U.S. patent application number 10/170221 was filed with the patent office on 2003-10-09 for production of recombinant polypeptides by bovine species and transgenic methods.
This patent application is currently assigned to Pharming B.V.. Invention is credited to DeBoer, Herman A., Heynecker, Herbert L., Krimpenfort, Paul J.A., Lee, Sang Ha, Pieper, Frank, Platenburg, Gerard, Strijker, Rein.
Application Number | 20030192068 10/170221 |
Document ID | / |
Family ID | 27536159 |
Filed Date | 2003-10-09 |
United States Patent
Application |
20030192068 |
Kind Code |
A1 |
DeBoer, Herman A. ; et
al. |
October 9, 2003 |
Production of recombinant polypeptides by bovine species and
transgenic methods
Abstract
Transgenes for producing recombinant polypeptides transgenic
bovine species. A transgene for producing recombinant polypeptides
in the milk of transgenic bovine species comprises at least one
expression regulation sequence, a secretory DNA sequence encoding a
secretory signal sequence which is functional in mammary secretory
cells of the bovine species and a recombinant DNA sequence encoding
the recombinant polypeptide. Also included are methods for
producing transgenic bovine species. The method includes
introducing the above transgene into an embryonal target cell of a
bovine species, transplanting the transgenic embryonic target cell
formed thereby into a recipient bovine parent and identifying at
least one female offspring which is capable of producing the
recombinant polypeptide in its milk. The invention also includes
transgenic bovine species capable of producing recombinant
polypeptides in transgenic milk as well as the milk from such
transgenic bovine species and food formulations containing one or
more recombinant polypeptide. Methods are also provided for
producing transgenic non-human mammals having a desirable
phenotype. The method comprises first methylating a transgene
followed by introduction into fertilized oocytes. The oocytes are
then cultured to form pre-implantation embryos. Thereafter, at
least one cell is removed from each of the pre-implantation embryos
and the DNA digested with a restriction endonuclease capable of
cleaving the methylated transgene but incapable of cleaving the
unmethylated form of the transgene. Those pre-implantation embryos
which have integrated the transgene contain DNA which is resistant
to cleavage by the restriction endonuclease in the region
containing the transgene.
Inventors: |
DeBoer, Herman A.;
(Roelofarendsveen, NL) ; Strijker, Rein;
(Oegstgeest, NL) ; Heynecker, Herbert L.;
(Hillsborough, CA) ; Platenburg, Gerard;
(Voorschoten, NL) ; Lee, Sang Ha; (Leiden, NL)
; Pieper, Frank; (Utrecht, NL) ; Krimpenfort, Paul
J.A.; (Heemstede, NL) |
Correspondence
Address: |
TOWNSEND AND TOWNSEND AND CREW, LLP
WO
TWO EMBARCADERO CENTER
EIGHTH FLOOR
SAN FRANCISCO
CA
94111-3834
US
|
Assignee: |
Pharming B.V.
Leiden
NL
|
Family ID: |
27536159 |
Appl. No.: |
10/170221 |
Filed: |
June 11, 2002 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10170221 |
Jun 11, 2002 |
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09426591 |
Oct 26, 1999 |
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09426591 |
Oct 26, 1999 |
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08476798 |
Jun 7, 1995 |
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6140552 |
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08476798 |
Jun 7, 1995 |
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08154019 |
Nov 16, 1993 |
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5633076 |
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08154019 |
Nov 16, 1993 |
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08077788 |
Jun 15, 1993 |
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08077788 |
Jun 15, 1993 |
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07898956 |
Jun 15, 1992 |
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07898956 |
Jun 15, 1992 |
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07619131 |
Nov 27, 1990 |
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07619131 |
Nov 27, 1990 |
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07444745 |
Dec 1, 1989 |
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Current U.S.
Class: |
800/15 ;
435/320.1; 435/325; 435/455; 435/69.1; 536/23.2 |
Current CPC
Class: |
A01K 2217/05 20130101;
A01K 2227/105 20130101; A01K 67/0275 20130101; A01K 2217/072
20130101; A01K 2207/15 20130101; C12N 2800/30 20130101; A01K
67/0278 20130101; A01K 2227/101 20130101; C12N 15/8509 20130101;
A23C 9/20 20130101; C12N 2830/15 20130101; C12N 2840/44 20130101;
A01K 2217/00 20130101; A01K 2267/01 20130101; C12N 15/85 20130101;
C12N 2830/85 20130101; C12Y 304/21069 20130101; C12N 9/2462
20130101; C12N 2830/42 20130101; C07K 14/765 20130101; C07K 14/4732
20130101; C12N 9/6464 20130101; C12N 2830/008 20130101; C07K 14/79
20130101 |
Class at
Publication: |
800/15 ;
435/69.1; 435/320.1; 435/455; 435/325; 536/23.2 |
International
Class: |
A01K 067/027; C07H
021/04; C12P 021/02; C12N 005/06; C12N 015/85 |
Foreign Application Data
Date |
Code |
Application Number |
Nov 30, 1990 |
WO |
PCT/US90/06874 |
Nov 30, 1990 |
AU |
69608/91 |
Nov 30, 1990 |
CA |
2,075,206 |
Nov 30, 1990 |
EP |
91901026.4 |
Nov 30, 1990 |
FI |
923485 |
Nov 30, 1990 |
LK |
10403 |
Nov 30, 1990 |
NO |
P922996 |
Nov 30, 1990 |
OA |
PV60265 |
Nov 30, 1990 |
RU |
5052392.13 |
Dec 1, 1990 |
EP |
90109733.0 |
Jan 21, 1992 |
ID |
P-001607 |
Dec 18, 1990 |
IN |
1037/CAL/90 |
Nov 30, 1990 |
MY |
PI 9002116 |
Dec 3, 1990 |
NZ |
236310 |
Dec 1, 1990 |
PK |
498/90 |
May 30, 1990 |
PH |
41666 |
Dec 5, 1990 |
TW |
79110238 |
Jun 15, 1993 |
WO |
PCT/US93/05724 |
Claims
What is claimed is:
1. A transgene for producing a recombinant polypeptide in
transgenic bovine species comprising at least one expression
regulation DNA sequence functional in at least one cell-type of
said bovine species operably linked to a recombinant DNA encoding a
recombinant polypeptide, wherein said transgene is capable of
directing the expression of said recombinant DNA sequence in at
least said one cell-type of a bovine species containing said
transgene to produce said recombinant polypeptide.
2. The transgene of claim 1 wherein said expression regulation
sequences comprise 5' and 3' expression regulation sequences from a
serum albumin, said cell-type is liver cell, said recombinant
polypeptide is human serum albumin and said transgene further
comprises a secretory DNA sequence functional in said liver cells
and operably linked to the recombinant DNA encoding said human
serum albumin.
3. A transgene for producing a recombinant polypeptide in the milk
of transgenic bovine species comprising at least one expression
regulation DNA sequence functional in the mammary secretory cells
of said bovine species, a secretory DNA sequence encoding a
secretory signal sequence also functional in the mammary secretory
cells of said bovine species and a recombinant DNA sequence
encoding a recombinant polypeptide, wherein said secretory DNA
sequence is operably linked to said recombinant DNA sequence and to
form a secretory-recombinant DNA sequence said at least one
expression regulation sequence operably linked to said
secretory-recombinant DNA sequence, such that said transgene is
capable of directing the expression of said secretory-recombinant
DNA sequence in mammary secretory cells of bovine species
containing said transgene to produce a form of recombinant
polypeptide which when secreted from said mammary secretory cells
produces recombinant polypeptide in the milk of said bovine
species.
4. The transgene of claim 1 or 3 further comprising a recombinant
intervening sequence.
5. The transgene of claim 4 wherein said recombinant intervening
sequence is a hybrid intervening sequence.
6. The transgene of claim 5 wherein said hybrid intervening
sequence contains a permissive RNA splice signal.
7. The transgene of claim 3 wherein said recombinant polypeptide is
a homologous polypeptide from bovine species.
8. The transgene of claim 7 wherein said homologous polypeptide is
selected from the group consisting of caseins, lactoferrin,
lysozyme, cholesterol hydrolase and serum albumin.
9. The transgene of claim 3 wherein said recombinant polypeptide is
a heterologous polypeptide.
10. The transgene of claim 9 wherein said heterologous polypeptide
is selected from the group consisting of human milk proteins, human
serum proteins, and industrial enzymes.
11. The transgene of claim 10 wherein said heterologous polypeptide
is a human milk protein.
12. The transgene of claim 11 wherein said human milk protein is
selected from the group consisting of secretory immunoglobulins,
lysozyme, lactoferrin, lactoglobulin, .alpha.-lactalbumin and bile
salt-stimulated lipase.
13. The transgene of claim 12 wherein said milk protein is
lactoferrin or lysozyme.
14. The transgene of claim 10 wherein said heterologous polypeptide
is a human serum protein.
15. The transgene of claim 14 wherein said human serum protein is
selected from the group consisting of albumin, immunoglobulin,
Factor VIII, Factor IX and Protein C.
16. The transgene of claim 15 wherein said serum protein is
albumin.
17. The transgene of claim 10 wherein said heterologous polypeptide
is an industrial enzyme selected from the group consisting of
proteases, lipases, chitinases and ligninases.
18. The transgene of claim 3 wherein said secretory DNA sequence is
selected from the group consisting of DNA sequences encoding
secretory signal sequences from human lactoferrin, human serum
albumin, human lysozyme and secretory signal sequences from bovine
.alpha.S1-casein, .alpha.S2-casein, .beta.-casein, .kappa.-casein,
.alpha.-lactalbumin, .beta.-lactoglobulin, and serum albumin.
19. The transgene of claim 18 wherein said secretory DNA sequence
is the DNA sequence encoding the signal secretion sequence of
bovine .alpha.S1 casein.
20. The transgene of claim 3 wherein said at least one expression
regulation sequence comprises 5' expression regulation DNA
sequences operably linked to the 5' end of said
secretory-recombinant DNA sequence.
21. The transgene of claim 20 wherein said 5' expression regulation
DNA sequence is selected from the group consisting of 5' expression
regulation sequence from bovine genes encoding .alpha.S1-casein,
.alpha.S2-casein, .beta.-casein, .kappa.-casein,
.alpha.-lactalbumin, and .beta.-lactoglobulin.
22. The transgene of claim 21 wherein said 5' expression regulation
DNA sequence is a proximal 5' expression regulation sequence
comprising the promoter of bovine .alpha.S1-casein.
23. The transgene of claim 22 wherein said 5' expression regulation
DNA sequence further comprises a distal 5' expression regulation
sequence comprising 5'-flanking DNA sequence from bovine
.alpha.S1-casein.
24. The transgene of claim 20 further comprising 3' expression
regulation sequences operably linked to the 3' end of said
secretory-recombinant DNA sequence.
25. The transgene of claim 24 wherein said 3' expression regulation
sequence comprise 3' expression regulation sequence from bovine
genes encoding .alpha.S1-casein, .alpha.S2-casein, .beta.-casein,
.kappa.-casein, .alpha.-lactalbumin, and .beta.-lactoglobulin.
26. The transgene of claim 25 wherein said 3' expression regulation
DNA sequence comprises a 3' proximal expression regulation sequence
from bovine .alpha.S1-casein.
27. The transgene of claim 26 wherein said 3' expression regulation
DNA sequence further comprises a 3' distal expression regulation
sequence from bovine .alpha.S1-casein.
28. The transgene of claim 27 wherein said distal 5' expression
regulation DNA sequence comprises about a 30 kb 5'-flanking region
of bovine .alpha.S1-casein,and said distal 3' expression regulation
DNA sequence comprises about a 15 kb 3'-flanking region of bovine
.alpha.S1-casein.
29. A transgenic bovine species capable of producing a recombinant
polypeptide in at least one cell type of said animal.
30. A transgenic bovine species capable of producing recombinant
polypeptide in the milk of said transgenic species.
31. The transgenic bovine species of claim 30 wherein said
recombinant polypeptide is a homologous polypeptide from bovine
species.
32. The transgenic bovine species of claim 30 wherein said
recombinant polypeptide is a heterologous polypeptide.
33. The transgenic bovine species of claim 32 wherein said
heterologous polypeptide is selected from the group consisting of
human milk proteins, human serum proteins, and industrial
enzymes.
34. The transgenic bovine species of claim 33 wherein said
heterologous polypeptide is a human milk protein.
35. The transgenic bovine species of claim 34 wherein said human
milk protein is selected from the group consisting of secretory
immunoglobulins, lysozyme, lactoferrin, lactoglobulin,
.alpha.-lactalbumin and bile salt-stimulated lipase.
36. The transgenic bovine species of claim 35 wherein said milk
protein is lactoferrin or lysozyme.
37. The transgenic bovine species of claim 33 wherein said
heterologous polypeptide is a human serum protein.
38. The transgenic bovine species of claim 37 wherein said human
serum protein is selected from the group consisting of albumin,
immunoglobulin, Factor VIII, Factor IX and Protein C.
39. The transgenic bovine species of claim 38 wherein said serum
protein is albumin.
40. The transgenic bovine species of claim 33 wherein said
heterologous polypeptide is an industrial enzyme selected from the
group consisting of proteases, lipases, chitinases and
ligninases.
41. Milk from transgenic bovine species containing a recombinant
polypeptide.
42. The milk of claim 41 wherein said recombinant polypeptide is a
homologous polypeptide from bovine species.
43. The milk of claim 41 wherein said recombinant polypeptide is a
heterologous polypeptide.
44. The milk of claim 43 wherein said heterologous polypeptide is
selected from the group consisting of human milk proteins, human
serum proteins, and industrial enzymes.
45. The milk of claim 44 wherein said heterologous polypeptide is a
human milk protein.
46. The milk of claim 45 wherein said human milk protein is
selected from the group consisting of secretory immunoglobulins,
lysozyme, lactoferrin, lactoglobulin, .alpha.-lactalbumin and bile
salt-stimulated lipase.
47. The milk of claim 46 wherein -said milk protein is lactoferrin
or lysozyme.
48. The milk of claim 43 wherein said heterologous polypeptide is a
human serum protein.
49. The milk of claim 48 wherein said human serum protein is
selected from the group consisting of albumin, immunoglobulin,
Factor VIII, Factor IX and Protein C.
50. The milk of claim 49 wherein said serum protein is albumin.
51. A food formulation comprising transgenic milk containing a
recombinant polypeptide.
52. The food formulation of claim 51 wherein said recombinant
polypeptide is at least partially purified from said transgenic
milk.
53. The food formulation of claim 51 formulated with nutrients
appropriate for infant formula.
54. A method for producing a transgenic bovine species capable of
producing a recombinant polypeptide in the milk of said bovine
species, said method comprising: introducing the transgene of claim
1 into an embryonal target cell of a bovine species; transplanting
the transgenic embryonal target cell formed thereby or the embryo
obtained herefrom into a recipient female bovine parent; and
identifying at least one female offspring which is capable of
producing said recombinant polypeptide in the milk of said
offspring.
55. A method for producing a transgenic non-human mammal having a
desirable phenotype comprising: (a) methylating a transgene capable
of conferring said phenotype when incorporated into the cells of
said transgenic non-human animal; (b) introducing said methylated
transgene into fertilized oocytes of said non-human mammal to
permit integration of said transgene into the genomic DNA of said
fertilized oocytes; (c) culturing the individual oocytes formed
hereby to pre-implantation embryos thereby replicating the genome
of each of said fertilized oocytes; (d) removing at least one cell
from each of said pre-implantation embryos and lysing said at least
one cell to release the DNA contained therein; (e) contacting said
released DNA with a restriction endonuclease capable of cleaving
said methylated transgene but incapable of cleaving the
unmethylated form of said transgene formed after integration into
and replication of said genomic DNA; and (f) detecting which of
said cells from said pre-implantation embryos contain a transgene
which is resistant to cleavage by said restriction endonuclease as
an indication of which pre-implantation embryos have integrated
said transgene.
56. The method of claim 55 wherein said removal of at least one
cell forms a first and second hemi-embryo for each of said
pre-implementation embryos and each of said first hemi-embryos is
lysed and analyzed according to steps (d) through (f), said method
further comprising; (g) cloning at least one of said second
hemi-embryos; and (h) to form a multiplicity of transgenic
embryos.
57. The method of claim 56 further comprising transplanting more
than one of said transgenic embryos into recipient female parents
to produce a population containing at least two transgenic
non-human animals having the same genotype.
58. The method of claim 55 further comprising transplanting the
remainder of said pre-implantation embryo containing a genomically
integrated transgene into a recipient female parent and identifying
at least one offspring having said phenotype.
59. The method of claim 55 wherein said restriction endonuclease is
DPNI and said transgene is methylated at N6 of the adenine of the
sequence GATC contained within said transgene.
60. The method of claim 59 wherein said detection utilizes a
polymerase chain reaction using extension primers complementary to
sequences upstream and downstream to said GATC sequence.
61. The method of claim 59 wherein said non-human transgenic mammal
is bovine species, said transgene encodes a recombinant polypeptide
and said desired phenotype is the ability to produce said
recombinant polypeptide in the milk of said bovine species.
62. The method of claim 61 wherein said transgene is the transgene
of claim 3.
63. A transgene for producing a recombinant polypeptide in the milk
of transgenic bovine species comprising: (i) a bovine 5' expression
regulation sequence; (ii) a secretory DNA sequence encoding a
secretory signal sequence functional in the mammary secretory cells
of the bovine species; (iii) a recombinant DNA sequence encoding a
recombinant polypeptide, said secretory DNA sequence being operably
linked to said recombinant DNA sequence, wherein a
secretory-recombinant DNA sequence is formed, said
secretory-recombinant DNA sequence being operably linked to said
bovine expression regulation sequence; (iv) a 3' untranslated
sequence; (v) a 3' flanking sequence of a bovine gene; and wherein
said transgene is capable of directing the expression of said
secretory-recombinant DNA sequence in mammary secretory cells of
bovine species containing said transgene to produce a form of
recombinant polypeptide which when secreted from said mammary
secretory cells produces recombinant polypeptide in the milk of
said bovine species.
64. The transgene of claim 63, further comprising a recombinant
intervening sequence.
65. The transgene of claim 64 wherein the recombinant intervening
sequence is a hybrid intervening sequence.
66. The transgene of claim 65 wherein the hybrid intervening
sequence comprises a 5' portion of an intervening sequence from
bovine .alpha.-S.sub.1-casein and a 3' sequence portion of an IgG
heavy chain intervening sequence.
67. The transgene of claim 66 wherein the 3' sequence portion is a
3' splice signal sequence associated with the J-C segment
rearrangement of an IgG heavy chain.
68. The transgene of claim 63, wherein the bovine expression
regulation sequence and the 3' flanking sequence are derived from
the same bovine gene.
69. The transgene of claim 63, wherein the bovine expression
regulation sequence, the 3' untranslated sequence, and the 3'
flanking sequence are derived from the same bovine gene.
70. The transgene of claim 68 or claim 69 wherein the bovine gene
is .alpha.-S.sub.1-casein.
71. The transgene of claim 70 wherein the bovine expression
regulation sequence comprises about a 30 kb 5'-flanking region of
bovine .alpha.S1-casein and the 3'-flanking sequence comprises
about a 15 kb 3'-flanking region of bovine .alpha.S1-casein.
72. The transgene of claim 3 or claim 63 wherein the milk comprises
greater than 50 micrograms of the recombinant polypeptide per
milliliter.
73. A transgenic bovine species capable of producing a recombinant
polypeptide in saliva.
74. The semen of a transgenic bovine.
75. A transgene for producing a recombinant polypeptide in the milk
of transgenic bovine species comprising: (i) a 5' expression
regulation sequence; (ii) a secretory DNA sequence encoding a
secretory signal sequence functional in the mammary secretory cells
of the bovine species; (iii) a recombinant DNA sequence encoding a
recombinant polypeptide, said secretory DNA sequence being operably
linked to said recombinant DNA sequence, wherein a
secretory-recombinant DNA sequence is formed, said
secretory-recombinant DNA sequence being operably linked to the 5'
expression regulation sequence; (iv) a 3' untranslated sequence;
and, (v) a 3' flanking sequence from a human gene; wherein the
transgene is capable of directing the expression of the
secretory-recombinant DNA sequence in mammary secretory cells of
bovine species containing the transgene to produce a form of
recombinant polypeptide which when secreted from the mammary
secretory cells produces recombinant polypeptide in the milk of the
bovine species.
76. The transgene of claim 75, wherein the 5' expression regulatory
sequence is a bovine sequence.
77. The transgene of claim 75 or claim 76, wherein the 3' flanking
sequence is from the human lactoferrin (hLF) gene.
78. The transgene of claim 77, wherein the 3' flanking sequence is
9 kilobase pairs in length.
79. The transgene of claim 77, wherein the recombinant polypeptide
is human lactoferrin.
80. The transgene of claim 75, wherein the 5' expression regulation
sequence, the secretory DNA sequence, the recombinant DNA sequence
encoding a recombinant polypeptide, the 3' untranslated sequence;
and the 3' flanking sequence are from a human gene.
81. The transgene of claim 80, wherein the human gene is the
lactoferrin gene.
82. A method for expressing a human polypeptide in the milk of a
bovine comprising: introducing a human genomic fragment encoding
the human polypeptide into an embryonal target cell of a bovine
species; transplanting the transgenic embryonal target cell formed
thereby or the embryo obtained therefrom into a recipient female
bovine parent; and identifying at least one female offspring which
is capable of producing the recombinant polypeptide in the milk of
the offspring.
83. The method of claim 82, wherein the human polypeptide is
lactoferrin.
84. A transgene for producing a recombinant polypeptide in the milk
of transgenic bovine species, said transgene comprising: (i) a 5'
expression regulation sequence from a first milk protein gene; (ii)
a recombinant DNA sequence from a second milk protein gene encoding
a recombinant polypeptide; (iii) a secretory DNA sequence from said
first or second milk protein gene, said secretory DNA sequence
encoding a secretory signal sequence functional in the mammary
secretory cells of the bovine species, and said secretory operably
linked to said recombinant DNA sequence and to said 5' expression
sequence; (iv) a 3' untranslated sequence from said first or second
milk protein gene; and (v) a 3' flanking sequence from said first
or second milk protein gene; wherein the transgene is capable of
directing the expression of the secretory-recombinant DNA sequence
in mammary secretory cells of bovine species containing the
transgene to produce a form of recombinant polypeptide which when
secreted from the mammary secretory cells produces recombinant
polypeptide in the milk of the bovine species.
85. The transgene of claim 84, further comprising: a 5'
untranslated sequence from said first or second milk protein
gene.
86. The transgene of claim 85, wherein said recombinant DNA
sequence is a genomic sequence comprising at least one intronic
sequence.
87. The transgene of claim 86, wherein said first gene is a bovine
.alpha.S1-casein gene.
88. The transgene of claim 86, wherein said first gene is a bovine
.beta.-lactoglobulin gene.
89. The transgene of claim 87, wherein said second gene is a human
lactoferrin gene.
90. The transgene of claim 88, wherein said second gene is a human
lactoferrin gene.
91. The transgene of claim 87, wherein the second gene is a human
lysozyme gene.
92. The transgene of claim 89, wherein said 3' untranslated
sequence and said 3' flanking sequence are from said second
gene.
93. The transgene of claim 89, wherein said 3' untranslated
sequence and said 3' flanking sequence are from said first
gene.
94. The transgene of claim 90, wherein said 3' untranslated
sequence and said 3' flanking sequence are from said second
gene.
95. The transgene of claim 91, wherein said 3' untranslated
sequence and said 3' flanking sequence are from said first
gene.
96. A method of producing a transgenic bovine species of claim 29,
said method comprising: obtaining a plurality of ova from bovine
ovaries; fertilizing said ova in vitro to form zygotes; introducing
a transgene, capable of being expressed in at least one cell type
of said transgenic bovine species to produce said recombinant
polypeptide, into said zygotes; propagating said zygotes to form
embryos; transplanting said embryos into a recipient female bovine
parent; identifying at least one offspring containing said
transgene; and breeding said offspring to produce said transgenic
bovine species.
97. The method of claim 96, wherein in said introducing step, said
zygotes are substantially synchronous.
Description
[0001] This a continuation-in-part of U.S. patent application Ser.
No. 08/077,788, filed Jun. 15, 1993, which is a
continuation-in-part of U.S. patent application Ser. No.
07/898,956, filed Jun. 15, 1992, which is a continuation-in-part of
U.S. patent application Ser. No. 07/619,131 filed Nov. 27, 1990,
which is a continuation-in-part of U.S. patent application Ser. No.
07/444,745 filed Dec. 1, 1989 (now abandoned). Each of the above
applications is incorporated by reference in its entirety for all
purposes.
FIELD OF THE INVENTION
[0002] The invention relates to the production of recombinant
polypeptides by transgenic bovine species and to methods for
producing transgenic non-human mammals having a desired
phenotype.
BACKGROUND OF THE INVENTION
[0003] There is a plethora of literature relating to the expression
of heterologous genes in lower organisms such as unicellular
bacteria, yeast and filamentous fungi, and in higher cell types
such as mammalian cells. There are also numerous reports on the
production of transgenic animals, most of which relate to the
production of transgenic mice. See, e.g., U.S. Pat. No. 4,736,866
(transgenic mice containing activated oncogene); Andres, A., et al.
(1987) Proc. Natl. Acad. Sci. USA 84:1299-1303 (HA-RAS oncogene
under control of whey acid protein promoter); Schoenberger, C. A.,
et al. (1987) Experientia 43:644 and (1988) EMBO J. 7:169-175
(C-myc oncogene under control of whey acid protein promoter); and
Muller, W. J., et al. (1988) Cell 54:105-115 (C-myc oncogene under
control of the mouse mammary tumor virus promoter). Several
laboratories have also reported the production of transgenic
porcine species (Miller, K. F., et al. (1989) J. Endocrin.
120:481-488 (expression of human or bovine growth hormone gene in
transgenic swine); Vize, P. D., et al. (1988) J. Cell Sci.
