U.S. patent application number 11/337302 was filed with the patent office on 2006-08-17 for avians that produce eggs containing exogenous proteins.
This patent application is currently assigned to AviGenics, Inc.. Invention is credited to Alex J. Harvey, Robert D. Ivarie, Guodong Liu, Julie A. Morris, Jeffrey C. Rapp.
Application Number | 20060185029 11/337302 |
Document ID | / |
Family ID | 22040673 |
Filed Date | 2006-08-17 |
United States Patent
Application |
20060185029 |
Kind Code |
A1 |
Ivarie; Robert D. ; et
al. |
August 17, 2006 |
Avians that produce eggs containing exogenous proteins
Abstract
This invention provides for proteins which are expressed in the
avian oviduct, packaged into eggs laid by the avian.
Inventors: |
Ivarie; Robert D.;
(Watkinsville, GA) ; Harvey; Alex J.; (Athens,
GA) ; Morris; Julie A.; (Watkinsville, GA) ;
Liu; Guodong; (Mississauga, CA) ; Rapp; Jeffrey
C.; (Athens, GA) |
Correspondence
Address: |
AVIGENICS, INC.
111 RIVERBEND ROAD
ATHENS
GA
30605
US
|
Assignee: |
AviGenics, Inc.
University of Georgia Research Foundation, Inc.
|
Family ID: |
22040673 |
Appl. No.: |
11/337302 |
Filed: |
January 23, 2006 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
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11274674 |
Nov 15, 2005 |
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11337302 |
Jan 23, 2006 |
|
|
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10696671 |
Oct 28, 2003 |
|
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11274674 |
Nov 15, 2005 |
|
|
|
09173864 |
Oct 16, 1998 |
6730822 |
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10696671 |
Oct 28, 2003 |
|
|
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60062172 |
Oct 16, 1997 |
|
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|
Current U.S.
Class: |
800/19 ;
800/7 |
Current CPC
Class: |
C07K 16/00 20130101;
C12N 2830/008 20130101; C12N 2740/11043 20130101; C12N 2830/90
20130101; A01K 2227/30 20130101; C12N 2800/90 20130101; A01K
2267/01 20130101; A01K 2217/072 20130101; A01K 2217/05 20130101;
C12N 15/8509 20130101; C12N 2830/00 20130101; C12N 9/00 20130101;
C12N 15/86 20130101; C12N 2830/40 20130101; C12N 2800/30 20130101;
C12N 2830/42 20130101; C07K 14/77 20130101; C12N 2840/44 20130101;
A01K 67/0275 20130101; C12N 15/902 20130101; C12N 2840/203
20130101; A01K 2267/025 20130101; C12N 2799/027 20130101; C12N
2840/20 20130101 |
Class at
Publication: |
800/019 ;
800/007 |
International
Class: |
A01K 67/027 20060101
A01K067/027 |
Claims
1. An avian which lays an egg containing a protein wherein the
protein is exogenous to the egg.
2. The avian of claim 1 wherein the egg is a chicken egg.
3. The avian of claim 1 wherein the exogenous protein is a
pharmaceutical protein.
4. The avian of claim 1 wherein the exogenous protein is selected
from the group consisting of antitrypsin, antithrombin III,
collagen, factors VIII, IX, X, fibrinogen, hyaluronic acid,
insulin, lactoferrin, protein C, tissue-type plasminogen activator,
somatotrophin, cytokine antibody, human growth hormone, immunotoxin
and chymotrypsin.
5. The avian of claim 1 wherein the exogenous protein is present in
egg white of the egg.
6. An avian which lays an egg containing a pharmaceutical protein
wherein the pharmaceutical protein is exogenous to the egg.
7. The avian of claim 6 wherein the egg is a chicken egg.
8. The avian of claim 6 wherein the pharmaceutical protein is
present in egg white of the egg.
9. An avian which produces egg white comprising a pharmaceutical
protein exogenous to the egg white.
10. The avian of claim 9 wherein the egg white is from a chicken
egg.
11. The avian of claim 10 wherein the protein is selected from the
group consisting of antitrypsin, antithrombin III, collagen,
factors VIII, IX, X, fibrinogen, hyaluronic acid, insulin,
lactoferrin, protein C, tissue-type plasminogen activator,
somatotrophin, cytokine antibody, human growth hormone, immunotoxin
and chymotrypsin.
12. An avian that lays an egg containing an antibody exogenous to
the egg.
13. The avian of claim 12 wherein the egg is a chicken egg.
14. The avian of claim 12 wherein the antibody is present in egg
white of the egg.
15. An avian that lays an egg containing a cytokine wherein the
cytokine is exogenous to the egg.
16. The avian of claim 15 wherein the egg is a chicken egg.
17. The avian of claim 15 wherein the cytokine is GM-CSF.
18. The avian of claim 15 wherein the cytokine is G-CSF.
19. The avian of claim 15 wherein the cytokine is
erythropoietin.
20. The avian of claim 15 wherein the cytokine is interferon.
21. An avian that lays an egg containing an immunotoxin wherein the
immunotoxin is exogenous to the egg.
22. The avian of claim 21 wherein the egg is a chicken egg.
Description
[0001] This application is a continuation of application Ser. No.
11/274,674, Filed Nov. 15, 2005, which is a continuation of
application Ser. No. 10/696,671, Filed Oct. 28, 2003, the
disclosures of which are incorporated in their entirety herein by
reference, which is a continuation of application Ser. No.
09/173,864, filed Oct. 16, 1998, now U.S. Pat. No. 6,730,822,
issued May 4, 2004, the disclosure of which is incorporated in its
entirety herein by reference, which claims the benefit of U.S.
Provisional Application No. 60/062,172, filed Oct. 16, 1997.
BACKGROUND OF THE INVENTION
[0002] a) Field of the Invention
[0003] The present invention relates to vectors and methods for the
introduction of exogenous genetic material into avian cells and the
expression of the exogenous genetic material in the cells. The
invention also relates to transgenic avian species, including
chickens, and to avian eggs which contain exogenous protein.
[0004] b) Description of Related Art
[0005] Numerous natural and synthetic proteins are used in
diagnostic and therapeutic applications; many others are in
development or in clinical trials. Current methods of protein
production include isolation from natural sources and recombinant
production in bacterial and mammalian cells. Because of the
complexity and high cost of these methods of protein production,
however, efforts are underway to develop alternatives. For example,
methods for producing exogenous proteins in the milk of pigs,
sheep, goats, and cows have been reported. These approaches suffer
from several limitations, including long generation times between
founder and production transgenic herds, extensive husbandry and
veterinary costs, and variable levels of expression because of
position effects at the site of the transgene insertion in the
genome. Proteins are also being produced using milling and malting
processes from barley and rye. However, plant post-translational
modifications differ from vertebrate post-translational
modifications, which often has a critical effect on the function of
the exogenous proteins.
[0006] Like tissue culture and mammary gland bioreactors, the avian
oviduct can also potentially serve as a bioreactor. Successful
methods of modifying avian genetic material such that high levels
of exogenous proteins are secreted in and packaged into eggs would
allow inexpensive production of large amounts of protein. Several
advantages of such an approach would be: a) short generation times
(24 weeks) and rapid establishment of transgenic flocks via
artificial insemination; b) readily scaled production by increasing
flock sizes to meet production needs; c) post-translational
modification of expressed proteins; 4) automated feeding and egg
collection; d) naturally sterile egg-whites; and e) reduced
processing costs due to the high concentration of protein in the
egg white.
[0007] The avian reproductive system, including that of the
chicken, is well described. The egg of the hen consists of several
layers which are secreted upon the yolk during its passage through
the oviduct. The production of an egg begins with formation of the
large yolk in the ovary of the hen. The unfertilized oocyte is then
positioned on top of the yolk sac. Upon ovulation or release of the
yolk from the ovary, the oocyte passes into the infundibulum of the
oviduct where it is fertilized if sperm are present. It then moves
into the magnum of the oviduct which is lined with tubular gland
cells. These cells secrete the egg-white proteins, including
ovalbumin, lysozyme, ovomucoid, conalbumin, and ovomucin, into the
lumen of the magnum where they are deposited onto the avian embryo
and yolk.
[0008] The ovalbumin gene encodes a 45 kD protein that is
specifically expressed in the tubular gland cells of the magnum of
the oviduct (Beato, Cell 56:335-344 (1989)). Ovalbumin is the most
abundant egg white protein, comprising over 50 percent of the total
protein produced by the tubular gland cells, or about 4 grams of
protein per large Grade A egg (Gilbert, "Egg albumen and its
formation" in Physiology and Biochemistry of the Domestic Fowl,
Bell and Freeman, eds., Academic Press, London, New York, pp.
1291-1329). The ovalbumin gene and over 20 kb of each flanking
region have been cloned and analyzed (Lai et al., Proc. Natl. Acad.
Sci. USA 75:2205-2209 (1978); Gannon et al., Nature 278:428-424
(1979); Roop et al., Cell 19:63-68 (1980); and Royal et al., Nature
279:125-132 (1975)).
[0009] Much attention has been paid to the regulation of the
ovalbumin gene. The gene responds to steroid hormones such as
estrogen, glucocorticoids, and progesterone, which induce the
accumulation of about 70,000 ovalbumin MRNA transcripts per tubular
gland cell in immature chicks and 100,000 ovalbumin mRNA
transcripts per tubular gland cell in the mature laying hen
(Palmiter, J. Biol. Chem. 248:8260-8270 (1973); Palmiter, Cell
4:189-197 (1975)). DNAse hypersensitivity analysis and
promoter-reporter gene assays in transfected tubular gland cells
defined a 7.4 kb region as containing sequences required for
ovalbumin gene expression. This 5' flanking region contains four
DNAse I-hypersensitive sites centered at -0.25, -0.8, -3.2, and
-6.0 kb from the transcription start site. These sites are called
HS-I, -II, -III, and -IV, respectively. These regions reflect
alterations in the chromatin structure and are specifically
correlated with ovalbumin gene expression in oviduct cells (Kaye et
al., EMBO 3:1137-1144 (1984)). Hypersensitivity of HS-II and -III
are estrogen-induced, supporting a role for these regions in
hormone-induction of ovalbumin gene expression.
[0010] HS-I and HS-II are both required for steroid induction of
ovalbumin gene transcription, and a 1.4 kb portion of the 5' region
that includes these elements is sufficient to drive
steroid-dependent ovalbumin expression in explanted tubular gland
cells (Sanders and McKnight, Biochemistry 27: 6550-6557 (1988)).
HS-I is termed the negative-response element ("NRE") because it
contains several negative regulatory elements which repress
ovalbumin expression in the absence of hormone (Haekers et al.,
Mol. Endo. 9:1113-1126 (1995)). Protein factors bind these
elements, including some factors only found in oviduct nuclei
suggesting a role in tissue-specific expression. HS-II is termed
the steroid-dependent response element ("SDRE") because it is
required to promote steroid induction of transcription. It binds a
protein or protein complex known as Chirp-I. Chirp-I is induced by
estrogen and turns over rapidly in the presence of cyclohexamide
(Dean et al., Mol. Cell. Biol. 16:2015-2024 (1996)). Experiments
using an explanted tubular gland cell culture system defined an
additional set of factors that bind SDRE in a steroid-dependent
manner, including a NF.kappa.B-like factor (Nordstrom et al., J.
