010] In preferred embodiments: elements a, b, c (if present), and
d are from the same gene; the elements a, b, c (if present), and d
are from two or more genes.
[0011] In preferred embodiments the secretion is into the milk of a
transgenic mammal.
[0012] In preferred embodiments: the signal sequence is the
.beta.-casein signal sequence; the promoter is the .beta.-casein
promoter sequence.
[0013] In preferred embodiments the non-secreted protein-coding
sequence: is of human origin; codes for a truncated, nuclear, or a
cytoplasmic polypeptide; codes for glutamic acid decarboxylase or
myelin basic protein.
[0014] In preferred embodiments, the protein is a mutant protein
which lacks a biological activity of the wild type protein.
[0015] In another aspect, the invention features, a nucleic acid
construct, preferably an isolated nucleic acid construct, which
includes:
[0016] (a) a promoter, e.g., a mammary epithelial specific
promoter, e.g., a milk protein promoter;
[0017] (b) a signal sequence which can direct the secretion of a
protein, e.g., a signal sequence from a milk specific protein;
[0018] (c) optionally, a sequence which encodes a sufficient
portion of the amino terminal coding region of a secreted protein,
e.g., a protein secreted into milk, to allow secretion, e.g., in
the milk of a transgenic mammal, of the non-secreted protein;
and
[0019] (d) a sequence which encodes a non-secreted protein,
[0020] wherein elements (a), (b), optionally (c), and (d) are
preferably coupled in the order recited.
[0021] In preferred embodiments: elements a, b, c (if present), and
d are from the same gene; the elements a, b, c (if present), and d
are from two or more genes.
[0022] In preferred embodiments the secretion is into the milk of a
transgenic mammal.
[0023] In preferred embodiments: the signal sequence is the
.beta.-casein signal sequence; the promoter is the .beta.-casein
promoter sequence.
[0024] In preferred embodiments the non-secreted protein-coding
sequence: is of human origin; codes for a truncated, nuclear, or a
cytoplasmic polypeptide; codes for glutamic acid decarboxylase or
myelin basic protein.
[0025] In preferred embodiments, the protein is inactive, e.g., it
is a mutant protein which lacks a biological activity of the wild
type protein.
[0026] In another aspect, the invention features, a method for
providing a non-secreted protein, e.g., a heterologous non-secreted
polypeptide, in the milk, of a transgenic mammal. The method
includes obtaining milk from a transgenic mammal having introduced
into its germline a nucleic acid construct described herein, e.g.,
a heterologous non-secreted protein-coding sequence operatively
linked to a signal and a promoter sequence that result in the
preferential expression of the protein-coding sequence in mammary
gland epithelial cells, thereby secreting the heterologous
non-secreted polypeptide in the milk of the mammal.
[0027] In preferred embodiments, the protein is inactive, e.g., it
is a mutant protein which lacks a biological activity of the wild
type protein.
[0028] In preferred embodiments the transgenic mammal is selected
from the group consisting of sheep, mice, pigs, cows and goats. The
preferred transgenic mammal is a goat.
[0029] In preferred embodiments, the promoter is selected from the
group consisting of the beta lactoglobulin promoter, whey acid
protein promoter, .beta.-casein promoter and the lactalbumin
promoter. The preferred promoter is the .beta.-casein promoter.
[0030] In preferred embodiments, the signal sequence is
.beta.-casein signal sequence.
[0031] In preferred embodiments, the non-secreted protein-coding
sequence: is of human origin; codes for a truncated, nuclear, or a
cytoplasmic polypeptide; codes for glutamic acid decarboxylase or
myelin basic protein; codes for an inactive form of glutamic acid
decarboxylase.
[0032] In preferred embodiments, the protein is fused to other
sequences, e.g., to one or both of: a signal sequence, e.g., the
signal sequence of .beta.-casein and/or a sequence which encodes a
sufficient portion of the amino terminal coding region of a
secreted protein, e.g., a protein secreted into milk, to allow
secretion, e.g., in the milk of a transgenic mammal, of the
non-secreted protein.
[0033] In other preferred embodiments the non-secreted polypeptide
is purified from the milk of a transgenic mammal.
[0034] In another aspect, the invention features, a method of
inducing tolerance in a subject to an antigen. The antigen can be a
xenoantigen, an alloantigen or a autoantigen. The antigen can be a
protein, e.g., a non-secreted protein.
[0035] The method includes:
[0036] providing a tolerogen expressed in a transgenic mammal which
includes the antigen;
[0037] and administering the tolerogen to the subject in an amount
sufficient to induce tolerance to said protein antigen.
[0038] In preferred embodiments: the tolerogen is administered,
preferably orally, to the subject in the milk of a transgenic
mammal, e.g., a transgenic dairy mammal, e.g., a goat, sheep, or
cow. Other mammals, e.g., pigs, can also be used.
[0039] In preferred embodiments, the tolerogen is or includes: a
protein, e.g., an inactive protein; a non-secreted protein; a
fusion of a non-secreted protein and another peptide sequence,
e.g., a protein antigen fused to all or part of a secreted
protein.
[0040] Preferably, the transgenically produced product, is in
inactive form, e.g., it is a mutant which lacks an activity of the
wild type protein.
[0041] In preferred embodiments, the autoantigen is fused to other
sequences, e.g., to one or both of: a signal sequence, e.g., the
signal sequence of .beta.-casein and/or a sequence which encodes a
sufficient portion of the amino terminal coding region of a
secreted protein, e.g. a protein secreted into milk, to allow
secretion, e.g., in the milk of a transgenic mammal, of the
non-secreted protein.
[0042] In preferred embodiments, the antigen is: an antigen which
is characteristic of an autoimmune disorder, e.g., diabetes, lupus,
multiple sclerosis, rheumatoid arthritis; GAD; MBP; a transcription
factor.
[0043] In preferred embodiments the subject is at risk of
developing, or has, an anutoimmune disorder, e.g., diabetes, lupus,
multiple sclerosis, of rheumatoid arthritis.
[0044] In yet another aspect, the invention features, a method of
treating insulin-dependent diabetes mellitus (IDDM) in a subject.
The method includes administering to the subject therapeutically
effective amount of a transgenically produced tolerogen which
includes an autoantigen, e.g., glutamic acid decarboxylase, or an
effective amount of a fusion protein which includes an autoantigen,
e.g., glutamic acid decarboxylase.
[0045] In preferred embodiments, the subject is orally administered
milk from a transgenic mammal which expresses the transgenically
produced tolerogen which includes an autoantigen, e.g., glutamic
acid decarboxylase, or an effective amount of a fusion protein
which includes an autoantigen, e.g., glutamic acid decarboxylase.
Preferably, the transgenically produced tolerogen, e.g., glutamic
acid decarboxylase is in inactive form, e.g., it is a mutant which
lacks an activity of the wild type protein.
[0046] In preferred embodiments, the autoantigen is fused to other
sequences, e.g., to one or both of: a signal sequence, e.g., the
signal sequence of .beta.-casein and/or a sequence which encodes a
sufficient portion of the amino terminal coding region of a
secreted protein, e.g., a protein secreted into milk, to allow
secretion, e.g., in the milk of a transgenic mammal, of the
non-secreted protein.
[0047] In still another aspect, the invention features treating
multiple sclerosis in a subject. The method includes administering
to the subject therapeutically effective amount of a transgenically
produced tolerogen which includes an autoantigen, e.g., myelin
basic protein, or an effective amount of a fusion protein which
includes an autoantigen, e.g., myelin basic protein (MBP).
[0048] In preferred embodiments, the subject is orally administered
milk from a transgenic mammal which expresses the transgenically
produced myelin basic protein, or an effective amount of a fusion
protein which includes an autoantigen, e.g., myelin basic protein.
Preferably, the transgenically produced tolerogen, e.g., MBP is in
inactive form, e.g., it is a mutant which lacks an activity of the
wild type protein.