90:295-300 (porcine growth hormone fusion gene in transgenic pigs);
and Ebert, K. et al. (1988) Mol. Endocrin. 2:277-283 (MMLV-rat
somatotropin fusion gene in transgenic pigs)), transgenic sheep
(Nancarrow, et al. (1987) Theriogenology 27:263 (transgenic sheep
containing bovine growth hormone gene) Clark, A. J. et al. (1989)
Bio/Technology 7:487-482 and Simons, J., et al. (1988)
Bio/Technology 6:179-183 (human factor IX and .alpha.-1 antitrypsin
CONA in ovine species), and rabbit (Hanover, S. V., et al. (1987)
Deutche Tierarztliche Wochenschrift 94,:476-478 (production of
transgenic rabbits by injection of uteroglobin-promoter-CAT fusion
gene into fertilized rabbit oocytes). A number of reports have also
suggested the production of transgenic cattle (Wagner, et al.
(1984) Theriogenology 21:29-44) with one reporting some progress in
microinjection techniques (Lohse, J. K., et al. (1985)
Theriogenology 23:205). However, little, if any, success has been
achieved in producing transgenic cows. Scientific articles which
clearly demonstrate the actual production of a transgenic cow
capable of producing a heterologous protein are presently unknown.
This, despite the statements that one transgenic cow was produced
in Canada which expressed human .beta.-interferon (Van Brunt, J.
(1988) Bio/Technology 6:1149-1155) and that transient expression of
human .alpha.-fetoprotein in liver and blood was obtained on one
occasion (Church, R. B. (1986) Biotechnology News Watch 6 (15), 4).
One reference reports that bovine papilloma virus was apparently
integrated but not expressed in a transgenic cow (Roschlau, et al-
(1988) Arch. Tierz., Berlin 31:3-8). A recent article has
summarized the genetic engineering of livestock. (Pursel, V. G. et
al. (1989) Science 244:1281-1288)
[0004] A number of laboratories have reported tissue-specific
expression of DNA encoding various proteins in the mammary gland or
the production of various proteins in the milk of transgenic mice
and sheep. For example, Simmons, J. P., et al. (1987) Nature
328:530-532 report the microinjection of a 16.2 kb genomic fragment
encoding .beta.-lactoglobulin (BLG) including 4 kb of 5' sequence,
4.9 kb of the BLG transcription unit and 7.3 kb of 3' flanking
sequence into fertilized mouse eggs. According to these authors,
the sheep BLG was expressed in mammary tissue and produced BLG in
the milk of the transgenic mice at concentrations ranging from
about 3.0 to about 23 mg/ml. When, however, cDNA encoding human
factor IX or human .alpha.1-antitrypsin was inserted into the 5'
untranslated region of the BLG gene and microinjected into sheep
(Simmons, J. P., et al. (1988) Bio/Technology 6:179-183) the
production of factor IX or .alpha.1-antitrypsin was significantly
reduced (25ng/ml for factor IX and 10mg/ml for
.alpha.1-antitrypsin; see Clark, A. J., et al. (1989)
Bio/Technology 7:487-492).
[0005] In a similar approach, a 14 kb genomic clone containing the
entire 7.5 kb rat .beta.-casein together with 3.5 kb of 5' and 3.0
kb of 3' flanking DNA was reportedly microinjected into fertilized
mouse oocytes. Lee, et al. (1988) Nucl. Acids Res. 16:1027-1041.
Yet, in this case, the level of expression of the rat
.beta.-transgene in the lactating mammary gland of transgenic mice
was reported to be at a level of 0.01-1% of the endogenous mouse
.beta.-casein gene.
[0006] Human tissue plasminogen activator (t-PA) reportedly was
produced in transgenic mouse milk at the levels between 0.2 and
about 0.4 .mu.g/ml when a cDNA encoding a human t-PA with its
endogenous secretion sequence was expressed under control of a 2.6
kb 5' sequence of the murine whey acid protein gene. Gordon, K., et
al. (1987) Bio/Technology 5:1183-1187. Subsequent experiments using
the same or similar construction reportedly produced t-PA in
different mouse lines arranging from less than 20 ng of t-PA per ml
of milk to about 50 .mu.g/ml. Pittius, C. W., et al. (1988) Proc.
Natl. Acad. Sci. USA 85:5874-5878.
[0007] U.S. Pat. No. 4,873,316 issued Oct. 10, 1989, discloses the
use of 9 kb of 5' sequence from the bovine .alpha.S1 casein gene
including the casein signal peptide and several casein codons fused
to a mature t-PA sequence. The transgenic mice obtained with this
construct reportedly produced about 0.2-0.5 .mu.g/ml of a t-PA
fusion protein in their milk.
[0008] In addition, a number of patent publications purportedly
describe the production of specific proteins in the milk of
transgenic mice and sheep. See, e.g. European Patent Publication
No. 0 264 166 published Apr. 20, 1988 (hepatitis B surface antigen
and t-PA genes under control of the whey acid promoter protein for
mammary tissue specific expression in mice); PCT Publication No.
W088/00239 published Jan. 14, 1988 (tissue specific expression of a
transgene encoding factor IX under control of a whey protein
promoter in sheep); PCT Publication No. W088/01648 published Mar.
10, 1988 (transgenic mouse having mammary secretory cells
incorporating a recombinant expression system comprising a bovine
.alpha.-lactalbumin gene fused to interleukin-2); European Pat.
Pub. No. 0 279 582 published Aug. 24, 1988 (tissue-specific
expression of chloramphenicol acetyltransferase under control of
rat .beta.-casein promoter in transgenic mice); and PCT Pub. No.
W088/10118 published Dec. 29, 1988 (transgenic mice and sheep
containing transgene encoding bovine .alpha.S1 casein promoter and
signal sequence fused to t-PA).
[0009] Given the state of the transgenic art, it is apparent that a
need exists for methods which enable the efficient production of
transgenic mammals, especially transgenic mammals other than
transgenic mice.
[0010] Further, it is apparent that a need exists for methods for
producing transgenic bovine species which are capable of producing
recombinant polypeptides such as human milk proteins and human
serum proteins in the milk of such transgenic mammals.
[0011] Accordingly, it is an object herein to provide methods for
detecting the transgenesis of fertilized oocytes prior to
implantation.
[0012] In addition, it is an object herein to provide transgenic
bovine species which are capable of producing recombinant
polypeptides which are maintained intracellularly or are secreted
extracellularly.
[0013] It is also an object herein to provide transgenic bovine
species which are capable of producing recombinant polypeptides
such as human milk proteins and human serum proteins in the milk of
such transgenic animals.
[0014] Further, it is an object herein to provide milk from a
transgenic bovine species containing such recombinant
polypeptides.
[0015] Still further, it is an object herein to provide food
formulations supplemented with recombinant polypeptides from such
transgenic milk such as human infant formula supplemented with
human lactoferrin.
[0016] Further, it is an object herein to provide transgenes which
are capable of directing the production of recombinant polypeptides
in the milk of transgenic bovine species.
[0017] The references discussed herein are provided solely for
their disclosure prior to the filing date of the present
application. Nothing herein is to be construed as an admission that
the inventors are not entitled to antedate such disclosure by
priority based on earlier filed applications.
SUMMARY OF THE INVENTION
[0018] In accordance with the above objects, the invention includes
transgenes for producing recombinant polypeptides in the milk of
transgenic bovine species. The production of such transgenic bovine
milk containing one or more recombinant polypeptides is desirable
since it provides a matrix wherein little or no purification is
necessary for human consumption- The transgene comprises a
secretory DNA sequence encoding a secretory signal sequence which
is functional in mammary secretory cells of the bovine species of
interest and a recombinant DNA sequence encoding the recombinant
polypeptide. These sequences are operably linked to form a
secretory-recombinant DNA sequence. At least one expression
regulation sequence, functional in the mammary secretory cells of
the bovine species, is operably linked to the secretory-recombinant
DNA sequence. The transgene so constructed is capable of directing
the expression of the secretory-recombinant DNA sequence in mammary
secretory cells of bovine species containing the transgene. Such
expression produces a form of recombinant polypeptide which is
secreted from the mammary secretory cells into the milk of the
transgenic bovine species.
[0019] In addition, the invention includes methods for producing
such transgenic bovine species. The method includes introducing the
above transgene into an embryonal target cell of a bovine species,
transplanting the transgenic embryonic target cell formed thereby
into a recipient bovine parent and identifying at least one female
offspring which is capable of producing the recombinant polypeptide
in its milk.
[0020] The invention also includes transgenic bovine species
capable of producing recombinant polypeptides in the milk of
lactating females of said species, the milk from such transgenic
bovine species containing such recombinant polypeptides and food
formulations containing the transgenic milk in liquid or dried
form, as well as food formulations supplemented with one or more
recombinant polypeptides from such transgenic milk.
[0021] In addition to the foregoing, the invention includes
transgenes and transgenic bovine species containing transgenes that
are capable of producing a recombinant polypeptide. Such transgenes
are similar to the aforementioned transgenes for milk secretion and
are characterized by having an expression regulation sequence which
targets the expression of the DNA encoding the recombinant
polypeptide to a particular cell or tissue type, e.g. expression of
human serum albumin in the liver of a transgenic bovine species.
When the recombinant polypeptide is to be secreted from such
targeted cells or tissues, a secretory DNA sequence encoding a
secretory signal sequence functional in the particular targeted
cell or tissue is operably linked to the recombinant DNA sequence
encoding the recombinant polypeptide, e.g. secretion of human serum
albumin from bovine liver into the bovine circulatory system.
[0022] Further, the invention includes methods for producing
transgenic non-human mammals having a desirable phenotype. The
method comprises first causing the methylation of a transgene
capable of conferring the desirable phenotype when incorporated
into the cells of a transgenic non-human animal, e.g., by
transforming an appropriate bacterium, such as E. coli MM 294, with
a plasmid containing the transgene. The methylated transgene is
then excised and introduced into fertilized oocytes of the
non-human animal to permit integration into the genome. The oocytes
are then cultured to form pre-implantation embryos thereby
replicating the genome of each of the fertilized oocytes.
Thereafter, at least one cell is removed from each of the
pre-implantation embryos and treated to release the DNA contained
therein. Each of the released DNAs are then digested with a
restriction endonuclease capable of cleaving the methylated
transgene but incapable of cleaving the unmethylated form of the
transgene formed after integration into and replication of the
genomic DNA. Those pre-implantation embryos which have integrated
the transgene contain DNA which is resistant to cleavage by the
restriction endonuclease in the region containing the transgene.
This resistance to digestion, which can be detected by
electrophoresis of the digest after PCR amplification of the DNA
and hybridization with a labelled probe for the transgene,
facilitates the identification of successful transgenesis.
[0023] The invention also includes a method to produce a population
of transgenic offspring having the same genotype. This method
utilizes a specific embodiment of the above method for detecting
early transgenesis. In this method, a methylated transgene is
introduced into fertilized oocytes which are cultured to
pre-implantation embryos. Thereafter, each pre-implantation embryo
is divided to form first and second hemi-embryos. Each of the first
hemi-embryos are then analyzed for transgenesis as described above.
After identifying successful transgenesis in at least one first
hemi-embryo, the second untreated hemi-embryo which contains the
integrated transgene, is cloned to form a multiplicity of clonal
transgenic blastocysts or hemi-blastocysts, each of which have the
same genotype. The transgenic embryos are thereafter transplanted
into one or more recipient female parents to produce a population
of transgenic non-human mammals having the same genotype.
BRIEF DESCRIPTION OF THE DRAWINGS
[0024] The accompanying drawings, which are incorporated in and
form a part of the specification, illustrate embodiments of the
present invention and, together with the description, serve to
explain the principles of the invention. In the drawings:
[0025] FIG. 1 depicts the DNA (Seq. ID No.: 1) and amino acid (Seq.
ID No.: 2) sequence for a human lactoferrin clone derived from a
human mammary cDNA library as described herein except that the
sequence between nucleotides 1557-1791 and 2050-2119 corresponds to
the previously published sequence (Rado et al. (1987) Blood
70:989-993).
[0026] FIG. 2 depicts the complete DNA (Seq. ID No.: 3) and amino
acid (Seq. ID No.: 4) sequence of human lactoferrin including 5,'
and 3' untranslated sequence as well as the complete human
lactoferrin signal sequence.
[0027] FIG. 3 is a restriction map of a clone of a 5'-flanking
region of bovine .alpha.S1 casein gene.
[0028] FIG. 4 is a restriction map of a clone of a 3'-flanking
region of bovine .alpha.S1 casein gene
[0029] FIGS. 5A, 5B and 5C depict the construction of pSI3'5'CAT
and pSI5'CAT.
[0030] FIG. 6 depicts pMH-1.
[0031] FIGS. 7A through 7F depict the construction of expression
vectors containing sequences encoding human lactoferrin.
[0032] FIG. 8 depicts the genome of human serum albumin, the
fragments used to generate transgenic mice contained in this
genomic DNA and the identification of the fragment sizes which
would be obtained upon the digestion of genomic DNA from a
transgenic mouse with the restriction enzymes BstE-II and Nco-I or
with Nco-I and Hindi-III.
[0033] FIG. 9 depicts an alternate pathway for the construction of
a transgene of the invention encoding human lactoferrin.
[0034] FIG. 10 depicts the construction of a plasmid pPC containing
a transgene encoding Protein C.
[0035] FIG. 11 depicts the DNA sequence for a hybrid intervening
sequence used in a preferred embodiment of the invention. The
predicted intervening sequence (shown in lower case) consists of
the 5'-end of IVS-1 from bovine .alpha.S1 casein (from position +54
to +180 with respect to the start of transcription) fused to the
3'-end of a human IgG splice sequence. The Hind III site (in bold
type and underlined) derives from the IgG sequence and marks the
junction between the .alpha.S1 and IgG splice sequences. The 5'-end
upper case sequence depicts the complete exon one of the bovine
.alpha.S1 casein gene. The 3'-end upper case sequence represents
the splice junction of the IgG gene through to the Pst I site
(CTGCAG) incorporated in the cloning vector, pMH1.
[0036] FIG. 12A is a restriction map or a bovine .alpha.S1 casein
promoter hLF cDNA transgene.
[0037] FIG. 12B shows a Southern blot analysis of DNA isolated from
various bovine and murine tissues using an hLF cDNA probe.
[0038] FIG. 13 depicts restriction maps of hLF genomic clones 13.1
and 13.2.
[0039] FIG. 14 depicts the BamHI fragment from genomic hLF
subcloned into plasmid pUC19.
[0040] FIG. 15A depicts a restriction map of the 8hLFgen9k or
16hLFgen9k construct containing the 8 or 16 kb .alpha.S1 casein
promoter, a ClaI-ApaI synthetic linker and the 9 kb (i.e., 8.9 kb)
ApaI-SalI genomic hLF fragment.
[0041] FIG. 15B depicts the DNA sequence of the ClaI-ApaI synthetic
sequence shown in FIG. 15A.
[0042] FIG. 15C depicts the IVS and the structure of exon 1 and
part of exon 2 of the genomic hLF construct shown in FIG. 15A
through FIG. 17.
[0043] FIG. 16 depicts the coinjection of the NotI-SalI fragment
from the 8hLFgen9k or 16hLFgen9k construct (as shown in FIG. 15A)
with the 3' ClaI fragment of genomic hLF.
[0044] FIG. 17 depicts the generation of a genomic 8hLF transgene
by linking the NotI-MluI fragment from the 8hLFgen9k construction
(shown in FIG. 15A), the MluI-ClaI fragment from clone 13.2
depicted in FIG. 13 and a ClaI-NotI linker. FIG. 17 also depicts
the DNA sequence of the ClaI-NotI linker.
[0045] FIGS. 18-20 depict the generation of the .beta.LG-hLFgen and
.beta.LG-hLFgen37 constructs.
[0046] FIG. 21 depicts the design of the 16,8hLZ expression
vector.
[0047] FIG. 22 depicts the design of the 16,8hLZ3 expression
vector.
[0048] FIGS. 23A-23E depict the pathway for the construction of
plasmid p16,8hLZ.
[0049] FIG. 24 depicts a comparison between the DNA of bovine
.beta.LG and sheep .beta.LG. The top sequence represents the bovine
sequence.
[0050] FIG. 25 shows the linker GP 278/279.
[0051] FIG. 26 depicts the p16,8A hLZ3 expression vector.
[0052] FIG. 27 depicts the 16,A hLZ3 expression vector.
DETAILED DESCRIPTION OF THE INVENTION
[0053] The "non-human mammals" of the invention comprise all
non-human mammals capable of producing a "transgenic non-human
mammal" having a "desirable phenotype". Such mammals include
non-human primates, murine species, bovine species, canine species,
etc. Preferred non-human animals include be, porcine and ovine
species, most preferably bovine species.
[0054] Desirable phenotypes for transgenic non-human mammals
include, but are not limited to, the production of recombinant
polypeptides in the milk of female transgenic non-human mammals,
the production of animal models for the study of disease, the
production of animals with higher resistance to disease (e.g.
diseases of the mammary gland such as mastitis) and the production
of recombinant polypeptides in the blood, urine or other suitable
body fluid or tissue of the animal. In the preferred
embodiments,-transgenic bovine species are disclosed which are
capable of producing recombinant human lactoferrin, human serum
albumin and human Protein C in the milk of lactating females or
human serum albumin in the liver of the transgenic animal.
[0055] The transgenic non-human mammals of the invention are
produced by introducing a "transgene" into an embryonal target cell
of the animal of choice. In one aspect of the invention, a
transgene is a DNA sequence which is capable of producing a
desirable phenotype when contained in the genome of cells of a
transgenic non-human mammal. In specific embodiments, the transgene
comprises a "recombinant DNA sequence" encoding a "recombinant
polypeptide". In such cases, the transgene is capable of being
expressed to produce the recombinant polypeptide.
[0056] As used herein, a "recombinant polypeptide" (or the
recombinant DNA sequence encoding the same) is either a
"heterologous polypeptide" or a "homologous polypeptide".
Heterologous polypeptides are polypeptides which are not normally
produced by the transgenic animal. Examples of heterologous
polypeptides include human milk proteins such as lactoferrin,
lysozyme, secreted immunoglobulins, lactalbumin, bile
salt-stimulated lipase, etc., human serum proteins such as albumin,
immunoglobulins, Factor VIII, Factor IX, protein C, etc. and
industrial enzymes such as proteases, lipases, chitinases, and
liginases from procaryotic and eucaryotic sources. The recombinant
DNA sequences include genomic and cDNA sequences encoding the
recombinant polypeptide.
[0057] When recombinant DNA sequences encoding a heterologous
polypeptide are used, the transgene may be integrated in a random
manner into the genome of the species used for transgenesis. As
disclosed in the Examples, transgenes encoding human lactoferrin,
human serum albumin and human Protein C in conjunction with a
.alpha.S1 casein secretory signal sequence under control of
.alpha.S1 casein expression regulation sequences are designed to
produce and secrete these heterologous polypeptides from the
mammary gland of a lactating transgenic mammal into its milk.
[0058] As used herein, a homologous polypeptide is one which is
endogenous to the particular transgenic species. Examples of
endogenous polypeptides from bovine species include bovine milk
proteins such as .alpha.S1, .alpha.S2, .beta.- and .kappa.-casein,
.beta.-lactoglobulin lactoferrin, lysozyme, cholesterol hydrolase,
serum proteins such as serum albumin and proteinaceous hormones
such as growth hormones. When recombinant DNA sequences encoding a
homologous polypeptide are used, the transgene is preferably
integrated in a random manner into the genome of the species used
for transgenesis. Such random integration results in a transgenic
animal which contains not only the transgene encoding the
endogenous polypeptide but also the corresponding endogenous
genomic DNA sequence. Accordingly, such transgenic non-human
mammals are readily characterized by an increase in the copy number
of genes encoding the endogenous polypeptide. Further, the
transgene will generally be located at a position which is
different from the endogenous gene.
[0059] When DNA encoding a homologous polypeptide is expressed, for
example, in bovine species, the transgenic animal is characterized
by an increase in the amount of the homologous polypeptide in
either the endogenous tissue or fluid in which it is normally found
and/or by its presence in a tissue and/or body fluid which either
does not normally contain the homologous polypeptide or produces it
at significantly lower levels.
[0060] Thus, for example, bovine cholesterol hydrolase is normally
present in the colostrum for about the first 15-20 days of
lactation. This naturally,occurring endogenous polypeptide
increases calf weight. This protein, however, is also a homologous
polypeptide when, for example, its expression in mammary secretory
cells is placed under the control of expression regulation
sequences, such as those obtained from bovine casein genes, which
facilitate the expression of the homologous polypeptide beyond the
lactation period that it is normally present. Thus, according to
one aspect of the invention, bovine cholesterol hydrolase
expression is maintained in transgenic bovine milk by placing the
expression of cholesterol hydrolase recombinant DNA (either cDNA or
genomic) under the control of bovine .alpha.S1 casein expression
regulation sequences. When a genomic recombinant DNA is used, it is
engineered such that it has appropriate restriction sites (e.g.
ClaI and SalI) at the 5' and 3' end of the structural gene such
that it is capable of being inserted into an appropriate transgene
genomic cassette (e.g. p-16 kb, CS which is described in Example
15). Alternatively, a recombinant DNA encoding bovine cholesterol
hydrolase derived from cDNA may be placed under control of bovine
.alpha.S1 casein expression regulation sequence by substituting the
human lactoferrin sequences in a plasmid such as p16, 8HLF3
(containing a hybrid intervening sequence) or p16, 8HLF4
(containing a homologous .alpha.S1 casein intervening sequence).
When these particular plasmids are used, the cDNA clone is
engineered such that it has appropriate ClaI and SalI restriction
sites at the ends of the recombinant DNA.
[0061] By way of further example, bovine lactoferrin is normally
present in only trace amounts in cow's milk. When, however, bovine
lactoferrin is expressed under control of other regulatory
sequences, for example, obtained from an .alpha.S1 casein gene,
higher amounts of lactoferrin in the milk of transgenic bovine
species are obtained. In another example, a transgene comprising
DNA encoding homologous bovine growth hormone is incorporated into
the bovine genome to confer superior growth characteristics to the
transgenic animal. In other instances, homologous polypeptides
include, for example, a polypeptide which normally is maintained
intracellularly in a particular species but which is secreted into
the milk or other extracellular compartment of the transgenic
species, such as the circulatory system.
[0062] Each of the heterologous or homologous polypeptides are
characterized by specific amino acid and nucleic acid sequences. It
is to be understood, however, that such sequences include naturally
occurring allelic variations thereof and variants produced by
recombinant methods wherein such nucleic acid and polypeptide
sequences have been modified by the substitution, insertion and/or
deletion of one or more nucleotides in such nucleic acids to cause
the substitution, insertion or deletion of one ore more amino acid
residues in the recombinant polypeptide.
[0063] When expression of the DNA of the transgene is necessary to
generate a desired phenotype, e.g. to produce a recombinant
polypeptide, the transgene typically includes at least a 5' and
preferably additional 3' "expression regulation sequences" each
operably linked to a recombinant or secretory-recombinant DNA as
defined hereinafter. Such expression regulation sequences in
addition to controlling transcription also contribute to RNA
stability and processing, at least to the extent they are also
transcribed.
[0064] Such expression regulation sequences are chosen to produce
tissue-specific or cell type-specific expression of the recombinant
or secretory-recombinant DNA. Once a tissue or cell type is chosen
for expression, 5' and optional 3' expression regulation sequences
are chosen. Generally, such expression regulation sequences are
derived from genes that are expressed primarily in the tissue or
cell type chosen. Preferably, the genes from which these expression
regulation sequences are obtained are expressed substantially only
in the tissue or cell type chosen, although secondary expression in
other tissue and/or cell types is acceptable if expression of the
recombinant DNA in the transgene in such tissue or cell type is not
detrimental to the transgenic animal. Particularly preferred
expression regulation sequences are those endogenous to the species
of animal to be manipulated. However, expression regulation
sequences from other species such as those from human genes may
also be used. Particularly preferred expression regulation
sequences from human genes are human lactoferrin (hLF) sequences.
In some instances, the expression regulation sequences and the
recombinant DNA sequences (either genomic or CDNA) are from the
same species, e.g., each from bovine species or from a human
source. In such cases, the expression regulation sequence and the
recombinant DNA sequence are homologous to each other.
Alteratively, the expression regulation sequences and recombinant
DNA sequences (either cDNA or genomic) are obtained from different
species, e.g., an expression regulation sequence from bovine
species and a recombinant DNA sequence from a human source). In
such cases, the expression regulation and recombinant DNA sequence
are heterologous to each other. The following defines expression
regulation sequences from endogenous genes. Such definitions are
also applicable to expression regulation sequences from
non-endogenous, heterologous genes.
[0065] In general, the 5' expression regulation sequence includes
the transcribed portion of the endogenous gene upstream from the
translation initiation sequence (the 5' untranslated region or 5'
UTR) and those flanking sequences upstream therefrom which comprise
a functional promoter. As used herein, a "functional promoter"
includes those necessary untranscribed DNA sequences which direct
the binding of RNA polymerase to the endogenous gene to promote
transcription. Such sequences typically comprise a TATA sequence or
box located generally about 25 to 30 nucleotides from the
transcription initiation site. The TATA box is also sometimes
referred to the proximal signal. In many instances, the promoter
further comprises one or more distal signals located upstream from
the proximal signal (TATA box) which are necessary to initiate
transcription. Such promoter sequences are generally contained
within the first 100 to 200 nucleotides located upstream from the
transcription initiation site, but may extend up to 500 to 600
nucleotides from the transcription initiation site. Such sequences
are either readily apparent to those skilled in the art or readily
identifiable by standard methods. Such promoter sequences alone or
in combination with the 5' untranslated region are referred to
herein as "proximal 5' expression regulation sequences".
[0066] In addition to such proximal 5' expression regulation
sequences, it is preferred that additional 5' flanking sequences
(referred to herein as "distal 5' expression regulation sequences")
also be included in the transgene. Such distal 5' expression
regulation sequences are believed to contain one or more enhancer
and/or other sequences which facilitate expression of the
endogenous gene and as a consequence facilitate the expression of
the recombinant or secretory-recombinant DNA sequence operably
linked to the distal and proximal 5' expression regulation
sequences. The amount of distal 5' expression regulation sequence
depends upon the endogenous gene from which the expression
regulation sequences are derived. In general, however, such
sequences comprise 5' flanking regions of approximately 1 kb, more
preferably 16 kb and most preferably about 30 kb of 5' flanking
sequence. The determination of the optimal amount of distal 5'
expression regulation sequence used from any particular endogenous
gene is readily determined by varying the amount of distal 5'
expression regulation sequence to obtain maximal expression. In
general, the distal 5' expression regulation sequence will not be
so large as to extend into an adjacent gene and will not include
DNA sequences which adversely effect the level of transgene
expression.