Biol. Chem. 268:13193-13202 (1993); Schweers and Sanders, J. Biol.
Chem. 266: 10490-10497 (1991)).
[0011] Less is known about the function of HS-III and -IV. HS-III
contains a functional estrogen response element, and confers
estrogen inducibility to either the ovalbumin proximal promoter or
a heterologous promoter when co-transfected into HeLa cells with an
estrogen receptor cDNA. These data imply that HS-III may play a
functional role in the overall regulation of the ovalbumin gene.
Little is known about the function of HS-IV, except that it does
not contain a functional estrogen-response element (Kato et al.,
Cell 68: 731-742 (1992)).
[0012] There has been much interest in modifying eukaryotic genomes
by introducing foreign genetic material and/or by disrupting
specific genes. Certain eukaryotic cells may prove to be superior
hosts for the production of exogenous eukaryotic proteins. The
introduction of genes encoding certain proteins also allows for the
creation of new phenotypes which could have increased economic
value. In addition, some genetically-caused disease states may be
cured by the introduction of a foreign gene that allows the
genetically defective cells to express the protein that it can
otherwise not produce.
[0013] Finally, modification of animal genomes by insertion or
removal of genetic material permits basic studies of gene function,
and ultimately may permit the introduction of genes that could be
used to cure disease states, or result in improved animal
phenotypes.
[0014] Transgenesis has been accomplished in mammals by several
different methods. First, in mammals including the mouse, pig,
goat, sheep and cow, a transgene is microinjected into the
pronucleus of a fertilized egg, which is then placed in the uterus
of a foster mother where it gives rise to a founder animal carrying
the transgene in its germline. The transgene is engineered to carry
a promoter with specific regulatory sequences directing the
expression of the foreign protein to a particular cell type. Since
the transgene inserts randomly into the genome, position effects at
the site of the transgene's insertion into the genome may variably
cause decreased levels of transgene expression. This approach also
requires characterization of the promoter such that sequences
necessary to direct expression of the transgene in the desired cell
type are defined and included in the transgene vector (Hogan et al.
Manipulating the Mouse Embryo, Cold Spring Harbor Laboratory, NY
(1988)).
[0015] A second method for effecting animal transgenesis is
targeted gene disruption, in which a targeting vector bearing
sequences of the target gene flanking a selectable marker gene is
introduced into embryonic stem ("ES") cells. Via homologous
recombination, the targeting vector replaces the target gene
sequences at the chromosomal locus or inserts into interior
sequences preventing expression of the target gene product. Clones
of ES cells bearing the appropriately disrupted gene are selected
and then injected into early stage blastocysts generating chimeric
founder animals, some of which bear the transgene in the germ line.
In the case where the transgene deletes the target locus, it
replaces the target locus with foreign DNA borne in the transgene
vector, which consists of DNA encoding a selectable marker useful
for detecting transfected ES cells in culture and may additionally
contain DNA sequences encoding a foreign protein which is then
inserted in place of the deleted gene such that the target gene
promoter drives expression of the foreign gene (U.S. Pat. Nos.
5,464,764 and 5,487,992 (M. P. Capecchi and K. R. Thomas)). This
approach suffers from the limitation that ES cells are unavailable
in many mammals, including goats, cows, sheep and pigs.
Furthermore, this method is not useful when the deleted gene is
required for survival or proper development of the organism or cell
type.
[0016] Recent developments in avian transgenesis have allowed the
modification of avian genomes. Germ-line transgenic chickens may be
produced by injecting replication-defective retrovirus into the
subgerminal cavity of chick blastoderms in freshly laid eggs (U.S.
Pat. No. 5,162,215; Bosselman et al., Science 243:533-25 534
(1989); Thoraval et al., Transgenic Research 4:369-36 (1995)). The
retroviral nucleic acid carrying a foreign gene randomly inserts
into a chromosome of the embryonic cells, generating transgenic
animals, some of which bear the transgene in their germ line.
Unfortunately, retroviral vectors cannot harbor large pieces of
DNA, limiting the size and number of foreign genes and foreign
regulatory sequences that may be introduced using this method. In
addition, this method does not allow targeted introduction or
disruption of a gene by homologous recombination. Use of insulator
elements inserted at the 5' or 3' region of the fused gene
construct to overcome position effects at the site of insertion has
been described (Chim et al, Cell 74:504-514 (1993)).
[0017] In another approach, a transgene has been microinjected into
the germinal disc of a fertilized egg to produce a stable
transgenic founder bird that passes the gene to the F1 generation
(Love et al Bio/Technology 12:60-63 (1994)). This method has
several disadvantages, however. Hens must be sacrificed in order to
collect the fertilized egg, the fraction of transgenic founders is
low, and injected eggs require labor intensive in vitro culture in
surrogate shells.
[0018] In another approach, blastodermal cells containing
presumptive primordial germ cells ("PGCs") are excised from donor
eggs, transfected with a transgene and introduced into the
subgerminal cavity of recipient embryos. The transfected donor
cells are incorporated into the recipient embryos generating
transgenic embryos, some of which are expected to bear the
transgene in the germ line. The transgene inserts in random
chromosomal sites by nonhomologous recombination. This approach
requires characterization of the promoter such that sequences
necessary to direct expression of the transgene in the desired cell
type are defined and included in the transgene vector. However, no
transgenic founder birds have yet been generated by this
method.
[0019] Lui, Poult. Sci. 68:999-1010 (1995), used a targeting vector
containing flanking DNA sequences of the vitellogenin gene to
delete part of the resident gene in chicken blastodermal cells in
culture. However, it has not been demonstrated that these cells can
contribute to the germ line and thus produce a transgenic embryo.
In addition, this method is not useful when the deleted gene is
required for survival or proper development of the organism or cell
type.
[0020] Thus, it can be seen that there is a need for a method of
introducing foreign DNA which is operably linked to a magnum-active
promoter into the avian genome. There is also a need for a method
of introducing foreign DNA into nonessential portions of a target
gene of the avian genome such that the target gene's regulatory
sequences drive expression of the foreign DNA, preferably without
disrupting the function of the target gene. The ability to effect
expression of the integrated transgene selectively within the avian
oviduct is also desirable. Furthermore, there exists a need to
create germ-line modified transgenic birds which express exogenous
genes in their oviducts and secrete the expressed exogenous
proteins into their eggs.
SUMMARY OF THE INVENTION
[0021] This invention provides methods for the stable introduction
of exogenous coding sequences into the genome of a bird and
expressing those exogenous coding sequences to produce desired
proteins or to alter the phenotype of the bird. Synthetic vectors
useful in the methods are also provided by the present invention,
as are transgenic birds which express exogenous protein and avian
eggs containing exogenous protein.
[0022] In one embodiment, the present invention provides methods
for producing exogenous proteins in specific tissues of avians. In
particular, the invention provides methods of producing exogenous
proteins in an avian oviduct. Transgenes are introduced into
embryonic blastodermal cells, preferably near stage X, to produce a
transgenic bird, such that the protein of interest is expressed in
the tubular gland cells of the magnum of the oviduct, secreted into
the lumen, and deposited onto the egg yolk. A transgenic bird so
produced carries the transgene in its germ line. The exogenous
genes can therefore be transmitted to birds by both artificial
introduction of the exogenous gene into bird embryonic cells, and
by the transmission of the exogenous gene to the bird's offspring
stably in a Mendelian fashion.
[0023] The present invention provides for a method of producing an
exogenous protein in an avian oviduct. The method comprises as a
first step providing a vector that contains a coding sequence and a
promoter operably linked to the coding sequence, so that the
promoter can effect expression of the nucleic acid in the tubular
gland cells of the magnum of an avian oviduct. Next, the vector is
introduced into avian embryonic blastodermal cells, either freshly
isolated, in culture, or in an embryo, so that the vector sequence
is randomly inserted into the avian genome. Finally, a mature
transgenic avian which expresses the exogenous protein in its
oviduct is derived from the transgenic blastodermal cells. This
method can also be used to produce an avian egg which contains
exogenous protein when the exogenous protein that is expressed in
the tubular gland cells is also secreted into the oviduct lumen and
deposited onto the yolk of an egg.
[0024] In one embodiment, the production of a transgenic bird by
random chromosomal insertion of a vector into its avian genome may
optionally involve DNA transfection of embryonic blastodermal cells
which are then injected into the subgerminal cavity beneath a
recipient blastoderm. The vector used in such a method has a
promoter which is fused to an exogenous coding sequence and directs
expression of the coding sequence in the tubular gland cells of the
oviduct.
[0025] In an alternative embodiment, random chromosomal insertion
and the production of a transgenic bird is accomplished by
transduction of embryonic blastodermal cells with
replication-defective or replication-competent retroviral particles
carrying transgene RNA between the 5' and 3' LTRs of the retroviral
vector. For instance, in one specific embodiment, an avian leukosis
virus (ALV) retroviral vector is used which comprises a modified
pNLB plasmid containing an exogenous gene that is inserted
downstream of a segment of the ovalbumin promoter region. An RNA
copy of the modified retroviral vector, packaged into viral
particles, is used to infect embryonic blastoderms which develop
into transgenic birds. Alternatively, helper cells which produce
the retroviral transducing particles are delivered to the embryonic
blastoderm.
[0026] In one embodiment, the vector used in the methods of the
invention contains a promoter which is magnum-specific. In this
embodiment, expression of the exogenous coding sequence occurs only
in the oviduct. Optionally, the promoter used in this embodiment
may be a segment of the ovalbumin promoter region. One aspect of
the invention involves truncating the ovalbumin promoter and/or
condensing the critical regulatory elements of the ovalbumin
promoter so that it retains sequences required for high levels of
expression in the tubular gland cells of the magnum of the oviduct,
while being small enough that it can be readily incorporated into
vectors. For instance, a segment of the ovalbumin promoter region
may be used. This segment comprises the 5'-flanking region of the
ovalbumin gene. The total length of the ovalbumin promoter segment
may be from about 0.88 kb to about 7.4 kb in length, and is
preferably from about 0.88 kb to about 1.4 kb in length. The
segment preferably includes both the steroid-dependent regulatory
element and the negative regulatory element of the ovalbumin gene.
The segment optionally also includes residues from the 5'
untranslated region (5' UTR) of the ovalbumin gene. In an
alternative embodiment, the magnum-specific promoter may be a
segment of the promoter region of the conalbumin, ovomucoid, or
ovomucin genes.
[0027] In another embodiment of the invention, the vectors
integrated into the avian genome contain constitutive promoters
which are operably linked to the exogenous coding sequence.
Alternatively, the promoter used in the expression vector may be
derived from that of the lysozyme gene, a gene expressed in both
the oviduct and macrophages.