[0049] In preferred embodiments, the autoantigen is fused to other
sequences, e.g., to one or both of: a signal sequence, e.g., the
signal sequence of .beta.-casein and/or a sequence which encodes a
sufficient portion of the amino terminal coding region of a
secreted protein, e.g. a protein secreted into milk, to allow
secretion, e.g., in the milk of a transgenic mammal, of the
non-secreted protein.
[0050] In another aspect, the invention features, a therapeutic
composition which includes a therapeutically effective amount of
the transgenically produced glutamic acid decarboxylase and a
pharmaceutically-acceptable carrier or diluent, e.g., the milk of a
transgenic mammal. Preferably, the transgenically produced glutamic
acid decarboxylase is in inactive form.
[0051] In another aspect, the invention features, a therapeutic
composition which includes a therapeutically effective amount of
the transgenically produced myelin basic protein and a
pharmaceutically-acceptable carrier or diluent, e.g., the milk of a
transgenic mammal.
[0052] In another aspect, the invention features, a fusion protein
which includes:
[0053] a non-secreted protein, e.g., an inactive non-secreted
protein,
[0054] a signal sequence which directs the secretion of the
protein, e.g., a signal from a secreted protein; and
[0055] (optionally) a sequence which encodes a sufficient portion
of the amino terminal coding region of a secreted protein, e.g., a
protein secreted into milk, to allow secretion, e.g., in the milk
of a transgenic mammal, of the non-secreted protein.
[0056] In preferred embodiments, the fusion protein includes: the
signal sequence and sufficient residues from the amino terminal end
of a secreted protein, to allow secretion of the fusion protein in
milk, fused to a non-secreted protein. Preferred embodiments
include the signal sequence and sufficient residues from the amino
terminal end of beta casein, e.g., goat beta casein, fused to a non
secreted protein.
[0057] In another aspect, the invention features, a composition
which includes a tolerogen or other trangenic protein described
herein and milk. In preferred embodiments, the milk is the milk of
a transgenic mammal which secretes the tolerogen or other
protein.
[0058] In another aspect, the invention features, a method of
inducing tolerance in a transgenic mammal to an antigen. The
antigen can be a xenoantigen, an alloantigen or a autoantigen. The
antigen can be a protein, e.g., a non-secreted protein, e.g., an
inactive protein.
[0059] The method includes:
[0060] expressing a tolerogen in the milk of the transgenic mammal
at a level sufficient to induce tolerance.
[0061] In preferred embodiments: the transgenic mammal is a
transgenic dairy mammal, e.g., a goat, sheep, or cow. Other
mammals, e.g., pigs, can also be used.
[0062] In preferred embodiments the tolerogen is or includes: a
protein; a protein described herein; a non-secreted protein; a
fusion of a non-secreted protein and another peptide sequence,
e.g., a protein antigen fused to all or part of a secreted
protein.
[0063] In preferred embodiments the antigen is: an antigen which is
characteristic of an autoimmune disorder, e.g., diabetes, lupus,
multiple sclerosis, rheumatoid arthritis; GAD; MBP; a transcription
factor.
[0064] In another aspect, the invention features, a preparation of
milk from a transgenic mammal, which milk includes a protein, e.g.,
a protein described herein, not normally secreted into the milk of
mammals of the species of the transgenic mammal. The transgenic
mammal can be a transgenic dairy mammal, e.g., a goat, sheep, or
cow. Other mammals, e.g., pigs, can also be used.
[0065] In another aspect, the invention features, a transgenically
produced non-secreted polypeptide, wherein the polypeptide is
secreted.
[0066] In preferred embodiments, the transgenically produced
non-secreted polypeptide is secreted into the milk of a transgenic
mammal.
[0067] In preferred embodiments, the transgenically produced
non-secreted polypeptide is selected from the group consisting of
truncated, nuclear, and cytoplasmic polypeptides. Examples of
cytoplasmic polypeptides include, but are not limited to, glutamic
acid decarboxylase and myelin basic protein. Preferably, the
protein, e.g., glutamic acid decarboxylase, is expressed in an
inactive form.
[0068] In other preferred embodiments, the transgenic mammal is
selected from the group consisting of sheep, mice, pigs, cows and
goats. The preferred transgenic mammal is a goat.
[0069] In methods herein which induce tolerance by the use of a
tolerogen which include a non-secreted protein fragments of the
non-secreted protein can be used. It is often desirable that the
non-secreted protein lack an activity possessed by the wild type
non-secreted protein.
[0070] A "signal" or "signal sequence," as used herein, refers to
an amino terminal sequence which directs the expression of a
protein to the exterior of the cell or into a membrane. Preferred
signal sequences are those from secreted proteins, more preferably
protein which are secreted into the milk of a mammal.
[0071] GAD, as used herein, refers to glutamic acid decarboxylase.
GAD produced by the GAD65 gene is preferred for use herein.
[0072] An "isolated" nucleic acid, as used herein, refers to a
nucleic acid molecule which is free of sequences which naturally
flank the nucleic acid (i.e., sequences located at the 5' and 3'
ends of the nucleic acid) in the genomic DNA of the organism from
which the nucleic acid is derived. Moreover, an "isolated" nucleic
acid, such as a cDNA molecule, can be free of other cellular
material.
[0073] As used herein, the phrase "non-secreted polypeptide,"
refers to a protein which is normally found in the nucleus or the
cytoplasm of a cell and which is not normally found as a membrane
protein or secreted through the membrane to be released outside the
cell. Examples of such proteins include, but are not limited to,
enzymes, transcription factors, cell cycle regulatory proteins,
oncoproteins, ribosomal proteins, structural proteins, and cellular
signal transduction proteins.
[0074] The terms "peptides", "proteins", and "polypeptides" are
used interchangeably herein.
[0075] As used herein, a tolerogen, is a molecule which presents an
epitope to an organism such that immunological tolerance is induced
to the epitope. Tolerogens can be proteins.
[0076] As used herein, the term "operatively linked," refers to a
DNA segment which is placed into a functional relationship with
another DNA segment. For example, DNA for a signal sequence is
operatively linked to DNA encoding a polypeptide if it participates
in the secretion of the polypeptide; a promoter or enhancer is
operatively linked to a coding sequence it is promotes the
transcription of the sequence. Generally, DNA sequences that are
operatively linked are contiguous, and in the case of a signal
sequence both contiguous and in reading phase. However, enhancers
need not be contiguous with the coding sequences whose
transcription they control. Linking is accomplished by ligation at
convenient restriction sites or at adapters or linkers inserted in
lieu thereof.
[0077] As used herein, the term "heterologous polypeptide," refers
to a protein or peptide coded for by a DNA sequence which is not
endogenous to the native genome of the organism in which it is
produced, e.g., a mammal in whose milk it is produced. The term
also includes a protein or peptide coded for by a DNA sequence
which if endogenous to the native genome of the mammal in whose
milk it is produced does not lead to the natural production of that
protein or peptide in its milk.
[0078] The term "subject," as used herein, is intended to include
mammals having or being susceptible to an unwanted disease or a
condition. Examples of such subjects include humans, dogs, cats,
pigs, goats, cows, horses, rats and mice.
[0079] The term "treating a condition" is intended to include
preventing, inhibiting, reducing, or delaying the progression of
the condition.
[0080] The transgenically produced non-secreted polypeptides
produced according to the invention find use in a wide variety of
therapeutic procedures, such as in preparation of pharmaceutical
compositions for administration to patients or in diagnosis of
diseases. For example, transgenically produced GAD can be used for
diagnosis and treatment of diabetes and transgenically produced MBP
can be used in the treatment and diagnosis of multiple
sclerosis.
[0081] The application of transgenic technology to the commercial
production of recombinant proteins in the milk of transgenic
animals using milk protein specific signal and promoter sequences
offers significant advantages over traditional methods of
non-secreted protein production. These advantages include a
reduction in the total amount of required capital expenditures,
elimination of the need for capital commitment to build facilities
early in the product development life cycle, and lower direct
production cost per unit for complex proteins. Of key importance is
the likelihood that, for certain non-secreted proteins, transgenic
production may represent the only technologically and economically
feasible method of commercial production.