[0067] In addition, it is preferred that 3' expression regulation
sequences also- be included to supplement tissue or cell-type
specific expression. Such 3' expression regulation sequences
include 3' proximal and 3' distal expression regulation sequences
from an appropriate endogenous gene. The 3' proximal expression
regulation sequences include transcribed but untranslated DNA
positioned downstream from the translation stop signal in the
recombinant DNA sequence (also referred to as the 3' untranslated
region or 3' UTR). Such sequences generally terminate at a
polyadenylation sequence (either from the endogenous gene or from
other sources such as SV40) and sequences that may affect RNA
stability. Generally, 3' UTR's comprise about 100 to 500
nucleotides downstream from the translation stop signal in the gene
from which the 3' regulation sequence is derived. Distal 3'
expression regulation sequences include flanking DNA sequences
downstream from the proximal 3' expression regulation sequence.
Some of these distal sequences are transcribed, but do not form
part of the mRNA while other sequences in this distal 3' expression
regulation sequence are not transcribed at all. Such distal 3'
expression regulation sequences are believed to contain enhancer
and/or other sequences which enhance expression. Such sequences are
believed to be necessary for efficient polydenylation and contain
transcription termination sequences Preferably, such sequences
comprise about 2 kb, more preferably 8 kb and most preferably about
15 kb of 3' flanking sequence.
[0068] A preferred 3' flanking sequence is the 3' flanking sequence
of the human lactoferrin (hLF) gene. Transgenic animals containing
transgenes that include about 9 kb of hLF 3' flanking sequences
show enhanced expression of recombinant polypeptides in milk
compared to animals containing transgenes that include 1 kb or less
of hLF 3' flanking sequence, due to an enhancer or other enhancing
sequence located in this region. Usually the human lactoferrin 3'
flanking sequence will be at least 1 kb in length up to about 9 kb
in length or longer, typically 3 to 7 kb, more typically 4 to 5 kb.
It will also be possible, and sometimes desirable, to use standard
methods (e.g., deletion analysis) to identify regions contained
within the 9 kb 3' flanking sequence that enhance mammary gland
expression of recombinant polypeptides. These enhancers or
enhancing sequences can be isolated and used in combination with
various amounts of homologous or heterologous sequences. Typically
the enhancing sequences can range in length from about 50 basepairs
to about 2 kb, more typically from about 100 basepairs to about 500
basepairs.
[0069] It will often be desirable to use a transgene having a 5'
expression regulation sequence and a 3' flanking sequence that
originate from the same gene. In a preferred embodiment, the 5'
expression regulation sequence and 3' flanking sequence are from
the bovine .alpha.S1-casein gene.
[0070] In an alternative embodiment a genomic sequence, such as a
human genomic clone or clones, can be introduced into an animal to
produce a transgenic animal containing a transgene that has the
sequence of the human gene, including all or part of the 5'
expression regulation sequences, coding sequences, introns, and 3'
untranslated and flanking sequences. In a preferred embodiment, the
human lactoferrin genomic sequence is used in its entirety, but
various components can be substituted with components from other
mammary gland specific genes.
[0071] Although the use of both 5' and 3' expression regulation
sequences are preferred, in some embodiments of the invention,
endogenous 3' regulation sequences are not used. In such cases, the
3' proximal expression regulation sequences normally associated
with the genomic DNA encoded by the recombinant DNA sequence are
used to direct polyadenylation. In addition, distal 3' regulation
sequences from the genomic DNA encoding the recombinant polypeptide
may also be employed preferably in the same amounts as set forth
for endogenous 3' expression regulation sequences. In such cases,
it is to be understood that the recombinant polypeptide encoded by
the transgene may comprise either genomic DNA or a double stranded
DNA derived from cDNA. As with the 5' expression regulation
sequences, the optimal amount of 3' expression regulation sequence
may be readily determined by varying the amount of 3' flanking
sequence to obtain maximal expression of the recombinant
polypeptide. In general, the distal 3' regulation sequence, be it
from an endogenous gene or a heterologous gene, will not extend
into the adjacent gene from which is derived and will exclude any
sequences which adversely effect the level of transgene
expression.
[0072] Examples of expression regulation sequences are provided in
Table I.
1 TABLE 1 Expression Regulation Tissue Animal Sequence Specificity
Species 16 kb of bovine .alpha.S1 Mammary bovine casein 5' to
structural secretory gene and 8 kb 3' to cells structural gene
.apprxeq.15 kb 5' to albumin Liver murine gene .apprxeq.15 kb 5' to
.alpha.-actin Muscle murine gene .apprxeq.15 kb upstream of
Spermatids murine protamine gene
[0073] In addition to the 5' and 3' expression regulation sequences
and the recombinant DNA (either genomic or derived from cDNA) the
transgenes of the invention preferably also comprise a "recombinant
intervening sequence" which interrupts the transcribed but
untranslated 5' region of the transgene. Such intervening sequences
can be derived, for example, from bovine .alpha.S1 casein and from
human lactoferrin. Such sequences as used herein are "homologous
recombinant intervening sequences" in that the 5' and 3' RNA splice
signals in such recombinant intervening sequences are those
normally found in an intervening sequence from an endogenous or
heterologous gene. Recombinant intervening sequences may, however,
also comprise a "hybrid intervening sequence". Such hybrid
intervening sequences comprise a 5' RNA splice signal and 3' RNA
splice signal from intervening sequences from different sources. In
some aspects of the invention, such hybrid intervening sequences
comprise at least one "permissive RNA splice sequence". As used
herein, a permissive RNA splice signal is an RNA splice signal
sequence, preferably a 3' RNA splice signal, from an intron
contained within a repertoire of germ line DNA segments which
undergo rearrangement during cell differentiation. Examples of such
gene repertoires include the immunoglobulin super gene family,
including the immunoglobulins and T-cell antigen receptors as well
as the repertoire of the major histocompatibility complex (MHC)
genes and others. Particularly preferred permissive splice
sequences are those obtained from the immunoglobulin repertoire,
preferably of the IgG class, and more preferably those 3' splice
signal sequences associated with the J-C segment rearrangement of
the Ig heavy and light chain, most preferably the heavy chain. A
particularly preferred permissive splice sequence comprises that
portion of the sequence as shown downstream of the HindIII site in
FIG. 11. A particularly preferred hybrid intervening sequence
comprises the entire sequence shown in FIG. 11 which includes a 5'
portion of an intervening sequence from bovine .alpha.S1 casein and
a 3' sequence portion of an IgG heavy chain intervening
sequence.
[0074] Such hybrid intervening sequences containing permissive RNA
splice signals are preferably used when the recombinant DNA
corresponds to a cDNA sequence. As indicated in the Examples, when
16 kb of 5' expression regulation sequence from the .alpha.S1
casein gene was used in conjunction with an .alpha.S1 casein-IgG
hybrid intervening sequence to express human lactoferrin cDNA
operably linked to the .alpha.S1 casein secretory signal sequence a
transgenic mouse was obtained which produced approximately 1330
.mu.g/ml of hLF in the transgenic milk. This amount of recombinant
polypeptide far exceeds the previously reported amounts for
production of various protein in transgenic mouse milk of generally
less than 10 .mu.g/ml and in one case approximately 50 .mu.g/ml. It
also exceeds the maximum of 8.mu.g/ml of hLF produced herein when
the same transgene was used that contained a homologous bovine
intervening sequence rather than the hybrid intervening
sequence.
[0075] However, such hybrid intervening sequences are not limited
to transgenes utilizing cDNA sequence. Rather, hybrid intervening
sequences are also useful when the recombinant polypeptide is
encoded by a genomic sequence. Based on the results obtained with
the cDNA recombinant DNA and the general expectation that genomic
DNA sequences express at higher levels than sequences derived from
cDNA, it is expected that such hybrid intervening sequences used in
conjunction with genomic recombinant DNA will further enhance
expression levels above that which would otherwise be obtained with
genomic sequence alone.
[0076] Based on the foregoing, it is apparent that preferred
transgenes include large amounts of 5' and 3' expression regulation
sequences. Further, the recombinant DNA is preferably derived from
genomic clones which may be tens to hundreds of kilobases in
length. Based on the present technology for cloning and
manipulating DNA, the construction and microinjection of transgenes
is practically limited to linearized DNA having a length not
greater than about 50 kb. However, the transgenes of the invention,
especially those having a length greater than about 50 kb, may be
readily generated by introducing two or more overlapping fragments
of the desired transgene into an embryonal target cell. When so
introduced, the overlapping fragments undergo homologous
recombination which results in integration of the fully
reconstituted transgene in the genome of the target cell. In
general, it is preferred that such overlapping transgene fragments
have 100% homology in those regions which overlap. However, lower
sequence homology may be tolerated provided efficient homologous
recombination occurs. If non-homology does exist between the
homologous sequence portions, it is preferred that the non-homology
not be spread throughout the homologous sequence portion but rather
be located in discrete areas. Although as few as 14 base pairs at
100% homology are sufficient for homologous recombination in
mammalian cells (Rubnitz, J. and Subramani, S. (1984) Mol. Cell.
Biol. 4:2253-2258), longer homologous sequence portions are
preferred, e.g. 500 bp, more preferably 1000 bp, next most
preferably 2000 bp and most preferably greater than 2000 bp for
each homologous sequence portion.
[0077] As indicated in the examples, three overlapping fragments of
the human serum albumin gene were microinjected into the pronuclei
of mouse zygotes in approximately equal molar portions. These
fragments successfully recombined and integrated into the mouse
genome as confirmed by analysis of the integrated DNA by Southern
blotting procedures and by detection of RNA transcript and human
serum albumin in the serum of the transgenic mouse. Although the
transgene so generated has a unit length of 38 kb, there is no
known practical limit to the size of the transgene which may be
formed using larger and/or greater numbers of overlapping transgene
fragments. In particular, it is expected that transgenes may be
formed by this approach having lengths between about 50 to 1000 kb
and more preferably between 50 and 500 kb. Further, the use of
homologous recombination of overlapping fragments is expected to be
fruitful in the generation of larger transgenic animals, such as
transgenic bovine species, containing transgenes incorporating
recombinant DNA comprising genomic DNA which otherwise could not be
incorporated into a pronucleus to form a transgenic animal. Such
genomic transgenes are expected to produce higher expression levels
in transgenic cows as compared to that which is produced by
transgenes encoding recombinant cDNA.
[0078] When, the ultimate object is to secrete a recombinant
polypeptide, a "secretory DNA sequence" encoding a functional
secretion signal peptide is also operably linked within the
transgene to direct secretion of the recombinant polypeptide from
one or more cell types within the transgenic animal. Secretory DNA
sequences in general are derived from genes encoding secreted
proteins of the same species of the transgenic animal. Such
secretory DNA sequences are preferably derived from genes encoding
polypeptides secreted from the cell type targeted for
tissue-specific expression, e.g. secreted milk proteins for
expression in and secretion from mammary secretory cells. Secretory
DNA sequences, however, are not limited to such sequences.
Secretory DNA sequences from proteins secreted from other cell
types within the species of transgenic animal may also be used,
e.g., the native signal sequence of a homologous gene encoding a
protein secreted other than in the mammary glands. In addition,
"heterologous secretory DNA sequences" which encode signal
secretion peptides from species other than the transgenic animals
my also be used e.g., human t-PA, human serum albumin human
lactoferrin and human lactalbumin and secretion signals from
microbial genes encoding secreted polypeptides such as from yeast,
filamentous fungi, and bacteria. In general, a secretory DNA
sequence may be defined functionally as any DNA sequence which when
operably linked to a recombinant DNA sequence encodes a signal
peptide which is capable of causing the secretion of the
recombinant polypeptide.
[0079] In one of the preferred embodiments, a secretory DNA
sequence encoding a secretory signal sequence functional in the
mammary secretory cells of bovine species is used to cause
secretion of recombinant polypeptide from bovine mammary secretory
cells. The secretory DNA sequence is operably linked to the
recombinant DNA sequence. Examples of such secretory DNA sequences
include DNA sequences encoding signal secretion sequences for
bovine .alpha.S1 casein, murine lactoferrin and human transferrin.
The preferred secretory DNA sequence is that encoding the secretory
sequence of .alpha.S1 casein from bovine species. The use of this
secretory DNA sequence is described in more detail in the
Examples.
[0080] "Operably linked" in the context of linking a secretory DNA
sequence to a recombinant DNA sequence means that the secretory DNA
sequence (comprising codons encoding the secretory signal peptide
sequence) is covalently coupled to the recombinant DNA sequence so
that the resultant secretory-recombinant DNA sequence encodes 5' to
3' for the secretory signal sequence and recombinant polypeptide.
Accordingly, the reading frame for the secretory sequence and the
recombinant DNA sequence must be covalently combined such that an
open reading frame exists from the 5' end of the mRNA sequence
formed after transcription and processing of the primary RNA
transcript. This open reading frame in the RNA contains a 5'
sequence portion encoding the secretory signal peptide and a 3'
sequence portion encoding the recombinant polypeptide. When so
constructed, the recombinant polypeptide produced upon expression
of the secretory-recombinant DNA sequence is of a form which is
capable of being secreted from targeted cells which express the DNA
sequence. The signal peptide generally is removed in vivo during
secretion to produce an extracellular form of the recombinant
polypeptide.
[0081] In the preferred embodiments of the invention, a
secretory-recombinant DNA sequence is expressed predominantly in
the mammary secretory cells of transgenic bovine species. Such
tissue-specific expression is obtained by operably linking mammary
specific expression regulation DNA sequences to the above
secretory-recombinant DNA sequence. Such mammary specific
regulation sequences include the aforementioned regulation
sequences contained in various bovine genes preferentially
expressed in the mammary secretory cells of the species. Such
mammary specific genes include .alpha.S1 casein; .alpha.S2-casein;
.beta.-casein; K-casein; .alpha.-lactalbumin; and
.beta.-lactoglobulin. Preferred expression regulation sequences are
derived from .alpha.S1 casein as described more in detail in the
Examples.
[0082] In general, the transgenes of the invention that are
designed to secrete the recombinant polypeptide into transgenic
bovine milk are capable of causing such secretion at levels
significantly higher than that previously reported for transgenic
mice and sheep. When the recombinant polypeptide is encoded by a
recombinant DNA corresponding to, or derived from, cDNA, the molar
concentration of the recombinant polypeptide is preferably greater
than about 1.0 .mu.M, more preferably greater than about 100 .mu.M,
and most preferably greater than 100 .mu.M. When viewed from the
perspective of the level of recombinant polypeptide present in the
transgenic milk, the amount of recombinant polypeptide is
preferably greater than 50 .mu.g/mi, more preferably greater than
about 500 .mu.g/ml and most preferably greater than about 1000
.mu.g/ml (1 mg/ml).
[0083] When the transgene of the invention encodes a recombinant
polypeptide that is encoded by recombinant DNA derived from or
corresponding to genomic DNA (or comprised substantially of such
genomic sequences, e.g. greater than about 50%, more preferably
greater than about 75%, most preferably greater than 90% of the
codons encoding the recombinant polypeptide are from genomic
sequences), the molar concentrations and protein levels in bovine
transgenic milk are the same as for cDNA or higher. In general, the
molar concentration of the recombinant polypeptide in such
transgenic milk is preferably greater than about 50 .mu.M, more
preferably greater than about 50 .mu.M, most preferably greater
than about 500 .mu.M. When viewed from the level of protein in the
transgenic milk, the levels are preferably greater than about 10
mg/ml, more preferably greater than about 2.5 mg/ml, most
preferably greater than 5 mg/ml.
[0084] The foregoing molar concentration and protein levels in
bovine transgenic milk will vary depending upon the molecular
weight of the particular recombinant polypeptide. A particular
advantage of producing a recombinant polypeptide in bovine
transgenic milk is that relatively large molecular weight
polypeptides may be so produced which are otherwise difficult to
produce in large quantities in other systems such as prokaryotic
expression systems. Although any recombinant polypeptide may be
produced in bovine transgenic milk according to the invention, it
is generally preferred that such recombinant polypeptides have a
molecular weight greater than about 10,000 Daltons. However, other
recombinant polypeptides having molecular weights of greater than
15,000, greater than 20,000 and greater than 60,000 Daltons may
also be expressed in transgenic bovine milk. For example, human
lysozyme having a molecular weight of 17,000 Daltons and
lactoferrin having a molecular weight of 79,000 Daltons may be
readily produced in the transgenic milk of bovine species according
to the disclosure of the invention. Thus, the recombinant
polypeptides of the invention have a wide range of molecular
weights.
[0085] As a consequence, the foregoing preferred molar
concentrations of recombinant polypeptides are adjusted when higher
molecular weight recombinant polypeptides are produced. Such
adjustment is made by converting the molar concentration to the
amount of protein produced and adjusting the molar concentrations
so that the recombinant protein level is within the following
preferred concentrations.
[0086] Most of the previous reports relating to the production of
polypeptides in transgenic milk involve transgenic mice. The mouse,
however, normally produces between 55 to 80 milligrams of protein
per ml of milk. A cow, on the other hand, normally produces between
30 to 34 milligrams of protein per ml. Since exceptionally high
levels of recombinant polypeptide production may adversely affect
the production of endogenous milk protein and/or have adverse
effects upon the mammary secretory gland, it is preferred that the
recombinant polypeptide concentration be between about 3 and 50% of
the normal bovine milk protein concentration (i.e., between about 1
and 17 milligrams of recombinant polypeptide per ml of transgenic
milk), more preferably between 10 to 20% (i.e., between 3 to about
7 milligrams per ml) and most preferably between 10 and 15% (i.e.,
between about 3 and 5 milligrams per ml) of the normal amount of
protein produced in bovine milk. Such preferred ranges also provide
a preferred maximum limit to the aforementioned levels of protein
produced in transgenic bovine milk.
[0087] The above described linking of various DNA sequences to form
the transgene of the invention are performed by standard methods
known to those skilled in the art or as described herein. Once the
transgene or overlapping homologous fragments encoding the
transgene are constructed as described they are used to make
transgenic non-human animals.
[0088] Methods of introducing transgenes or overlapping transgene
fragments into embryonal target cells include microinjection of the
transgene into the pronuclei of fertilized oocytes or nuclei of ES
cells of the non-human animal. Such methods for murine species are
well known to those skilled in the art. Alternatively, the
transgene may be introduced into an animal by infection of zygotes
with a retrovirus containing the transgene (Jaenisch, R. (1976)
Proc. Natl. Acad. Sci. USA 73:1260-1264). The preferred method is
microinjection of the fertilized oocyte. In this preferred
embodiment, the fertilized oocytes are first microinjected by
standard techniques. They are thereafter cultured in vitro until a
"pre-implantation embryo" is obtained. Such pre-implantation
embryos preferably contain approximately 16 to 150 cells. The 16 to
32 cell stage of an embryo is commonly referred to as a morula.
Those pre-implantation embryos containing more than 32 cells are
commonly referred to as blastocysts. They are generally
characterized as demonstrating the development of a blastocoel
cavity typically at the 64 cell stage. Methods for culturing
fertilized oocytes to the pre-implantation stage include those
described by Gordon, et al. (1984) Methods in Enzymology 101:414;
Hogan, et al. (1986) in Manipulating the Mouse Embryo, Cold Spring
Harbor Laboratory Press, Cold Spring Harbor, N.Y. (for the mouse
embryo); and Hammer, et al. (1985) Nature 315:680 (for rabbit and
porcine embryos) Gandolfi, et al. (1987) J. Reprod. Fert. 81:23-28;
Rexroad, et al. (1988) J. Anim. Sci. 66:947-953 (for ovine embryos)
and Eyestone, W. H. et al. (1989) J. Reprod. Fert. 85:715-720;
Camous., et al. (1984) J. Reprod. Fert. 72:779-785; and Heyman, Y.,
et al. (1987) Theriogenology 27:5968 (for bovine embryos). Such
pre-implantation embryos are thereafter transferred to an
appropriate female by standard methods to permit the birth of a
transgenic or chimeric animal depending upon the stage of
development when the transgene is introduced. As is well known,
mosaic animals can be bred to form true germline transgenic
animals.
[0089] Since the frequency of transgene incorporation is often low,
the detection of-transgene integration in the pre-implantation
embryo is highly desirable. In one aspect of the invention methods
are provided for identifying embryos wherein transgenesis has
occurred and which permit implantation of transgenic embryos to
form transgenic animals. In this method, one or more cells are
removed from the pre-implantation embryo. When equal division is
used, the embryo is preferably not cultivated past the morula stage
(32 cells). Division of the pre-implantation embryo (reviewed by
Williams et al. (1986) Theriogenology 22:521-531) results in two
"hemi-embryos" (hemi-morula or hemi-blastocyst) one of which is
capable of subsequent development after implantation into the
appropriate female to develop in utero to term. Although equal
division of the pre-implantation embryo is preferred, it is to be
understood that such an embryo may be unequally divided either
intentionally or unintentionally into two hemi-embryos which are
not necessarily of equal cell number. Essentially, all that is
required is that one of the embryos which is not analyzed as
hereinafter described be of sufficient cell number to develop to
full term in utero. In a specific embodiment, the hemi-embryo which
is not analyzed as described herein, if shown to be transgenic, is
used to generate a clonal population of transgenic non-human
animals.
[0090] One of each of the hemi-embryos formed by division of
pre-implantation embryos is analyzed to determine if the transgene
has been integrated into the genome of the organism. Each of the
other hemi-embryos is maintained for subsequent implantation into a
recipient female of the species. A preferred method for detecting
transgenesis at this early stage in the embryo's development uses
these hemi-embryos in connection with a unique property of the
restriction endonuclease Dpn I. This enzyme recognizes the sequence
GATC in double-stranded DNA but only when the adenine in each
strand within this sequence is methylated at N-6. When using this
preferred method, the transgene containing the sequence GATC is
methylated prior to microinjection either by transferring the
transgene on an appropriate plasmid through a DAM.sup.+ strain of
microorganisms such as E. coli MM294 or by directly methylating the
transgene with dam methylase. The methylated transgene (preferably
without any exogenous sequences such as plasmid vector) is then
microinjected into fertilized oocytes (approximately 10 to 500
copies per pronucleus, more preferably 50 to 100 copies per
pronucleus). The fertilized oocytes so obtained =re cultured in
vitro to the pre-implantation stage During this early growth and
cell division phase, the genomic DNA is replicated. Accordingly,
those copies of the methylated transgene integrated into the genome
of the fertilized oocyte are unmethylated after replication whereas
any non-integrated transgenes which may still exist after
replication will remain methylated. (Lacks, S., et al. (1977) J.
Mol. Biol. 114:153.) This differential methylation pattern for
integrated versus non-integrated transgene permits the
identification of which fertilized oocytes have integrated the
transgene into the genome.
[0091] The identification of the pre-implantation embryos
containing the integrated transgene is achieved by analyzing the
DNA from each of the hemi-embryos. Such DNA is typically obtained
by lysing the hemi-embryo and analyzing the thus released DNA after
treatment as described by Ninomiy, T. et al. (1989) Molecular
Reproduction and Development 1:242-248. Each of the DNA samples is
treated with Dpn I. Thereafter, a polymerase chain reaction (Saiki,
et al. (1985) Science 230:1350-1354) is preformed to amplify all or
part of the transgene. When the entire transgene is amplified, two
extension primers each complimentary to opposite strands at
opposing ends of the transgene are used for amplification. When,
however, less than the entire transgene is amplified, such
extension primers are chosen such that the amplified gene product
spans the Dpn I site in the transgene. If Dpn I cleavage has not
occurred, PCR amplification results in amplified sequences having a
predetermined size whereas primer extension for those transgenes
which have been cleaved will not result in exponential
amplification. Generally, the Dpn I/PCR amplified DNA from the
hemi-embryo is subjected to electrophoresis followed by
hybridization with labeled probe complimentary to the region of the
transgene between the two extension primers. This facilities the
determination of the size of the amplified DNA sequences, if any,
and provides an indication of whether the transgene has been
integrated into the pre-implantation embryo from which the
hemi-embryo was obtained (now called a "transgenic hemi-embryo").
If it has, the remaining untreated transgenic hemi-embryo is
transplanted into a recipient parent. After in utero development,
the transgenic non-human animal having the desired phenotype
conferred by the integrated transgene is identified by an
appropriate method in utero or after birth. Of course, other
restriction endonucleases capable of cleaving a methylated DNA
sequence but incapable of cleaving the unmethylated form of a
recognition sequence may be used in the aforementioned method.
[0092] The above described method using Dpn I requires that the
sequence GATC be present in the transgene of interest. In those
cases when such a sequence is not present, it may be readily
introduced into the transgene by site directed mutagenesis (Kunkel,
T. A. (1985) Proc. Natl. Acad. Sci. 82:488) or cassette mutagenesis
(Wells, J. A., et al. (1985) Gene 34:315) provided such mutagenesis
does not change the amino acid sequence encoded by the transgene
(or causes an inconsequential change in amino acid sequence) and
that any codons so generated are functional in the transgenic
non-human animal of interest.
[0093] The above described methods for the detection of
transgenesis in pre-implantation embryos provide economical and
time saving method for generating transgenic non-human animals
since they significantly decrease the number of pregnancies
required to produce a transgenic animal and substantially increase
the likelihood that an implanted embryo will produce a transgenic
non-human animal. Such methods are especially important for those
animals for which very low or non-existent frequencies of
transgenesis have been obtained, e.g. bovine species.
[0094] In an alternate embodiment, the above described method for
detecting transgenesis in pre-implantation embryos is combined with
embryonic cloning steps to generate a clonal population of
transgenic embryos which may thereafter be implanted into recipient
females to produce a clonal population of transgenic non-human
animals also having the same genotype. In this regard, it is to be
understood that transgenic embryos and/or non-human transgenic
animals having the same "genotype" means that the genomic DNA is
substantially identical between the individuals of the embryo
and/or transgenic animal population. It is to be understood,
however, that during mitosis various somatic mutations may occur
which may produce variations in the genotype of one or more cells
and/or animals. Thus, a population having the same genotype may
demonstrate individual or subpopulation variations.