[0028] If a constitutive promoter is operably linked to an
exogenous coding sequence which is to be expressed in the oviduct,
then the methods of the invention may also optionally involve
providing a second vector which contains a second coding sequence
and a magnum-specific promoter operably linked to the second coding
sequence. This second vector is also expressed in the tubular gland
cells of the mature transgenic avian. In this embodiment,
expression of the first coding sequence in the magnum is directly
or indirectly dependent upon the cellular presence of the protein
expressed by the second vector. Such a method may optionally
include the use of a Cre-loxP system.
[0029] In an alternative embodiment, the production of the
transgenic bird is accomplished by homologous recombination of the
transgene into a specific chromosomal locus. An exogenous
promoter-less minigene is inserted into the target locus, or
endogenous gene, whose regulatory sequences then govern the
expression of the exogenous coding sequence. This technique,
promoter-less minigene insertion (PMGI), is not limited to use with
target genes directing oviduct-specific expression, and may
therefore be used for expression in any organ when inserted into
the appropriate locus. In addition to enabling the production of
exogenous proteins in eggs, the promoter-less minigene insertion
method is amenable to applications in the poultry production and
egg-laying industries where gene insertions may enhance critical
avian characteristics such as muscling, disease resistance, and
livability or to reduce egg cholesterol.
[0030] One aspect of the present invention provides for a targeting
vector which may be used for promoter-less minigene insertion into
a target endogenous gene in an avian. This vector includes a coding
sequence, at least one marker gene, and targeting nucleic acid
sequences. The marker gene is operably linked to a constitutive
promoter, such as the Xenopus laevis ef-1.alpha. a promoter, the
HSV tk promoter, the CMV promoter, and the .beta.-actin promoter,
and can be used for identifying cells which have integrated the
targeting vector. The targeting nucleic acid sequences correspond
to the sequences which flank the point of insertion in the target
gene, and then direct insertion of the targeting vector into the
target gene.
[0031] The present invention provides for a method of producing an
exogenous protein in specific cells in an avian. The method
involves providing a targeting vector containing the promoter-less
minigene. The targeting vector is designed to target an endogenous
gene that is expressed in the specific cells into avian embryonic
blastodermal cells. The transgenic embryonic blastodermal cells are
then injected into the subgerminal cavity beneath a recipient
blastoderm or otherwise introduced into avian embryonic
blastodermal cells. The targeting vector is integrated into the
target endogenous gene. The resulting bird then expresses the
exogenous coding sequence under the control of the regulatory
elements of the target gene in the desired avian cells. This method
may also be used for producing an avian egg that contains exogenous
protein if a mature transgenic bird is ultimately derived from the
transgenic embryonic blastodermal cells. In the transgenic bird,
the coding sequence is expressed in the magnum under the control of
the regulatory sequences of a target gene, and the exogenous
protein is secreted into the oviduct lumen, so that the exogenous
protein is deposited onto the yolk of an egg laid by the bird.
[0032] In one embodiment of the invention, the targeted endogenous
gene is a gene expressed in the tubular gland cells of the avian
oviduct. A preferred target endogenous gene for selective
expression in the tubular gland cells is the ovalbumin gene (OV
gene). While the invention is primarily exemplified via use of the
ovalbumin gene as a target endogenous gene, other suitable
endogenous genes may be used. For example, conalbumin, ovomucoid,
ovomucin, and lysozyme may all be used as target genes for the
expression of exogenous proteins in tubular gland cells of an avian
oviduct in accordance with the invention.
[0033] The point of insertion in a method involving promoter-less
minigene insertion may be in the 5' untranslated region of the
target gene. Alternatively, if the targeting vector used for the
insertion contains an internal ribosome entry element directly
upstream of the coding sequence, then the point of insertion may be
in the 3' untranslated region of the target gene.
[0034] Another aspect of the invention provides for an avian egg
which contains protein exogenous to the avian species. Use of the
invention allows for expression of exogenous proteins in oviduct
cells with secretion of the proteins into the lumen of the oviduct
magnum and deposition upon the yolk of the avian egg. Proteins thus
packaged into eggs may be present in quantities of up to one gram
or more per egg.
[0035] Other embodiments of the invention provide for transgenic
birds, such as chickens or turkeys, which carry a transgene in the
genetic material of their germ-line tissue. In one embodiment, the
transgene comprises an exogenous gene operably linked to a promoter
which optionally may be magnum-specific. In this transgenic bird
the exogenous gene is expressed in the tubular gland cells of the
oviduct. In an alternative embodiment, the transgene instead
comprises an exogenous gene which is positioned in either the 5'
untranslated region or the 3' untranslated region of an endogenous
gene in a manner that allows the regulatory sequences of the
endogenous gene to direct expression of the exogenous gene. In this
embodiment, the endogenous gene may optionally be ovalbumin,
lysozyme, conalbumin, ovomucoid, or ovomucin.
BRIEF DESCRIPTION OF THE DRAWINGS
[0036] FIGS. 1(a) and 1(b) illustrate ovalbumin promoter expression
vectors comprising ovalbumin promoter segments and a coding
sequence, gene X which encodes an exogenous protein X.
[0037] FIGS. 2(a), 2(b), 2(c) and 2(d) illustrate retroviral
vectors of the invention comprising an ovalbumin promoter and a
coding sequence, gene X, encoding an exogenous protein X.
[0038] FIG. 2(e) illustrates a method of amplifying an exogenous
gene for insertion into the vectors of 2(a) and 2(b).
[0039] FIG. 2(f) illustrates a retroviral vector comprising an
ovalbumin promoter controlling expression of a coding sequence,
gene X, and an internal ribosome entry site (IRES) element enabling
expression of a second coding sequence, gene Y.
[0040] FIGS. 3(a) and 3(b) show schematic representations of the
ALV-derived vectors pNLB and pNLB-CMV-BL, respectively. The vectors
are both shown as they would appear while integrated into the
chicken genome.
[0041] FIG. 4 shows a graph showing the amount of .beta.-lactamase
found in the egg white of eggs from hens transduced with
NLB-CMV-BL, as determined by the .beta.-lactamase activity
assay.
[0042] FIG. 5 shows a western blot indicating the presence of
.beta.-lactamase in the egg white of eggs from hens transduced with
NLB-CMV-BL.
[0043] FIGS. 6(a) and 6(b) illustrate magnum-specific,
recombination-activated gene expression. Schematic cre and
.beta.-lactamase transgenes are shown integrated into the genome of
a hen in a non-magnum cell in FIG. 6(a). In FIG. 6(b), schematic
cre recombinase and .beta.-lactamase transgenes are shown
integrated into the genome of a hen in a magnum cell.
[0044] FIG. 7 illustrates an alternative method of silencing
.beta.-lactamase expression using loxP sites in which two loxP
sites flanking a stop codon (TAA) in frame with the first codon
(ATG) are inserted into the .beta.-lactamase signal peptide coding
sequence such that the signal peptide is not disrupted.
[0045] FIGS. 8(a) and 8(b) illustrate targeting vectors used for
insertion of a promoter-less minigene of the invention into a
target gene.
[0046] FIG. 9 illustrates a targeting vector used for detecting
correct homologous insertion of a promoter-less minigene of the
invention into a target gene.
DETAILED DESCRIPTION OF THE INVENTION
a) Definitions and General Parameters
[0047] The following definitions are set forth to illustrate and
define the meaning and scope of the various terms used to describe
the invention herein.
[0048] A "nucleic acid or polynucleotide sequence" includes, but is
not limited to, eucaryotic mRNA, cDNA, genomic DNA, and synthetic
DNA and RNA sequences, comprising the natural nucleoside bases
adenine, guanine, cytosine, thymidine, and uracil. The term also
encompasses sequences having one or more modified bases.
[0049] A "coding sequence" or "open reading frame" refers to a
polynucleotide or nucleic acid sequence which can be transcribed
and translated (in the case of DNA) or translated (in the case of
mRNA) into a polypeptide in vitro or in vivo when placed under the
control of appropriate regulatory sequences. The boundaries of the
coding sequence are determined by a translation start codon at the
5' (amino) terminus and a translation stop codon at the 3'
(carboxy) terminus. A transcription termination sequence will
usually be located 3' to the coding sequence. A coding sequence may
be flanked on the 5' and/or 3' ends by untranslated regions.
[0050] "Exon" refers to that part of a gene which, when transcribed
into a nuclear transcript, is "expressed" in the cytoplasmic mRNA
after removal of the introns or intervening sequences by nuclear
splicing.
[0051] Nucleic acid "control sequences" or "regulatory sequences"
refer to translational start and stop codons, promoter sequences,
ribosome binding sites, polyadenylation signals, transcription
termination sequences, upstream regulatory domains, enhancers, and
the like, as necessary and sufficient for the transcription and
translation of a given coding sequence in a defined host cell.
Examples of control sequences suitable for eucaryotic cells are
promoters, polyadenylation signals, and enhancers. All of these
control sequences need not be present in a recombinant vector so
long as those necessary and sufficient for the transcription and
translation of the desired gene are present.
[0052] "Operably or operatively linked" refers to the configuration
of the coding and control sequences so as to perform the desired
function. Thus, control sequences operably linked to a coding
sequence are capable of effecting the expression of the coding
sequence. A coding sequence is operably linked to or under the
control of transcriptional regulatory regions in a cell when DNA
polymerase will bind the promoter sequence and transcribe the
coding sequence into mRNA that can be translated into the encoded
protein. The control sequences need not be contiguous with the
coding sequence, so long as they function to direct the expression
thereof. Thus, for example, intervening untranslated yet
transcribed sequences can be present between a promoter sequence
and the coding sequence and the promoter sequence can still be
considered "operably linked" to the coding sequence.
[0053] The terms "heterologous" and "exogenous" as they relate to
nucleic acid sequences such as coding sequences and control
sequences, denote sequences that are not normally associated with a
region of a recombinant construct or with a particular chromosomal
locus, and/or are not normally associated with a particular cell.
Thus, a "heterologous" region of a nucleic acid construct is an
identifiable segment of nucleic acid within or attached to another
nucleic acid molecule that is not found in association with the
other molecule in nature. For example, a heterologous region of a
construct could include a coding sequence flanked by sequences not
found in association with the coding sequence in nature. Another
example of a heterologous coding sequence is a construct where the
coding sequence itself is not found in nature (e.g., synthetic
sequences having codons different from the native gene). Similarly,
a host cell transformed with a construct which is not normally
present in the host cell would be considered heterologous for
purposes of this invention.
[0054] "Exogenous protein" as used herein refers to a protein not
naturally present in a particular tissue or cell, a protein that is
the expression product of an exogenous expression construct or
transgene, or a protein not naturally present in a given quantity
in a particular tissue or cell.
[0055] "Endogenous gene" refers to a naturally occurring gene or
fragment thereof normally associated with a particular cell.
[0056] The expression products described herein may consist of
proteinaceous material having a defined chemical structure.
However, the precise structure depends on a number of factors,
particularly chemical modifications common to proteins. For
example, since all proteins contain ionizable amino and carboxyl
groups, the protein may be obtained in acidic or basic salt form,
or in neutral form. The primary amino acid sequence may be
derivatized using sugar molecules (glycosylation) or by other
chemical derivatizations involving covalent or ionic attachment
with, for example, lipids, phosphate, acetyl groups and the like,
often occurring through association with saccharides. These
modifications may occur in vitro, or in vivo, the latter being
performed by a host cell through posttranslational processing
systems. Such modifications may increase or decrease the biological
activity of the molecule, and such chemically modified molecules
are also intended to come within the scope of the invention.