[0082] Myelin basic protein (MBP) is membrane associated protein
synthesized by oligodendrocytes and Schwann cells. It is not
secreted in its natural environment. MBP is also an autoantigen of
the disease multiple sclerosis. Animal model studies and clinical
trial data have suggested that administrating MBP orally could
establish peripheral immune tolerance and thereby suppress the
symptoms of the disease.
[0083] Glutamic acid decarboxylase (GAD), another cytoplasmic
protein, is an enzyme that catalyzes the biosynthesis of the
neurotransmitter, .gamma.-aminobutyric acid. The human genome has
at least two homologous genes located on chromosomes 2 and 10. The
GAD65 and the GAD67 cDNA derived primary amino acid sequences are
65% identical, with the difference between the two isomers in the
first 250 amino acids being approximately 75%. GAD65 has been
recently identified as a critical .beta.-cell autoantigen in the
disease insulin-dependent diabetes mellitus. Experiments in the NOD
mouse model of insulin-dependent diabetes mellitus have shown an
early appearance of GAD65-reactive antibodies and T cells, and have
demonstrated protection of diabetes by early GAD treatment,
indicating that GAD65 is a key antigen in the disease process
(Kaufman et al. Nature 366:69-72, 1993; Tisch et al. Nature
366:72-5, 1993). The presence of anti-GAD antibodies in the sera of
prediabetic individuals can serve as reliable makers for
progression to overt diabetes. GAD is, therefore, thought to be a
candidate for tolerance therapy.
[0084] Current methods of obtaining glutamic acid decarboxylase
involve purification from a natural sources such as human or
non-human CNS or pancreatic cells (Ortel et al. Brain Res. Bull.
5(2):713-719, 1980; Ortel et al. J. Neurosci. 6:2689-2700, 1981);
by preparing synthetic proteins based on the sequence, or by
expression in cultured mammalian cells. However, the use of
conventionally obtained GAD presents various problems due to the
unavailability of large quantities of cells, expense associated
with producing synthetic peptides and inability to secrete the
recombinant protein in COS cells.
[0085] The methods described herein allow the production of high
levels of secreted proteins which are not normally secreted, e.g.,
glutamic acid decarboxylase (GAD), a protein that is a potential
therapy for insulin-dependent diabetes mellitus (Type 1 diabetes),
in the milk of a transgenic animal.
[0086] The expression of GAD, a non-secreted protein, is a
significant technical achievement. Proteins that are not normally
secreted by cells are extremely difficult to produce as they remain
with in the producing cell. In order to generate sufficient
quantities of a non-secreted protein, like GAD, the producing cells
must be harvested an the protein separated from the cells and other
proteins found within those cells.
[0087] In a transgenic mammal, the protein is secreted by the
mammary glad cells into the milk of the mammal.
[0088] The work herein shows the ability of transgenic technology
to produce a broad and important class of proteins difficult to
express in any other system.
[0089] The transgenic production of a non-secreted protein in milk
provides a cost-effective method of producing high volumes needed
for potential commercialization. In addition, since milk from dairy
animals can be ingested by humans, this method is especially
appropriate for use in oral tolerization.
[0090] Oral tolerance is a mechanism that allows the human body to
regulate the immune system so that it can absorb foreign materials
as nourishment. Preclinical and clinical observations of oral
tolerance show that digesting certain proteins can suppress
autoimmune diseases such as rheumatoid arthritis and multiple
sclerosis.
[0091] The practice of the present invention will employ, unless
otherwise indicated, conventional techniques of cell biology, cell
culture, molecular biology, transgenic biology, microbiology,
recombinant DNA, and immunology, which are within the skill of the
art. Such techniques are described in the literature. See, for
example, Molecular Cloning A Laboratory Manual, 2nd Ed., ed. by
Sambrook, Fritsch and Maniatis (Cold Spring Harbor Laboratory
Press: 1989); DNA Cloning, Volumes I and II (D. N. Glover ed.,
1985); Oligonucleotide Synthesis (M. J. Gait ed., 1984); Mullis et
al. U.S. Pat. No: 4,683,195; Nucleic Acid Hybridization (B. D.
Hames & S. J. Higgins eds. 1984); Transcription And Translation
(B. D. Hames & S. J. Higgins eds. 1984); Culture Of Animal
Cells (R. I. Freshney, Alan R. Liss, Inc., 1987); Immobilized Cells
And Enzymes (IRL Press, 1986); B. Perbal, A Practical Guide To
Molecular Cloning (1984); the treatise, Methods In Enzymology
(Academic Press, Inc., N.Y.); Gene Transfer Vectors For Mammalian
Cells (J. H. Miller and M. P. Calos eds., 1987, Cold Spring Harbor
Laboratory); Methods In Enzymology, Vols. 154 and 155 (Wu et al.
eds.), Immunochemical Methods In Cell And Molecular Biology (Mayer
and Walker, eds., Academic Press, London, 1987); Handbook Of
Experimental Immunology, Volumes I-IV (D. M. Weir and C. C.
Blackwell, eds., 1986); Manipulating the Mouse Embryo, (Cold Spring
Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1986).
[0092] Other features and advantages of the invention will be
apparent from the following detailed description, and from the
claims.
DESCRIPTION OF THE INVENTION
[0093] The drawings will first be briefly described.
DRAWINGS
[0094] FIG. 1 is a schematic diagram of the construction of the
.beta.-casein/MBP fusion gene.
[0095] FIG. 2 is a schematic diagram of the construction of the
hGAD-17 fusion gene.
[0096] FIG. 3 is a schematic diagram of the construction of the
hGAD-18 fusion gene.
[0097] This invention is based upon the discovery that naturally
non-secreted polypeptides, e.g., GAD or MBP, produced by the
transgenic method of the present invention can be secreted in the
milk of transgenic mammals.
[0098] The method of the invention demonstrates a strategy that
leads to the efficient secretion of normally non-secreted proteins,
e.g., truncated, cytoplasmic or nuclear proteins, in the milk of
transgenic mammals. It has been demonstrated herein that adding a
goat .beta.-casein signal peptide, or .beta.-casein signal peptide
and the N-terminal portion of .beta.-casein, to the N-terminal
portion of MBP and GAD is sufficient to secrete these normally
cytoplasmic proteins in the milk of transgenic mice. Thus, the
method of the invention facilitates the production of normally
non-secreted proteins, as well as truncated polypeptides, in the
milk of transgenic mammals.
[0099] Milk Specific Promoters
[0100] The transcriptional promoters useful in practicing the
present invention are those promoters that are preferentially
activated in mammary epithelial cells, including promoters that
control the genes encoding milk proteins such as caseins, beta
lactoglobulin (Clark et al., (1989) Bio/Technology 7: 487-492),
whey acid protein (Gorton et al. (1987) Bio/Technology 5:
1183-1187), and lactalbumin (Soulier et al., (1992) FEBS Letts.
297: 13). Casein promoters may be derived from the alpha, beta,
gamma or kappa casein genes of any mammalian species; a preferred
promoter is derived from the goat beta casein gene (DiTullio,
(1992) Bio/Technology 10:74-77). The milk-specific protein promoter
or the promoters that are specifically activated in mammary tissue
may be derived from either cDNA or genomic sequences. Preferably,
they are genomic in origin.
[0101] DNA sequence information is available for all of the mammary
gland specific genes listed above, in at least one, and often
several organisms. See, e.g., Richards et al., J. Biol. Chem. 256,
526-532 (1981) (.alpha.-lactalbumin rat); Campbell et al., Nucleic
Acids Res. 12, 8685-8697 (1984) (rat WAP); Jones et al., J. Biol.