[0095] After a hemi-embryo is identified as a transgenic
hemi-embryo, it is cloned. Such embryo cloning may be performed by
several different approaches. In one cloning method, the transgenic
hemi-embryo is cultured in the same or in a similar media as used
to culture individual oocytes to the pre-implantation stage. The
"transgenic embryo" so formed (preferably a transgenic morula) is
then divided into "transgenic hemi-embryos" which can then be
implanted into a recipient female to form a clonal population of
two transgenic non-human animals. Alternatively, the two transgenic
hemi-embryos obtained may be again cultivated to the
pre-implantation stage, divided, and recultivated to the transgenic
embryo stage. This procedure is repeated until the desired number
of clonal transgenic embryos having the same genotype are obtained.
Such transgenic embryos may then be implanted into recipient
females to produce a clonal population of transgenic non-human
animals.
[0096] In a preferred cloning method, the transgenic embryo is
cloned by nuclear transfer according to the techniques of Prather,
et al. (1988) Biol. Reprod. 37:59-86; Roble, et al. (1987) J. Anim.
Sci. 64:642-664. According to this method, nuclei of the transgenic
embryo are transplanted into enucleated oocytes, each of which is
thereafter cultured to the blastocyst stage. At this point, the
transgenic embryos may be resubjected to another round of cloning
by nuclear transplantation or may be transferred to a recipient
parent for production of transgenic offspring having the same
genotype.
[0097] In addition to the foregoing methods for detecting early
transgenesis, other methods may be used to detect transgenesis.
Such methods include in utero and post partum analysis of tissue.
In utero analysis is performed by several techniques. In one,
transvaginal puncture of the amniotic cavity is performed under
echoscopic guidance (Bowgso, et al. (1975) Bet. Res. 96:124-127;
Rumsey, et al. (1974) J. Anim. Sci. 39:386-391). This involves
recovering about 15 to 20 milliliters of amniotic fluid between
about day 35 and day 100 of gestation. This volume of amniotic
fluid contains about 1000 to 12,000 cells per ml originating from
the urogenital tract, the skin and possibly the lungs of the
developing embryo. Most of these cells are dead. Such cells,
however, contain genomic DNA which is subjected to PCR analysis for
the transgene as an indication of a successful transgenesis.
Alternatively, fetal cells may be recovered by chorion puncture.
This method also may be performed transvaginally and under
echoscopic guidance. In this method, a needle is used to puncture
the recipient animal's placenta, particularly the placentonal
structures, which are fixed against the vaginal wall. Such sampling
may be performed around day 60 of gestation in bovine species.
Chorion cells, if necessary, are separated from maternal tissue and
subjected to PCR analysis for the transgene as an indication of
successful transgenesis.
[0098] Transgenesis may also be detected after birth. In such
cases, transgene integration can be detected by taking an
appropriate tissue biopsy such as from the ear or tail of the
putative transgenic animal. About one to two centimeters of tail or
about five to ten square millimeters of ear are obtained followed
by southern blotting with a probe for the transgene according to
the method of Hogan, et al. (1986) Manipulating the Mouse Embryo,
Cold Spring Harbor Laboratory.
[0099] Transgenesis can also be determined by using the southern
blot technique with DNA obtained from other tissues. In particular,
semen from a recombinant bull will be useful for identifying
transgenic animals.
[0100] Transgenesis may also by detected by assaying for expression
of the recombinant polypeptide in a tissue, secretion (e.g.,
saliva), or other body fluid. In the case where the goal is
expression of a recombinant polypeptide in milk of cows it will be
especially useful to assay the saliva of bulls for expression
levels. This is because some mammary specific promoters may also
cause salivary gland expression, albeit at low levels. See, e.g.,
Archibald et al. (1990) Proc. Nat. Acad. Sci. USA
87Z:5178-5182.
[0101] In those embodiments where a recombinant polypeptide is
expressed and secreted into the milk of transgenic bovine species,
the transgenic milk so obtained may be either used as is or further
treated to purify the recombinant polypeptide. This depends, in
part, on the recombinant polypeptide contained in the transgenic
milk and the ultimate use for that protein. Thus, when the
recombinant polypeptide is secreted into transgenic milk to
increase the nutritional value of the bovine milk, no further
purification is generally necessary. An example of such a situation
involves one of the preferred embodiments wherein human lactoferrin
is produced in the milk of bovine species as a supplement to
control intestinal tract infections in newborn human infants and to
improve iron absorption. In other situations, a partial
purification may be desired to isolate a particular recombinant
polypeptide for its nutritional value. Thus, for example, human
lactoferrin produced in transgenic bovine milk may be partially
purified by acidifying the milk to about pH 4-5 to precipitate
caseins. The soluble fraction (the whey) contains the human
lactoferrin which is partially purified.
[0102] The recombinant polypeptide contained in bovine transgenic
milk may also be used in food formulations. A particularly useful
food formulation comprises an infant formula containing one or more
recombinant polypeptides from transgenic bovine milk which have
either nutritional or other beneficial value. For example, an
infant formula containing human lactoferrin from transgenic bovine
milk made according to the present invention provides a
bacteriostatic effect which aids in controlling diarrhea in
newborn. Similarly, recombinant polypeptides such as human casein
and human lysozyme may also be generated in transgenic bovine milk
to provide nutritional value. Table 2 sets forth the constituents
of a typical infant formula. As indicated therein, the protein
content varies between about 1.8 and 4.5 grams of protein per 100
kilocalories of formula. Thus, the total protein including
recombinant polypeptide should lie between the values at least
based on regulatory requirements in the United States from which
the formulation in Table 2 is based. The amount of total protein
including recombinant polypeptide, of course, may vary from the
foregoing depending upon the local regulations where the particular
formula is intended to be used.
2TABLE 2 Nutrient Minimum.sup.a Maximum.sup.b Protein (gm).sup.a
1.8.sup.b 4.5 Fat: gm 3.3 6.0 percent cal 30.0 54.0 Essential fatty
acids (linoleate): percent cal 2.7 mg 300.0 Vitamins: (A) (IU)
250.0 (75 .mu.g).sup.a 750.0 (225 .mu.g).sup.c D (IU) 40.0 100.0 K
(.mu.g) 4.0 E (IU) 0.7 (with 0.7 IU/gm lineoleic acid) C (ascorbic
acid (mg) 8.0 B.sub.1 (thiamine (.mu.g) 40.0 B.sub.2 (riboflavin)
(.mu.g) 60.0 B.sub.4 (pyridoxine) (.mu.g) 35.0 (with 15 .mu.g/gm of
protein in formula) B.sub.12 (.mu.g) 0.15 Niacin (.mu.g) 250.0
Folic acid (.mu.g) 4.0 Pantothenic acid (g) 300.0 Biotin (.mu.g)
1.5.sup.a Choline (mg) 7.0.sup.b Inositol (mg) 4.0.sup.c Minerals:
Calcium (mg) 50.0.sup.a Phosphorus (mg) 25.0.sup.b Magnesium (mg)
6.0 Iron (mg) 0.15 Iodine (.mu.g) 5.0 Zinc (mg) 0.5 Copper (.mu.g)
60.0 Manganese (.mu.g) 5.0 Sodium (mg) 20.0 60.0 Potassium (mg)
80.0 200.0 Chloride (mg) 55.0 150.0 .sup.aStated per 100
kilocalories. .sup.bThe source of protein shall be at least
nutritionally equivalent to casein. .sup.cRetinol equivalents.
.sup.dRequired to be included in this amount only in formulas which
are not milk-based. .sup.eCalcium to phosphorus ratio must be no
less than 1.1 nor more than 2.0. .sup.fIncludes recombinant protein
according to the invention or recombinant proteins and other
proteins.
[0103] In addition to infant formulas, other food formulations may
also be supplemented with recombinant polypeptides from transgenic
bovine milk. For example, such recombinant polypeptides may be used
to supplement common diet formulations.
[0104] When the recombinant polypeptide is intended to be used
pharmaceutically, purification methods consistent with such an
application are called for. Such purification methods will depend
on the particular recombinant polypeptide to be purified and are
generally known to those skilled in the art. Such methods typically
include a partial purification by casein fractionation followed by
chromatography of the appropriate fraction containing the
recombinant polypeptide. Such chromotography includes affinity
chromatography, ion exchange chromotography, gel filtration and
HPLC.
[0105] In a specific embodiment of the invention, transgenes are
provided for producing human lactoferrin in the milk of transgenic
bovine species. Human lactoferrin (HLF) is a single chain
glycoprotein which binds two ferric ions. Secreted by exocrine
glands (Mason, et al. (1978) J. Clin. Path. 31:316-327; Tenovuo, et
al. (1986) Infect. Immun. 51:49-53) and polymorphonuclear
neutrophil granulocytes (Mason, et al. (1969) J. Exp. Med.
130:643-658), this protein functions as part of a host non-specific
defense system by inhibiting the growth of a diverse spectrum-of
bacteria. HLF exhibits a bacteriostatic effect by chelation of the
available iron in the media, making this essential metal
inaccessible to the invading microorganisms (Bullen, et al. (1972)
Br. Med. J. 1:69-75; Griffiths, et al. (1977) Infect. Immun.
15:396-401; Spik, et al. (1978) Immunoloy 8:663-671; Stuart, et al.
(1984) Int. J. Biochem. 16:1043-1947). This effect is blocked if
the protein is saturated with ferric ions. Several studies suggest
that HLF displays a direct bacteriocidal effect on certain
microorganisms (Arnold, et al. (1980) Infect. Immun. 28:893-698;
Arnold, et al. (1977) Science 197:263-265; Arnold, et al. (1981)
Infect. Immun. 32:655-660; Arnold, et al. (1982) Infect. Immun.
35:792-797; Bortner, et al. (1986) Infect. Immun. 51:373-377). The
bacteriocidal effect is also inhibited by iron saturation of the
protein. No mechanism for the bactericidal effect of HLF has been
postulated, although it has been demonstrated that it can damage
the outer membrane and alter outer membrane permeability in
gram-negative bacteria (Ellison, et al. (1988) Infect. Immun.
56:2774-2781).
[0106] Lactoferrin is the major iron binding protein in human milk
(present at a concentration of about 1.5-1.7 mg/ml) and may play a
role in the absorption of iron by the small intestine. All of the
iron present in breast milk is thought to be bound to hLF and is
taken up at very high efficiencies compared to formula (Hide, D.
W., et al. (1981) Arch. Dis. Child. 56:172). It has been postulated
that the high uptake of the hLF bound iron is due to a receptor in
the jejunum and data has been presented suggesting existence of
receptors in Rhesus monkeys (Cox, et al. (1979) BBA 588:120;
Davidson, L. A., et al. (1985) Fed. Proc. 18:901). There is also
evidence for specific lactoferrin receptors on mucosal cells of the
small intestine of human adults (Cox, et al. (1979) Biochem.
Biophys. Acta. 588:120-128). Free iron levels have been implicated
in the control of the intestinal flora (Mevissen-Verhage, et al.
(1985) Eur. J. Clin. Microbiol. 4:14). Breast fed infants, compared
with infants fed cow's milk, with and without added iron, were
shown to have substantially reduced coliform and, elevated
bifidobacteria and clostridia counts in fecal samples. In in vitro
studies, human milk has been shown to have a specific inhibitory
effect on E. coli (Brock, et al. (1983) Infect. and Immunit.
40:453). Human milk has also been shown to have a specific
inhibitory effect on E. coli in small intestine due to its high
content of iron binding protein, predominantly hLF (Bullen, et al.
(1972) British Med. J. i:69).
[0107] Thus, the production of human lactoferrin in the milk of
transgenic bovine species provides a source of human lactoferrin.
Such lactoferrin may be purified from the transgenic milk for
formulation purposes. Alternatively, the whole transgenic milk may
be used, preferably after pasteurization, in either liquid or dried
form. In addition, the beneficial action of human lactoferrin may
be potentiated by combining the human lactoferrin or the transgenic
milk containing it with human lysozyme. The human lysozyme may be
simultaneously produced in the transgenic cow by introducing a
second transgene simultaneously with the HLF transgene to produce a
transgenic cow capable of producing more than one recombinant
polypeptide in the transgenic milk. Alternatively, the transgenes
may be sequentially introduced into bovine species. When such is
the case, a transgenic bovine species is obtained containing one of
the transgenes. Thereafter, embryonic cells, such as eggs, are
obtained from the transgenic female and treated so as to
incorporate the second transgene encoding the second polypeptide.
Preferably, the egg is fertilized, followed by microinjection of
the pronucleus of the zygote so obtained. It is to be understood
that the foregoing combination of more than two recombinant
polypeptides in transgenic bovine milk is not limited to the
aforementioned human lactoferrin and lysozyme combination. Thus,
the invention contemplates the production of transgenic bovine
species and transgenic milk wherein more than one recombinant
polypeptide is produced by such a transgenic animal in the
transgenic milk.
[0108] The complete amino acid sequence of HLF has been determined
(Metz-Boutigue et al. (1984) Eur. J. Biochem. 1451:659-676). HLF
comprises two domains, each containing one iron-binding site and
one N-linked glycosylation site. These domains show homology
between each other, indicative of an ancestral gene duplication and
fusion event. In addition, HLF shares extensive homology with other
members of the transferrin family (Metz-Boutigue, supra; Pentecost,
et al. (1987) J. Biol. Chem. 262:10134-10139). Location of the
amino acids involved in the iron-binding sites has been determined
by X-ray crystallography (Anderson et al. (1987) Proc. Natl. Acad.
Sci. 84:1769-1773). A partial cDNA sequence for neutrophil HLF was
published by Rado, et al. (1987) Blood 70:989-993. There was a
>98% agreement between the amino acid sequence deduced from the
cDNA and that which was determined by direct analysis of
lactoferrin from human milk. The structure of the iron-saturated
and iron-free form of human lactoferrin have recently been
published. (Anderson, et al., (1989) J. Mol. Biol. 209:711-734;
Anderson, et al. (1990) Nature:784-787.)
[0109] As used herein, "human lactoferrin" comprises a polypeptide
having the amino acid sequence substantially as described by
Metz-Boutigue, et al. (1984) Eur. J. Biochem. 1451:659-676 and as
set forth in FIG. 2. It is noted, however, that an earlier partial
sequence of the human lactoferrin sequence disclosed a number of
discrepancies between the published sequence and that obtained
herein. Specifically, the following discrepancies exist (amino acid
numbering is from the sequence in FIG. 1 with DNA position in
parenthesis):
3 Amino Acid Position In Metz-Boutique Arg 122 (418) Absent Thr 130
(442) Ile Gln 151 (505) Arg Ser 184 (604) Leu Tyr 189 (619) Lys Ser
372 (1169) TrP between Ala 391 (1122) 13 amino acids and Met Cys
403 (1225) Gly Gln 512 (1588) Glu Lys 675 (2077) Arg
[0110] Accordingly, human lactoferrin is also defined by the
sequence shown in FIG. 1 which combines the sequence differences
obtained herein with the published sequence. The term human
lactoferrin also includes allelic variations of either of these
sequences or recombinant human lactoferrin variants wherein one or
more amino acids have been modified by the substitution, insertion
or deletion of one or more amino acid residues. In some instances
human lactoferrin may be produced in milk with all or part of a
secretory signal sequence covalently attached thereto.
[0111] As used herein, a "human lactoferrin DNA sequence" is a DNA
sequence which encodes human lactoferrin as defined above. Such a
human lactoferrin DNA sequence may be obtained from a human mammary
gland cDNA library or may be derived from the human genome. Example
2 herein describes the cloning and nucleotide sequence of human
lactoferrin derived from a human mammary gland cDNA library. The
DNA sequence of this human lactoferrin is shown in FIG. 1 and FIG.
2 and is substantially the same as that described by Rado, et al.
(1987) Blood 70:989-993. The construction of plasmids containing an
expressible transgene encoding hLF is described in the examples.
One of these plasmids is cGP1HLF also sometimes referred to as
16,8HLF3) contains a transgene designed for tissue-specific
expression in bovine mammary secretory cells.
[0112] In a second embodiment of the invention, transgenes are
provided for producing human serum albumin in the milk of
transgenic bovine species. Human serum albumin is a serum protein
which contains 584 amino acid residues (Minghetti, et al. (1986) J.
Biol. Chem. 261:6747). It is the most abundant protein in human
serum and performs two very important physiological functions.
Serum albumin is responsible for about 80% of the total osmolarity
of blood and it transports fatty acids between adipose tissues.
[0113] Human serum albumin is used primarily to expand plasma
volume by restoring osmotic pressure in the circulatory system.
Currently, a heat treated serum derived hSA fraction is infused in
most shock and trauma victims, including most of the patients
undergoing extensive surgery. HSA is presently derived from human
blood plasma as a by-product from blood fractionation processes to
obtain rare blood proteins such as factor VIII and IX. The recently
developed technology of producing such factors by biotechnological
means, however, threatens the source of human serum albumin.
[0114] As used herein "human serum albumin" comprises a polypeptide
having the amino acid sequence substantially as that described by
Minghetti, et al., ibid; Lawn, et al. (1981) Nucl. Acids Res.
9:6103. Also included are variations thereof including recombinant
human serum albumin variants wherein one or more amino acids have
been modified by the substitution, insertion or deletion of one or
more amino acid residues (Minghetti, et al. (1986) J. Biol. Chem.
261:6747-6757). In some instances, human serum albumin may be
produced in milk by expressing a transgene which contains DNA
encoding the secretory signal sequence of hSA. Alternatively, human
serum albumin may be produced in and secreted from liver cells of a
transgene animal utilizing a completely heterologous transgene
comprising human genomic DNA encoding 5' expression regulation
sequences, the human serum albumin secretion signal and structural
gene and 3' expression regulation sequences. As indicated in the
Examples, transgenes containing this heterologous sequence were
formed by in vivo homologous recombination of overlapping transgene
fragments to reconstitute the hSA gene in the transgenic animal.
The so formed transgenic animal produced human serum albumin in its
circulatory system.
[0115] As used herein, a "human serum albumin DNA sequence" is a
DNA sequence which encodes human serum albumin as defined above.
Such a human serum albumin DNA sequence may be obtained from
.lambda.HAL-HAI, .lambda.HAL-3W and .lambda.HAL-HI4 as described by
Urano, et al. (1986) J. Biol. Chem. 261:3244-3251 and Urano, et al.
(1984) Gene 32:255-261 and in the Examples herein.
[0116] The human serum albumin DNA sequence was cloned as described
in Example 10 herein and subsequently manipulated to substitute for
the human lactoferrin gene encoded in plasmid cGP1HLF (also
referred to as p16,8HLF4). From this plasmid a transgene is
obtained containing 16 kb of the 5' expression regulation sequence
of the bovine .alpha.S1 casein gene, human serum albumin DNA
sequence and approximately 8 kb of the 3'-flanking region of the
.alpha.S1 casein bovine gene. This transgene is used to microinject
fertilized oocytes from bovine species. After early detection of
transgenesis, blastocysts containing the hSA transgene are
implanted into a recipient female bovine species and brought to
term. The following is presented by way of example and is not to be
construed as any limitation on the scope of the invention.
EXAMPLE 1
[0117] Construction of a Probe Specific for Bovine .alpha.S1 Casein
Sequences
[0118] A. Isolation of Chromosomal DNA
[0119] Placental tissue was obtained from the slaughterhouse.
Surrounding connective tissue was removed and pieces of about 30
grams were quickly frozen in liquid N.sub.2. Chromosomal DNA was
isolated as follows: 30 grams of tissue was homogenized (on ice)
with 35 ml of Buffer 1 containing 300 mM Sucrose; 60 mM KCl; 15 mM
NaCl; 60 mM Tris.HCl pH 8.2; 0.5 mM spermidine; 0.15 mM spermine; 2
mM EDTA; 0.5 mM EGTA. 65 ml of icecold buffer 1 containing 1% NP40
was added and the mixture was incubated for five minutes on ice.
After centrifugation for five minutes at 3000 xg the pellet was
rinsed with buffer 1 containing 1% NP40. After repeating the
centrifugation step the pellet was resuspended in 5 ml of buffer 1.
5 ml 0.5 M EDTA was quickly added. Final volume was now 15 ml. 0.15
ml of a 10% SDS solution was added. After mixing, RNAse A and T1
were added to final concentrations of 0.4 mg/ml and 6 u/ml
respectively. After incubation at 37.degree. C. for three hours,
Proteinase K was added to a final concentration of 0.1 mg/ml. This
mixture was incubated for 15 hours at 37.degree. C. The mixture was
then carefully extracted with phenol. The aqueous phase was
isolated and {fraction (1/30)} volume of 3M NaOAc pH 5.2 and one
volume of isopropylalcohol was added. The precipitate (DNA) was
rinsed with 70% ethanol and slowly dissolved in 0.5 ml of 10 mM
Tris.HCl pH 8.0; 1 mM EDTA, at 4.degree. C.
[0120] B. Amplification of Sequences from the 5'-Flanking Region of
the .alpha.S1-Casein Gene
[0121] Two DNA-primers were synthesized based on the sequence
published by Yu-Lee et al., (1986) Nucl. Acids Res. 14, 1883-1902.
Primer 1 was located at position-681 relative to the major
transcription initiation site and had the following sequence:
[0122] 5'-TCC ATG GGG GTC ACA AAG AAC TGG AC-3'. (Seq. ID No.:
5)
[0123] Primer #2 was located at position +164 relative to the major
transcription initiation site and had the following sequence:
5'-TGA AGC TTG CTA ACA GTA TAT CAT AGG-3' (Seq. ID. No.: 6). The
first eight nucleotides of this primer are not encoded by the
bovine genome, but contain a HindIII restriction site to facilitate
subsequent cloning steps. These primers were annealed to the
chromosomal DNA and extended in the presence of deoxynucleotides by
TAQ-polymerase. After three minutes the mixture was denatured for
one minute at 92.degree. C., reannealed at 50.degree. C. for 1.5
minutes and again incubated at extension temperature (68.degree.
C.) for 2 minutes. This cycle was repeated 30 times. After the last
cycle DNA was checked for the presence of the expected EcoRI sites.
Both the size of the fragment and the presence of EcoRI sites was
as expected. The fragment was then treated with Klenow enzyme to
repair any overhanging ends, treated with kinase to attach
phosphate groups at the ends of the fragment, incubated at
65.degree. C. for 10 minutes to inactivate the kinase and klenow
enzymes and finally digested with HindIII. This fragment was then
subcloned in pUC19 (Yanisch-Perron, et al. (1985), Gene, 33,
103-109) digested with SmaI and HindIII. Formal proof of the
identity of this fragment was obtained by sequencing parts of this
subclone (after re-cloning into M13 vector). The determined
sequence was identical to the published sequence. This probe was
then used to screen a bovine genomic library to obtain clones
specific for the 5'-flanking region of the .alpha.S1-casein
gene.
[0124] C. Amplification of Sequences from the 3'-Flanking Region of
the .alpha.S1-Casein Gene
[0125] A similar approach was taken as described above. Two primers
were designed based on the sequence published by Stewart et al
(1984) Nucl. Acids Res. 12, 3895-3907. The 5'-primer was located
just downstream of the coding sequence starting at position 713 of
the cDNA sequence. It had the following sequence:
[0126] 5'-GAG GGA CTC CAC AGT TAT GG-3' (Seq. ID No.: 7).
[0127] The other primer was located at position 1070 of the cDNA
sequence and had the following sequence: 5'-GCA CAC AAT TAT TTG ATA
TG-3' (Seq. ID No.: 8). These primers were annealed to the
chromosomal DNA and the region between these primers was amplified
as described above. The resulting fragment was .apprxeq.900 bp
longer then expected. Sequence analysis showed that an intervening
sequence of this size was present between nucleotide 737 and 738 of
the cDNA. The amplified fragment was treated with Klenow-polymerase
to repair any overhanging ends and treated with kinase to attach
phosphate groups to the ends of the fragment. The fragment was then
ligated into pUC19 previously cut with SmaI.
[0128] D. Screening of a Bovine Phage Library for .alpha.S1-Casein
Flanking Sequences
[0129] A bovine genomic library, constructed in EMBL3, was obtained
from Dr. M. Groenen, Agricultural University Wageningen,
Netherlands, and was screened in the following way. The
bacteriophage particle titre was determined on Escherichia coli
MB406 a permissive host strain (Stratagene Inc.). For this, several
dilutions of the phage stock were made in SM buffer (50 mM Tris.HCl
pH 7.5, 100 mM NaCl, 10 mM MgSO4, 0.01% gelatin) and mixed with 200
.mu.l MB406 (O.D..sub.550=0.9); after 20 minutes at 37.degree. C.,
3 ml top agarose (Luria-Bertani medium, 0.8% agarose, 10 mM
MgCl.sub.2) was added and this was plated on LB plates and
incubated overnight at 37.degree. C.
[0130] Approximately 600,000 phages were then plated by adding the
required amount of phage stock to 400 .mu.l MB406. The subsequent
plating was as described as above. The next step was transfer of
the phage to nitrocellulose filters. Plates were placed at
4.degree. C. for one hour. Nitrocellulose filters (S&S) were
placed on the top agarose layer and exact position was marked.
After lifting, the filters were soaked for (1) 30 minutes in
denaturation buffer (1.5M NaCl, 0.5M NaOH); (2) 5 minutes in
neutralizing buffer (1.5M NaCl, 0.5M Tris.HCl pH 8.0). After
rinsing with 2.times.SSPE (360 mM NaCl, 20 mM NaH.sub.2PO.sub.4, 2
mM EDTA), the filters were baked under vacuum at 80.degree. C. for
two hours.
[0131] Prehybridization of the filters was performed in a buffer
containing 50% formamide, 5.times. Denhardt's solution (0.1%
Ficoll, 0.1% polyvinylpydrolidone, 0.1% bovine serum albumin),
5.times.SSPE, 0.1% SDS and 100 .mu.g/ml denatured salmon sperm DNA
at 42.degree. C. for two hours. Hybridization was performed in same
buffer at 42.degree. C. overnight in a shaking waterbath. The
probe, generated as previously described, was labelled using the
Random Primed labelling kit from Boehringer Mannheim. After
overnight hybridization the filters were washed three times with
2.times.SSC, 0.1% SDS at room temperature.
[0132] Overnight exposure of Kodak XAR films was performed with
amplifying screens (Dupont) at -70.degree. C. Putative positives
were plugged out of the plates and put overnight in SM buffer at
4.degree. C. These were plated out as described above and DNA was
isolated following the plate lysate method (Maniatis, T., et al.