[0057] Alternative methods of cloning, amplification, expression,
and purification will be apparent to the skilled artisan.
Representative methods are disclosed in Sambrook, Fritsch, and
Maniatis, Molecular Cloning, a Laboratory Manual, 2nd Ed., Cold
Spring Harbor Laboratory (1989).
[0058] "PMGI" refers to promoter-less minigene insertion, a method
in which a gene lacking a promoter is inserted via homologous
recombination into a target gene such that the target gene's
regulatory sequences govern the expression of the inserted gene in
an appropriate tissue. A minigene is a modified version of a gene,
often just a cDNA with an appropriate polyadenylation signal and
sometimes an intron. A minigene usually lacks all of the introns of
the genomic gene.
[0059] "Vector" means a polynucleotide comprised of single strand,
double strand, circular, or supercoiled DNA or RNA. A typical
vector may be comprised of the following elements operatively
linked at appropriate distances for allowing functional gene
expression: replication origin, promoter, enhancer, 5' mRNA leader
sequence, ribosomal binding site, nucleic acid cassette,
termination and polyadenylation sites, and selectable marker
sequences. One or more of these elements may be omitted in specific
applications. The nucleic acid cassette can include a restriction
site for insertion of the nucleic acid sequence to be expressed. In
a functional vector the nucleic acid cassette contains the nucleic
acid sequence to be expressed including translation initiation and
termination sites. An intron optionally may be included in the
construct, preferably .gtoreq.100 bp 5' to the coding sequence.
[0060] In some embodiments the promoter will be modified by the
addition or deletion of sequences, or replaced with alternative
sequences, including natural and synthetic sequences as well as
sequences which may be a combination of synthetic and natural
sequences. Many eukaryotic promoters contain two types of
recognition sequences: the TATA box and the upstream promoter
elements. The former, located upstream of the transcription
initiation site, is involved in directing RNA polymerase to
initiate transcription at the correct site, while the latter
appears to determine the rate of transcription and is upstream of
the TATA box. Enhancer elements can also stimulate transcription
from linked promoters, but many function exclusively in a
particular cell type. Many enhancer/promoter elements derived from
viruses, e.g. the SV40, the Rous sarcoma virus (RSV), and CMV
promoters are active in a wide array of cell types, and are termed
"constitutive" or "ubiquitous." The nucleic acid sequence inserted
in the cloning site may have any open reading frame encoding a
polypeptide of interest, with the proviso that where the coding
sequence encodes a polypeptide of interest, it should lack cryptic
splice sites which can block production of appropriate mRNA
molecules and/or produce aberrantly spliced or abnormal mRNA
molecules.
[0061] The termination region which is employed primarily will be
one of convenience, since termination regions appear to be
relatively interchangeable. The termination region may be native to
the intended nucleic acid sequence of interest, or may be derived
from another source.
[0062] A vector is constructed so that the particular coding
sequence is located in the vector with the appropriate regulatory
sequences, the positioning and orientation of the coding sequence
with respect to the control sequences being such that the coding
sequence is transcribed under the "control" of the control or
regulatory sequences. Modification of the sequences encoding the
particular protein of interest may be desirable to achieve this
end. For example, in some cases it may be necessary to modify the
sequence so that if may be attached to the control sequences with
the appropriate orientation; or to maintain the reading frame. The
control sequences and other regulatory sequences may be ligated to
the coding sequence prior to insertion into a vector.
Alternatively, the coding sequence can be cloned directly into an
expression vector which already contains the control sequences and
an appropriate restriction site which is in reading frame with and
under regulatory control of the control sequences.
[0063] A "marker gene" is a gene which encodes a protein that
allows for identification and isolation of correctly transfected
cells. Suitable marker sequences include, but are not limited to
green, yellow, and blue fluorescent protein genes (GFP, YFP, and
BFP, respectively). Other suitable markers include thymidine kinase
(tk), dihydrofolate reductase (DHFR), and aminoglycoside
phosphotransferase (APH) genes. The latter imparts resistance to
the aminoglycoside antibiotics, such as kanamycin, neomycin, and
geneticin. These, and other marker genes such as those encoding
chloramphenicol acetyltransferase (CAT), .beta.-lactamase,
.beta.-galactosidase (.beta.-gal), may be incorporated into the
primary nucleic acid cassette along with the gene expressing the
desired protein, or the selection markers may be contained on
separate vectors and cotransfected.
[0064] A "reporter gene" is a marker gene that "reports" its
activity in a cell by the presence of the protein that it
encodes.
[0065] A "retroviral particle", "transducing particle", or
"transduction particle" refers to a replication-defective or
replication-competent virus capable of transducing non-viral DNA or
RNA into a cell.
[0066] The terms "transformation", "transduction" and
"transfection" all denote the introduction of a polynucleotide into
an avian blastodermal cell.
[0067] "Magnum" is that part of the oviduct between the infudibulum
and the isthmus containing tubular gland cells that synthesize and
secrete the egg white proteins of the egg.
[0068] A "magnum-specific" promoter, as used herein, is a promoter
which is primarily or exclusively active in the tubular gland cells
of the magnum.
b) Transgenesis of Blastodermal Cells
[0069] By the methods of the present invention, transgenes can be
introduced into avian embryonic blastodermal cells, to produce a
transgenic chicken, or other avian species, that carries the
transgene in the genetic material of its germ-line tissue. The
blastodermal cells are typically stage VII-XII cells, or the
equivalent thereof, and preferably are near stage X. The cells
useful in the present invention include embryonic germ (EG) cells,
embryonic stem (ES) cells & primordial germ cells (PGCs). The
embryonic blastodermal cells may be isolated freshly, maintained in
culture, or reside within an embryo.
[0070] A variety of vectors useful in carrying out the methods of
the present invention are described herein. These vectors may be
used for stable introduction of an exogenous coding sequence into
the genome of a bird. In alternative embodiments, the vectors may
be used to produce exogenous proteins in specific tissues of an
avian, and in the oviduct in particular. In still further
embodiments, the vectors are used in methods to produce avian eggs
which contain exogenous protein.
[0071] In some cases, introduction of a vector of the present
invention into the embryonic blastodermal cells is performed with
embryonic blastodermal cells that are either freshly isolated or in
culture. The transgenic cells are then typically injected into the
subgerminal cavity beneath a recipient blastoderm in an egg. In
some cases, however, the vector is delivered directly to the cells
of a blastodermal embryo.
[0072] In one embodiment of the invention, vectors used for
transfecting blastodermal cells and generating random, stable
integration into the avian genome contain a coding sequence and a
magnum-specific promoter in operational and positional relationship
to express the coding sequence in the tubular gland cell of the
magnum of the avian oviduct. The magnum-specific promoter may
optionally be a segment of the ovalbumin promoter region which is
sufficiently large to direct expression of the coding sequence in
the tubular gland cells. For instance, the promoter may be derived
from the promoter regions of the ovalbumin, lysozyme, conalbumin,
ovomucoid, or ovomucin genes. Alternatively, the promoter may be a
promoter that is largely, but not entirely, specific to the magnum,
such as the lysozyme promoter.
[0073] FIGS. 1(a) and 1(b) illustrate examples of ovalbumin
promoter expression vectors. Gene X is a coding sequence which
encodes an exogenous protein. Bent arrows indicate the
transcriptional start sites. In one example, the vector contains
1.4 kb of the 5' flanking region of the ovalbumin gene (FIG. 1(a)).
The sequence of the "-1.4 kb promoter" of FIG. 1(a) corresponds to
the sequence starting from approximately 1.4 kb upstream (-1.4 kb)
of the ovalbumin transcription start site and extending
approximately 9 residues into the 5' untranslated region of the
ovalbumin gene. The approximately 1.4 kb-long segment harbors two
critical regulatory elements, the steroid-dependent regulatory
element (SDRE) and the negative regulatory element (NRE). The NRE
is so named because it contains several negative regulatory
elements which block the gene's expression in the absence of
hormone. A shorter 0.88 kb segment also contains both elements. In
another example, the vector contains approximately 7.4 kb of the 5'
flanking region of the ovalbumin gene and harbors two additional
elements (HS-III and HS-IV), one of which is known to contain a
functional region enabling induction of the gene by estrogen (FIG.
1(b)). A shorter 6 kb segment also contains all four elements and
could optionally be used in the present invention.
[0074] Each vector used for random integration according to the
present invention preferably comprises at least one 1.2 kb element
from the chicken .beta.-globin locus which insulates the gene
within from both activation and inactivation at the site of
insertion into the genome. In a preferred embodiment, two insulator
elements are added to one end of the ovalbumin gene construct. In
the .beta.-globin locus, the insulator elements serve to prevent
the distal locus control region (LCR) from activating genes
upstream from the globin gene domain, and have been shown to
overcome position effects in transgenic flies, indicating that they
can protect against both positive and negative effects at the
insertion site. The insulator element(s) are only needed at either
the 5' or 3' end of the gene because the transgenes are integrated
in multiple, tandem copies effectively creating a series of genes
flanked by the insulator of the neighboring transgene. In another
embodiment, the insulator element is not linked to the vector but
is cotransfected with the vector. In this case, the vector and the
element are joined in tandem in the cell by the process of random
integration into the genome.
[0075] Each vector may optionally also comprise a marker gene to
allow identification and enrichment of cell clones which have
stably integrated the expression vector. The expression of the
marker gene is driven by a ubiquitous promoter that drives high
levels of expression in a variety of cell types. In a preferred
embodiment the green fluorescent protein (GFP) reporter gene
(Zolotukhin et al., J. Virol 70:4646-4654 (1995)) is driven by the
Xenopus elongation factor 1-60 (ef-1.alpha.) promoter (Johnson and
Krieg, Gene 147:223-26 (1994)). The Xenopus ef-1.alpha. promoter is
a strong promoter expressed in a variety of cell types. The GFP
contains mutations that enhance its fluorescence and is humanized,
or modified such that the codons match the codon usage profile of
human genes. Since avian codon usage is virtually the same as human
codon usage, the humanized form of the gene is also highly
expressed in avian blastodermal cells. In alternative embodiments,
the marker gene is operably linked to one of the ubiquitous
promoters of HSV tk, CMV, or .beta.-actin.
[0076] While human and avian codon usage is well matched, where a
nonvertebrate gene is used as the coding sequence in the transgene,
the nonvertebrate gene sequence may be modified to change the
appropriate codons such that codon usage is similar to that of
humans and avians.
[0077] Transfection of the blastodermal cells may be mediated by
any number of methods known to those of ordinary skill in the art.
The introduction of the vector to the cell may be aided by first
mixing the nucleic acid with polylysine or cationic lipids which
help facilitate passage across the cell membrane. However,
introduction of the vector into a cell is preferably achieved
through the use of a delivery vehicle such as a liposome or a
virus. Viruses which may be used to introduce the vectors of the
present invention into a blastodermal cell include, but are not
limited to, retroviruses, adenoviruses, adeno-associated viruses,
herpes simplex viruses, and vaccinia viruses.
