U.S. patent application number 12/148398 was filed with the patent office on 2008-09-11 for in vivo transfection in avians.
This patent application is currently assigned to AviGenics, Inc.. Invention is credited to Leandro Christmann, Jeffrey C. Rapp.
Application Number | 20080222744 12/148398 |
Document ID | / |
Family ID | 46330249 |
Filed Date | 2008-09-11 |
United States Patent
Application |
20080222744 |
Kind Code |
A1 |
Christmann; Leandro ; et
al. |
September 11, 2008 |
In vivo transfection in avians
Abstract
The present invention provides for methods of producing
transgenic avians which may include delivering a heterologous
nucleic acid to oviduct tissue of an avian wherein the nucleic acid
enters a cell of the oviduct tissue and is expressed.
Inventors: |
Christmann; Leandro;
(Watkinsville, GA) ; Rapp; Jeffrey C.; (Athens,
GA) |
Correspondence
Address: |
AVIGENICS, INC.
111 RIVERBEND ROAD
ATHENS
GA
30605
US
|
Assignee: |
AviGenics, Inc.
|
Family ID: |
46330249 |
Appl. No.: |
12/148398 |
Filed: |
April 18, 2008 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10842606 |
May 10, 2004 |
7381712 |
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12148398 |
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10790455 |
Mar 1, 2004 |
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10842606 |
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60469527 |
May 9, 2003 |
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Current U.S.
Class: |
800/21 |
Current CPC
Class: |
C12N 2800/30 20130101;
A01K 67/0275 20130101; C12N 15/8509 20130101; A01K 2227/30
20130101; A01K 2267/02 20130101; C12N 15/87 20130101; A01K 2217/05
20130101 |
Class at
Publication: |
800/21 |
International
Class: |
A01K 67/027 20060101
A01K067/027 |
Claims
1. A method comprising: injecting a solution containing nucleic
acid into a blood vessel of an avian wherein the nucleic acid
enters a cell of the oviduct tissue and is expressed.
2. The method of claim 1 wherein the avian is selected from the
group consisting of chicken, quail, turkey, duck, goose, pheasant,
parrot, finche, hawk, crow, ratite, ostrich, emu and cassowary.
3. The method of claim 1 wherein the avian is a chicken.
4. The method of claim 1 wherein the nucleic acid is RNA.
5. The method of claim 1 wherein the nucleic acid is DNA.
6. The method of claim 1 wherein the nucleic acid is delivered
under pressure to the oviduct tissue of the avian.
7. The method of claim 1 wherein the cell is a tubular gland
cell.
8. The method of claim 1 wherein the injecting is into a blood
vessel having no organs between the oviduct and point of
injection.
9. The method of claim 1 wherein the injecting introduces pressure
into the blood vessel such that the blood vessel diameter is
enlarged as a result of pressure incurred by the injecting.
10. The method of claim 1 wherein the blood vessel is selected form
the group consisting of an anterior oviductal vein; a middle
oviductal vein; a hypogastric vein; an anterior oviductal artery; a
middle oviductal artery; a superior uterine artery; inferior
oviductal artery; a middle uterine artery; an inferior uterine
artery; a renal portal vein; a renal rehevens vein and a dorsal
arota artery.
11. The method of claim 1 wherein a polypeptide encoded by a coding
sequence of the nucleic acid is present in egg white produced by
the avian.
12. The method of claim 11 wherein the polypeptide is a
pharmaceutical protein.
13. The method of claim 1 wherein the polypeptide is an
antibody.
14. The method of claim 1 wherein the polypeptide is a
cytokine.
15. A method comprising: injecting a nucleic acid containing
solution into a blood vessel which delivers blood to or delivers
blood away from oviduct tissue of an avian wherein the blood vessel
intersects no organs between the oviduct and where the nucleic acid
enters the blood vessel wherein the nucleic acid enters a cell of
the oviduct tissue and is expressed.
16. The method of claim 15 wherein the injecting introduces
pressure into the blood vessel such that the blood vessel diameter
is enlarged as a result of pressure incurred by the injecting.