(1982), Molecular Cloning: A Laboratory Manual, Cold Spring Harbor,
N.Y.). 5 ml SM buffer was added to the top agarose layer; after two
hours gentle shaking buffer was removed and spun at 4000 rpm at
4.degree. C. for 10 minutes. Supernatant was transferred to sterile
tubes and RNase A and DNaseI (both final concentration 1 .mu.g/ml)
was added, this was incubated at 37.degree. C. for 30 minutes. One
volume of a 20% polyethyleneglycol, 2.5 M NaCl solution was added
and put on ice for one hour. Centrifugation at 4000 rpm for 30
minutes at 4.degree. C. left precipitated bacteriophage particles.
These were resuspended in 500 ml SM buffer, SDS (final
concentration 0.1%) and EDTA (final concentration 5 mM) was added,
this was incubated at 68.degree. C. for 15 minutes. Protein was
removed with one phenol and one chloroform extraction step.
Precipitation of phage DNA was performed with one volume
isopropanol. Phage DNA was washed once with 70% ethanol and
dissolved in 50 ml Tris.HCl pH 7.5, 1 mM EDTA buffer.
[0133] Restriction enzyme analysis, agarose gel electrophoresis,
transfer of DNA from gel to nitrocellulose filter and Southern
blotting were all done according to standard procedures (Maniatis
(1982), Molecular Cloning: A Laboratory Manual). Hybridization with
probes (described hereinafter) was performed according to the same
procedure as the screening conditions described above.
[0134] E. Isolation of Clones Containing 5'-Flanking Region of
Bovine S1-Casein
[0135] Three putative clones were identified using the probe and
procedures as described above. After another round of screening,
clean recombinant bacteriophage was analyzed. Digestion of cloned
DNA with SalI, EcoRI and SalI/EcoRI (double digestion) and
hybridization with the probe described above showed identical
inserts in all three clones. The insert consisted of an 18 kb
(partial Sau3A fragment excised with SalI). Transciptional
orientation in the clone was determined with hybridization of above
described restriction fragments with (1) probe 1 described above,
and (2) the NcoI-NsiI fragment of probe 1. This showed a region of
about 16 kb upstream of transcription start. Downstream from the
transcription start was another 1.9 kbp. Sequencing of part of the
latter region showed the presence of exon 2 and part of intron 2 of
the bovine .alpha.S1-casein gene. Additional sequencing of the
region--103--+300 confirmed the identity of the clone. The
ethidium-bromide pattern of the described restriction fragments
also showed the orientation of the clone in the EMBL vector.
Subsequent analysis of the clone with the following restriction
enzymes (NcoI, PstI, KpnI, BamHI, HindIII, BaqlII) resulted in the
restriction map of 5' flanking region of bovine S1-casein gene as
shown in FIG. 3.
[0136] F. Isolation of Clones Containing 3'-Flanking Region of
Bovine .alpha.S1-Casein
[0137] Duplicate nitrocellulose filters from the initial phage
plating used for isolating 5' clones were screened with the 3'
.alpha.S1-casein probe using the same hybridization conditions
previously described. Eight positive clones were identified after
two rounds of screening. Phage DNA was prepared as described.
Subsequent restriction digests with SaII, EcoRI and SaI/EcoRI and
Southern hybridization with the 3' .alpha.S1 probe showed identical
inserts in seven of the eight clones. One clone containing an 18.5
kb EcoRI insert was further analyzed with the restriction enzymes
BsteII and BamHI. A restriction map of that clone is shown in FIG.
4.
EXAMPLE 2
[0138] Cloning of Human Lactoferrin Gene
[0139] A. Materials
[0140] Restriction endonucleases, T4 ligase, and T7 polynucleotide
kinase were obtained from Boehringer-Mannheim, New England Biolabs,
or Bethesda Research Laboratories. Radio-isotopes were purchased
from Amersham. A human mammary gland cDNA library in bacteriophage
.lambda.gt11 was obtained from Clontech, Inc., Palo Alto,
Calif.
[0141] B. Isolation of the Human Lactoferrin Gene
[0142] The human mammary gland library was screened by standard
plaque hybridization technique (Maniatis, et al. (1982) Molecular
Cloning: A Laboratory Manual) with three synthetic oligomers. Two
of the oligomers were 30-mers corresponding to the cDNA sequence of
Rado et al., supra, at amino acid positions 436-445 and 682-691.
The third was a 21-mer "best guess" probe based on human codon bias
and coding for amino acid sequence of HLF between amino acid
residues 18 and 24. Respectively, they were:
4 (Seq. ID No.: 9) (1) 5'-CTTGCTGTGGCCGTGGTTAGGAGATCAGAC-- 3' (Seq.
ID No.: 10) (2) 5'-CTCCTGGAAGCCTGTGAATTCCTCAGGAAG-3', and (Seq. ID
No.: 11) (3) 5'-ACCAAGTGCTTCCAGTGGCAG-3'.
[0143] The probes were radiolabeled (Crouse et al. (1983) Methods
Enzymol. 101, 78-98) and used to screen duplicate filters. Filters
were washed at a final stringency of 2 .times.SSC, 37.degree.
C.
[0144] C. Nucleotide Sequence Analysis
[0145] DNA fragments were isolated by use of low-melting agarose
(Crouse et al, supra) and subcloned into bacteriophase M13mp18 or
M13mp19 (Messing et al. (1983) Methods Enzymol. 101, 20-78). The
sequence was determined using the Sequenase enzyme (modified T7 DNA
polymerase) (Tabor et al. (1987) Proc. Natl. Acad. Sci. USA 84,
4767-4771). All reactions were carried out according to -the
manufacturer's specifications (US Biochemicals). The sequence is
shown in FIG. 1. The hLF sequence was digested with HindIII and
EcoRI (present in the surrounding phage sequences) and subcloned
into the HindIII and EcoRI site of pUC19 to form pUS119 Lacto 4.1.
This clone contained the entire coding sequence of the mature form
of hLF, but lacked the complete signal sequence.
EXAMPLE 3
[0146] Construction of Bovine .alpha.S1-Casein CAT Vectors
[0147] In order to determine whether the .alpha.S1 casein fragments
obtained in Example 1 had promoter and other properties needed to
express a heterologous gene, expression plasmids were constructed
containing variable amounts of 5-' and 3'-flanking regions from the
.alpha.S1-casein gene. The chloramphenicol Acetyl transferase gene
(CAT) was used as a heterologous gene in these vector constructs.
The CAT gene is useful to detect the expression level for a
heterologous gene construct since it is not normally present in
mammalian cells and confers a readily detectable enzymatic activity
(see Gorman, C. N., et al. (1983), Mol. Cell. Biol., 2, 1044-1051)
which can be quantified in the cells or animals containing an
expressible gene.
[0148] A. DNA Sequences
[0149] 681 bp of a .alpha.S1-casein promoter plus the first
non-coding exon plus approximately 150 bp of the first intervening
sequence (IVS) were isolated from a 5'-flanking genomic clone from
Example 1 by PCR amplification as an NcoI-HindIII fragment
(approximately 830 bp). This fragment is identified as fragment 1
in FIG. 5A. The primer sequences consisted of:
5 (Seq. ID No.: 12) 5'-TCCATGGGGGTCACAAAGAACTGGAC-3' and (Seq. ID
No.: 13) 5'-TGAAGCTTGCTAACAGTATATCATA- GG-3'
[0150] that were designed from a sequence published by Yu-Lee et
al. (1986) Nuc. Acids Res. 14, 1883-1902.
[0151] Approximately 1.6 kb (fragment 2, FIG. 5A) of
.alpha.S1-casein 3'-flanking sequence was isolated by PCR
amplification from a bovine 3'-flanking genomic clone from Example
1. This region contained the previously described splice within the
3' untranslated region of .alpha.S1-casein gene. Fragment 2 was
subcloned into the SmaI site of pUC19. The primer sequences
consisted of:
6 5'-GAGGGACTCCACAGTTATGG-3' and (Seq. ID No.: 14)
5'-GCACACAATTATTTGATATG-3' (Seq. ID No.: 15)
[0152] that were designed from a sequence published by Stewart et
al. (1984) Nucl. Acids Res. 12, 3895-3907.
[0153] A hybrid splicing signal comprising the 3' splice site of an
immunoglobulin gene (Bothwell et al. (1981) Cell, 24, 625-637) was
synthetically prepared and inserted into pUC18 along with unique
restriction sites flanking either side to produce pMH-1. This
plasmid is shown in FIG. 6. NcoI and HindIII sites were designed
such that ligation with fragment 1 from the bovine 5' genomic clone
would result in the functional hybrid splice sequence. See FIG.
11.
[0154] A polyadenylation sequence was obtained from SV40 virus as a
BamHI-DraI fragment (fragment 3 in FIG. 5A) isolated from pRSVcat
(Gorman, C. M., et al. (1982), Proc. Natl. Acad. Sci., 79,
6777-6781).
[0155] A bacterial CAT coding sequence was subcloned into pUC19 as
a PstI-BamHI fragment.
[0156] B. Construction of pS13' 5.degree. CAT
[0157] Fragment 1 of .alpha.S1-casein promoter was subcloned into
pMH-1 (FIG. 6) between the NcoI and HindIII sites to form
pMHS15'flank.
[0158] The SV40 polyadenylation sequence (fragment 3) was subcloned
as a BamHI-DraI fragment into pUC19 immediately 3' to the 3'
.alpha.S1-casein flanking sequence (fragment 2) to form pUC19 3'
UTR/SV40. This allowed for the removal of a continuous EcoRI-SalI
fragment (containing the 3'-flanking sequence and poly (A)
sequence) that was subcloned into pMH-1 to derive pMHS13'UTR (FIG.
5B) which was used later to construct pMHSI 3'UTR hlf which
contains sequences encoding human lactoferrin.
[0159] The EcoRI-SalI sequence (fragments 2 and 3) were subcloned
into the EcoRI-SalI sites of pMHS15'flank to form pS13'5'flank.
[0160] The PstI-BamHI CAT fragment (fragment 4 in FIG. 5B), after
blunting the BamHI site with Klenow, was subcloned into
pS13'5'flank (FIG. 5B) between the PstI and SmaI sites to form
pS13'5.degree.CAT.
[0161] C. Construction of pS15'CAT
[0162] The CAT fragment (fragment 4 in FIG. 5B, PstI-BamHI) and
SV40 polyadenylation fragment (fragment 3 in FIG. 5A, BamHI-DraI)
were subcloned into the PstI and SmaI sites of pMHS15'flank to form
pS15'CAT (FIG. 5C).
[0163] D. Assay for CAT Production
[0164] Each of these CAT plasmids were transfected into human 293S
cells (Graham, F. L., et al. (1977), J. Gen. Virol., 36, 59-72) by
the calcium phosphate co-precipitation method (Gorman, C. M., et
al. (1983), Science, 221, 551; Graham, F. L., et al. (1973),
Virology, 52, 456-467). Cells were harvested 44 hours after
transfection and cell extracts were assayed for CAT activity
(Gorman, C. M., et al. (1982), Mol. Cell. Biol., 2, 1011;
deCrombrugghe, B., et al. (1973), Nature [London], 241, 237-251, as
modified by Nordeen, S. K., et al. (1987), DNA, 6, 173-178). A
control plasmid expressing CAT driven by the Cytomegalovirus
Immediate early promoter (Boshart, M., et al. (1985), Cell, 41,
521) was transfected into human 293 S cells to assay for
transfected efficiency.
[0165] pS13'5'CAT was expressed in these cells at a level which was
approximately 30-100 fold lower than the control plasmid, but
significantly higher than background. Primer extension analysis
indicated that transcription had initiated predominantly in the
expected region.
[0166] When pS15'CAT was transfected into 293S cells, expression
was also detected.
EXAMPLE 4
[0167] Bovine .alpha.S1-Casein/Human Lactoferrin Expression Cosmid
cGP1HLF
[0168] A. Construction of DNA Sequences.
[0169] 16 kb of bovine .alpha.S1-casein 5'-flanking sequence from
Example 1 was isolated from the bovine genomic library (phage GP1)
as a SalI-BglII fragment. The BglII site lies at the junction of
the first intron and second exon of the .alpha.S1-casein gene.
[0170] Bovine .alpha.S1-casein signal sequence (Stewart et al.
(1984) Nucl. Acids Res. 12, 3895) was prepared from synthetic DNA
synthesized on a Cylone Plus.RTM. DNA Synthesizer
(Millgen/Biosearch I) and contained the entire signal sequence plus
XhoI and Cla I sites attached to the 5'-end, and NaeI to the 3'-end
(fragment 8, FIG. 7B).
[0171] Cleavage of pUC119 Lacto 4.1 with EaeI precisely opened the
plasmid at the codon for the first amino acid of mature hLF.
Treatment with Klenow was used to fill in the overhanging 5'-end.
Further digestion with AccI and EcoRI gave two fragments: (a) an
EaeI-AccI fragment containing the first 243 bp of mature hLF
(fragment 5, FIG. 7C), and (b) a contiguous AccI-EcoRI fragment
(fragment 6, FIG. 7C) of 1815 bp that contained all but five
terminal codons of the remaining coding sequence.
[0172] A synthetic linker was prepared that contained the last five
codons of hLF beginning at the EcoRI site and extending for four
bases beyond the stop codon. A KpnI site was added to the 3'-end
(fragment 7 in FIG. 7C).
[0173] An 8.5 kb EcoRI 3'-fragment was isolated from the bovine
genomic library (FIG. 4) containing sequences beginning just
downstream of the coding region of .alpha.S1-casein and a BstEII
site approximately 350 bp from the 5'-end. This fragment was
subcloned into pMH-1 at the EcoRI site to form pMH3'E10 (FIG. 7A).
A SalI site is adjacent to the 3'-EcoRI site in pMH3'E10.
[0174] B. Construction of cGP1HLF
[0175] The hLF 3'-linker (fragment 7, FIG. 7C) was subcloned into
the EcoRI-KpnI sites of pMH3'UTR (FIG. 7A) to produce
pMH3'UTRhLF2linker (FIG. 7A).
[0176] The synthetic bovine .alpha.S1-casein signal sequence
(fragment 8) was then subcloned into the XhoI and SmaI sites of
pMH3'UTRhLF2linker to make pS13'hLF1/2L (FIG. 7B).
[0177] The two hLF coding fragments (fragments 5 and 6 in FIG. 7C)
were subcloned into the NaeI and EcoRI sites of pS13'hLF1/2L (FIG.
7B) to make pS13'UTRhLF (FIG. 7C).
[0178] The large .alpha.S1-casein 3'UTR fragment from pMH3'E10
(FIG. 7A) was isolated as a BstEII-SalI fragment and subcloned into
the same sites of pS13'UTRhLF to form phLF3'10 kb (FIG. 7D).
[0179] Cosmid cGP1HLF was prepared from a 3-way ligation (FIG.
7F):
[0180] (1) the 16 kb 5'-flanking sequence from phage GP1 (Example
1, FIG. 3) was modified by attaching two linker adapters. The SalI
site at the 5'-end was ligated to a NotI-SalI linker. The BglII
site at the 3'-end was ligated to a BglII-XhoI linker;
[0181] (2) the hLF coding region, flanked on the 5'-end by the
.alpha.S1-casein signal sequence and on the 3'-end by approximately
8.5 kb of .alpha.S1-casein 3'-flanking sequence, was isolated as a
XhoI-SalI fragment from phLF3'10 kb. The SalI site at the 5'-end
was ligated to a SalI-NotI linker;
[0182] (c) Cosmid pWE15 (Stratagene, Inc.) was linearized with
NotI.
[0183] Fragments from (a), (b), and (c) were ligated together and
transfected into bacteria using commercial lambda packaging
extracts (Stratagene, Inc.) to produce cGP1HLF.
EXAMPLE 5
[0184] Bovine .alpha.S1-Casein/hLF Expression Plasmids.
[0185] A. Construction of pS13'5'hLF
[0186] The HindIII-SalI fragment of pS13'UTRhLF was subcloned into
the same sites in pMHS15'flank to form pS13'5'hLF (FIG. 7E). This
plasmid contains 681 bp of bovine .alpha.S1-casein promoter
sequence, the .alpha.S1-casein/IgG hybrid intron, the
.alpha.S1-casein signal sequence, the hLF coding region,
approximately 1.6 kb of .alpha.S1-casein 3'-flanking sequence, and
the SV40 late region polyadenylation sequence.
[0187] B. pS15'hLF
[0188] Plasmid pS13'5'hLF (FIG. 7E) was cut with KpnI and BamHI
which border the .alpha.S1-casein 1.6 kb 3 '-flanking sequence. The
larger vector fragment was purified, made blunt ended with Klenow,
and self-ligated to form pS15'hLF.
[0189] C. Radioimmunoassay for hLF
[0190] An immunoglobulin-enriched fraction of ascites fluid of a
monoclonal antibody against human lactoferrin, which does not
cross-react with the bovine or murine protein, was prepared by 50%
ammonium sulfate precipitation and coupled to CNBr-activated
Sepharose 4B (20 mg of protein to 1 g of Sepharose). The Sepharose
beads were suspended (2 mg/ml) in phosphate-buffered saline (PBS;
10 mM sodium phosphate, 0.14 M NaCl containing 10 mM EDTA, 0.1%
(.sup.w/v) Polylorene and 0.02% (.sup.w/v) NaN.sub.3, pH 7.4.
Sepharose suspensions (0.3 ml) were incubated for five hours at
room temperature by head-over-head rotation with samples (usually
50 .mu.l) in 2-ml polystyrene tubes. Sepharose beads were then
washed with saline (five times with 1.5 ml) and incubated for 16
hours at room temperature with 50 .mu.l (1 kBq) of
.sup.125I-labeled-affinity-purified polyclonal rabbit anti human
lactoferrin antibodies, together with 0.5 ml of PBS, 0.1%
(.sup.w/v) Tween-20. Thereafter the Sepharose was washed again with
saline (four times with 1.5 ml) and bound radio activity was
measured. Results were expressed as percent binding of the labelled
antibodies added. Levels of lactoferrin in test samples were
expressed in nanomolar, using purified human milk lactoferrin as a
standard (serial dilutions in PBS, 10 mM EDTA, 0.1% (.sup.w/v)
Tween-20.
[0191] Repeated testing of standard on separate occasions revealed
that this RiA was highly reproducible, intra- and inter assay
coefficients of variation ranged from 5-10%. As little as 0.1
nanogram human lactoferrin is easily detected by this RIA.
[0192] D. Expression in 293S Cells
[0193] 293S cells were transfected with the above hLF plasmids as
described (1 .mu.g of a CMV-CAT plasmid was co-transfected as
control for transfection efficiency). Forty-four hours after
transfection medium was removed from the cells and assayed for hLF
as described supra, RNA was isolated as described by Stryker, et
al. (1989) EMBO J. 8, 2669. The results can be summarized as
follows:
[0194] 1. Transfection efficiencies are identical for the two hLF
plasmids;
[0195] 2. hLF is expressed in the cells and secreted into the
medium. In both cases, the levels are about 0.4 .mu.g/ml medium
using about 3.times.10.sup.6 cells
[0196] 3. The proteins behave identical to hLF in a human milk
sample in a dose response assay measuring the amount of
.sup.125I-anti-lactoferrin bound as a function of the amount of
sample used.
[0197] 4. The protein has about the same size (.sup.-80 kD) as in a
human milk sample as judged by Western blotting.
[0198] 5. The hLF RNA produced in the cells has the correct size
and its level is similar for both plasmids as judged by
Northern-blotting.
[0199] These data indicate that these two expression plasmids are
able to express hLF. By all standards used so far, the protein is
identical to hLF present in human milk. The heterologous signal
sequence is functional in that it promotes secretion of the protein
from the cells into the medium. Further, the casein regulatory
sequences used in these plasmids are able to promote expression of
a heterologous gene.
EXAMPLE 6
[0200] In vitro Maturation, Fertilization and Culture of Bovine
Oocytes
[0201] Immature oocytes are obtained in large quantity
(400-600/day) by aspirating follicles of ovaries obtained at
abbatoirs. Immature oocytes are cultured for a period in vitro
before they are competent to be fertilized. Once "matured", oocytes
are fertilized with sperm which has also been matured, or
"capacitated" in vitro. The pronuclei of the fertilized oocyte (or
zygote) is then injected with the transgene encoding for the
expression and secretion of human lactoferrin. Preferably the
zygotes are substantially synchronous such that greater than about
30, 50, 70, 90 or 95% of zygotes are in S-phase at the time of
injection. Zygotes resulting from this in vitro fertilization and
microinjection are then cultured to the late morula or blastocyst
stage (5-6 days) in medium prepared, or "conditioned" by oviductal
tissue. Blastocysts are then transferred non-surgically to
recipient cattle for the balance of gestation or analyzed for
integration of the transgene as described herein.
[0202] In vitro maturation (IVM). Ovaries are obtained immediately
after slaughter at local abbatoirs and oocytes are recovered.
Alternatively, oocytes are obtained from living cattle by surgical,
endoscopic, or transvaginal ultrasonic approaches. In all cases,
oocytes are aspirated from ovarian follicles (2-10 mm diameter).
After washing, oocytes are placed in a maturation medium consisting
of M199 supplemented with 10% fetal calf serum, and incubated for
24 hours at 39.degree. C. Sirard et al. (1988) Biol. Reprod. 39,
546-552.
[0203] In vitro fertilization (IVF). Matured oocytes are fertilized
with either fresh or thawed sperm. Sperm are prepared for
fertilization by first obtaining a population of sperm enriched for
motility by a "swim-up" separation technique (Parrish et al. (1986)
Theriogenology 25, 591-600). Motil sperm are then added to a
fertilization media, consisting of a modified Tyrode's solution
(Parrish et al. (1986) supra.) supplemented with heparin to induce
sperm capacitation (Parrish et al. (1988) Biol. Reprod. 38,
1171-1180). Capacitation constitutes the final sperm maturation
process which is essential for fertilization. Sperm and oocytes are
co-cultured for 18 hours. A useful feature of this IVF method is
that (in the case of frozen sperm) consistent, repeatable results
are obtained once optimal fertilization conditions for a particular
ejaculate have been defined (Parrish et al. (1986) supra.).
[0204] In vitro culture (IVC). Conventional culture systems, which
support development of murine, rabbit, or human ova, do not support
development of bovine embryos past the 8-16 cell stage. This
problem has been overcome by pre-conditioning culture media with
oviductal tissue. Oviduct-conditioned medium will support bovine
embryos past the 8-16 cell stage to the blastocyst stage in vitro
(Eyestone and First (1989) J. Reprod. Fert. 85, 715-720).
[0205] Bovine embryos have proved refractory to in vitro culture.
This in part stems from the existence of a "block" to cleavage in
vitro at the 8-16 cell stage. This block may be alleviated by
culturing embryos in the oviducts of rabbits (reviewed by Boland
(1984) Theriogenology 21, 126-137) or sheep (Willadeen (1982) in:
Mammalian Egg Transfer, (E. Adams, ed., pp. 185-210)); Eyestone et
al. (1987) Theriogenology 28, 1-7). However, these in vivo
alternatives have been less than ideal, in that: (1) they require
the maintenance of large numbers of recipient animals, (2) they
require surgery to gain access to the oviducts for transfer, and a
second surgery (or sacrifice) to recover the embryos, (3) all
transferred embryos are seldom recovered, and (4) access to embryos
during culture for observation or treatment is entirely precluded.
The lack of in vitro culture systems has hampered the development
of various manipulation techniques (such as gene transfer by
pronuclear injection) by preventing accumulation of basic
information of the chronology and ontogeny of bovine development,
and by complicating the process of culturing embryos to a stage
compatible with non-surgical embryo transfer and cryopreservation
techniques (e.g., late blastocyst stages).
[0206] Bovine embryos did not yield to attempts to culture them in
vitro past the 8-16 cell "block" until Camous et al. (1984) J.
Reprod. Fert. 72, 479-485 demonstrated cleavage to 216 cells when
embryos were co-cultured with trophoblastic tissue.
[0207] The co-culture procedure was extended to oviductal tissue,
based on the ability of homo- or hetero-oviducts to support
development from zygote to blastocyst. Thus, bovine embryos
co-cultured with oviductal tissue, or in medium conditioned by
oviductal tissue, developed from zygote to blastocyst in vitro
(Eyestone and First, (1989) J. Reprod. Fert. 85, 715-720; Eyestone
W. H. (1989) "Factors affecting the development of early bovine
embryos in vivo and in vitro." Ph.D. Thesis, University of
Wisconsin). Blastocysts have been produced in this system after
superovulation and artificial insemination, or by in vitro
maturation (IVM), and fertilization (IVF) of immature oocytes.
Blastocysts produced in this fashion resulted in pregnancies and
live calves after transfer to recipient animals. The results
obtained were as follows:
7 Efficiency Number Step (%) (per 100) IVM 90 90 IVF 80 72 IVC 30
22 Embryo transfer 50 11 (% pregnant)
[0208] Therefore, from an initial daily harvest of 500 oocytes, it
is expected the approximately 55 pregnancies will result.
[0209] Preparation of Oviduct Tissue Co-Culture and Conditioned
Medium
[0210] 1. Obtain bovine oviducts after slaughter or by
salpingectomy.
[0211] 2. Harvest lumenal tissue by scraping intact oviduct gently
with a glass slide.
[0212] 3. Wash tissue 5 times in 10 ml modified tyrodes-hepes
solution (Parrish et al. (1988) Biol. Reprod. 38, 1171-1180).
[0213] 4. Resuspend final tissue pellet in M199+10% fetal calf
serum at a ratio of 1 volume tissue:50 volumes of media.
[0214] 5. Tissue suspension can be used for embryo-co-culture.
[0215] 6. Alternatively, media may be conditioned for 48 h; after
centrifuging the suspension, the supernatant may be used as embryo
culture medium. Conditioned medium may be stored at -70.degree. C.,
if desired. Conditioned medium should be used at full strength for
embryo culture (no dilution) (Eyestone (1989) ibid).
EXAMPLE 7
[0216] Microinjection of hLF Transgene into Bovine Pronuclei
[0217] The DNA fragment containing the hLF expression unit is
excised from the vector by digestion with the appropriate
restriction enzyme(s) and separated on agarose gels. The fragment
is purified by electroelution, phenol and chloroform extraction and
ethanol precipitation (Maniatis et al.). The DNA fragment is
dissolved in and dialyzed in 10 mM tris, 0.1 mM EDTA pH 7.2 at a
concentration of 1 to 2 .mu.g/ml. Microinjection needles are filled
with the dialyzed DNA solution.