[0078] In one method of transfecting blastodermal cells, a packaged
retroviral-based vector is used to deliver the vector into
embryonic blastodermal cells so that the vector is integrated into
the avian genome.
[0079] As an alternative to delivering retroviral transduction
particles to the embryonic blastodermal cells in an embryo, helper
cells which produce the retrovirus can be delivered to the
blastoderm.
[0080] A preferred retrovirus for randomly introducing a transgene
into the avian genome is the replication-deficient ALV retrovirus.
To produce an appropriate ALV retroviral vector, a pNLB vector is
modified by inserting a region of the ovalbumin promoter and one or
more exogenous genes between the 5' and 3' long terminal repeats
(LTRs) of the retrovirus genome. Any coding sequence placed
downstream of the ovalbumin promoter will be expressed at high
levels and only in the tubular gland cells of the oviduct magnum
because the ovalbumin promoter drives the high level of expression
of the ovalbumin protein and is only active in the oviduct tubular
gland cells.
[0081] While a 7.4 kb ovalbumin promoter has been found to produce
the most active construct when assayed in cultured oviduct tubular
gland cells, the ovalbumin promoter must be shortened for use in
the retroviral vector. In a preferred embodiment, the retroviral
vector comprises a 1.4 kb segment of the ovalbumin promoter; a 0.88
kb segment would also suffice.
[0082] Any of the vectors of the present invention may also
optionally include a coding sequence encoding a signal peptide that
will direct secretion of the protein expressed by the vector's
coding sequence from the tubular gland cells of the oviduct. This
aspect of the invention effectively broadens the spectrum of
exogenous proteins that may be deposited in avian eggs using the
methods of the invention. Where an exogenous protein would not
otherwise be secreted, the vector bearing the coding sequence is
modified to comprise a DNA sequence comprising about 60 bp encoding
a signal peptide from the lysozyme gene. The DNA sequence encoding
the signal peptide is inserted in the vector such that it is
located at the N-terminus of the protein encoded by the cDNA.
[0083] FIGS. 2(a)-2(d), and 2(f) illustrate examples of suitable
retroviral vectors. The vector is inserted into the avian genome
with 5' and 3' flanking LTRs. Neo is the neomycin
phosphotransferase gene. Bent arrows indicate transcription start
sites. FIGS. 2(a) and 2(b) illustrate LTR and oviduct transcripts
with a sequence encoding the lysozyme signal peptide (LSP), whereas
FIGS. 2(c) and 2(d) illustrate transcripts without such a sequence.
There are two parts to the retroviral vector strategy. Any protein
that contains a eukaryotic signal peptide may be cloned into the
vectors depicted in FIGS. 2(b) and 2(d). Any protein that is not
ordinarily secreted may be cloned into the vectors illustrated in
FIGS. 2(a) and 2(b) to enable its secretion from the tubular gland
cells.
[0084] FIG. 2(e) illustrates the strategy for cloning an exogenous
gene into a lysozyme signal peptide vector. The polymerase chain
reaction is used to amplify a copy of a coding sequence, gene X,
using a pair of oligonucleotide primers containing restriction
enzyme sites that enable the insertion of the amplified gene into
the plasmid after digestion with the two enzymes. The 5' and 3'
oligonucleotides contain the Bsu361 and Xbal restriction sites,
respectively.
[0085] Another aspect of the invention involves the use of internal
ribosome entry site (IRES) elements in any of the vectors of the
present invention to allow the translation of two or more proteins
from a di- or polycistronic mRNA. The IRES units are fused to 5'
ends of one or more additional coding sequences which are then
inserted into the vectors at the end of the original coding
sequence, so that the coding sequences are separated from one
another by an IRES. Pursuant to this aspect of the invention,
post-translational modification of the product is facilitated
because one coding sequence may encode an enzyme capable of
modifying the other coding sequence product. For example, the first
coding sequence may encode collagen which would be hydroxylated and
made active by the enzyme encoded by the second coding
sequence.
[0086] For instance, in the retroviral vector example of FIG. 2(f),
an internal ribosome entry site (IRES) element is positioned
between two exogenous coding sequences (gene X and gene y). The
IRES allows both protein X and protein Y to be translated from the
same transcript directed by the ovalbumin promoter. Bent arrows
indicate transcription start sites. The expression of the protein
encoded by gene X is expected to be highest in tubular gland cells,
where it is specifically expressed but not secreted. The protein
encoded by gene Y is also expressed specifically in tubular gland
cells but because it is efficiently secreted, protein Y is packaged
into the eggs.
[0087] In another aspect of the invention, the coding sequences of
vectors used in any of the methods of the present invention are
provided with a 3' untranslated region (3' UTR) to confer stability
to the RNA produced. When a 3' UTR is added to a retroviral vector,
the orientation of the fused ovalbumin promoter, gene X and the 3'
UTR must be reversed in the construct, so that the addition of the
3' UTR will not interfere with transcription of the full-length
genomic RNA. In a presently preferred embodiment, the 3' UTR may be
that of the ovalbumin or lysozyme genes, or any 3' UTR that is
functional in a magnum cell, i.e. the SV40 late region.
[0088] In an alternative embodiment of the invention, a
constitutive promoter is used to express the coding sequence of a
transgene in the magnum of a bird. In this case, expression is not
limited to only the magnum; expression also occurs in other tissues
within the avian. However, the use of such a transgene is still
suitable for effecting the expression of a protein in the oviduct
and the subsequent secretion of the protein into the egg white if
the protein is non-toxic to the avian in which it is expressed.
[0089] FIG. 3(a) shows a schematic of the replication-deficient
avian leukosis virus (ALV)-based vector pNLB, a vector which is
suitable for use in this embodiment of the invention. In the pNLB
vector, most of the ALV genome is replaced by the neomycin
resistance gene (Neo) and the lacZ gene, which encodes
b-galactosidase. FIG. 3(b) shows the vector pNLB-CMV-BL, in which
lacZ has been replaced by the CMV promoter and the .beta.-lactamase
coding sequence (.beta.-L.alpha. or BL). Construction of the vector
is reported in the specific example, Example 1, below.
.beta.-lactamase is expressed from the CMV promoter and utilizes a
poly adenylation signal (pA) in the 3' long terminal repeat (LTR).
.beta.-Lactamase has a natural signal peptide; thus, it is found in
blood and in egg white.
[0090] Avian embryos have been successfully transduced with
pNLB-CMV-BL transduction particles (see specific examples, Example
2 and 3, below). The egg whites of eggs from the resulting stably
transduced hens were found to contain up to 20 mg of secreted,
active .beta.-lactamase per egg (see specific examples, Example 4
and 5, below).
[0091] In an alternative embodiment of the invention, transgenes
containing constitutive promoters are used, but the transgenes are
engineered so that expression of the transgene effectively becomes
magnum-specific. Thus, a method for producing an exogenous protein
in an avian oviduct provided by the present invention involves
generating a transgenic avian that bears two transgenes in its
tubular gland cells. One transgene comprises a first coding
sequence operably linked to a constitutive promoter. The second
transgene comprises a second coding sequence that is operably
linked to a magnum-specific promoter, where expression of the first
coding sequence is either directly or indirectly dependent upon the
cellular presence of the protein expressed by the second coding
sequence.
[0092] Optionally, site-specific recombination systems, such as the
Cre-loxP or FLP-FRT systems, are utilized to implement the
magnum-specific activation of an engineered constitutive promoter.
In one embodiment, the first transgene contains an FRT-bounded
blocking sequence which blocks expression of the first coding
sequence in the absence of FTP, and the second coding sequence
encodes FTP. In another embodiment, the first transgene contains a
loxP-bounded blocking sequence which blocks expression of the first
coding sequence in the absence of the Cre enzyme, and the second
coding sequence encodes Cre. The loxP-bounded blocking sequence may
be positioned in the 5' untranslated region of the first coding
sequence and the loxP-bounded sequence may optionally contain an
open reading frame.
[0093] For instance, in one embodiment of the invention,
magnum-specific expression is conferred on a constitutive
transgene, by linking a cytomegalovirus (CMV) promoter to the
coding sequence of the protein to be secreted (CDS) (FIGS. 6(a) and
6(b)). The 5' untranslated region (UTR) of the coding sequence
contains a loxP-bounded blocking sequence. The loxP-bounded
blocking sequence contains two loxP sites, between which is a start
codon (ATG) followed by a stop codon, creating a short, nonsense
open reading frame (ORF). Note that the loxP sequence contains two
start codons in the same orientation. Therefore, to prevent them
from interfering with translation of the coding sequence after loxP
excision, the loxP sites must be orientated such that the ATGs are
in the opposite strand.
[0094] In the absence of Cre enzyme, the cytomegalovirus promoter
drives expression of the small open reading frame (ORF) (FIG.
6(a)). Ribosomes will initiate at the first ATG, the start codon of
the ORF, then terminate without being able to reinitiate
translation at the start codon of the coding sequence. To be
certain that the coding sequence is not translated, the first ATG
is out of frame with the coding sequence's ATG. If the Cre enzyme
is expressed in cells containing the CMV-cDNA transgene, the Cre
enzyme will recombine the loxP sites, excising the intervening ORF
(FIG. 6(b)). Now translation will begin at the start codon of the
coding sequence, resulting in synthesis of the desired protein.
[0095] To make this system tissue specific, the Cre enzyme is
expressed under the control of a tissue-specific promoter, such as
the magnum-specific ovalbumin promoter, in the same cell as the
CMV-loxP-coding sequence transgene (FIG. 6(b)). Although a
truncated ovalbumin promoter may be fairly weak, it is still
tissue-specific and will express sufficient amounts of the Cre
enzyme to induce efficient excision of the interfering ORF. In
fact, low levels of recombinase should allow higher expression of
the recombinant protein since it does not compete against coding
sequence transcripts for translation machinery.
[0096] Alternate methods of blocking translation of the coding
sequence include inserting a transcription termination signal
and/or a splicing signal between the loxP sites. These can be
inserted along with the blocking ORF or alone. In another
embodiment of the invention, a stop codon can be inserted between
the loxP sites in the signal peptide of the coding sequence (see
FIG. 7). Before recombinase is expressed, the peptide terminates
before the coding sequence. After recombinase is expressed (under
the direction of a tissue specific promoter), the stop codon is
excised, allowing translation of the coding sequence. The loxP site
and coding sequence are juxtaposed such that they are in frame and
the loxP stop codons are out of frame. Since signal peptides are
able to accept additional sequence (Brown et al., Mol. Gen. Genet.
197:351-7 (1984)), insertion of loxP or other recombinase target
sequences (i.e. FRT) is unlikely to interfere with secretion of the
desired coding sequence. In the expression vector shown in FIG. 7,
the loxP site is present in the signal peptide such that the amino
acids encoded by loxP are not present in the mature, secreted
protein. Before Cre enzyme is expressed, translation terminates at
the stop codon, preventing expression of .beta.-lactamase. After
recombinase is expressed (only in magnum cells), the loxP sites
recombine and excise the first stop codon. Therefore,
.beta.-lactamase is expressed selectively only in magnum cells.