Chem. 260, 7042-7050 (1985) (rat .beta.-casein); Yu-Lee &
Rosen, J. Biol. Chem. 258, 10794-10804 (1983) (rat .gamma.-casein);
Hall, Biochem. J. 242, 735-742 (1987) (.alpha.-lactalbumin human);
Stewart, Nucleic Acids Res. 12, 389 (1984) (bovine .alpha.s1 and
.kappa. casein cDNAs); Gorodetsky et al., Gene 66, 87-96 (1988)
(bovine .beta. casein); Alexander et al., Eur. J. Biochem. 178,
395-401 (1988) (bovine .kappa. casein); Brignon et al., FEBS Lett.
188, 48-55 (1977) (bovine .alpha.S2 casein); Jamieson et al., Gene
61, 85-90 (1987), Ivanov et al., Biol. Chem. Hoppe-Seyler 369,
425-429 (1988), Alexander et al., Nucleic Acids Res. 17, 6739
(1989) (bovine .beta. lactoglobulin); Vilotte et al., Biochimie 69,
609-620 (1987) (bovine .alpha.-lactalbumin). The structure and
function of the various milk protein genes are reviewed by Mercier
& Vilotte, J. Dairy Sci. 76, 3079-3098 (1993) (incorporated by
reference in its entirety for all purposes). To the extent that
additional sequence data might be required, sequences flanking the
regions already obtained could be readily cloned using the existing
sequences as probes. Mammary-gland specific regulatory sequences
from different organisms are likewise obtained by screening
libraries from such organisms using known cognate nucleotide
sequences, or antibodies to cognate proteins as probes.
[0102] Signal Sequences
[0103] Among the signal sequences that are useful in accordance
with this invention are milk-specific signal sequences or other
signal sequences which result in the secretion of eukaryotic or
prokaryotic proteins. Preferably, the signal sequence is selected
from milk-specific signal sequences, i.e., it is from a gene which
encodes a product secreted into milk. Most preferably, the
milk-specific signal sequence is related to the milk-specific
promoter used in the expression system of this invention. The size
of the signal sequence is not critical for this invention. All that
is required is that the sequence be of a sufficient size to effect
secretion of the desired recombinant protein, e.g., in the mammary
tissue. For example, signal sequences from genes coding for
caseins, e.g., alpha, beta, gamma or kappa caseins, beta
lactoglobulin, whey acid protein, and lactalbumin are useful in the
present invention. The preferred signal sequence is the goat
.beta.-casein signal sequence.
[0104] Signal sequences from other secreted proteins, e.g.,
proteins secreted by liver cells, kidney cell, or pancreatic cells
can also be used.
[0105] Amino-Terminal Regions of Secreted Proteins
[0106] The efficacy with which a non-secreted protein is secreted
can be enhanced by inclusion in the protein to be secreted all or
part of the coding sequence of a protein which is normally
secreted. Preferably the entire sequence of the protein which is
normally secreted is not included in the sequence of the protein
but rather only a portion of the amino terminal end of the protein
which is normally secreted. For example, a protein which is not
normally secreted is fused (usually at its amino terminal end) to
an amino terminal portion of a protein which is normally
secreted.
[0107] Preferably, the protein which is normally secreted is a
protein which is normally secreted in milk. Such proteins include
proteins secreted by mammary epithelial cells, milk proteins such
as caseins, beta lactoglobulin, whey acid protein, and lactalbumin.
Casein proteins include alpha, beta, gamma or kappa casein genes of
any mammalian species. A preferred protein is beta casein, e.g., a
goat beta casein. The sequences which encode the secreted protein
can be derived from either cDNA or genomic sequences. Preferably,
they are genomic in origin, and include one or more introns.
[0108] DNA Constructs
[0109] The expression system or construct, described herein, can
also include a 3' untranslated region downstream of the DNA
sequence coding for the non-secreted protein. This region
apparently stabilizes the RNA transcript of the expression system
and thus increases the yield of desired protein from the expression
system. Among the 3' untranslated regions useful in the constructs
of this invention are sequences that provide a poly A signal. Such
sequences may be derived, e.g., from the SV40 small t antigen, the
casein 3' untranslated region or other 3' untranslated sequences
well known in the art. Preferably, the 3' untranslated region is
derived from a milk specific protein. The length of the 3'
untranslated region is not critical but the stabilizing effect of
its poly A transcript appears important in stabilizing the RNA of
the expression sequence.
[0110] Optionally, the expression system or construct includes a 5'
untranslated region between the promoter and the DNA sequence
encoding the signal sequence. Such untranslated regions can be from
the same control region from which promoter is taken or can be from
a different gene, e.g., they may be derived from other synthetic,
semi-synthetic or natural sources. Again their specific length is
not critical, however, they appear to be useful in improving the
level of expression.
[0111] The construct can also include about 10%, 20%, 30%, or more
of the N-terminal coding region of the gene preferentially
expressed in mammary epithelial cells. For example, the N-terminal
coding region can correspond to the promoter used, e.g., a goat
.beta.-casein N-terminal coding region.
[0112] The above-described expression systems may be prepared using
methods well known in the art. For example, various ligation
techniques employing conventional linkers, restriction sites etc.
may be used to good effect. Preferably, the expression systems of
this invention are prepared as part of larger plasmids. Such
preparation allows the cloning and selection of the correct
constructions in an efficient manner as is well known in the art.
Most preferably, the expression systems of this invention are
located between convenient restriction sites on the plasmid so that
they can be easily isolated from the remaining plasmid sequences
for incorporation into the desired mammal.
[0113] Prior art methods often include making a construct and
testing it for the ability to produce a product in cultured cells
prior to placing the construct in a transgenic animal.
Surprisingly, the inventors have found that such a protocol may not
be of predictive value in determining if a normally non-secreted
protein can be secreted, e.g., in the milk of a transgenic animal.
Therefore, it may be desirable to test constructs directly in
transgenic animals, e.g., transgenic mice, as some constructs which
fail to be secreted in CHO cells are secreted into the milk of
transgenic animals.
[0114] Transgenic Mammals
[0115] The DNA constructs of the invention are introduced into the
germ line of a mammal. For example, one or several copies of the
construct may be incorporated into the genome of a mammalian embryo
by standard transgenic techniques.
[0116] Any non-human mammal can be usefully employed in this
invention. Mammals are defined herein as all animals, excluding
humans, that have mammary glands and produce milk. Preferably,
mammals that produce large volumes of milk and have long lactating
periods are preferred. Preferred mammals are cows, sheep, goats,
mice, oxen, camels and pigs. Of course, each of these mammals may
not be as effective as the others with respect to any given
expression sequence of this invention. For example, a particular
milk-specific promoter or signal sequence may be more effective in
one mammal than in others. However, one of skill in the art may
easily make such choices by following the teachings of this
invention.
[0117] One technique for transgenically altering a mammal is to
microinject the construct into the pronuclei of the fertilized
mammalian egg(s) to cause one or more copies of the construct to be
retained in the cells of the developing mammal(s). Usually, at
least 40% of the mammals developing from the injected eggs contain
at least one copy of the cloned construct in somatic tissues and
these "transgenic mammals" usually transmit the gene through the
germ line to the next generation. The progeny of the transgenically
manipulated embryos may be tested for the presence of the construct
by Southern blot analysis of the segment of tissue. If one or more
copies of the exogenous cloned construct remains stably integrated
into the genome of such transgenic embryos, it is possible to
establish permanent transgenic mammal lines carrying the
transgenically added construct.
[0118] The litters of transgenically altered mammals may be assayed
after birth for the incorporation of the construct into the genome
of the offspring. Preferably, this assay is accomplished by
hybridizing a probe corresponding to the DNA sequence coding for
the desired recombinant protein product or a segment thereof onto
chromosomal material from the progeny. Those mammalian progeny
found to contain at least one copy of the construct in their genome
are grown to maturity. The female species of these progeny will
produce the desired protein in or along with their milk.
Alternatively, the transgenic mammals may be bred to produce other
transgenic progeny useful in producing the desired proteins in
their milk.
[0119] Transgenic females may be tested for protein secretion into
milk, using any of the assay techniques that are standard in the
art (e.g., Western blots or enzymatic assays).
[0120] Pharmaceutical Compositions
[0121] Preferred pharmaceutical compositions for inducing tolerance
to proteins include the appropriate tolerogen in the milk of a
transgenic animal.