[0218] Before in vitro fertilization, cumulus cells are removed
from the egg by either vortexing at maximal speed for 2 minutes or
pipetting the eggs up and down several times in a standard
micropipet. Bovine pronuclei are injected in principle as murine
pronuclei (Hogan, B. et al. (1986) in: Manipulating the mouse
embryo, Cold Spring Harbor Laboratory) with an additional
centrifugation step in order to visualize the pronuclei. The
injection takes place 18-24 hours after fertilization. The time
varies depending on the bull used as a source of semen. Different
batches of semen cause the nuclei to become visible at different
times.
[0219] Bovine oocytes, matured and fertilized in vitro, are spun in
an eppendorf tube in 1 ml of tyrodes-hepes solution (Parrish
(1987)) at 14500 g for eight minutes (Wall et al. (1985) Biol.
Reprod. 32, 645-651). The embryos are transferred to a drop of
tyrodes-hepes solution on a microscope slide covered with paraffin
oil. Using a hydraulic system the oocytes are fixed to the egg
holder in such a way that both the pronuclei are visible (using
interference-contrast or phase contrast optics). If necessary, the
oocytes are rolled to change their position on the egg holder to
visualize the pronuclei. The injection needle is brought into the
same sharp focus of one of the pronuclei. The needle is then
advanced through the zona pellucida, cytoplasm into the pronucleus.
A small volume of 1-3 pl is injected (containing 20-100 DNA copies)
into the pronucleus either by using a constant flow or a pulse flow
(using a switch) of DNA solution out of the needle. Alternatively,
two cell stage embryos are spun as described and the nuclei of both
blastomers are injected as described. The injected embryos are then
transferred to a drop of co-culture medium as described in Example
6 in order to develop to the morula or blastocyst stage.
EXAMPLE 8
[0220] Early Detection of Transgenesis with hLF Transgene
[0221] Upon the microinjection of a construct, the oocyte is
cultured. A proper site of each embryo is cleaved and subjected to
lysis (King, D. et al. (1988) Molecular Reproduction and
Development 1, 57-62), proteolysis (Higuchi, R., (1989)
"Amplifications (A forum for PCR Users." 2, 1-3) and DPNI
digestion. PCR is performed as described previously (Ninomiy, T. et
al. (1979) Molecular Reprod. and Devel. 1, 242-248) with sets of
two primers, one in .alpha.S1 and the other in hLF cDNA sequence.
For example, in a PCR where the forward primer (30 mer) .alpha.S1
sequence is
[0222] ATG AAA CTT ATC CTC ACC TGT CTT GTG (Seq. ID No.: 16)
[0223] and the reverse primer (30 mer) in hLF sequence is GGG TTT
TCG AGG GTG CCC CCG AGG ATG GAT (Seq. ID No.: 17); 971-1000 of FIG.
1), a 990 bp fragment will be generated. This fragment contains the
hitherto inactivated DpNI site by loss of adenosine-methylation, at
934 bp away from the start of the forward primer.
EXAMPLE 9
[0224] Production of hLF in Milk of Bovine Species
[0225] Bovine morula developed from microinjected oocytes are split
according to the method of Donahue (Donahue, S. (1986) Genetic
Engineering of Animals, ed. J. Warren Evans et al., Plenum). One
half of the morula is kept in culture to develop into blastocysts.
The other half is subjected to the DNA analysis as described in
Example 8. When the result of this analysis is known, the morula
kept in culture are developed into a blastocyst or as a source for
nuclear transfer into enucleated zygotes. Blastocyst transfer into
synchronized cows is performed according to the method of
Betteridge (Betteridge, K. J. (1977) in: Embryo transfer in farm
animals: a review of techniques and applications).
[0226] hLF is detected in the milk of lactating transgenic
offspring using the RIA of Example 5.
EXAMPLE 10
[0227] Bovine .alpha.S1 Casein/hSA Expression Plasmids
[0228] Three overlapping phage clones that contain the complete hSA
gene are used to construct an expression vector for hSA. They are
designated .lambda.HAL-HA1, .lambda.HAL-3W and .lambda.HAL-H14.
They are described in Urano, et al. (1986), J. Biol. Chem., 261,
3244-3251; and Urano, et al. (1984), Gene, 32, 255-261. The
sequence of the gene plus some surrounding regions is published in
Minghetti, et al. (1986), J. Biol. Chem., 261, 6747-6757. A single
phage containing the complete hSA gene is constructed as
follows:
[0229] Clone HA-1 is cut with BstEII and AhaII. The .apprxeq.1400
bp fragment running from position 1784 (in the first exon, just
downstream of the ATG) to 3181 is isolated and a synthetic linker
is attached to the BstEII site at the 5' end containing the first
few amino acids that are cut off with BstEII as well as the
sequence surrounding the ATG as well as a few convenient
restriction sites. This fragment is called fragment #1.
[0230] Clone 3W is cut with AhaII and SacI the .apprxeq.13.1 kb
fragment running from position 3181 to 16322 is isolated and a
synthetic linker is attached to the SacI site to facilitate cloning
in phage EMBL3. This fragment is called fragment #2.
[0231] These two fragments are ligated and cloned in phage EMBL3.
After identification of the correct phage, a fragment running from
just upstream of the BstEII site (where unique restriction sites
have been introduced) to the SacI site are isolated and ligated
from a SacI to SalI fragment (running from position 16322 to
[0232] .apprxeq.21200 isolated from clone H-14. These two fragments
are then ligated and cloned in EMBL4.
[0233] After cutting with ClaI (just upstream of the BstEII site,
newly introduced) and BamHI (just downstream of the SalI site in
the phage DNA) this new clone yields a fragment containing the
complete hSA gene with about 2.5 kb 3'-flanking sequence.
[0234] To construct an expression vector for hSA cosmid cGP1HLF is
partially digested with ClaI and BamHI. This removes the signal
sequence, the coding sequence of hLF, the 3'-UTR and poly(A)
addition region of .alpha.S1-casein as well as a small region 3' of
the casein gene.
[0235] This is ligated to the hSA fragment described above and the
resulting cosmid is called cGP1HSA.
[0236] The expression vector so formed contains, (1) 16 kb of
promoter sequences derived from the .alpha.S1-casein gene, (2) the
first exon and intervening sequence of this gene both present in
GP1, (3) the signal sequence of the hSA gene the complete genomic
gene coding for hSA including 2.5 kb downstream of that gene, and
(4) .apprxeq.8 kb of 3'-flanking sequence derived from the
.alpha.S1-casein gene.
[0237] This transgene is used to produce transgenic bovine species
producing hSA in their milk in a manner analogous to that used to
produce hLF in the milk of bovine species.
EXAMPLE 11
[0238] Purification of HSA from the Milk of Bovine Species
[0239] Purification of heterologous proteins from milk is
facilitated by the fact that, following casein precipitation, those
proteins, for the most part, are found in the whey fraction which
is less contaminated than the production media used in microbial or
cell-based systems.
[0240] Chromatographic techniques are preferred for the
purification of hSA from cow milk. This approach produces a better
recovery and higher albumin purity as well as a lower content of
albumin polymers as compared with ethanol fractionation (Curling
(1980) in: "Methods of Plasma Protein Fractionation", Curling, ed.,
Academic Press London, UK; Curling et al. (1982) J. Parenteral Sci.
Technol. 36, 59; Berglof et al and Martinache et al. (1982) Joint
Meeting IHS-ISBT, Budapest). The specific transport role of hSA as
well as its major role in maintaining intravascular osmotic
pressure may also be better preserved upon chromatographic
purification (Steinbruch (1982), Joint Meeting ISH-ISBT,
Budapest).
[0241] The following steps are used to recover hSA produced in the
milk of transgenic cows:
[0242] 1. Precipitation of caseins (about 80% of milk protein) and
essentially all the milk fat at pH 4.5 and/or by adding chymosin.
The whey fraction contains the albumin;
[0243] 2. Affinity-chromatography of albumin on Cibacron blue
3GA-Sepharose CL-6B (Harvey (1980) in: Methods of Plasma Protein
Fractionation, op. cit.) This step serves both to remove proteins
other than albumin and to decrease the volume to be handled about
30-fold. Albumin is eluted from this matrix with 0.15 M NaCl and 20
mM sodium salicylate at pH 7.5;
[0244] 3. Buffer-exchange on Sephadex G-25: desalting into 0.025 M
sodium acetate, adjustment to pH 5.2, followed by filtration;
[0245] 4. Anion-exchange chromatography on DEAE-Sepharose CL-6B.
Desorption of albumin at pH 4.5;
[0246] 5. Cation-exchange chromatography on CM-Sepharose CL-6B.
Albumin elution with 0.11 M sodium acetate, pH 5.5 and
concentration of albumin at a 6% (w/v) solution by ultrafiltration;
and
[0247] 6. Gel filtration on Sephacryl S-200. Fraction of
high-molecular weight protein (e.g. albumin polymers, pyrogens) is
discarded. The main fraction (albumin monomers) is concentrated by
ultrafiltration and formulated.
[0248] It is to be noted that steps 3-6 are essentially identical
to the method described by Curling and others (Curling (1980) op.
cit.; Curling et al. (1982) op. cit.; Berglof et al. (1982) op.
cit.) for the purification of hSA from plasma.
EXAMPLE 12
[0249] Transgenic Mice Containing the Human Serum Albumin (hSA)
Transgene Generated by Homologous Recombination
[0250] Three overlapping genomic hSA clones were used to generate
the hSA gene in transgenic mice, .lambda.HAL-HA1, .lambda.HAL-H14
and .lambda.HAL-3W, are shown in FIG. 8 as reported by Urano, et
al. (1984), Gene. 32, 255-261 and Urano, et al. (1986), J. Biol.
Chem., 261 3244-3251. Briefly, a genomic library was constructed
from a partial EcoRI digest of human fibroblast DNA. For the clones
.lambda.HAL-H14 and .lambda.HAL-3W, this library was screened with
.sup.32P-labeled human albumin genomic clones by hybridization in 1
M NaCl, 50 mM Tris-HCl (pH 8.0), 10 mM EDTA, 0.1% SDS, 100 ug/ml of
sheared salmon sperm DNA and 10.times. Denhardt's solution at
65.degree. C. overnight after prehybridization in 3.times.SSC and
10.times. Denhardt's solution. Following hybridization, filters
were washed in 0.2.times.SSC and 0.1% SDS at 65.degree. C. The
isolation of the .lambda.HAL-HA1 clone was identical except that a
0.9 kb BglII-EcoRI fragment from the 5' end of .lambda.HAL-3W was
used to screen the human fibroblast library.
[0251] These three hSA phage clones were used to generate three
overlapping linear DNA fragments, which in composite comprised the
whole HSA gene and flanking regions. The 5' most fragment I was a
EcoRI-EcoRI fragment isolated from .lambda.HAL-HA1; the middle
fragment II was a Acyl (=AhaII)-SacI fragment of .lambda.HAL-3W;
and the 3' most fragment III was a XhoI-SalI fragment of
.lambda.HAL-H14 (FIG. 7). The fragments were treated with klenow
DNA polymerase and dNTP's to fill in overhanging sticky ends. In
some experiments, the blunt ended fragments were then treated with
bacterial alkaline phosphatase to remove the 5' phosphate groups
from each fragment. The overlapping DNA fragments were next
concentrated then coinjected into the male pronuclei of fertilized
mouse eggs according to published methods (Hogan, et al. (1986) in
"Manipulating the Mouse Embryo: A Laboratory Manual", Cold Spring
Harbor Laboratory). While the number of molecules injected varied
from .apprxeq.25 to .apprxeq.100 per egg cell, the ratio of the
individual fragments was approximately 1:1:1. Embryos were
implanted into the uteri of pseudo pregnant female mice according
to the methods of Hogan, et al., supra.
[0252] To assay correct homologous recombination of the three
overlapping fragments and integration of the nascent transgene into
the mouse genome, genomic DNA from the newborn pups was subject to
the following specific digestions followed by Southern
hybridization with HSA cDNA probes:
[0253] Bst EII: cuts outside the HSA gene region and yields an 18
kb band if correct recombination occurred;
[0254] Nco I: cuts outside the overlapping regions and yields bands
of 8.0 and 9.3 kb if correct recombination occurred;
[0255] Nco I+Hind III: cuts at several positions outside the region
of overlap, indicative of the presence of intact fragments;
[0256] Hinc II: cuts in the overlapping regions, yielding several
bands indicative of correct arrangement in these regions.
[0257] In an initial experiment of 28 transgenic animals born, 22
had correctly recombined all three fragments. From 20 out of those
22 animals blood was collected and assayed for the presence of hSA
protein using a radio immuno assay. 15 out of those 20 animals
showed hSA expression at levels between 0.5 and 5 .mu.g/mL. None of
the animals that had no recombination or that were not transgenic
showed any expression. Using RNA blots, only two (the two with the
highest protein level) showed a band. We are currently performing
blots on RNA that has been enriched for the presence of mRNA (i.e.,
poly(A)+RNA). Using reverse transcriptase to synthesize cDNA,
followed by PCR, we have observed a perfect relationship between
the presence of RNA and protein. However, in this experiment we
could not determine the size(s) of the RNA.
EXAMPLE 13
[0258] Alternate Construction of Transgenes Encoding hLF
[0259] This example describes the construction of two hLF
transgenes wherein the first contains approximately 16 kb of
.alpha.S1 casein 5' expression regulation sequence (pGP1hLF (16 kb)
also referred to as p16,8HLF4) and the second contains
approximately 7.9 kb of .alpha.S1 casein 5' expression regulation
sequence (pGP1hLF (8 kb) also referred to as p8.8HLF4). The overall
strategy for these constructions is depicted in FIG. 9.
[0260] A 1.8 kb EcoRI-BglII fragment (fragment C in FIG. 9) was
isolated from phage clone GP1. This fragment runs from position
-100 of the transcription start site into the second exon of the
.alpha.S1 casein gene. The BglII site lies at the junction of the
first entron and second exon of the .alpha.S1 casein gene. The 3'
end containing the BglII site was ligated to a synthetic BglII-ClaI
linker and subcloned into the plasmid pUC19. The resulting plasmid
is designated pEBS.
[0261] Fragment B in FIG. 9 was isolated as an EcoRI fragment and
cloned into the EcoRI site of pEBS. Fragment B includes sequences
from position -7500 to position -100 of the transcription start
site in the .alpha.S1 casein gene. The plasmid so formed is
designated pEB3S and contains the combination of fragments B and C
is the 8.9 kb EcoRI-ClaI fragment running from position -7500 to
position +1400 of the transcription start site. The 8.9 kb
EcoRI-ClaI fragment from pEB3, obtained by complete digestion with
ClaI and partial digestion with EcoRI was isolated and subcloned
into EcoRI-ClaI cut pKUN2 (a derivative of pKUN; Gene (1986) 46,
269-276 containing a NotI restriction site) to form pNE3BS.
[0262] An 8.5 kb ClaI-EcoRI fragment (fragment A in FIG. 9) running
from position -16000 to position -7500 of the transcription start
site was isolated from phage GP1. It was thereafter subcloned into
pUC19 to form pSE. Using synthetic oligonucleotide, a unique NotI
site was introduced into the ClaI site thereby destroying it. The
resulting plasmid is designated pNE.
[0263] The insert from pNE was isolated as a NotI-EcoRI fragment
and together with the EcoRI-ClaI insert from pNE3BS was ligated
into the cloning vector pKUN2. The resulting plasmid pGP1
(.DELTA.2ex) contains 16 kb of .alpha.S1 casein promoter plus the
5' end of the gene to the BglII site at the border of the second
exon.
[0264] The final plasmid (16,8HLF4) containing the transgene was
assembled using the NotI-ClaI fragment from clone pGPI (.DELTA.2ex)
and the Xho-NotI fragment from clone pHLF 3' 10 kb. The structure
of this transgene is the same as previously described herein.
[0265] As a minor modification to this plasmid the SalI site of
this plasmid was removed by cutting with SalI and inserting a
linker that contains a NotI site, but not a SalI site.
Subsequently, a SalI site was introduced just downstream of the hLF
sequence by cutting the KpnI site as that position adding the
following linker:
8 5'-CGTCGACAGTAC-3' (Seq. ID No.: 18) CATGGCAGCTGT-5' (sEQ. id
nO.: 19)
[0266] In effect, the hLF sequence is now surrounded by two unique
restriction sites (ClaI and SalI) and can be replaced by any
recombinant ANA sequence that has a ClaI-site at the 5'-end and a
SalI-site at the 3'-end.
[0267] Another transgene was constructed that is identical to the
foregoing except that it contains only about 8 kb of 5' .alpha.S1
casein expression regulation sequence. It was constructed by taking
the NotI-ClaI fragment from pNE3BS and fusing it directly into
Xho-otI fragment from clone pHLF 3' 10 kb. The resulting plasmid
was designated pGPIhLF (7 kb) (also referred to as p8.8HLF4).
[0268] Plasmid 16,8hLF4 was modified to contain a hybrid splice
signal (.alpha.S1 casein-IgG) described in examples 3 and 5. The
resulting plasmid was designated 16,8hLF3 and is identical to
16,8hLF4 except for the presence of a hybrid intron versus a
"natural" casein intron in the 5'-UTR.
[0269] The hLF signal sequence can also be used in all of the cDNA
constructs disclosed herein instead of the casein signal sequence.
This can be done in the following way: A synthetic oligo was made
that contains the complete hLF signal sequence (see FIG. 2) plus a
ClaI restriction site at the 5'-end and an EagI restriction site at
the 3'-end. These restriction sites also border the casein-signal
sequence in the other plasmids (e.g., p16,8hLF4). A fragment
containing the hLF-cDNA surrounded by ClaI and SalI sites was
cloned in pGEM7 (Stratagene, Inc.) containing a ClaI and SalI site.
The resulting plasmid was digested with ClaI and EagI and used as a
vector to accommodate the ClaI-EagI fragment containing the hLF
sequence. From the positive clones, the cDNA, with its own
sequence, was excised as a ClaI-SalI fragment and inserted in
ClaI-SalI digested p16,8hLF4 to generate p16,8hLF5. Similarly, this
ClaI-SalI fragment containing the hLF-cDNA plus hLF signal sequence
can be inserted in any hLF cDNA vector.
EXAMPLE 14
[0270] Production of Recombinant Human Lactoferrin and Human Serum
Albumin in the Milk of Transgenic Mice
[0271] Transgenic mice were generated utilizing several of the
transgenes identified in the examples herein. The transgenes used
are identified in Tables 3 and 4. In each case, the 5' and 3'
expression regulation sequences were from the bovine .alpha.S1
casein gene, the RNA splice signal in the 5' untranslated region
was either homologous from the .alpha.S1 casein gene or a hybrid
casein-IgG intervening sequence. The recombinant DNA in each case
was derived from cDNA clones.
[0272] The transgene containing 26 kb of 5' .alpha.S1 casein
expression regulation sequence was generated by in vivo homologous
recombination of overlapping fragments. Briefly, a phage clone
containing an approximately 14 kb SalI insert was identified. This
insert contains about 11.5 kb of sequence upstream from the 5'
casein sequence contained in 16,8hLF4 and about 2.5 kb of
overlapping sequence. The NotI insert from 16,8hLF4 and the SalI
phage insert were coinjected to produce the 26.8hLF4 mice.
9TABLE 3* Length Plasmid of 5' - Length Maximum Range from
expression of 3' - expression of mean which regulator expression
Number levels levels transgene segment regulator of recorded
recorded excised (kb) segment IVS strains (.mu.g/ml) (.mu.g/ml)
p0.7,8 0.68 8 homologous 6 0.0-0.8 0.0-0.1 hLF4 p8,8 6.2 8
homologous 6 5-36 2.5-16 hLF4 .dagger-dbl.p16,8 14.5 8 homologous 5
0.3-3.6 0.0-1.8 hLF4 p26,8 26 8 homologous 5 0.6-10 0.2-1.7 hLF4
p16,8 14.5 8 heterologous 13 0.0-708 0.0-200 hLF3 *The number in
the plasmid designations before the comma represents the
approximate length in kbp in the 5' sequence from the bovine
.alpha.S1 casein promoter/flanking region while the number after
the comma represents the approximate length in kbp in the 3'
flanking sequence of the .alpha.S1 gene. Note the actual number of
bases for the 8 kb and 16 kb promoter (5' flanking region) is 6.2
and 14.5 kbp, respectively. .dagger-dbl.Exception: An additional
p16,8hLF4 transgenic mouse (line 145) not included in the data in
Table 3 gave a maximum expression level of 224 .mu.g/ml and a mean
of 112 .mu.g/ml.
[0273]
10TABLE 4 16,8hLF 3 Expression Data Mean level of Maximum level of
Mouse expression range expression range line No. (.mu.g/ml)
(.mu.g/ml) 5/13 - High Expressors: 27 33.5 97.5 29 37.5 66.0 32
21.2 148.0 33 200.0 708.0 38 25.0 126.0 8/13 - Low Expressors:
0.0-1.7 0.2-18
[0274] The data in Tables 3 and 4 demonstrates that the hybrid
intron+heterologous splice acceptor site dramatically increases
expression levels in a significant number of cases (5/13).
[0275] The construct 16,8hLF4 is expressed at high level (in same
range as 16,8hLF3). However, (in mice) this only occurs in a small
number of cases and 1/16 when 8,8hLF4 and 26,8hLF4 are included).
Similar results were obtained using a hSA cDNA.
[0276] Briefly, the 16,8hSA4 transgene was constructed by digesting
p16,8hLF4 with ClaI and SalI to remove the hLF cDNA sequence. hSA
cDNA was excised from a clone with EcoRI. A ClaI synthetic linker
was added to the 5' (upstream) end and a SalI linker to the 3'
(downstream) end. After insertion into the ClaI/SalI digested
16,8hLF4 vector, 16,8hSA4 was formed from which the NotI insert was
excised and used for microinjection.
[0277] The 16,8hSA4 construct yield 9 lines. One of the 9 lines
gave high level expression (100 .mu.g/ml)), while the remaining 8
of 9 gave low expression (0.01-0.05 .mu.g/ml). This indicates that
the level and the frequency of hLF expression in the mouse mammary
gland are not determined by the particular cDNA used, but are an
inherent characteristic of the 16,8.times.4 construct (i.e., the 16
kb 5' and 8 kb 3' flanking regions of the .alpha.-S1 casein gene
combined with the heterologous IVS).
[0278] The data also show that 0.7 kb of 5' .alpha.-51 casein
flanking sequence does not drive high level expression and that 8
(6.2), 16 (14.5) and 26 kb are more effective. In this respect, 8
kb is slightly more effective than 16 or 26 kb of 5' flanking
sequence.
[0279] Also, RNA analysis has shown that expression of the cDNA
constructs is tissue-specific and stage-specific (i.e., 1s
expression is only observed in the lactating mammary gland), that
the transcripts are correctly sized and that RNA and protein levels
correlate.
EXAMPLE 15
[0280] Generation of hLF Transgenic Cattle
[0281] Transgenesis in the bovine system was obtained utilizing the
p16,8hLF4 transgene described in Example 13.
[0282] Oocyte Maturation and Fertilization
[0283] Bovine oocytes were collected by aspiration of follicles
present on ovaries obtained from slaughterhouses and transported in
an insulated container at 30-32.degree. C. Oocytes, together with
follicular fluid, were aspirated from 2-8 mm diameter follicles and
pooled into 50 ml conical tubes. Cumulus-oocyte complexes (COC)
were allowed to settle into a pellet, after which the supernatant
was discarded and the pellet washed in 50 ml TL-Hepes (Vander
Shaws, et al. (1991) Theriogenology 35, 288 (Abstr.). COC,
containing several intact, unexpanded cumulus cell layers, were
selected and isolated under a dissecting microscope at 15.times.
magnification, washed four times in 10 ml TL-Hepes, once in 2-3 ml
TCM199+10% fetal calf serum (M199) and then paraffin oil (20
COC/droplet). COC were incubated for 23 h in a humidified
atmosphere of 5% CO.sub.2 in air at 39.degree. C.
[0284] A total of about 2500 oocytes were used. On average, two
aspiration sessions occurred per week. The yield of aspirated
oocytes was highly variable from day to day, with a mean daily
number of about 150. Maturation and fertilization were analyzed by
cytological analysis. Maturation was defined as the breakdown
of-the nuclear membrane, the appearance of the first polar body and
a metaphase plate. Oocytes were fertilized in vitro with frozen
thawed-sperm obtained from three different bulls with excellent
characteristics with respect to genetic background, field
performance and ease of calving. Sperm capacitation was facilitated
with heparin. Parrish, J. et al. (1986) Theriogenology 25:591-600.
Since sperm from individual bulls respond differently to specific
fertilization conditions, semen from each lot was tested in advance
to determine optimal heparin and sperm concentration required to
maximize normal fertilization frequency and to minimize polyspermy.
Fertilization conditions for a given bull were selected after
screening at heparin concentrations of 0.0, 1.0 and 10.0 mg
heparin/ml, and at 1.0, 2.0 and 4.0.times.10.sup.6 motile sperm/ml.
Since the proportion of sperm that survives freezing and thawing
varies from bull to bull (approximately 30-60% for the bulls was
used here) sperm preparations were enriched for live, motile sperm
by a "swim-up" procedure (Parrish, J. et al. Ibid), alternatively,
sperm were centrifuged through a percoll gradient. After isolation
of the motile portion, sperm were counted on a hemocytometer,
diluted to an appropriate concentration to yield a 25-fold
concentrated stock. The fertilization medium consisted of TALP
medium (Banister, Bethal Biol. Reprod. 28:235-247) supplemented
with 2.0-10.0 mg/ml heparin (from porcine intestinal mucosa, 177
IU/mg; Sigma) and if the cumulus was removed prior to
fertilization, 1 mM hypotaurine, 10 mM penicillamine, 20 mM
epinephrine and 2 mM sodium metabisulfite. Matured COC were
selected on the basis of expanded cumulus masses for fertilization,
washed once in 10 ml fertilization medium, and either added
directly to fertilization droplets, or first stripped of their
cumulus investment by gentle pipetting through a small-bore,
fire-polished pipet and then added to the droplets. Finally, sperm
cells were added to a final concentration of
1.times.10.sup.6-2.0.times.10.sup.- 6/ml. After 16.about.24 h,
presumptive zygotes were removed from fertilization droplets. At
this point, 20.about.30 zygotes for each experiment were fixed in
3:1 ethanol:acetic acid for 24 h, stained with 1% aceto-orcein (in
40% acetic acid), and examined to determine fertilization frequency
(percentage of sample with 2 pronuclei and a sperm tail). For each
batch of semen, the `in vitro` fertilization conditions (heparin
concentration and sperm number) were optimized to obtain normal
fertilization rates ranging from 50 to 70% as determined by the
presence of two pronuclei and a sperm tail as described above.