[0097] In the aforementioned embodiments, the blocking ORF can be
any peptide that is not harmful to chickens. The blocking ORF can
also be a gene that is useful for production of the
ALV-transduction particles and/or transgenic birds. In one
embodiment, the blocking ORF is a marker gene.
[0098] For instance, the blocking ORF could be the neomycin
resistance gene, which is required for production of transduction
particles. Once the transgene is integrated into the chicken
genome, the neomycin resistance gene is not required and can be
excised.
[0099] Alternatively, .beta.-lactamase can be used as the blocking
ORF as it is an useful marker for production of transgenic birds.
(For specific examples of the use of .beta.-lactamase as a marker
in transgenic birds, see Example 4, below.) As an example, the
blocking ORF in FIG. 6(a) is replaced by .beta.-lactamase and the
downstream coding sequence now encodes a secreted
biopharmaceutical. .beta.-Lactamase will be expressed in blood and
other tissues; it will not be expressed in the magnum after
magnum-specific expression of Cre and recombination-mediated
excision of .beta.-lactamase, allowing expression of the desired
protein.
[0100] The Cre and loxP transgenes could be inserted into the
chicken genome via mediated transgenesis either simultaneously or
separately. Any method of transgenesis that results in stable
integration into the chicken genome is suitable. Both the ovalbumin
promoter-recombinase and CMV-loxP-CDS transgenes could be placed
simultaneously into chickens. However, the efficiencies of
transgenesis are low and therefore the efficiency of getting both
transgenes into the chicken genome simultaneously is low. In an
alternative and preferred method, one flock is produced that
carries the magnum-specific promoter/recombinase transgene and a
second is produced that carries the CMV-loxP-CDS transgene. The
flocks would then be crossed to each other. Hens resulting from
this outbreeding will express the coding sequence and only in their
magnum.
[0101] In an alternative method of transfecting blastodermal cells
to produce a transgenic chicken, a targeting vector is used for
promoter-less minigene insertion (PMGI) into a target gene. The
targeting vector comprises a coding sequence, at least one marker
gene which is operably linked to a constitutive promoter, and
targeting nucleic acid sequences which match the sequence flanking
the desired point of insertion in the desired target gene. The
targeting nucleic acid sequences direct insertion of the targeting
vector into the target gene. The length of these targeting
sequences will vary. Each targeting sequence is typically at least
about 1 kb in length, although longer sequences (up to 10 kb, for
instance) may be preferred in some cases and shorter sequences may
be required in others. The marker gene allows for the
identification of cells which have integrated the targeting
vector.
[0102] In one embodiment, the target gene is an endogenous gene
that is expressed in the avian oviduct. For instance, the target
gene may be selected from the group consisting of ovalbumin,
lysozyme, conalbumin, ovomucoid, and ovomucin. (It should be noted
that because the lysozyme gene is expressed in macrophages in
addition to the oviduct cells, it is not a suitable target gene
when expression is desired to be restricted only to oviduct
cells.)
[0103] PMGI may be used with target genes other than those
expressed in the avian oviduct, and in species other than the avian
species.
[0104] The point of insertion to which the vector is directed may
be in either the 5' or 3' untranslated region of the target gene.
If the 3' untranslated region is targeted, then the targeting
vector further comprises an internal ribosome entry site element
positioned directly upstream of the coding sequence on the
vector.
[0105] FIGS. 8(a) and 8(b) illustrate the insertion of PMGI into
the 5' or 3' untranslated region (UTR) of the ovalbumin target
gene, respectively. In the embodiment illustrated in FIG. 8(a), a
promoter-less minigene (PMG) is inserted into the 5' UTR of the
ovalbumin target gene. A dicistronic mRNA encoding both the
exogenous protein and ovalbumin is transcribed from the
transcription start site depicted by the arrow. Ribosomes bind to
the 5' end of the dicistronic mRNA, translate the exogenous gene,
then terminate before translating the ovalbumin coding region. Note
that the ovalbumin portion of the polycistronic transcript is not
translated. Thus, the level of ovalbumin protein produced will be
about half of the normal level, as translation of one copy of the
ovalbumin gene is disrupted. In the embodiment illustrated in FIG.
8(b), the PMG is inserted into the 3' UTR. Translation of the
exogenous gene is initiated by the presence of an IRES element to
which the ribosome binds and translates the downstream coding
region.
[0106] In either case, the targeting vectors contain a marker gene
to enable identification and enrichment of cell clones and
populations which have stably integrated the targeting vectors.
Suitable identification genes include but are not limited to neo,
which encodes a protein conferring resistance to G418, or GFP,
which encodes the green fluorescent protein (GFP). In a preferred
embodiment, GFP expression is used to identify clones uniformly
fluorescing green and, therefore, containing a stably integrated
targeting vector. The marker gene is expressed from a ubiquitous
promoter such as but not limited to the promoters of HSV tk,
.beta.-actin, CMV, or ef-1.alpha.. In a presently preferred
embodiment, the ef-1 .alpha. promoter drives expression of GFP.
[0107] The present invention also provides for a vector which may
be used for insertion of a promoter-less minigene into a target
gene, which comprises the elements of the targeting vector
described above but also includes a second marker gene which is
operably linked to a second constitutive promoter. The second
marker gene is positioned outside the targeting nucleic acid
sequences of the targeting vector, so that upon insertion of the
promoter-less minigene into the target gene, the second marker gene
will not be inserted.
[0108] For instance, one embodiment of the invention involves use
of marker genes encoding blue fluorescent protein (BFP) and GFP in
the PMGI targeting vector (FIG. 9). This strategy is a variation of
the positive-negative selection strategy (U.S. Pat. Nos. 5,464,764
and 5,487,992 (Capecchi et al.), in which BFP is used to identify
the rare cells in which the promoter-less minigene (PMG) has
correctly inserted into the target gene. The BFP gene is inserted
on the 3' end of the original targeting vector (See FIG. 9). When
the targeting vector and target correctly undergo homologous
recombination, only the GFP gene is inserted. Thus, colonies
containing a correctly inserted PMG will fluoresce green. By
contrast, in the majority of cells, the entire vector, including
the BFP gene, will insert at random spots in the genome. Colonies
in which random insertion has taken place will fluoresce blue and
green due to the presence of GFP and BFP.
[0109] Although FIG. 9 illustrates use of this vector in the 5'
UTR, this vector is suitable for use in either the 5' or 3'
UTR.
[0110] As mentioned above, the vectors produced according to the
methods of the invention may optionally be provided with a 3' UTR
containing a polyadenylation site to confer stability to the RNA
produced. In a preferred embodiment, the 3' UTR may be that of the
exogenous gene, or selected from the group consisting of the
ovalbumin, lysozyme, or SV40 late region. However, the ovalbumin 3'
UTR is not suitable in a PMGI vector that is to be inserted into
the endogenous ovalbumin gene because the addition of ovalbumin
sequences to the PMGI vector will interfere with proper
targeting.
c) Production of Exogenous Protein
[0111] Methods of the invention which provide for the production of
exogenous protein in the avian oviduct and the production of eggs
which contain exogenous protein involve an additional step
subsequent to providing a suitable vector and introducing the
vector into embryonic blastodermal cells so that the vector is
integrated into the avian genome. The subsequent step involves
deriving a mature transgenic avian from the transgenic blastodermal
cells produced in the previous steps. Deriving a mature transgenic
avian from the blastodermal cells optionally involves transferring
the transgenic blastodermal cells to an embryo and allowing that
embryo to develop fully, so that the cells become incorporated into
the bird as the embryo is allowed to develop. The resulting chick
is then grown to maturity. In an alterantive embodiment, the cells
of a blastodermal embryo are transfected or transduced with the
vector directly within the embryo. The resulting embryo is allowed
to develop and the chick allowed to mature.
[0112] In either case, the transgenic bird so produced from the
transgenic blastodermal cells is known as a founder. Some founders
will carry the transgene in the tubular gland cells in the magnum
of their oviducts. These birds will express the exogenous protein
encoded by the transgene in their oviducts. If the exogenous
protein contains the appropriate signal sequences, it will be
secreted into the lumen of the oviduct and onto the yolk of an
egg.
[0113] Some founders are germ-line founders. A germ-line founder is
a founder that carries the transgene in genetic material of its
germ-line tissue, and may also carry the transgene in oviduct
magnum tubular gland cells that express the exogenous protein.
Therefore, in accordance with the invention, the transgenic bird
will have tubular gland cells expressing the exogenous protein and
the offspring of the transgenic bird will also have oviduct magnum
tubular gland cells that express the exogenous protein.
(Alternatively, the offspring express a phenotype determined by
expression of the exogenous gene in a specific tissue of the
avian.)
[0114] The invention can be used to express, in large yields and at
low cost, a wide range of desired proteins including those used as
human and animal pharmaceuticals, diagnostics, and livestock feed
additives. Proteins such as human growth hormone, interferon,
lysozyme, and .beta.-casein are examples of proteins which are
desirably expressed in the oviduct and deposited in eggs according
to the invention. Other possible proteins to be produced include,
but are not limited to, albumin, .alpha.-1 antitrypsin,
antithrombin III, collagen, factors VIII, IX, X (and the like),
fibrinogen, hyaluronic acid, insulin, lactoferrin, protein C,
erythropoietin (EPO), granulocyte colony-stimulating factor
(G-CSF), granulocyte macrophage colony-stimulating factor (GM-CSF),
tissue-type plasminogen activator (tPA), feed additive enzymes,
somatotropin, and chymotrypsin. Genetically engineered antibodies,
such as immunotoxins which bind to surface antigens on human tumor
cells and destroy them, can also be expressed for use as
pharmaceuticals or diagnostics.
d) EXAMPLES
[0115] The following specific examples are intended to illustrate
the invention and should not be construed as limiting the scope of
the claims.
Example 1
Vector Construction
[0116] The lacZ gene of pNLB, a replication-deficient avian
leukosis virus (ALV)-based vector (Cosset et al., 1991), was
replaced with an expression cassette consisting of a
cytomegalovirus (CMV) promoter and the reporter gene,
.beta.-lactamase (.beta.-L.alpha. or BL). The pNLB and pNLB-CMV-BL
vector constructs are diagrammed in FIGS. 3(a) and 3(b),
respectively.