17. The method of claim 16 wherein the blood vessel diameter is
enlarged by more than about 5%.
18. The method of claim 16 wherein the blood vessel diameter is
enlarged by between about 20% and about 200%.
19. The method of claim 16 wherein the blood vessel diameter is
enlarged for between about 2 seconds and 20 minutes.
20. The method of claim 16 wherein the blood vessel diameter is
enlarged for between about 5 seconds and 5 minutes.
21. The method of claim 15 wherein the avian is a chicken.
22. The method of claim 15 wherein the avian is selected from the
group consisting of chicken, quail, turkey and duck.
23. The method of claim 15 wherein the nucleic acid is RNA
24. The method of claim 15 wherein the nucleic acid is DNA.
25. The method of claim 15 wherein the cell is a tubular gland
cell.
26. The method of claim 15 wherein the blood vessel is selected
form the group consisting of an anterior oviductal vein; a middle
oviductal vein; a hypogastric vein; an anterior oviductal artery; a
middle oviductal artery; a superior uterine artery; inferior
oviductal artery; a middle uterine artery; an inferior uterine
artery; a renal portal vein; a renal rehevens vein and a dorsal
arota artery.
27. The method of claim 15 wherein the nucleic acid encodes a
therapeutic protein.
28. A method comprising: injecting a nucleic acid into an artery
which provides blood to oviduct tissue of an avian wherein the
artery intersects no organs between the oviduct and point of
injection into the artery wherein the nucleic acid enters a cell of
the oviduct tissue and is expressed.
Description
[0001] This application is a continuation-in-part of U.S. patent
application Ser. No. 10/842,606, filed May 10, 2004, the disclosure
of which is incorporated by reference in its entirety herein, which
is a continuation-in-part of U.S. patent application Ser. No.
10/790,455, filed Mar. 1, 2004, now abandoned, the disclosure of
which is incorporated by reference in its entirety herein and
claims priority from U.S. provisional patent application No.
60/469,527, filed May 9, 2003, the disclosure of which is
incorporated in its entirety herein by reference.
FIELD OF THE INVENTION
[0002] The present invention relates to the field of biotechnology,
and more specifically to the field of avian genome modification.
Disclosed herein are methods for the generation of genetically
transformed avians.
BACKGROUND
[0003] The present invention is based, in part, on the discovery
that transgenic avians may be produced by in vivo transfection. In
particular, the invention provides methods for producing transgenic
avians by delivering nucleic acid (e.g., heterologous nucleic acid)
to tissue, such as the oviduct tissue, of a live avian. The present
invention provides for, among other things, producing transgenic
avians by in vivo electroporation of nucleic acid into avian tissue
and by applying nucleic acid to avian tissue under pressure.
[0004] Transgenic technology to convert animals into "bioreactors"
for the production of specific proteins or other substances of
pharmaceutical interest (Gordon et al., 1987, Biotechnology 5:
1183-1187; Wilmut et al., 1990, Theriogenology 33: 113-123) offers
significant advantages over more conventional methods of protein
production by gene expression. Recombinant nucleic acid molecules
have been engineered and incorporated into transgenic animals so
that an expressed heterologous protein may be joined to a protein
or peptide that allows secretion of the transgenic expression
product into milk or urine, from which the protein may then be
recovered. These procedures, however, may require lactating
animals, with the attendant costs of maintaining individual animals
or herds of large species, such as cows, sheep, or goats.
[0005] Historically, transgenic animals have been produced mostly
by microinjection of the fertilized egg. The pronuclei of
fertilized eggs are microinjected in vitro with heterologous DNA or
hybrid DNA molecules. The microinjected fertilized eggs are then
transferred to the genital tract of a pseudopregnant female (e.g.,
Krimpenfort et al., U.S. Pat. No. 5,175,384).
[0006] The production of an avian egg begins with formation of a
large yolk in the ovary of the hen. The unfertilized oocyte or ovum
is positioned on top of the yolk sac. After ovulation, the ovum
passes into the infundibulum of the oviduct where it is fertilized,
if sperm are present, and 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. The hen oviduct has
shown to be an outstanding protein bioreactor because of the high
levels of protein production, proper folding and post-translation
modification of the target protein, ease of product recovery, and
the shorter developmental period of chickens compared to other
potential animal species.