Either one of two techniques were used for selection of motile
sperm: the swim-up technique and centrifugation through a Percoll
gradient. No significant differences in fertilization rates between
these methods were recorded. The efficiencies of these and the
following steps are shown in Table 5. The remaining oocyte were
then prepared for microinjection.
11TABLE 5 Efficiencies of the steps involved in the process from
immature bovine oocytes to transqenic calves Step Total No.
Percent* oocytes 2470 -- matured 2297 93 fertilized 1358 61
injected 1154 85 survival 981 85 cleavage 687 70 transferred
129.sup..dagger-dbl. 1.9 pregnant 21 2.1 integration 2 1.0
*Percentages indicate the proportion of embryos or cells that
successfully complete each step. .sup..dagger-dbl.Sixty-nine
transfers of single blastocytes resulting in 7 pregnancies; 30
transfers of twinned embryos, resulting in 14 pregnancies.
[0285] Microinjection. The 26 kbp casein-hLF transgene (from
p16,8hLF4) used for microinjection was released by NotI digestion
and purified by agarose gel electrophoresis and electroelution. The
final DNA concentration was adjusted to 2.5 .mu.g/ml. Batches of 50
cumulus-intact fertilized oocytes were stripped either as described
above or by vortexing 2 minutes in 2ml TL-hepes medium in a 10 ml
conical tube. In order to visualize the pronuclei, cumulus free
oocytes were centrifuged in 1 ml TL-hepes medium 8 minutes at
14,500.times.g in an Eppendorf centrifuge. Wall, R. et al. Biol.
Reprod. 32:645-651. Microinjection was performed essentially as
described by Hogan B. et al. (1985) Manipulating the Mouse Embryo:A
Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring
Harbor, N.Y..
[0286] Embryo culture. Embryos were cultured from the zygote to the
compact morula or blastocyst stage in oviductal-tissue conditioned
medium. Eyestone et al., (1991) J. Reprod. Fert. 92:59-64. Oviducts
were obtained at slaughter and transported at ambient temperature.
Luminal tissue from 2-4 oviducts (1-2 cows) was harvested by gently
scraping intact oviducts on the outside with a glass slide. The
extruded material was washed 5 times in 10 ml TALP-Hepes and
diluted in M199 to a tissue:media ratio of 1:50. Media were
conditioned in 50 ml "T" flasks containing five ml of oviduct
tissue suspension. Conditioned media frequently contained a
proteinaceous precipitate after thawing, which was removed by
centrifugation. Droplets were covered with paraffin oil and were
incubated for 2 h to permit pH to equilibrate prior to adding
zygotes. Zygotes were placed in culture droplets within 2 h after
microinjection. Initial cleavage (>2 cells) was assessed 42 h
after adding sperm. Media were not changed during the course of
incubations. Criteria for normal development consisted of
attainment of the compact morula or blastocyst stage.
[0287] Embryo transfer. The synchronization schedule was set up so
that recipients started estrous on the same day at which oocytes
were aspirated from slaughterhouse ovaries (i.e., start of
maturation is day 1). Estrous in recipient cattle was synchronized
with a 9-day Norgestamet (Intervert, Boxmeer, The Netherlands)
treatment (administered in an ear implant according to the
manufacturer), and a 500 .mu.g dose of cloprostanol given on day 7
of the Norgestamet treatment. Estrous occurred within 2-3 days
after implant removal. Embryos were transferred non-surgically to
recipient heifers 5-7 days after estrous (1-2 embryos/uterine
horn). Recipients received 9-day old embryos, at which time they
have developed to the compact morula or early blastocyst stage.
These embryos are one day ahead in development compared to the
stage of the estrous cycle of the recipients. In case of two
microinjection sessions on subsequent days, one group of recipients
was used that were in synchrony with the first batch of oocytes
collected. Transfers of embryos that developed from oocytes
aspirated on the day of the start of estrous gave better results
than embryos from oocytes obtained one day later. Due to the
somewhat delayed development of microinjected embryos, there
appeared to be a better synchrony between the recipients and the
first group of embryos. Recipients received two embryos when the
quality grade (according to Linder and Wright, Theriogenology
20:407-416) was fair to poor and only one single embryo when the
quality grade was excellent to good. Each pregnant recipient that
received 2 embryos carried only one fetus to term. The overall
pregnancy rate was 21%, which is significantly less than the rates
reported by others with non-microinjected embryos which had
developed in vivo (Linder and Wright, Ibid and Massy et al. (1984)
Theriogenology 21:196-217). In the experiments described here, no
transfers with non-injected embryos were performed.
[0288] Pregnancy was determined by rectal palpation at 45 to 60
days of gestation. A total of 21 pregnancies were established
(confirmed by rectal palpation 45-460 days after transfer). During
pregnancy, 2 fetuses were lost. One recipient aborted spontaneously
for unknown reasons at 7.5 months of gestation. The second fetus,
collected at slaughter of the recipient at 3 weeks after the
calculated day of parturition, was a full grown dead calf having an
abnormal embryonic development called `schistosoma reflexum`. In
both cases, no intact DNA could be isolated for analysis. Nineteen
calves were born after normal pregnancies. One of these calves died
during parturition, and a second, 24 hours after birth, because of
pneumonia following accidental inhalation of milk. A third calf,
born after a pregnancy of 10 months and with a body weight of 70 kg
was euthanized at an age of 3 weeks. Pathological analysis
indicated that the animal was suffering from sepsis due to chronic
omphalophlebitis. Tissues that could be analyzed from the three
dead calves contained no integrated human lactoferrin (hLF)
sequences. Therefore, the cause of their death is unlikely to be
related to transaene integration. The remaining 16 calves are in
excellent health.
[0289] Structure of the transgene. In FIG. 12A, the coding sequence
of the hLF cDNA is depicted by a hatched box. The position of the
translational start and stop codon is indicated. The 5' and 3'
untranslated regions are encoded by .alpha.S1 casein exons (open
boxes). Intervening sequences interrupting these exons are
represented by a single line. The expression unit is surrounded by
flanking sequences derived from the bovine .alpha.S1 casein gene
(indicated by a double line). Positions of restriction enzyme sites
are indicated by the following symbols: R, EcoRI; A, Asp718; N,
NotI. The NotI sites is are not present at the indicated positions
in the bovine .alpha.S1 casein gene itself, but were introduced by
synthetic linkers. The black bar represents the position of the
probe used to detect the presence of the transgene. Sizes of the
fragments (in kbp) obtained after digestion with EcoRI or Asp718
are shown at the bottom.
[0290] DNA analysis. DNA was isolated from placenta, blood and ear
tissue from all calves. A Southern blot analysis of the extracted
DNA is shown in FIG. 12B. Ten .mu.g of DNA was loaded per lane.
Fragment size markers are in kbp (HindIII digest of lamda DNA) are
indicated on the left. Lane 1, EcoRI digested human DNA (isolated
from blood), lane 2, Eco RI digested DNA from calf #4 isolated from
blood; lane 3, Asp718 digested DNA from calf #4 isolated from
blood; lane 4, EcoRI digested placental DNA from calf #4; lane 5,
Asp718 digested placental DNA from calf 14; lane 6, EcoRI digested
DNA from calf #15 isolated from blood; lane 7, Asp718 digested DNA
from calf #15 isolated from blood; lane 8, EcoRI digested DNA from
calf #15 isolated from ear tissue; lane 9, Asp718 digested DNA from
calf #15 isolated from ear tissue; lane 10, EcoRI digested
placental DNA from calf #15; lane 11, Asp718 digested placental DNA
from calf #15; lane 12, EcoRI digested DNA isolated from the tail
of a transgenic mouse harboring the same construct. DNA extraction,
Southern blot analysis and hybridization were performed according
to standard procedures. The probe used in the Southern blotting
experiment was a 758 bp EcoRV-EcoRI fragment covering the 3' part
of the hLF cDNA of FIG. 2. Southern blot analysis using hLF cDNA as
a probe indicated that in tissues of two calves (#4 and #15)
transgene sequences had been integrated into the host genome. Calf
#15 (a female) was mosaic for integration of the transgene.
Placental tissue was positive, is whereas in blood and ear tissue
no hLF sequences could be detected. The copy number in the placenta
was 1-2. The restriction enzyme map of the transgene was different
from that expected based on the map of the casein-hLF plasmid (FIG.
12A) and based on the pattern obtained in many individual
transgenic mice (data not shown). Apparently, a rearrangement had
occurred involving a deletion of part of the DNA construct. It is
not clear whether this rearrangement event is related to the fact
that the transgene could not be detected in all tissues. In mice,
it has been shown that over 30% of all transgenic animals born are
mosaic.
[0291] Calf #4 (a male) showed, in all three tissues, the same
hybridization pattern that was identical to the expected one.
Restriction digestions with different enzymes indicated that
head-to-tail concatamers of intact copies had integrated and there
was no indication of rearrangements. Copy numbers were estimated by
comparing the intensities of the transgenic band with bands
resulting from hybridization of the hLF probe to human DNA (FIG.
12B). In calf #4, between 5 and 10 copies of the transgene had
integrated in all three tissues examined.
[0292] An analysis of sperm producted by calf #4 detected no
abnormalities. DNA was subsequently isolated from the sperm and
analyzed for the presence of the hLF-transgene. It appeared that
the copynumber of the transgene (2-3) was the same in sperm as in
other tissues indicating that calf #4 is not mosaic and should be
able to transmit the transgene to 50% of his offspring.
EXAMPLE 16
[0293] Construction of Transgene Cassette for Genomic Recombinant
DNA
[0294] The plasmids described so far all contain regions derived
from the bovine .alpha.S1-casein untranscribed regions (including
intervening sequences). When a genomic gene is to be expressed that
already contains untranslated regions and intervening sequences
permissive for high expression, it is preferable to use expression
cassettes where the flanking regions including the transcription
initiation site of the .alpha.S1 casein gene are operably linked to
the untranslated regions of the gene to be expressed. Such an
expression cassette is p-16 kb, CS and was constructed as follows:
plasmid pS1 3'5'hLF was used as a template in a PCR experiment.
This plasmid contains 680 bp of promoter sequence of the .alpha.S1
casein gene as well as its first exon. The rest of this plasmid is
not relevant for this experiment. The upstream primer was located
just upstream of the insert in the plasmid moiety (just upstream of
a NotI restriction site). Its sequence is: 5'-CGA CGT TGT AAA ACC
ACGG-3'.
[0295] The downstream primer was located in exon 1. Its sequence
matches the first 19 bp of the exon exactly and also has a
non-hydridizing region of 17 bp containing a ClaI and a SalI site.
It has the following sequence:
[0296] 5'-ATTGTCGACTTATCGATGGGTTGATGATCAAGGTGA-3'
[0297] The amplified fragment was digested with NotI and SalI and
ligated into pKUN2 (see Example 13). The resulting plasmid
(p-680CS) therefore harbors a proximal promoter fragment from -680
to +19, plus two restriction sites just downstream of those 19
bp
[0298] This plasmid was digested with NotI (just upstream of -680)
and NsiI (at-280) and used as a vector to ligate to a fragment
running from a NotI site (just upstream of -16 kb) to NsiI (-280)
isolated from p16,8hLF4 (Example 13). This plasmid (p-16 kb, CS)
therefore harbors a promoter fragment from .apprxeq.-16,000 to +19.
It can be used to insert genomic genes that carry their own UTR's
and poly(A)-signal. After insertion of the genomic gene as a
ClaI-SalI fragment, the .alpha.S1 casein 3'-flanking region can be
inserted as a SalI-fragment.
EXAMPLE 17
[0299] Construction of Transgene for Production of Protein C
[0300] The genomic sequence of Protein C has been published.
Foster, et al. (1985) Proc. Natl. Acad. Sci. USA 82, 4673-4677.
This sequence, however, does not include the first exon which was
identified through the cDNA sequence published by Beckman, et al.
(1985) Nucl. Acids Res. 13, 5233-5247. The first exon of Protein C
is located at position -1499 to -1448 in the Foster sequence. The
transgene for expressing and secreting Protein C into the milk of
bovine species is shown in FIG. 10. This transgene was constructed
as follows.
[0301] A human genomic library in EMBL-3 (Clonotech) is probed with
a sequence specific for protein C. A purified phage DNA prep
containing the complete Protein C gene is isolated. The phage is
isolated from an E. coli strain having the Dam phenotype, such a
strain GM113. This results in cloned DNA which is not methylated
and as such all ClaI restriction sites can be cleaved.
[0302] A ClaI NheI fragment running from positions +1333 to 11483
is isolated. This is designated fragment I.
[0303] pGEM7 (Stratogene, Inc.) is digested with SphI and SmaI. The
region in between is replaced by the corresponding region of
plasmid pKUN (Gene (1986) 46, 269-276). The resulting plasmid is
designated pGEM7A and has the following restriction map in the
relevant region:
12 HindIII ClaI XbaI SalI SpeI
[0304] Two primers are synthesized. Primer GP125 has the following
sequence:
13 Primer GP125 has the following sequence: 5'-CAA ATC GAT TGA ACT
TGC AGT ATC TCC ACG AC-3' ClaI Primer GP 126 has the following
sequence: 5'-GGG ATC GAT CAG ATT CTG TCC CCC AT-3' ClaI
[0305] Primer GP125 has an overlap with exon 0 (position 654 to 675
of the Protein C gene) and introduces a ClaI site in the 5'
untranslated region. Exon 0 is the exon not identified by Foster,
et al. Primer GP126 overlaps the region from 1344 to 1315 in the
Protein C gene. This region contains a ClaI site.
[0306] The region between position 654 and 1344 is amplified using
either human DNA or phage DNA as a template. The so amplified
material is digested with ClaI and cloned in vector pGEN7a to form
pPCCC. This vector is propagated in a dam negative strain such as
GM113 and partially cut with ClaI (only the plasmids that are cut
once with ClaI at position 1340 are of interest) and completely
with XbaI. The ClaI NheI fragment (fragment 1) is cloned into this
vector. The resultant plasmid is designated pPC. Its structure is
shown in FIG. 10. From this plasmid, the Protein C transgene is
isolated as a ClaI-SalI fragment and ligated into p16 kb, CS (See
Example 15) to generate a transgene capable of expressing Protein C
in bovine milk, this plasmid is designated p16 kb, CS, PC.
[0307] The transgene contained within plasmid p 16 kb, CS, PC is
excised with NotI and used to generate transgenic bovine species as
previously described. Such transgenic animals are capable of
producing protein C in their milk.
EXAMPLE 18
[0308] Human Lactoferrin Transgene Formed by In Vivo Homologous
Recombination: Microinjection of Two Overlapping DNA Fragments
[0309] To obtain the entire hLF genomic clone, two human genomic
cosmid libraries were screened using an hLF cDNA clone described
herein as a probe. Of 14 clones isolated, 2 clones (designated 13.1
and 13.2; one from each human cosmid library) contained the entire
hLF gene as determined by hybridization with primers specific for
the first and last (17th) hLF exons and by DNA sequencing. The
insert sizes of these hLF genomic clones was 42 kbp for clone 13.1
and 43 kbp for clone 13.2. Clones 13.1 and 13.2 contain 5 kbp and
13 kbp of 5' flanking sequences, respectively. The 3' flanking
region of clone 13.2 is between 1 kbp and 3 kbp; clone 13.1
contains 7 kbp of additional 3' flanking sequence. The size of the
structural hLF gene (=introns+exons) is approximately 30 kb.
[0310] The identity of the hLF clones was confirmed by sequencing
several exons (incl. first and last) and comparing these sequences
and the promoter region to the hLF cDNA sequence shown in FIG. 2.
In addition, the clones were transfected into human kidney 293
cells and hLF expression was detected, indicating that both clones
were functional.
[0311] A comparison of the 13.1 and 13.2 clones (derived from
independent libraries) by restriction mapping and Southern blotting
revealed no differences in the corresponding regions (i.e. in the
structural hLF gene). Southern blotting experiments revealed that
the hLF gene is a single copy gene in the human genome. FIGS. 13-16
illustrate the overall procedure for generating the .alpha.S1
casein/genomic hLF transgene.
[0312] The most 5' ApaI site in the structural hLF gene is located
in exon I, in the hLF signal sequence. The 400 bp region
immediately 5' of exon I was sequenced. This region contains the
transcription initiation site of the hLF gene and a TATA-box. This
region also includes a BamHI restriction site.
[0313] To construct a mammary gland specific expression vector it
was necessary to fuse the 8 (6.2) kbp or 16 (14.5) kbp .alpha.S1
bovine casein promoter region to the genomic hLF clone. However,
the total size of such a construct, about 50 or 60 kb (6.2 or 14.5
kb from the casein gene promoter +8 kb from the cosmid vector and
35-40 kb from the hLF genomic clone, i.e., about 50-63 kb), renders
the use of conventional cloning vectors difficult. Therefore, the 8
kbp or 16 kbp .alpha.S1 5' casein promoter and flanking sequence
was fused to 9 kb of the 5' region of the structural hLF gene (FIG.
15A) and this fragment was coinjected with an overlapping hLF
fragment containing about 33 to 34 kbp of the 3' sequence of
generic hLF clone 13.1 obtained by ClaI digestion. See FIGS. 13 and
16.
[0314] The BamHI fragment (containing exon I) from clone 13.2 was
subcloned into the plasmid pUC19 (FIG. 14). From this clone, a 8.9
kbp ApaI-SalI fragment was isolated by ApaI (partial digest) and
SalI digestion. This fragment lacks most of the hLF signal sequence
and all of the hLF 5' UTR. A synthetic sequence (FIG. 15B)
representing this missing region was obtained by synthesizing 2
complementary DNA strands (a 68-mer and a 62-mer) which runs from
the 5' ApaI site into the downstream region from the hLF TATA-box.
After annealing these primers a DNA fragment was generated which
has a 5' ClaI overhang and a 3' ApaI overhang. Subsequent
sequencing of the cbI-ApcI fragment showed that it has the sequence
given in FIG. 15B, which differs at one position from the native
sequence. This synthetic ClaI-ApaI fragment and the 8.9 kbp
ApaI-SalI fragment described above were ligated into p-16 kbCS and
into a similar plasmid, containing 8 kbp instead of 16 kbp of the
.alpha.S1 casein promoter. This yields two plasmids, containing 16
kbp or 8 kbp of bovine .alpha.S1 casein promoter, fused to the 5'
part (9 kbp) of the hLF genomic gene. See FIG. 15A. These fragments
were cut out (NotI-SalI) and coinjected with the 3' 33 to 34 kbp
ClaI fragment from hLF cosmid clone 13.1. The coinjected fragments
had an overlap of 5.4 kbp.
[0315] Upon coinjection of the constructs containing the 8 kbp
.alpha.S1 casein promoter, 8 independent transgenic mice were
identified by tail-DNA blotting. To determine if homologous
recombination had occurred, chromosomal DNA (from tails of founders
and offspring) was digested with ApaI and analyzed by Southern
blotting. The 2.7 kb ClaI-MluI fragment (see FIG. 13 or FIG. 17)
that is located in the overlap was used as a probe. When homologous
recombination occurred, a band of 7.5+0.3=7.8 kb is generated and
detected with this probe. This band is also present in human
chromosomal DNA, which was used as a control in the analysis. If
homologous recombination has not occurred, the probe detects bands
of varying size, depending on the location of ApaI sites around the
site of integration.
[0316] The diagnostic 7.8 kb band was detected in all 8 transgenic
mouse lines, indicating that each transgenic mouse contained
recombined fragments. For these 8 mouse lines (founder no's: 936,
937, 950, 951, 982, 983, 984 and 985), milk was collected from
lactating females (founder and/or offspring) and assayed for hLF
protein expression. The data on 7 mouse lines is shown below.
14 Expression Level Mouse line (max.) (mghLF/ml) 936 4.5 937 6.0
950 0.003 951 0.010 982 5.9 983 similar to 982 and 937 on day 2 and
4 of lactation* 984 2.8 985 6.6
[0317] *: This mouse died (by accident) on day 4 of lactation. At
this time, hLF expression had reached a level of 0.3 mghLF/ml. This
is exactly the level found for other high expressors (e.g. lines
937, 982, 984) at this early stage of lactation. This phenomenon of
gradual increase of hLF expression at the beginning of, and in
particular, the- first lactation has been commonly observed by us
in the mice generated herein. Therefore, mouse 983 is classified as
a high level expresser.
[0318] The tissue-specificity of hLF expression was determined by
isolating total RNA from a large number of tissues and analyzing
for the presence and levels of transgene derived mRNA. Based on
this analysis, hLF mRNA only occurs in the lactating mammary gland
and expression is tissue- and stage-specific.
[0319] RNA levels were below the threshold of detection in lines
950 and 951, but were high in high expressing lines and correlated
with bovine .alpha.S1 casein expression levels. This was determined
by Northern blot analysis of both bovine lactating mammary gland
RNA and mammary gland RNA from lactating transgenic mice. A 24 bp
synthetic oligomer which hybridizes to exactly the same sequence in
the 5' UTR of bovine .alpha.S1 casein RNA and in the transgene RNA
was used as a probe. Expression levels were compared directly by
quantification of the amount of labelled probe hybridized to the
transgene- and .alpha.S1 RNA. When a correction was made for the
size difference between bovine .alpha.S1 casein (20 kD) and hLF (80
kD), the ratio of mRNA to protein was in the same range for bovine
.alpha.S1 casein and hLF. This indicates that translation and
secretion of the transgene derived hLF is not impaired. The length
of the hLF mRNA was as expected (about 2.5 kb) but in mouse line
937 a longer band (3-3.5 kb) of slightly less intensity was also
observed. The occurrence of this band may be related to the
homologous recombination process. It remains to be determined if
this RNA translates into bona fine hHL.
[0320] It has been suggested that casein promoters are less
favorable for obtaining high level expression than other milk
specific promoters. However, the present data show that this is not
the case. With respect to both expression level and percentage of
expressing animals, the transgenes containing .alpha.S1 casein
sequences perform better than any other mammary gland specific
transgene reported.
[0321] The above data compared to those obtained with constructs
containing hLF cDNA provide the following observation. The best
cDNA expression vector herein (16, 8hLF3) always expresses at much
lower levels as compared to the genomic hLF construct. Of 13 cDNA
lines generated, 8 expressed at very low levels (1-5 .mu.g/ml), 5
expressed from 40 to 200 .mu.g/ml. These relatively low levels
(although high for cDNA expression) as compared to that observed
for genomic hLF (containing the same flanking sequences) indicate
that genomic sequences produce consistently higher expression
levels.
EXAMPLE 19
[0322] Generation of Genomic Human Lactoferrin Transgenes by
Conventional Cosmid Ligation Techniques
[0323] hLF genomic transgenes have also been generated by
conventional ligations in cosmids. The first construct 8hLFgen is
similar to the transgene generated by coinjection, but contains the
3.degree. ClaI fragment from hLF clone 13.2. The size of this
fragment is about 26-27 kb. The second construct 16hLFgen is
identical to 8hLFgen, but contains a larger stretch of .alpha.S1
casein promoter sequences.
[0324] Construction Detail:
[0325] The NotI-MluI fragment from the construct depicted in FIG.
15A (referred to as 8hLFgen9k) was used to prepare the 8hLFgen
construct. This NotI-MluI fragment contains the synthetic ClaI-ApaI
fragment depicted in FIG. 15B. This synthetic sequence contains 24
bp of the hLF 5'-UTR and encodes for most of the hLF signal
sequence (see FIG. 15C). This NotI-MluI was ligated with the 3'
MluI-ClaI fragment from clone 13.2 and a ClaI-NotI linker as shown
in FIG. 17. The cloning vector was cosmid pWE15 cut with NotI, from
which the internal ClaI and SalI sites had been deleted.
[0326] The first intron of the hLF gene is located 4 bp downstream
of the ApaI site in the signal sequence. As a result, the DNA
sequence encoding the 19 aa signal sequence is partly located in
exon 1 (43 bp, encoding 14 aa and 1 codon partially) and in exon 2
(the first 14 bp, encoding 4 aa and 1 codon partially). The exact
position of hLF intron 1 was determined by DNA sequencing and
comparing the genomic sequence to the hLF cDNA sequence. The
sequence upstream of the translation initiation site (355 bp,
containing the hLF 5' UTR and .sub.5' flanking region) was also
sequenced.
[0327] The hLF transcription initiation site was not included in
the genomic hLF constructs as shown. Instead, they contain the
bovine .alpha.S1 casein gene transcription initiation site.
Although the exact position of the hLF `cap` site has not been
determined, it is probably located about 30 bp downstream of the
`TATA` box, as is the case for the vast majority of eukaryotic
genes. In addition, for the mouse LF gene the transcription
initiation site has been mapped (Shirsat, et al. (1992) Gene 110,
229-234; Liu and Teng (1991) J. of Biol. Chem. 32, 21880-21885). On
the basis of homology between the mLF and hLF 5' UTR, it is
concluded that genomic hLF constructs herein do not contain the hLF
transcription initiation site.
[0328] The cDNA contains a Thr codon (ACA) at aa position 130 (see
FIG. 2). The corresponding region in genomic hLF clones 13.1 and
13.2 (exon 4, plus parts of intron 3 and 4) have been sequenced.
These clones contain the sequence ATA, which encodes isoleucine.
The cDNA also contains a Cys codon (TGC) at position 404 (see FIG.
2). In hLF clones 13.1 and 13.2 this is a GGC, encoding
glycine.
[0329] By using the NotI-MluI fragment from 16hLFgen9k instead of
from 8hLFgen9k, 16hLFgen was generated.
[0330] Construction of 8hLFgen37:
[0331] The 5' NotI-MluI fragment from the construct depicted in
FIG. 15A (called 8hLFgen9k) was ligated to the 3' MluI-ClaI
fragment from clone 13.1, combined with a ClaI-NotI linker (compare
FIG. 17: read 13.1 instead of 13.2). The cloning vector was cosmid
pWE15, from which the internal ClaI and SalI sites had been
deleted, cut with NotI. Prior to microinjection, vector sequences
were removed via NotI digestion.