[0117] To efficiently replace the lacZ gene of pNLB with a
transgene, an intermediate adaptor plasmid was first created,
pNLB-Adapter. pNLB-Adapter was created by inserting the chewed back
ApaI/ApaI fragment of pNLB (Cosset et al., J. Virol. 65:3388-94
(1991)) (in pNLB, the 5' Apal resides 289 bp upstream of lacZ and
the 3' Apal resides 3' of the 3' LTR and Gag segments) into the
chewed-back KpnI/SacI sites of pBluescriptKS(-). The filled-in
MluI/XbaI fragment of pCMV-BL (Moore et al., Anal. Biochem. 247:
203-9 (1997)) was inserted into the chewed-back KpnI/NdeI sites of
pNLB-Adapter, replacing lacZ with the CMV promoter and the BL gene
(in pNLB, KpnI resides 67 bp upstream of lacZ and NdeI resides 100
bp upstream of the lacZ stop codon), thereby creating
pNLB-Adapter-CMV-BL. To create pNLB-CMV-BL, the HindIII/BlpI insert
of pNLB (containing lacZ) was replaced with the HindIII/BlpI insert
of pNLB-Adapter-CMV-BL. This two step cloning was necessary because
direct ligation of blunt-ended fragments into the HindIII/BlpI
sites of pNLB yielded mostly rearranged subclones, for unknown
reasons.
Example 2
Production of Transduction Particles
[0118] Sentas and Isoldes were cultured in F10 (Gibco), 5% newborn
calf serum (Gibco), 1% chicken serum (Gibco), 50 .mu.g/ml
phleomycin (Cayla Laboratories) and 50 .mu.g/ml hygromycin (Sigma).
Transduction particles were produced as described in Cosset et al.,
1993, herein incorporated by reference, with the following
exceptions. Two days after transfection of the retroviral vector
pNLB-CMV-BL (from Example 1, above) into 9.times.10.sup.5 Sentas,
virus was harvested in fresh media for 6-16 hours and filtered. All
of the media was used to transduce 3.times.10.sup.6 Isoldes in 3
100 mm plates with polybrene added to a final concentration of 4
.mu.g/ml. The following day the media was replaced with media
containing 50 .mu.g/ml phleomycin, 50 .mu.g/ml hygromycin and 200
.mu.g/ml G418 (Sigma). After 10-12 days, single G418.sup.r colonies
were isolated and transferred to 24-well plates. After 7-10 days,
titers from each colony were determined by transduction of Sentas
followed by G418 selection. Typically 2 out of 60 colonies gave
titers at 1-3.times.10.sup.5 . Those colonies were expanded and
virus concentrated to 2-7.times.10.sup.7 as described in Allioli et
al., Dev. Biol. 165:30-7 (1994), herein incorporated by reference.
The integrity of the CMV-BL expression cassette was confirmed by
assaying for .beta.-lactamase in the media of cells transduced with
NLB-CMV-BL transduction particles.
Example 3
Production of Transgenic Chickens
[0119] Stage X embryos in freshly laid eggs were transduced with
NLB-CMV-BL transduction particles (from Example 2, above) as
described in Thoraval et al., Transgenic Res. 4:369-377 (1995),
herein incorporated by reference, except that the eggshell hole was
covered with 1-2 layers of eggshell membrane and, once dry, Duco
model cement.
[0120] Approximately 120 White Leghorns were produced by
transduction of the stage X embryos with NLB-CMV-BL transduction
particles. These birds constitute chimeric founders, not fully
transgenic birds. Extensive analysis of DNA in the blood and sperm
from the transduced chickens indicates that 10-20% of the birds had
detectable levels of the transgene in any given tissue. Of those
birds carrying the transgene, approximately 2-15% of the cells in
any given tissue were actually transgenic.
Example 4
.beta.-lactamase Activity Assay in Blood and Egg White
[0121] When hens produced in Example 3, above, began to lay eggs,
the egg whites of those eggs were assayed for the presence of
.beta.-lactamase. The .beta.-lactamase assay was carried out as
described in Moore et al., Anal. Biochem. 247:203-9 (1997), herein
incorporated by reference, with the following modifications.
[0122] To assay blood from two to ten day old chicks, the leg vein
was pricked with a scalpel. 50 .mu.l of blood was collected in a
heparinized capillary tube (Fisher), of which 25 .mu.l was
transferred to 100 .mu.l phosphate-buffered saline (PBS) in a
96-well plate. Various dilutions of purified .beta.-lactamase
(Calbiochem) was added to some wells prior to addition of blood
from control (non-transduced) chicks to establish a
.beta.-lactamase standard curve. After one day at 4.degree. C., the
plate was centrifuged for 10 minutes at 730.times.5 g. 25 .mu.l of
the supernatant was added to 75 .mu.l of PBS. 100 .mu.l of 20 .mu.M
7-(thienyl-2-acetamido)-3-[2-(4-N,N-dimethylaminophenylazo)pyridinium-met-
hyl]-3-cephem-4-carboxylic acid (PADAC, from Calbiochem) in PBS was
added, and the wells were read immediately on a plate reader in a
10 minute kinetic read at 560 nm or left overnight in the dark at
room temperature. Wells were scored positive if the well had turned
from purple to yellow. To assay blood from older birds, the same
procedure was followed except that 200-300 .mu.l blood was drawn
from the wing vein using a syringe primed with 50 .mu.l of heparin
(Sigma).
[0123] Analysis of the NLB-CMV-BL transduced flock revealed nine
chickens that had significant levels of .beta.-lactamase in their
blood. Three of these chickens were males and these were the only
three males that had significant levels of the NLB-CMV-BL transgene
in their sperm as determined by PCR analysis (see Example 10,
below).
[0124] Thus, these are the males that are to be outbred to obtain
fully transgenic G.sub.1 offspring. The other six chickens were the
hens that expressed .beta.-lactamase in their magnum tissue (see
below). Other birds had low levels of .beta.-lactamase (just above
the level of detection) in their blood but did not have transgenic
sperm or eggs containing .beta.-lactamase. Thus .beta.-lactamase
expression in blood is a strong indicator of whether a chicken was
successfully transduced.
[0125] To assay .beta.-lactamase in egg white, freshly laid eggs
were transferred that day to a 4.degree. C. cooler, at which point
the .beta.-lactamase is stable for at least one month.
(Bacterially-expressed, purified .beta.-lactamase added to egg
white was determined to lose minimal activity over several weeks at
4.degree. C., confirming the stability of .beta.-lactamase in egg
white.) To collect egg white samples, eggs were cracked onto
plastic wrap. The egg white was pipetted up and down several times
to mix the thick and thin egg whites. A sample of the egg white was
transferred to a 96 well plate. 10 .mu.l of the egg white sample
was transferred to a 96-well plate containing 100 .mu.l of PBS
supplemented with 1.5 .mu.l of 1 M NaH.sub.2PO.sub.4, pH 5.5 per
well. After addition of 100 .mu.l of 20 .mu.M PADAC, the wells were
read immediately on a plate reader in a 10 minute or 12 hour
kinetic read at 560 nm. Various dilutions of purified
.beta.-lactamase was added to some wells along with 10 .mu.l of egg
white from control (non-transduced) hens to establish a
.beta.-lactamase standard curve. Egg white from both untreated and
NLB-CMV-BL transduced hens were assayed for the presence of
.beta.-lactamase. TABLE-US-00001 TABLE 1 Expression of
.beta.-lactamase in eggs of NLB-CMV-BL treated hens. Hen # Average
mg of .beta.-lactamase per egg # of eggs assayed 1 Control 0.1 .+-.
0.07 29 2 1522 0.31 .+-. 0.07 20 3 1549 0.96 .+-. 0.15 22 4 1581
1.26 .+-. 0.19 12 5 1587 1.13 .+-. 0.13 15 6 1790 0.68 .+-. 0.15 13
7 1793 1.26 .+-. 0.18 12 Control is eggs from untreated hens. The
low level of BL in these eggs is due to spontaneous breakdown of
PADAC during the course of the kinetic assay. The other hens were
transduced with NLB-CMV-BL as described in Example 3. Egg white
from each egg was assayed in triplicate.
[0126] Based on the .beta.-lactamase activity assay, the expression
levels of .beta.-lactamase appeared to range from 0.1 to 1.3 mg per
egg (assuming 40 milliliters of egg white per egg). However, these
quantities were significantly lower from the quantities obtained by
western blot assay (see Example 5, below) and were determined to be
deceptively lower than the true values. The difference in results
between the enzymatic activity assay and the western blot analysis
(Example 5) was found to be due to the presence of a
.beta.-lactamase inhibitor in egg white. The activity of purified
.beta.-lactamase was shown to be inhibited by egg white such that
50 ml of egg white in a 200 ml reaction resulted in nearly 100%
inhibition, whereas 10 ml of egg white in a 200 ml reaction
resulted in only moderate inhibition. Furthermore, spontaneous
breakdown of the enzymatic substrate, PADAC, during the course of
the assay also contributed to the erroneously low calculation of
.beta.-lactamase concentration.
Example 5
Western Blot of .beta.-Lactamase in Egg White
[0127] Western blot analysis of the same egg white as was assayed
in Example 4 confirmed the presence of .beta.-lactamase and
provided a more accurate measurement of the amount of
.beta.-lactamase present in the egg than the kinetic assay of
Example 4, above.
[0128] To perform the analysis, 10 .mu.l of egg white was added to
30 .mu.l of 0.5 M Tris-Cl, pH 6.8, 10% sodium dodecyl sulfate
(SDS), 10% glycerol, 1.43 M 2-mercaptoethanol, 0.001% bromophenol
blue. Samples were heated to 95.degree. C. for 5 min, separated on
12% SDS-PAGE and transferred to Immobilon P membranes (Millipore).
.beta.-lactamase was detected with 1:500 dilution of rabbit
anti-.beta.-lactamase (5 Prime-3 Prime) and 1:5000 dilution of goat
anti-rabbit IgG HRP conjugate (Promega). Immunoblots were
visualized with the Enhanced Chemiluminescence (ECL) Western
Blotting System (Amersham).
[0129] Various .beta.-lactamase samples were analyzed by western
blotting and anti-.beta.-lactamase antibody. The results are shown
in FIG. 5. Lanes 1-4 of the blot contain 5.2, 1.3, 0.325, and 0.08
.mu.g, respectively, of bacterially expressed, purified
.beta.-lactamase added to control egg white, forming a standard
curve. Lane 5 contains control egg white from an untreated hen. In
lane 6 is 2 .mu.l of egg white from Hen 1522 (Betty Lu). Lanes 7-8
contain 1 and 2 .mu.ls, respectively, of egg white from Hen 1790.
Lanes 9-10 contain 1 and 2 .mu.ls, respectively, of egg white from
Hen 1793. 1 and 2 .mu.ls aliquots of egg white from Hen 1549 was
run in lanes 11-12. Lanes 13-14 show 1 and 2 .mu.ls, respectively,
of egg white from Hen 1581. 2 .mu.ls of egg white from Hen 1587 is
shown in lane 15.
[0130] The position of molecular weight standards is noted in FIG.
5 to the left of the blot in kilodaltons (kDa). The band at 31 kDa
is .beta.-lactamase. The molecular weight of the .beta.-lactamase
in the egg white is similar to that of purified .beta.-lactamase.
The egg white .beta.-lactamase is also a single molecular species,
indicating that synthesis was faithful to the .beta.-lactamase
coding sequence and that .beta.-lactamase is very stable in magnum
cells as well as egg white. The band at 13 kDa is an egg white
protein that cross-reacts with the anti-.beta.-lactamase
antibody.