[0122] Transgenically produced non-secreted polypeptide of the
invention can be incorporated into pharmaceutical compositions
useful to attenuate, inhibit, or prevent a disease or a disorder,
e.g., the destruction of pancreatic .beta.-cells associated with
the onset of insulin-dependent diabetes mellitus, or the
autoimmunity associated with multiple sclerosis. The compositions
should contain a therapeutic or prophylactic amount of the
transgenically produced non-secreted polypeptide, e.g., GAD or MBP,
in a pharmaceutically-acceptable carrier or in the milk of the
transgenic animal. The pharmaceutical carrier can be any
compatible, non-toxic substance suitable to deliver the
polypeptides to the patient. Sterile water, alcohol, fats, waxes,
and inert solids may be used as the carrier.
Pharmaceutically-acceptable adjuvants, buffering agents, dispersing
agents, and the like, may also be incorporated into the
pharmaceutical compositions. The concentration of the
transgenically produced non-secreted peptide or other active agent
in the pharmaceutical composition can vary widely, i.e., from less
than about 0.1% by weight, usually being at least about 1% weight
to as much as 20% by weight or more.
[0123] For oral administration, the active ingredient can be
administered in solid dosage forms, such as capsules, tablets, and
powders, or in liquid dosage forms, such as elixirs, syrups, and
suspensions. Active component(s) can be encapsulated in gelatin
capsules together with inactive ingredients and powdered carriers,
such as glucose, lactose, sucrose, mannitol, starch, cellulose or
cellulose derivatives, magnesium stearate, stearic acid, sodium
saccharin, talcum, magnesium carbonate and the like. Examples of
additional inactive ingredients that may be added to provide
desirable color, taste, stability, buffering capacity, dispersion
or other known desirable features are red iron oxide, silica gel,
sodium lauryl sulfate, titanium dioxide, edible white ink and the
like. Similar diluents can be used to make compressed tablets. Both
tablets and capsules can be manufactured as sustained release
products to provide for continuous release of medication over a
period of hours. Compressed tablets can be sugar coated or film
coated to mask any unpleasant taste and protect the tablet from the
atmosphere, or enteric-coated for selective disintegration in the
gastrointestinal tract. Liquid dosage forms for oral administration
can contain coloring and flavoring to increase patient
acceptance.
[0124] For nasal administration, the polypeptides can be formulated
as aerosols. The term "aerosol" includes any gas-borne suspended
phase of the compounds of the instant invention which is capable of
being inhaled into the bronchioles or nasal passages. Specifically,
aerosol includes a gas-borne suspension of droplets of the
compounds of the instant invention, as may be produced in a metered
dose inhaler or nebulizer, or in a mist sprayer. Aerosol also
includes a dry powder composition of a compound of the instant
invention suspended in air or other carrier gas, which may be
delivered by insufflation from an inhaler device, for example. See
Ganderton & Jones, Drug Delivery to the Respiratory Tract,
Ellis Horwood (1987); Gonda (1990) Critical Reviews in Therapeutic
Drug Carrier Systems 6:273-313; and Raeburn et al. (1992) J.
Pharmacol. Toxicol. Methods 27:143-159.
[0125] The pharmaceutical compositions of the present invention are
usually administered intravenously or orally. Intradermal or
intramuscular administration is also possible in some
circumstances. The compositions can be administered for
prophylactic treatment of individuals suffering from, or at risk of
a disease or a disorder, e.g., IDDM or multiple sclerosis. For
therapeutic applications, the pharmaceutical compositions are
administered to a patient suffering from established diabetes in an
amount sufficient to inhibit or prevent further .beta.-cell
destruction. For individuals at risk of IDDM or multiple sclerosis,
the pharmaceutical compositions are administered prophylactically
in an amount sufficient to either prevent or inhibit immune
destruction. An amount adequate to accomplish this is defined as a
"therapeutically-effective dose."
[0126] Production of Non-Secreted Protein Variants (Non Wild-Type
Proteins)
[0127] Production of Altered DNA and Peptide Sequences: Random
Methods
[0128] Amino acid sequence variants of a protein can be prepared by
random mutagenesis of DNA which encodes a protein or a particular
domain or region of a protein. Useful methods include PCR
mutagenesis and saturation mutagenesis. A library of random amino
acid sequence variants can also be generated by the synthesis of a
set of degenerate oligonucleotide sequences.
[0129] PCR Mutagenesis
[0130] In PCR mutagenesis, reduced Taq polymerase fidelity is used
to introduce random mutations into a cloned fragment of DNA (Leung
et al., 1989, Technique 1:11-15). This is a very powerful and
relatively rapid method of introducing random mutations. The DNA
region to be mutagenized is amplified using the polymerase chain
reaction (PCR) under conditions that reduce the fidelity of DNA
synthesis by Taq DNA polymerase, e.g., by using a dGTP/dATP ratio
of five and adding Mn.sup.2+ to the PCR reaction. The pool of
amplified DNA fragments are inserted into appropriate cloning
vectors to provide random mutant libraries.
[0131] Saturation Mutagenesis
[0132] Saturation mutagenesis allows for the rapid introduction of
a large number of single base substitutions into cloned DNA
fragments (Mayers et al., 1985, Science 229:242). This technique
includes generation of mutations, e.g., by chemical treatment or
irradiation of single-stranded DNA in vitro, and synthesis of a
complimentary DNA strand. The mutation frequency can be modulated
by modulating the severity of the treatment, and essentially all
possible base substitutions can be obtained. Because this procedure
does not involve a genetic selection for mutant fragments both
neutral substitutions, as well as those that alter function, are
obtained. The distribution of point mutations is not biased toward
conserved sequence elements.
[0133] Degenerate Oligonucleotides
[0134] A library of homologs can also be generated from a set of
degenerate oligonucleotide sequences. Chemical synthesis of a
degenerate sequences can be carried out in an automatic DNA
synthesizer, and the synthetic genes then ligated into an
appropriate expression vector. The synthesis of degenerate
oligonucleotides is known in the art (see for example, Narang, S A
(1983) Tetrahedron 39:3; Itakura et al. (1981) Recombinant DNA,
Proc 3rd Cleveland Sympos. Macromolecules, ed. A G Walton,
Amsterdam: Elsevier pp 273-289; Itakura et al. (1984) Annu. Rev.
Biochem. 53:323; Itakura et al. (1984) Science 198:1056; Ike et al.
(1983) Nucleic Acid Res. 11:477. Such techniques have been employed
in the directed evolution of other proteins (see, for example,
Scott et al. (1990) Science 249:386-390; Roberts et al. (1992) PNAS
89:2429-2433; Devlin et al. (1990) Science 249: 404-406; Cwirla et
al. (1990) PNAS 87: 6378-6382; as well as U.S. Pat. Nos. 5,223,409,
5,198,346, and 5,096,815).
[0135] Production of Altered DNA and Peptide Sequences: Methods for
Directed Mutagenesis
[0136] Non-random or directed, mutagenesis techniques can be used
to provide specific sequences or mutations in specific regions.
These techniques can be used to create variants which include,
e.g., deletions, insertions, or substitutions, of residues of the
known amino acid sequence of a protein. The sites for mutation can
be modified individually or in series, e.g., by (1) substituting
first with conserved amino acids and then with more radical choices
depending upon results achieved, (2) deleting the target residue,
or (3) inserting residues of the same or a different class adjacent
to the located site, or combinations of options 1-3.
[0137] Alanine Scanning Mutagenesis
[0138] Alanine scanning mutagenesis is a useful method for
identification of certain residues or regions of the desired
protein that are preferred locations or domains for mutagenesis,
Cunningham and Wells (Science 244:1081-1085, 1989). In alanine
scanning, a residue or group of target residues are identified
(e.g., charged residues such as Arg, Asp, His, Lys, and Glu) and
replaced by a neutral or negatively charged amino acid (most
preferably alanine or polyalanine). Replacement of an amino acid
can affect the interaction of the amino acids with the surrounding
aqueous environment in or outside the cell. Those domains
demonstrating functional sensitivity to the substitutions are then
refined by introducing further or other variants at or for the
sites of substitution. Thus, while the site for introducing an
amino acid sequence variation is predetermined, the nature of the
mutation per se need not be predetermined. For example, to optimize
the performance of a mutation at a given site, alanine scanning or
random mutagenesis may be conducted at the target codon or region
and the expressed desired protein subunit variants are screened for
the optimal combination of desired activity.