[0332] All constructs were cut from the vector using NotI, and
microinjected.
[0333] Expression Data:
[0334] Three mice containing 8hLFgen and 5 mice containing 16hLFgen
were generated. Preliminary expression date in milk are as
follows:
15 Max. hLF expression Construct Line in milk (mg/ml) 8hLFgen 1089
0.95 1252 1.2 1401 1.4 16 hLFgen 1112 2.8 1113 ND 1134 0.3 1185 ND
1191 ND 8hLF37 1507 4.1 1556 8.7 ND = not done
EXAMPLE 20
[0335] Bovine .beta.LG/Human Lactoferrin Transgenes
[0336] The bovine .beta.LG-promoter (beta-lactoglobulin) was used
to construct a transgene encoding for the expression of hLF.
Briefly, the .alpha.S1 promoter in the genomic hLF constructs
8hLFgen and 8hLFgen37 were replaced with the bovine
.beta.LG-promoter. The resulting constructs are referred to as
.beta.LG-hLFgen and .beta.LG-hLFgen37. The overall strategy for
these constructions are depicted in FIGS. 18-20.
[0337] Isolation of the Bovine .beta.LG-Promoter
[0338] The charon 28 phage clone .lambda..beta.LG-13, described by
Silva et al., (1990) Nucl. Acids Res. 18:3051, was obtained from
Dr. Carl A. Batt. This clone was isolated from a bovine genomic
library by screening with a .beta.LG cDNA probe. It contains the
structural .beta.LG gene and about 8 kb of 5' flanking region. From
this clone, a 4.3 kb EcoRI fragment was isolated and subcloned into
plasmid pKUN5 using standard procedures (see FIG. 18).
[0339] From this plasmid, a 3.2 kb NotI-SacI fragment was isolated.
The NotI site was derived from the polylinker of the cloning
vector. The SacI site lies 15 bp downstream of the BLG
transcription initiation site. A PvuII site is located five bp
upstream of the translation initiation site. A fragment
representing the region between the SacI and PvuII sites (including
these sites) was generated by synthesizing and annealing the 30-mer
and 37-mer DNA oligomers depicted in FIG. 18. This fragment also
contains a ClaI and a SalI site directly downstream of the PvuII
site (FIG. 18). The 3.2 kb NotI-SacI fragment and the synthetic
SacI-SalI fragment were ligated into a pKUN plasmid (pKUN1deltaC),
from which the internal ClaI site had previously been removed by
cutting with ClaI and subsequent treatment of the cut vector with
Klenow enzyme. This ligation resulted in plasmid pBLG3.2.
[0340] The 734 bp region directly upstream of the translation
initiation site was sequenced and compared to the corresponding
region of the published sequence of the sheep BLG promoter (see
FIG. 24). Overall homology was 91%, indicating that the sheep- and
bovine BLG-promoters are very similar.
[0341] Generation of .beta.LG-HLF Constructs:
[0342] The 8.9 ClaI-SalI fragment from construct 8hLFgen9k (Example
15A) was isolated and cloned into p.beta.LG3.2 after cutting this
vector with ClaI and SalI. This ligation resulted in construct
p.beta.LGhLFgen9k (FIG. 19). From this construct the 9.4 kb
NotI-MluI fragment was isolated and, together with the 23-24 kb
MluI-NotI fragment isolated from 8hLFgen, ligated into a NotI cut
pWE15 cosmid, resulting in p.beta.LG-hLFgen (FIG. 19). The 34 kb
NotI insert was isolated from the cosmid by NotI digestion and
microinjected following standard procedures.
[0343] For the generation of p.beta.LG-hLFgen37 the 9.4 kb
NotI-MluI fragment from p.beta.LGhLFgen9k was ligated with the 30
kb 3' MluI-ClaI fragment from hLF clone 13.1, combined with a
ClaI-NotI linker into a NotI cut pWE15 cosmid vector.
[0344] The .beta.LG-hLFgen insert was isolated and microinjected
following standard procedure.
[0345] Expression data:
[0346] .beta.LG-hLFgen (the shorter of the 2 constructs) was
injected and 7 independent mouse lines were produced. Expression
data for hLF product in milk is available for the following
lines:
16 Max. hLF expression Construct Line in milk (mg/ml)
.beta.LG-hLFgen 1106 0.02 1107 1.9 1108 0.8 1110 6.2 1111 1.3 1155
2.1 1156 2.2 .beta.LG-hLFgen37 1591 0.05 1592 27 1593 5.9
EXAMPLE 21
[0347] Isolation of a Genomic hLF Fragment Containing Both the
Structural Gene and the hLF Promoter
[0348] HLF is normally expressed at relatively high levels (1-2
mg/ml) in human milk. To determine whether the hLF promoter can
drive high level hLF expression in the milk of transgenic animals,
the intact hLF gene under control of its own promoter was
microinjected using standard procedures.
[0349] Construction Details:
[0350] Two important points determined the construction route.
Since the cosmid vector C2RB (FIG. 13) containing the genomic hLF
clones does not contain unique restriction sites flanking the hLF
insert, the intact insert could not be isolated directly from this
cosmid. It was desirable to include all 5' and 3' flanking
sequences present in hLF clones 13.1 and 13.2 into the transgene.
Since clone 13.2 (FIG. 13) contains the most 5' flanking sequences
(13 kb) and clone 13.1 the most 3' flanking sequences (7 kb more
than 13.2), the 5' part of 13.2 was combined with the 3' part of
13.1.
[0351] The cosmid 13.2 was linearized at the PvuI site 0.5 to 0.8
kb upstream of the 5' region of the hLF insert (FIG. 20) and
subsequently treated with the exonuclease Bal31, thereby removing
approximately 1 kb of cosmid and 0.2 to 0.5 of 5' hLF sequence.
Subsequently, the DNA was treated with T4 polymerase to create
blunt ends and cut with MluI. The approximately 19 kb (12.5 5'
flanking sequences +6.2 kb hLF gene) blunt end-MluI cut plasmid
vector (pKUN6deltaCla, SmaI-MluI), resulted in plasmid phLF5'M gene
37. This plasmid contains a NotI site directly 5' of the SmaI site.
From this plasmid, the 19 kb NotI-MluI fragment was isolated and
ligated with the 30 kb MluI-NotI 3' fragment from construct
8hLFgen37 into a NotI cut pWE15 cosmid, resulting in p5'hLFgen37
(FIG. 20).
[0352] The 49 kb NotI insert was isolated and microinjected
following standard procedures.
[0353] Expression Data on Construct p5' h1LFgen37:
[0354] Eight independent founder mice have been generated for the
p5'hLFgen37 construct; expression data are available for 6
lines.
17 Max. hLF expression Construct Line in milk (mg/ml) p5"hLFgen37
1491 ND 1492 2.5 1493 4.2 1495 6.5 1496 18 1497 ND 1506 6.3 1551
6.4 ND = not done
EXAMPLE 22
[0355] Generation of Mammary Gland Specific hLZ Expression
Cassettes
[0356] The structure and sequence of the human lysozyme gene has
been described (Peters, et al. (1989) Eur. J. Biochem 182:507-516).
The structural hLZ gene contains 4 exons and is 5.3 kb in size.
[0357] Using a 91-mer synthetic DNA sequence complementary to part
of exon 2 of the hLZ gene as a probe, several independent hLZ
clones were isolated from a human genomic phage library. The clone
.lambda.7.2.1 contains 14 kb insert comprising 8.7 kb of 5'
flanking sequences and 5.3 kb of the genomic hLZ gene. Exon 4 is
only partly included: clone .lambda.7.2.1 stops at one of the Sau3A
sites at position 5333 and 5350 (numbering according to Peters, et
al., op. cit.). The region downstream of position 5333/5350 (532 or
549 bp of exon 4 sequences) is missing. These sequences are
non-coding and represent part of the 3' UTR of the hLZ gene. All
hLZ coding sequences are present in .lambda.7.2.1.
[0358] Expression Vector 16,8hLZ
[0359] The design of expression vector 16,8hLZ, shown in FIG. 21,
is as follows. The 5' flanking region (including the promoter) of
the hLZ gene was removed and replaced with the bovine .alpha.S1
casein gene promoter by subcloning into the plasmid p-16kbCS which
is described in Example 16. The fusion site is located in the 5'
UTR of the hLZ gene (exon 1), such that in addition to 23 bp of
casein 5' UTR most of the hLZ 5' UTR is present. All coding
sequences in this construct, including the signal sequence, are
derived from hLZ clone .lambda.7.2.1 (FIG. 23A).
[0360] The 3' UTR of the hLZ gene in clone .lambda.7.2.1 was fused
to the 3'UTR +flanking region of the bovine .alpha.S1 casein gene
described previously. The resulting 3'UTR of construct 16,8hLZ is
therefore derived partly from the hLZ gene (exon 4, running from bp
4761 to bp 5333/5350) and partly (including par of exon 8 and all
of exon 9) from the bovine .alpha.S1 casein gene. The 3' flanking
region (8 kb) is derived entirely from the bovine .alpha.S1 casein
gene.
[0361] Construction Details for 16,8hLZ: 16,hLZ:
[0362] The 6 bp directly 5' to the AUG codon in hLZ exon 1
constitute a HincII site. A SalI phage polylinker site is located
directly 3' of the .lambda.7.2.1 insert. These sites were used to
isolate a 5.3 kb HincII-SalI insert (FIG. 23). The sequence running
from +3 (relative to the transcription initiation site at .div.1)
to the HincII site, was synthesized by annealing the 31-mer and
35-mer depicted in FIG. 23A. The resulting synthetic DNA fragment
has artificial 5' KpnI-HincII fragment and the 5.3 kb HincII-SalI
fragment were subcloned into a KpnI-SalI cut pKUN-1 plasmid (FIG.
23A). From the resulting 9.3 kb plasmid (pKHLys3'5.3) the 5.3 kb
ClaI-SalI fragment was isolated and subcloned into a ClaI-SalI cut
p-0.7kbCS plasmid (the equivalent of p-16CS but containing less 5'
flanking sequences), resulting in pKhLZ0.7.
[0363] The 8 kb bovine .alpha.S1 casein gene EcoRI fragment
containing the 3' casein UTR and ca 6.6 kb of flanking sequences,
was isolated from plasmid pKE3' E10 (described previously) as an 8
kb 5'-XhoI-SalI-3' fragment (FIG. 23B). This fragment was subcloned
into the SalI site of pKhLZ0.7, resulting in p0.7,8hLZ. After this,
the SalI site of p0.7,8hLZ was replaced with a NotI site by
insertion of linker S1/S2 (FIG. 23C), yielding plasmid p0.7,8hLZNt
(FIG. 23D). From this plasmid, the 13.3 kb ClaI-NotI fragment was
isolated and ligated with the 14.5 kn NotI-ClaI fragment from
p-16CS into a NotI cut pWE15 cosmid (FIG. 23E). From the resulting
construct (named 16,8hLZ in FIG. 23E) the 27.8 kb NotI insert was
isolated, purified and microinjected into murine and bovine zygotes
following standard procedures.
[0364] Expression Vector 16,8hLZ3
[0365] The design of expression vector 16,8hLZ3, shown in FIG. 22,
is as follows. Previously described expression vector 16,8hLF3 was
used in the construction of 16,8hLZ3. The vector 16,8hLZ3 contains
not only the bovine .alpha.S1 casein gene promoter, but also the
complete first exon and part of the first intron of the bovine
.alpha.S1 gene. In addition, it contains part of the first intron
plus the splice acceptor site of an immunoglobulin gene. The signal
sequence and part of the 3' UTR and the complete 3' flanking region
are also derived from the bovine .alpha.S1 casein gene. The hLF
cDNA and the .alpha.S1 casein signal sequence are excised from this
vector by ClaI-SalI double digestion. The ClaI site is located 5 bp
5' to the translation initiation codon.
[0366] An 5.3 kb ClaI-SalI hLZ fragment was isolated from plasmid
pKhLZ0.7 and subcloned into a ClaI-SalI cut 16,8hLF3 vector from
which the hLF cDNA had been removed by ClaI-SalI double
digestion.
[0367] The 16,8hLZ expression cassette vector sequence was removed
by NotI digestion, subsequently purified according to standard
procedures and microinjected into mouse zygotes.
[0368] Expression Data:
[0369] Construct 16,8hLZ:
[0370] Seven transgenic mice were generated for construct 16,8hLZ.
Expression data are available for 6 independent mouse lines (data
from lactating offspring, using our standard hLZ assay on milk
samples).
18 Max. hLF expression Construct Line in milk (mg/ml) 16,8 hLZ 645
10 647 0.7 661 260 662 7.4 1069 60 1070 28
[0371] The above data illustrates that 16,8hLZ expresses at
relatively high levels. In human milk, hLZ levels are only 50
.mu.g/ml (max). Since hLZ is a 15 kD protein, a level of 0.26 mg/ml
hLZ compares to .about.1.3 mg/ml of hLF (hLF is 80 kD).
[0372] Construct 16,8hLZ3:
[0373] Four independent transgenic mice were generated for covalent
16,8hLZ3. The following expression data are available from mouse
lines 905 and 907.
19 Mouse line Expression (.mu.g/ml) (max) 905 475 907 10
[0374] The data show that 16,8hLZ3 can be expressed at relatively
high levels (0.36 mg/ml compares to .about.1.8 mg/ml hLF). However,
as also shown, 16,8hLZ3 does not always express at high levels.
Although the number of mice analyzed is very low, constructs
16,8hLZ and 16,8hLZ3 seem to behave more or less similar with
regard to frequency of expression and expression levels. It should
be noted, however, that another 7 lines of mice transgenic for
16,8hLZ also contain the 16,8hLF3 construct. (See below.) None of
these lines expressed as high as 0.36 mg/ml. Therefore, 16,8hLZ3
appears to be a more efficient construct then 16,8hLZ. This could
be caused by the heterologous splice site (which does enhance hLF
cDNA expression levels).
EXAMPLE 23
[0375] Transgenic Mice Containing Transgenes Encoding Genomic hLZ
and hLF cDNA
[0376] Coinjection of 16,8hLF3 and 16,8hLZ
[0377] To assess the feasibility of simultaneously expressing hLF
and hLZ in the milk of transgenic animals, the appropriate isolated
and purified 16,8hLF3 and 16,8hLZ constructs were coinjected into
murine zygotes.
[0378] Seven independent mouse lines transgenic for both constructs
were generated. The expression data available for each line are as
follows:
20 Mouse hLZ expression hLF expression line (.mu.g/ml) (.mu.g/ml)
649 150-250 (max: 311) 500-2000 (max: 2100) 650 10-30 1-9 651 1-2.5
1-4.3 657 1-6 1-15 658 0.5 1 659 <0.1 0.1 660 5-25 300-1260
[0379] Conclusions:
[0380] Only line 649 (1/7) expresses hLZ at relatively high levels.
Line 649 and mouse line 660 (2/7) express high levels of hLF.
[0381] Comparison to Data Obtained from Single Construct
Injections: for 16,8hLZ:
[0382] The hLZ expression level of mouse line 649, coinjected with
the 16,8hLF3 and 16,8hLZ expression cassettes is comparable to that
of line 661 injected only with 16,8hLZ.
[0383] In most cases, high level expression of hLZ is not obtained
upon coinjection (1/7: high expression (line 649); 2/7 (650 and
660): intermediate-low expression; 4/7: low expression). Upon
injection of single hLZ transgene, similar data are obtained (1/4:
high expression (line 661); 1/4: intermediate; 2/4: low
expression). Therefore, behavior of the 16,8hLZ transgene is not
measurably influenced by the presence of the 16,8hLF3
transgene.
[0384] Note that none of the 7 lines expressed as high as line 905
(construct 16,8hLZ3), although the level of 649 is in the same
range.
[0385] In conclusion, these constructs can be expressed at
relatively high levels (0.2-0.5 mg/ml range) with approximately
20-25% of the resulting transgenic mice expressing at these high
levels (3/13; 7 coinjections+6 single inj.). Also coinjection with
16,8hLF3 does not appear to influence expression of 16,8hLZ.
[0386] For 16,8hLF3:
[0387] The single injection of 16,8hLF3 resulted in 13 independent
transgenic mouse lines which can be divided into 2 groups:
[0388] (1) the low expressors which produced levels are from 0.1 to
5 .mu.g/ml (8/13) and
[0389] (2) the high expressors which produced levels from 40 to 200
.mu.g/ml (5/13).
[0390] Of the mice having the coinjected fragments, 2/7 express at
high levels. This is similar to the frequency of high level
expression observed upon injection of one fragment (5/13). However,
both 16,8hLF3/16,8hLZ mice lines (649 and 660) express hLF at much
higher levels than observed previously. This indicates that the
presence of the hLZ construct stimulates expression of the 16,8hLF3
construct. In line 649, the high hLF levels are accompanied by high
hLZ levels. For line 660, this is less clear as hLZ levels are
intermediate. However, as illustrated below, RNA analysis reveals
that the 16,8hLZ transgene in line 660 is transcriptionally at
least as active as the hLF transgene.
[0391] Results from Expression Analysis at the mRNA Level:
[0392] Northern blot analysis was performed on both bovine
lactating mammary gland total RNA and mammary gland total RNA from
lactating transgenic mice (including mice transgenic for genomic
hLF, 16,8hLF3 and 16,8hLF3+16,8hLz). A 24 bp synthetic oligomer
which hybridizes to exactly the same sequence in the 5' UTR of
bovine .alpha.S1 casein RNA and in all transgene derived RNA was
used as a probe. Expression levels were compared directly by
quantification of the amount of labelled probe hybridized to the
transgene- and bovine .alpha.S1 RNA.
[0393] It appeared that the ratio of hLZ- to hLF-mRNA and of hLZ-
to bovine .alpha.S1 mRNA was much higher than expected from the
hLZ- and hLF protein levels. For example, line 649 expressed
.about.0.2 mg/ml of hLZ and -1-2 mg/ml of hLF. After correcting for
protein size (factor 5), hLZ and hLF mRNA levels are expected to be
within the same range, with hLF levels about 2-fold higher than hLZ
RNA levels. However, in line 649 hLZ mRNA levels were 20-fold
higher than the hLF mRNA levels. Comparative RNA analysis of lines
650, 661, 662 and bovine mammary gland RNA confirmed these
data.
[0394] It can therefore be concluded that transcriptionally, very
high levels of hLZ expression are obtained using genomic hLZ
sequences and the bovine .alpha.S1 casein gene based expression
system of the invention. The genomic hLZ constructs are transcribed
at much higher levels than the hLF cDNA constructs, and expressed
in the same range as the genomic hLF transgenes.
[0395] To compare the performance of different hLF and hLZ
transgenes at the translational level, a 20-fold correction should
be made. The transcriptional activity of hLZ transgenes expressing
at 0.25 mg/ml is comparable to a protein level of 5 mg/ml, a level
of 50 .mu.g/ml equivalent to 1 mg/ml. In-addition, mouse line 649
hLZ mRNA levels exceeded bovine .alpha.S1 mRNA levels--which had
been 10-fold diluted--several fold. Since bovine .alpha.S-casein is
expressed at -12 mg/ml (and is of similar size as hLZ), these hLZ
RNA levels would be equivalent to an expression level of several
mg/ml.
EXAMPLE 24
[0396] Generation of 16,8 A hLZ:
[0397] Construct 16,8 A hLZ3 is a derivative of 16,8 hLZ3. In 16.8
A hLZ3 the hLZ 5' UTR sequences and the hLZ signal sequence have
been replaced with the corresponding sequences from the bovine
.alpha.S1-casein gene.
[0398] Construct 16,A hLZ3 is a derivative of 16,8 A hLZ3. In 16.A
hLZ3 the bovine .alpha.S1-casein gene 3' UTR and flanking sequences
have been replaced with the hLZ3' UTR and 4.5 kb of hLZ3" flanking
sequences.
[0399] Construction Details:
[0400] Vector p0.7.8hLZ (FIG. 23B) was digested with Cla1 and Sal1.
The 4.7 kb fragment (comprising 0.7 kb of the .alpha.S1-casein 5'
flanking sequences and the plasmid vector) was isolated and ligated
to linker GP 278/279 (FIG. 25). This DNA sequence comprises part of
the bovine .alpha.S1-casein 5' UTR, the complete bovine
.alpha.S1-casein signal sequence and 25 bp of hLZ sequence,
encoding the N-terminal region of adult hLZ. The ligation product
was isolated and ligated to a 5.3 kb Bal1-Sal1 fragment from
pKHLys3'5.3 (which is depicted in FIG. 23A). The resulting
constructs is pO.7AhLZ.DELTA.3'. From this construct the 5.3 kb
Cla1-Sal1 fragment was isolated and inserted into a Cla1-Sal1
vector, derived from p16, 8hLF3 (also used in construction of 16,8
hLZ3). The resulting construct is designated p16,8A hLZ3 (FIG.
26).
[0401] For construction of 16,A hLZ3 the vector p0.7AhLZ.DELTA.3'
was digested with Xba1 and Sal1, and a Xba1-Not1-Sal1 linker was
inserted (FIG. 27). This vector was linearized with Xba1, and the
6.5 kb Xba1 fragment from .lambda.HLYS1 (described by Peters et
al., Eur. J. Biochem. 182, 507-516, 1989) was inserted in the sense
orientation. This resulted in vector p0.7AhLZ. From this vector,
the 9.8 kb Cla1-Not1 hLZ fragment was isolated and, together with
the 14.5 kb Not1-Cla1 fragment from p16.8hLZ3, inserted into the
Not1 digested pWE15 cosmid vector.
[0402] In both cases, the transgene without plasmid sequences was
isolated as a Not1 fragment (16,8A hLZ3; 27.8 kb; 16,AhLZ3; 24.3
kb), purified and microinjected into fertilized mouse oocytes
following standard procedures.
[0403] Four independent transgenic founder mice have been generated
with construct 16,8 A hLZ3 and 6 mice were generated with construct
16 A hLZ3.
[0404] Preliminary Expression Data:
21 max. hLZ expression Mouse line (.mu.g/ml in milk) 16,8 A hLZ3:
1711 56 1783 20 16 A hLZ3: 1806 267 1809 2400
[0405] From these results it can be concluded that construct 16 A
hLZ3 yields much higher expression levels than any other hLZ
construct tested.
[0406] Preliminary quantitative Northern blotting data combined
with data on hLZ protein expression levels indicate that the
discrepancy between RNA and protein levels as observed for
constructs 16,8hLZ and 16,8hLZ3 does not occur with construct 16 A
hLZ3.
EXAMPLE 25
[0407] Transmission Experiments with "Calf #4
[0408] Three heifers were super-ovulated using normal procedures
used in cattle breeding (described in Diekman, S. J. et al. (1989)
Theriogenology 31:473-487). These animals were subsequently
inseminated with sperm from "calf #4" as described in Example 15.
Calf #4 was judged to be transgenic as described in Example 15. The
insemination resulted in two pregnancies.
[0409] These two animals were slaughtered four weeks after
insemination and the embryos recovered from the uterus. Total DNA
was isolated from these embryos following procedures as described
in Maniatis et al. (1982), digested with EcoRI, and analyzed by
"Southern Blot technique". The blot was hybridized to a probe
specific for the hLF gene (same protocol as in example 15). Of the
12 embryos recovered 6 (50%) showed an hLF-specific band. In all
cases the band was of the expected size and intensity. This
indicates that:
[0410] (a) the transgene transmits with an efficiency of appr.
50%
[0411] (b) the copy number is the same as in the founder
(.about.3)
[0412] (c) no gross rearrangements have occurred during
transmission of the six transgenic embryos, five were male and one
was female according to a PCR-analysis with primers specific for a
bovine Y-chromosome repeat. These data demonstrate that the
transgene can be transmitted to both males and females and has not
integrated in the Y-chromosome.
[0413] The sequences of the Y-chromosome specific primers are:
22 Forward primer: 5'-GGA TCC GAG ACA CAG AAC AGG-3' Reverse
primer: 5'-GCT AAT CCA TCC ATC CTA TAG-3'
EXAMPLE 26
[0414] Expression of Recombinant Proteins in Saliva of Calves
[0415] Ten animals were born from oocytes co- injected with the hLF
transgene (as described in Example 18) and a hLz transgene
(16,8hLZ; described in Example 22). None of these animals appeared
transgenic as judged by Southern Blot, but four of them (all males)
were judged to be mosaic based on PCR with 0.5 .mu.g DNA from blood
and ear. Primers for this PCR-experiment were located in exon 8 of
the hLF gene. The sequences of the primers are
[0416] 5'-TTT GGA AAG GAC AAG TCA CCG-3' and
[0417] 5'-CTC ACT TTT CCT CAA GTT CTG-3'
[0418] All ten animals were tested for hLF and hLZ expression in
saliva. Epithelial cells in the salivary gland are structurally and
functionally similar to such cells in the mammary gland, and some
milk protein genes may also be expressed in salivary gland (albeit
at much lower levels than in mammary gland).
[0419] Approximately 2 ml of saliva was collected from the mouth of
the animal and levels of protein were determined in these samples
using a radioimmunoassay as described in Example 5. Of the ten
animals, three showed expression of hLF above the lower limit of
detection. All three animals were part of the group of four animals
judged to be mosaic.
[0420] Expression levels were as follows:
23 animal sample 1 (ng/ml) sample 2 (ng/ml) 9772 25 18 9773 3 1.4
9774 1.2 nd nd = not determined
[0421] All 10 animals were also tested for hLZ expression. Only
animal 9772 showed expression of hLZ in saliva. The amount detected
was 2 ng/ml.
[0422] Of the 21 animals born in the experiment described in
Example 15, one animal (male) was judged to be mosaic based on the
fact that it was immunotolerant for hLF. This animal showed an hLF
expression in saliva of 100 ng/ml.
[0423] These data show that the transgenes used are capable of
expressing hLF (and hLZ) in bovines.
[0424] Having described the preferred embodiments of the present
invention, it will appear to those ordinarily skilled in the art
that various modifications may be made to the disclosed
embodiments, and that such modifications are intended to be within
the scope of the present invention.
[0425] All references cited herein are expressly incorporated in
their entirety by reference for all purposes.
Sequence CWU 1
1
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