[0131] Based on the western blot results, .beta.-lactamase in lane
6 (from Hen 1522, Betty Lu) is estimated at 120 ng, or 2.4 mg per
egg, assuming 40 mls of egg white per egg. .beta.-Lactamase in lane
9 (from Hen 1793) is estimated at 325 ng which corresponds to 13 mg
per egg. The .beta.-lactamase levels per egg as estimated by the
western blot analysis were considerably higher (up to 10-fold
higher) than the levels estimated by the .beta.-lactamase enzyme
assay of Example 4. As explained above, the discrepancy in the
protein estimates is believed to be caused by inhibition of enzyme
activity by egg white and breakdown of the substrate.
[0132] It should be noted that the up to 13 mg of .beta.-lactamase
per egg reported here was produced by chimeric founders, not fully
transgenic birds. As reported above, only 2-15% of the cells in any
given tissue of the chimeric founders were actually transgenic.
Assuming that this extent of mosaicism also applies to magnum
tissue, then the magnums of the six .beta.-lactamase egg-positive
hens were only partially transgenic. Therefore, fully transgenic
birds (G.sub.1 offspring) would be expected to express much higher
levels, possibly as high as 200 mg/egg. This estimate is
significant because it indicates that non-magnum specific promoters
such as CMV can effectively compete with magnum specific genes such
as ovalbumin and lysozyme for the egg-white protein synthesis
machinery.
Example 6
Isolation and Ex Vivo Transfection of Blastodermal Cells
[0133] In an alternative embodiment of the invention, blastodermal
cells are transfected ex vivo with an expression vector.
[0134] In this method, donor blastodermal cells are isolated from
fertilized eggs of Barred Plymouth Rock hens using a sterile
annular ring of Whatman filter paper which is placed over a
blastoderm and lifted after cutting through the yolk membrane of
the ring. The ring bearing the attached blastoderm is transferred
to phosphate-buffered saline (PBS) in a petri dish ventral side up,
and adhering yolk is removed by gentle pipetting. The area opaca is
dissected away with a hair loop and the translucent stage X
blastoderm is transferred via a large-bore pipette tip to a
microfuge tube. About 30,000-40,000 cells are isolated per
blastoderm and for a typical experiment 10 blastoderms are
collected.
[0135] Cells are dispersed by brief trypsin (0.2%) digestion,
washed once by low speed centrifugation in Dulbecco's modified
Eagle's medium (DMEM) and then transfected with linearized plasmids
via lipofectin (16 mg/200 ml, BRL) for 3 hours at room temperature.
The vectors shown in FIGS. 1, 3, or 4 would serve as suitable
expression constructs here. Cells are washed free of lipofectin
with medium and then 400-600 cells are injected into g-irradiated
(650 rads) recipient stage X embryos from the Athens-Canadian
randombred line (AC line). Injection is through a small window
(.about.0.5 cm) into the subgerminal cavity beneath the recipient
blastoderms. Windows are sealed with fresh egg shell membrane and
Duco plastic cement. Eggs are then incubated at 39.1.degree. C. in
a humidified incubator with 90.degree. rotation every 2 hr.
Example 7
Identification of Transgenic Mosaics by PCR Assay
[0136] Among the chicks which hatch from embryos containing
transfected or transduced blastodermal cells, only those exhibiting
Barred Plymouth Rock feather mosaicism are retained. Even if no
reporter gene is present in the transgene, transgenic mosaics can
be identified by PCR assay.
[0137] To identify transgenic mosaics, DNA blood and black feather
pulp of individual chicks are assayed by PCR for the presence of
the transgene using a primer pair specific to the transgene as
described by Love et al., Bio/Technology 12:60-63 (1994). Transgene
chimeras are induced, withdrawn and re-induced with
diethylstilbestrol (DES) pellets and excised magnums analyzed for
expression of reporter activity. Blood and liver are assayed to
monitor tissue specificity.
[0138] Male and female blood DNA was collected at 10 to 20 days
post-hatch. The DNA is extracted from the blood using a novel
high-throughput method of DNA extraction developed in our
laboratory. In this method, blood is drawn from a wing vein into a
heparinized syringe and one drop is immediately dispensed into one
well of a flat-bottom 96-well dish containing a buffer which lyses
cytoplasmic membranes exclusively. The plate is then briefly
centrifuged, which pellets the nuclei. The supernatant is removed
and a second lysis buffer is added which releases genomic DNA from
nuclei and degrades nucleases. The DNA is ethanol precipitated in
the plate, washed with 70% ethanol, dried and resuspended in 100
.mu.l of water per well. As much as 80 .mu.g of DNA can be obtained
from one drop (8 .mu.l) of chick blood. At least 768 samples can be
processed by one person in one day and the DNA is suitable for PCR
and Taqman.TM. (Perkin Elmer/Applied Biosystems) analysis.
[0139] The isolated DNA is then tested for the presence of the
transgenes using the Taqman.TM. sequence detection assay to
evaluate the efficiency of the embryo transduction process. The
Taqman.TM. sequence detection system allows the direct detection of
a specific sequence. A fluorescently-labeled oligonucleotide probe
complementary to an internal region of a desired PCR product only
fluoresces when annealed to the desired PCR product, which in this
case is complementary to the transgene. Because all of the
detection occurs in the PCR tube during the cycling process, the
Taqman.TM. system allows high-throughput PCR (no gel
electrophoresis is need) as well as sequence detection analogous to
and as sensitive as Southern analysis. 1 .mu.l of the isolated DNA,
which contains 600-800 ng of DNA, is used for the Taqman.TM.
reaction. Each reaction contains two sets of primer pairs and
Taqman.TM. probes. The first set detects the chicken glyceraldehyde
3-phosphate dehydrogenase gene (GAPDH) and is used as an internal
control for the quality of the genomic DNA and also serves as a
standard for quantitation of the transgene dosage. The second set
is specific for the desired transgene. Fluorescence is detected in
a dissecting stereomicroscope equipped with epifluorescence
detection. The two Taqman.TM. probes are attached to different dyes
which fluoresce at unique wavelengths: thus both PCR products are
detected simultaneously in an ABI/PE 7700 Sequence Detector. It is
estimated that up to 180 birds will hatch, and 20% (36 birds) will
contain the transgene in their blood.
Example 8
Identification of Blastodermal Cells with a Correctly Integrated
Promoter-Less Minigene (PMG)
[0140] Following transfection with a PMGI targeting vector such as
those shown in FIG. 8, cells are grown on a feeder line in
conditioned medium to produce colonies in which all or nearly half
of the cells are uniformly green in fluorescence. Fluorescence is
detected in a dissecting stereomicroscope equipped with
epifluorescence detection. Uniform fluorescence indicates that the
vector has stably integrated into the genome. Of these cell clones,
only a small subset actually have the PMG inserted correctly in the
target gene. The majority of the clones have PMG integrated
randomly into the genome. To identify clones containing a correctly
integrated PMG, colonies are screened using a Taqman.TM. PCR assay,
as described above. Two primers are used to amplify a segment of
the transgene at its site of integration. One primer lies in gene
X, the exogenous gene to be expressed in the oviduct, and the other
just outside the 5' targeting sequence, so that the fragment can
only be amplified by correct insertion into the target gene.
Colonies containing a correctly integrated transgene are subjected
to limited passage in culture on feeder cells in the presence of a
variety of cytokines that promote their growth in the absence of
differentiation. Cells are injected into recipient embryos.
Alternatively, green colonies are pooled and injected into
recipient embryos. Hatched chicks are screened subsequently for the
presence of the correctly inserted transgene.
Example 9
Blue/Green Detection for Promoter-Less Minigene Insertion
(PMGI)
[0141] Following transfection with a PMGI targeting vector like
that of FIG. 4, cells are grown for one day in the absence of a
feeder layer and green cells separated from blue/green cells using
a fluorescence-activated cell sorter the next day. Green cells are
then briefly passaged on feeder cells prior to injection into
recipient embryos. Green cells are also screened as above for
correct insertion.
Example 10
Production of Fully Transgenic G.sub.1 Chickens
[0142] Males are selected for breeding because a single male can
give rise to 20 to 30 G.sub.1 offspring per week as opposed to 6
G.sub.1 offspring per female per week, thereby speeding the
expansion of G.sub.1 transgenics. The feed of G.sub.0 males is
supplemented with sulfamethazine, which accelerates the sexual
maturation of males such that they can start producing sperm at
10-12 weeks of age instead of 20-22 weeks without influencing their
health or fertility (Speksnijder and Ivarie, unpublished data).
[0143] Sperm DNA of all males are screened for the presence of the
transgene. Sperm are collected and the DNA extracted using
Chelex-100. Briefly, 3 .mu.l of sperm and 200 .mu.l of 5%
Chelex-100 are mixed, followed by addition of 2 .mu.l of 10 mg/ml
proteinase K and 7 .mu.l of 2 M DTT. Samples are incubated at
56.degree. C. for 30-60 minutes. Samples are boiled for 8 minutes
and vortexed vigorously for 10 seconds. After centrifugation at 10
20 to 15 kG for 2-3 minutes, the supernatant is ready for PCR or
Taqman.TM. analysis. The DNAs are analyzed by the Taqman.TM. assay
using a Taqman.TM. probe and primers complementary to the
transgene. Of the 90 G.sub.0 males, it is estimated that 5%, or 4
to 5, will have the transgene in their sperm DNA.
[0144] As noted above in Example 4, the NLB-CMV-BL transduced flock
included three males that had significant levels of the NLB-CMV-BL
transgene in their sperm as determined by PCR analysis (see Example
10). Thus, these males are chosen for further breeding to obtain
fully transgenic G.sub.1 offspring.
[0145] By breeding germline transgenic males to 90 non-transgenic
White Leghorn females per week, it is estimated that 16 G.sub.1
offspring per week will be obtained. Hatched chicks are vent-sexed
and screened for the presence of the transgene in their blood DNA
by the Taqman.TM. assay. Twenty male and female G.sub.1 transgenics
will be obtained or 40 total, which will take up to 3 weeks.
[0146] Males will be kept for further breeding and females tested
for expression of transgenes in the egg.
[0147] All documents cited in the above specification are herein
incorporated by reference. Various modifications and variations of
the present invention will be apparent to those skilled in the art
without departing from the scope and spirit of the invention.
Although the invention has been described in connection with
specific preferred embodiments, it should be understood that the
invention as claimed should not be unduly limited to such specific
embodiments. Indeed, various modifications of the described modes
for carrying out the invention which are obvious to those skilled
in the art are intended to be within the scope of the following
claims.
Sequence CWU 1
1
2 1 69 DNA Lysozyme signal sequence CDS (6)..(59) 1 ccacc atg ggg
tct ttg cta atc ttg gtg ctt tgc ttc ctg ccg cta gct 50 Met Gly Ser
Leu Leu Ile Leu Val Leu Cys Phe Leu Pro Leu Ala 1 5 10 15 gcc tta
ggg ccctctagag 69 Ala Leu Gly 2 18 PRT Lysozyme signal sequence 2
Met Gly Ser Leu Leu Ile Leu Val Leu Cys Phe Leu Pro Leu Ala Ala 1 5
10 15 Leu Gly
* * * * *