[0139] Oligonucleotide-Mediated Mutagenesis
[0140] Oligonucleotide-mediated mutagenesis is a useful method for
preparing substitution, deletion, and insertion variants of DNA,
see, e.g., Adelman et al., (DNA 2:183, 1983). Briefly, the desired
DNA is altered by hybridizing an oligonucleotide encoding a
mutation to a DNA template, where the template is the
single-stranded form of a plasmid or bacteriophage containing the
unaltered or native DNA sequence of the desired protein. After
hybridization, a DNA polymerase is used to synthesize an entire
second complementary strand of the template that will thus
incorporate the oligonucleotide primer, and will code for the
selected alteration in the desired protein DNA. Generally,
oligonucleotides of at least 25 nucleotides in length are used. An
optimal oligonucleotide will have 12 to 15 nucleotides that are
completely complementary to the template on either side of the
nucleotide(s) coding for the mutation. This ensures that the
oligonucleotide will hybridize properly to the single-stranded DNA
template molecule. The oligonucleotides are readily synthesized
using techniques known in the art such as that described by Crea et
al. (Proc. Natl. Acad. Sci. USA, 75: 5765[1978]).
[0141] Cassette Mutagenesis
[0142] Another method for preparing variants, cassette mutagenesis,
is based on the technique described by Wells et al. (Gene,
34:315[1985]). The starting material is a plasmid (or other vector)
which includes the protein subunit DNA to be mutated. The codon(s)
in the protein subunit DNA to be mutated are identified. There must
be a unique restriction endonuclease site on each side of the
identified mutation site(s). If no such restriction sites exist,
they may be generated using the above-described
oligonucleotide-mediated mutagenesis method to introduce them at
appropriate locations in the desired protein subunit DNA. After the
restriction sites have been introduced into the plasmid, the
plasmid is cut at these sites to linearize it. A double-stranded
oligonucleotide encoding the sequence of the DNA between the
restriction sites but containing the desired mutation(s) is
synthesized using standard procedures. The two strands are
synthesized separately and then hybridized together using standard
techniques. This double-stranded oligonucleotide is referred to as
the cassette. This cassette is designed to have 3' and 5' ends that
are comparable with the ends of the linearized plasmid, such that
it can be directly ligated to the plasmid. This plasmid now
contains the mutated desired protein subunit DNA sequence.
[0143] Combinatorial Mutagenesis
[0144] Combinatorial mutagenesis can also be used to generate
mutants (Ladner et al., WO 88/06630). In this method, the amino
acid sequences for a group of homologs or other related proteins
are aligned, preferably to promote the highest homology possible.
All of the amino acids which appear at a given position of the
aligned sequences can be selected to create a degenerate set of
combinatorial sequences. The variegated library of variants is
generated by combinatorial mutagenesis at the nucleic acid level,
and is encoded by a variegated gene library. For example, a mixture
of synthetic oligonucleotides can be enzymatically ligated into
gene sequences such that the degenerate set of potential sequences
are expressible as individual peptides, or alternatively, as a set
of larger fusion proteins containing the set of degenerate
sequences.
[0145] This invention is further illustrated by the following
examples which in no way should be construed as being further
limiting. The contents of all cited references (including
literature references, issued patents, published patent
applications, and co-pending patent applications) cited throughout
this application are hereby expressly incorporated by
reference.
EXAMPLES
[0146] 1. Cloning of .beta.-casein-MBP Fusion Gene
[0147] Five plasmids containing the .beta.-casein/MBP fusing gene
were constructed. As depicted in FIG. 1, plasmid pBCMBP1 was
generated by ligating goat .beta.-casein signal sequences (oligos
OT1 AND OT2) to SalI/BglII sites of pMBP6. Cloning the HindIII-XhoI
fragment of pBCMBP1 into pCDNA3 generated pBCMBP101. Plasmid
pBCMBP102 was generated by inserting an additional NcoI fragment
pMBP6 into the NcoI site of pBCMBP101. SalI/XhoI fragments of
pBCMBP101, pBCMBP102 were inserted into BC157 to generated BC183
and BC172, respectively.
[0148] Plasmid pBCMBP2 was generated by ligating SalI-ApaI fragment
of pBC12 (which contains .beta.-casein signal sequence and 30% of
the N-terminal coding region of goat .beta.-casein) and a pair of
adapter oligos (oligos OT3 and OT4) into the SalI-BglII site of
pMBP6. The HindIII/EcoRI fragment of the pBCMBP2, which carries
sequences encoding the .beta.-casein signal and N terminal portion
followed by the entire MBP coding region was cloned into pCDNA3 to
yield pBCMBP201. Plasmid pBCMBP202 was generated by inserting an
additional NcoI fragment of pMBP6 into the NcoI site of pBCMBP201.
SalI/XhoI fragments of pBCMBP201, pBCMBP202 were inserted into
BC157 to generated BC94 and BC95, respectively.
[0149] To construct MBP and tPA signal sequence, polymerase chain
reaction was used to obtain a MBP cDNA fragment with oligos MBP1
and MBP2. The PCR fragment was digested with BglII/XhoI and cloned
into BglII/XhoI digested pUCsigtPA, which contains the human LatPA
signal sequence. The new plasmid, designated pMBP6, contained a
fusion gene with tPA signal fused to MBP coding region. SalI/XhoI
fragment from pMBP6 was cloned into the mammary expression vector
BC163 to generate BC60.
1 Oligos Used OT1, TCG ACG AGA GCC ATG AAG GTC CTC ATC CTT GCCT TGT
CTG GTG (SEQ ID NO:1) GCT CTG CGG ATT GCA AGA GAG CAG GAA GAA CTC
AAT GTA GTC GGT A OT2, GAT CTA CCG ACT ACA TT A GT TCT TCC TGC TCT
CTT GCA ATG (SEQ ID NO:2) GCC AGA GCC ACC AGA CAG GCA AGG ATG AGG
ACC TTC ATG GCT CTC G OT3, GAT CGG TGG TA (SEQ ID NO:3) OT4, GAT
CTA CCA CCG ATG GGC C (SEQ ID NO:4) MBP1, CCA TTG CAA GAT CTG CGT
CAC AGA AGA GAC CCT CC (SEQ ID NO:5) MBP2, TCA CCC ATG GCT AGA CGC
TGA CTC GAG GAT CCT TGT CA (SEQ ID NO:6)
[0150] 2. Secretion of MBP, MBP Fusion Proteins in Tissue
Culture
[0151] To determine if the cytoplasmic protein MBP can be secreted
by adding tPA or .beta.-casein signal peptides plasmids pMBP6,
PBCMBP101, pBCMBP102, pBCMBP201 and pBCMBP202 were first
transfected into COS-7 cells. Northern analysis revealed that mRNA
for all these MBP constructs was expressed in tissue culture
following transient transfection. When the transfected COS cells
were analyzed by immunoprecipitation for protein secretion, it was
found that only the tPA signal peptide sequence (pMBP6) failed to
secrete MBP. MBP with the .beta.-casein fusion proteins (pBCMBP201,
pBCMBP202) were all secreted in tissue culture.
[0152] 3. Cloning of the GAD-Casein Fusion Genes
[0153] To further demonstrate that fusion to .beta.-casein signal
peptide is a reliable method to direct the secretion of
non-secreted protein into the milk of the transgenic animals, the
similar strategy was employed to secrete another non-secreted
cytoplasmic protein, the glutamic acid decarboxylase (GAD).
[0154] To secrete GAD, the following fusion genes were
constructed:
[0155] 1. Human GAD cDNA fused to goat .beta.-casein signal
sequence. The fusion gene inserted in the expression vector pCDNA3
was designated as huGAD7. This plasmid was constructed by ligating
a pair of oligo (OT1 and OT2) that encode the .beta.-casein signal
sequence flanked by SalI/BglII sites, a BglII/NcoI adapter, and a
NcoI/XhoI fragment of huGAD1, which encode the wild type GAD, into.
the XhoI site of the expression vector pCDNA3.
[0156] 2. Human GAD cDNA with a point mutation (PLP mutation) that
knocks out the decarboxylase activity, was fused to goat
.beta.-casein signal sequence. The fusion gene inserted in the
expression vector pCDNA3 was designated as huGAD8. This plasmid was
constructed by ligating oligo OT1 and OT2, a BglII/NcoI adapter,
and a NcoI/XhoI fragment of huGAD2, which encode the mutated GAD,
into the XhoI site of the expression vector pCDNA3.
[0157] 3. hGAD cDNA was fused to goat .beta.-casein signal sequence
and an N-terminal portion of goat .beta.-casein coding sequence.
The fusion gene inserted in the expression vector pCDNA3 was
designated as huGAD17. The construction of this plasmid is
illustrated in FIG. 2.
[0158] 4. hGAD cDNA with PLP mutation was fused to goat b-casein
signal sequence and an N-terminal portion of goat b-casein coding
sequences. The fusion gene inserted in the expression vector pCDNA3
was designated as huGAD18. The construction of this plasmid is
illustrated in FIG. 3.
[0159] 5. A pair of control GAD constructs (both wild type and
mutant) that lack any signal sequences, were cloned into pCDNA3.
The plasmids were designated, respectively, as huGAD9, for wild
type GAD, and huGAD10, for GAD with the PLP mutation.
[0160] 4. Expression of GAD-Casein Fusion Protein in Tissue
Culture
[0161] Cultured COS-7 cells were transfected with huGAD7, huGAD8,
huGAD17, huGAD 18, huGAD9, huGAD10, and the expression of the GAD
or GAD-casein fusion proteins were examined by Northern blotting of
total RNA isolated from transfected COS cells and by
immunoprecipitation of .sup.35S labeled medium and lysates of the
transfected COS cells with a polyclonal anti-GAD antiserum. The
results from the Northern blotting indicated that the mRNA was
transcribed from all these constructs. Immunoprecipitation of the
metabolically labeled transfected COS cells indicated that 1) GAD
or GAD-casein fusion proteins were synthesized from all of the
constructs, because they can be readily detected in the lysate
fraction; 2) GAD or GAD-casein fusion protein was not detected in
the media fraction. Only residual amount of GAD or GAD-casein
fusion proteins was detected in the culture media, which was most
likely the result of nonspecific lysis of the COS cells. There was
no significant difference between the control constructs, huGAD9
and huGAD10, that lack the signal sequence and the constructs
huGAD7, huGAD8, huGAD17 and huGAD18 in the level and pattern of GAD
expression. These results suggested that the GAD or GAD-casein
fusion protein was not secreted by COS cells.
[0162] 5. Testing and Characterization of Gene Constructs in
Transgenic Mice
[0163] Transgene constructs are generally tested in a mouse model
system to assess their ability to direct high levels of expression
and their ability to express in a tissue-specific manner.
[0164] Transgenic mice were generated by injecting mouse embryos
with DNA fragments prepared by BC60, BC183, BC94, BC172, and BC192.
Analysis of the milk of the female transgenic mice revealed that
while the tPA signal peptide failed to secrete MBP into the milk
(BC60), MBP with the .beta.-casein signal peptide (BC183, BC172),
as well as MBP/.beta.-casein fusion protein (BC94) were expressed
by the mouse mammary epithelial cell and secreted into the milk.
When milk from the BC94 line (MBP/.beta.-casein fusion) was
analyzed by SDS PAGE followed by Coomassie staining, an additional
protein was found to be present in the milk of transgenic mouse
lines 94-1-106 and 94-2-167. Furthermore, when a duplicate SDS PAGE
was subjected to Western analysis, this additional protein was
found to be immunologically reactive to antibodies against MBP,
confirming that the MBP/.beta.-casein fusion protein is secreted
into the milk. The level of MBP fusion expression in the milk of
the BC94-1-106 line was estimated to be 4 mg/ml.
[0165] To access if GAD-casein fusion protein can be secreted by
the mammary epithelial cells, the coding sequence of hGAD 18 was
excised as a SalI/XhoI fragment and subcloned into the XhoI site of
the .beta.-casein expression vector BC350 to yield construct BC433.
Transgenic mice were generated by microinjecting the SalI/NotI
fragment of BC433 into the mouse embryos. Fourteen lines of
transgenic mice were established. Western analysis of the milk of
the GAD transgenic mice using monoclonal anti-GAD antibody showed
that five of the six lines expressed GAD-casein fusion protein in
the milk. The concentration of the GAD in milk of the highest
expressor, BC433-208, was 8 mg/ml.
[0166] To access if the wild type GAD-casein fusion protein can be
expressed in transgenic mice by mammary epithelial cells, the
coding sequence of hGAD 18 was excised as a Sal I/Xho I fragment
and subcloned into the Xho I site of .beta.-casein expression
vector BC350 to yield plasmid BC569. Transgenic mice were generated
by microinjecting the Sal I/Not I fragment of BC569 into the mouse
embryos. Eleven lines of transgenic mice were established. Western
analysis of the milk collected from these transgenic mice using
monoclonal anti-GAD antibody showed that the wild type GAD-casein
fusion protein was expressed at the levels of 4 mg/ml.
[0167] To access if the 15 amino acid .beta.-casein signal peptide
is by itself sufficient to secrete GAD into the milk of transgenic
mice, a plasmid was constructed by fusing oligos which encode the
15 amino acid .beta.-casein signal peptide directly to the GAD
cDNA. Consequently, if the signal peptide is recognized and
processed, this construct should lead to the secretion of natural
GAD into milk, instead of GAD with 67 extra amino acids fused to
its N-terminal, as is the case with BC569 construct. The plasmid
was constructed by ligating oligos OT9 (TCG AGC CCA CCA TGA AGG TCC
TCA TCC TTG CCT GTC TGG TGG CTC TGG CCA TTG (SEQ ID NO:7)) and OT10
(CAT GGC AAT GGC CAG AGC CAC CAG ACA GGC AAG GAT GAG GAC CTT CAT
GGT GGC (SEQ ID NO:8)) and a Sal I/Nco I GAD fragment of huGAD 17
into the Xho I site of the .beta.-casein expression vector BC350.
This yielded the BC577 construct. A Sal I/Xho I fragment of BC577
was microinjected into the mouse embryo to generate transgenic
mice. Twenty two transgenic lines were established. Western
analysis of the milk collected from the female transgenic mice
showed GAD expression of 4 mg/ml.
[0168] 6. Generation and Characterization of Transgenic Animals
[0169] A founder (F.sub.O) transgenic goat is defined as a viable
transgenic animal resulting from, embryo transfer of fertilized
goat eggs that have been microinjected with a specified construct
(e.g., BC433, BC569, BC577, or BC94). The general methodologies
that follow in this section can be used to generate all transgenic
goats.
[0170] Goat Species and Breeds:
[0171] The transgenic goats produced for non-secreted protein,
e.g., GAD or MBP, production can be of Swiss origin, and are the
Alpine, Saanen, and Toggenburg breeds.
[0172] Goat Superovulation:
[0173] The timing of estrus in the donors are synchronized on Day 0
by 6 mg subcutaneous norgestomet ear implants (Syncromate-B, CEVA
Laboratories, Inc., Overland Park, Kans.). Prostaglandin is
administered after the first seven to nine days to shut down the
endogenous synthesis of progesterone. Starting on Day 13 after
insertion of the implant, a total of 18 mg of follicle-stimulating
hormone (FSH-Schering Corp., Kenilworth, N.J.) is given
intramuscularly over three days in twice-daily injections. The
implant is removed on Day 14. Twenty-four hours following implant
removal the donor animals are mated several times to fertile males
over a two-day period (Selgrath, et al., Theriogenology, 1990. pp.
1195-1205).
[0174] Embryo Collection:
[0175] Surgery for embryo collection occurs on the second day
following breeding (or 72 hours following implant removal).
Superovulated does are removed from food and water 36 hours prior
to surgery. Does are administered 0.8 mg/kg Diazepam (Valium.RTM.),
IV, followed immediately by 5.0 mg/kg Ketamine (Keteset), IV.
Halothane (2.5%) is administered during surgery in 2 L/min oxygen
via an endotracheal tube. The reproductive tract is exteriorized
through a midline laparotomy incision. Corpora lutea, unruptured
follicles greater than 6 mm in diameter, and ovarian cysts are
counted to evaluate superovulation results and to predict the
number of embryos that should be collected by oviductal flushing. A
cannula is placed in the ostium of the oviduct and held in place
with a single temporary ligature of 3.0 Prolene. A 20 gauge needle
is placed in the uterus approximately 0.5 cm from the uterotubal
junction. Ten to twenty ml of sterile phosphate buffered saline
(PBS) is flushed through the cannulated oviduct and collected in a
Petri dish. This procedure is repeated on the opposite side and
then the reproductive tract is replaced in the abdomen. Before
closure, 10-20 ml of a sterile saline glycerol solution is poured
into the abdominal cavity to prevent adhesions. The linea alba is
closed with simple interrupted sutures of 2.0 Polydioxanone or
Supramid and the skin closed with sterile wound clips.
[0176] Fertilized goat eggs are collected from the PBS oviductal
flushings on a stereomicroscope, and are then washed in Ham's F12
medium (Sigma, St. Louis, Mo.) containing 10% fetal bovine serum
(FBS) purchased from Sigma. In cases where the pronuclei are
visible, the embryos is immediately microinjected. If pronuclei are
not visible, the embryos are placed in Ham's F12 containing 10% FBS
for short term culture at 37.degree. C. in a humidified gas chamber
containing 5% CO2 in air until the pronuclei become visible
(Selgrath, et al., Theriogenology, 1990. pp. 1195-1205).
[0177] Microinjection Procedure
[0178] One-cell goat embryos are placed in a microdrop of medium
under oil on a glass depression slide. Fertilized eggs having two
visible pronuclei are immobilized on a flame-polished holding
micropipet on a Zeiss upright microscope with a fixed stage using
Normarski optics. A pronucleus is microinjected with the DNA
construct of interesrt, e.g., BC433, BC569, BC577, or BC94, in
injection buffer (Tris-EDTA) using a fine glass microneedle
(Selgrath, et al., Theriogenology, 1990. pp. 1195-1205).
[0179] Embryo Development:
[0180] After microinjection, the surviving embryos are placed in a
culture of Ham's F12 containing 10% FBS and then incubated in a
humidified gas chamber containing 5% CO2 in air at 37.degree. C.
until the recipient animals are prepared for embryo transfer
(Selgrath, et al., Theriogenology, 1990. p. 1195-1205).
[0181] Preparation of Recipients:
[0182] Estrus synchronization in recipient animals is induced by 6
mg norgestomet ear implants (Syncromate-B). On Day 13 after
insertion of the implant, the animals are given a single
non-superovulatory injection (400 I.U.) of pregnant mares serum
gonadotropin (PMSG) obtained from Sigma. Recipient females are
mated to vasectomized males to ensure estrus synchrony (Selgrath,
et al., Theriogenology, 1990. pp. 1195-1205).
[0183] Embryo Transfer:
[0184] All embryos from one donor female are kept together and
transferred to a single recipient when possible. The surgical
procedure is identical to that outlined for embryo collection
outlined above, except that the oviduct is not cannulated, and the
embryos are transferred in a minimal volume of Ham's F12 containing
10% FBS into the oviductal lumen via the fimbria using a glass
micropipet. Animals having more than six to eight ovulation points
on the ovary are deemed unsuitable as recipients. Incision closure
and post-operative care are the same as for donor animals
(Selgrath, et al., Theriogenology, 1990. pp. 1195-1205).
[0185] Monitoring of Pregnancy and Parturition:
[0186] Pregnancy is determined by ultrasonography 45 days after the
first day of standing estrus. At Day 110 a second ultrasound exam
is conducted to confirm pregnancy and assess fetal stress. At Day
130 the pregnant recipient doe is vaccinated with tetanus toxoid
and Clostridium C&D. Selenium and vitamin E (Bo-Se) are given
IM and Ivermectin was given SC. The does are moved to a clean stall
on Day 145 and allowed to acclimatize to this environment prior to
inducing labor on about Day 147. Parturition is induced at Day 147
with 40 mg of PGF2a (Lutalyse.RTM.) purchased from Upjohn Company,
Kalamazoo Mich. This injection is given IM in two doses, one 20 mg
dose followed by a 20 mg dose four hours later. The doe is under
periodic observation during the day and evening following the first
injection of Lutalyse.RTM. on Day 147. Observations are increased
to every 30 minutes beginning on the morning of the second day.
Parturition occurred between 30 and 40 hours after the first
injection. Following delivery the doe is milked to collect the
colostrum and passage of the placenta is confirmed.
[0187] Verification of the Transgenic Nature of F.sub.0
Animals:
[0188] To screen for transgenic F.sub.0 animals, genomic DNA is
isolated from two different cell lines to avoid missing any mosaic
transgenics. A mosaic animal is defined as any goat that does not
have at least one copy of the transgene in every cell. Therefore,
an ear tissue sample (mesodern) and blood sample are taken from a
two day old F.sub.0 animal for the isolation of genomic DNA (Lacy,
et al., A Laboratory Manual, 1986, Cold Springs Harbor, N.Y.; and
Herrmann and Frischauf, Methods Enzymology, 1987. 152: pp.
180-183). The DNA samples are analyzed by the polymerase chain
reaction (Gould, et al., Proc. Natl. Acad. Sci, 1989. 86:pp.
1934-1938) using primers specific for GAD or MBP and by Southern
blot analysis (Thomas, Proc Natl. Acad. Sci., 1980. 77:5201-5205)
using a random primed GAD or MBP cDNA probe (Feinberg and
Vogelstein, Anal. Bioc., 1983. 132: pp. 6-13). Assay sensitivity is
estimated to be the detection of one copy of the transgene in 10%
of the somatic cells.
[0189] Generation and Selection of Production Herd
[0190] The procedures described above are utilized for production
of the transgenic founder (F.sub.0) goats, as well as other
transgenic goats. The transgenic F.sub.0 founder goats, for
example, are bred to produce milk, if female, or to produce a
transgenic female offspring if it is a male founder.
[0191] This transgenic founder male, is bred to non-transgenic
females, to produce transgenic female offspring.
[0192] Transmission of Transgene and Pertinent Characteristics
[0193] Transmission of the transgene of interest, e.g., GAD or MBP,
in the goat line is analyzed in ear tissue and blood by PCR and
Southern blot analysis. For example, Southern blot analysis of the
founder male and the three transgenic offspring shows no
rearrangement or change in the copy number between generations. The
Southern blots are probed with the non-secreted protein, e.g., GAD
or MBP, cDNA probe. The blots are analyzed on a Betascope 603 and
copy number determined by comparison of the transgene to the goat
beta casein endogenous gene.
[0194] Evaluation of Expression Levels
[0195] The expression level of the recombinant non-secreted
protein, e.g., hGAD or hMBP, in the milk of transgenic animals is
determined using enzymatic assays or Western blots.
[0196] Equivalents
[0197] Those skilled in the art will be able to recognize, or be
able to ascertain using no more than routine experimentation,
numerous equivalents to the specific procedures described herein.
Such equivalents are considered to be within the scope of this
invention and are covered by the following claims.
Sequence CWU 1
1
* * * * *