[0007] Various techniques have been used in efforts to create
transgenic chickens expressing heterologous proteins in the
oviduct.
[0008] Bosselman et al. in U.S. Pat. No. 5,162,215 describes a
method for introducing a replication-defective retroviral vector
into a pluripotent stem cell of an unincubated chick embryo, and
further describes chickens whose cells express a heterologous
vector nucleic acid sequence. Such retroviral vectors have
significant limitations, for example, only relatively small
fragments of nucleic acid can be inserted into the vectors
precluding, in most instances, the use of large portions of the
regulatory regions or introns of a genomic locus which can be
useful in obtaining significant levels of heterologous protein
expression.
[0009] In addition, previously described methods of producing
transgenic avians may not always allow for the targeting or
expression of nucleic acid of interest in the desired tissue in the
avian, for example, the oviduct tissue. Further, many techniques
currently available for producing a transgenic chicken require the
time necessary for a chicken to develop from a blastoderm to egg
laying maturity.
[0010] What is needed are methods of introducing large segments of
nucleic acid into avian cells and methods for the targeting nucleic
acid to specific tissue in the avian for example, the oviduct
tissue. Further, what is needed are methods of producing transgenic
avians for which a prolonged time necessary to achieve egg-laying
maturity is not required.
SUMMARY
[0011] The invention provides for methods of injecting a solution
containing nucleic acid into a blood vessel of an avian wherein the
nucleic acid enters a cell of the oviduct tissue of the avian and
is expressed. In one embodiment, the invention provides for methods
which include injecting a solution containing nucleic acid into a
blood vessel (e.g., an artery or vein) which delivers blood to or
delivers blood away from oviduct tissue of an avian wherein the
nucleic acid enters a cell of the oviduct tissue and is expressed.
In one particularly useful embodiment, the blood vessel does not
intersect any organs between the oviduct and point of injection
into the blood vessel (i.e., the blood vessel does not supply blood
to or receive blood from an organ between the point at the blood
vessel where the nucleic acid solution is injected and where the
blood carried by the vessel enters or leaves the oviduct).
[0012] In one particular embodiment, the injecting of the nucleic
acid solution introduces pressure into the blood vessel such that
the blood vessel diameter is enlarged as a result of the pressure
incurred by the injecting. For example, the blood vessel diameter
may be enlarged by more than about 5%, for example, the blood
vessel diameter may be enlarged by between about 5% and about 500%
or may be enlarged by between about 10% and about 300% or may be
enlarged by between about 10% and about 200% or may be enlarged by
between about 20% and about 200%. The invention contemplates the
blood vessel diameter being enlarged for any useful period of time.
For example, the blood vessel diameter may be enlarged for between
about 1 second and about 6 hours or for between about 5 seconds and
about 1 hour or for between about 5 seconds and about 15 minutes or
for between about 5 seconds and about 5 minutes.
[0013] The invention contemplates the injection of nucleic acid
solution into any blood vessel which may be useful in accordance
with the invention. For example, the invention contemplates the
injection of the nucleic acid solution into an anterior oviductal
vein; a middle oviductal vein; a hypogastric vein; an anterior
oviductal artery; a middle oviductal artery; a superior uterine
artery; inferior oviductal artery; a middle uterine artery; an
inferior uterine artery; a renal portal vein; a renal rehevens vein
and a dorsal arota artery. In addition, it is understood that
injecting into a blood vessel where the blood in that blood vessel
can travel to or from oviduct tissue through a second blood vessel
which is named in FIG. 1 is the same, for purposes of this
invention, as injecting into the second blood vessel. That is,
injecting into a certain blood vessel does not require that, for
example, a needle be introduced into that certain blood vessel. For
example, a needle can be inserted into the vein "16" of FIG. 1 and
a solution injected into the vein and since the solution will pass
through veins 13 and 14 en route to oviduct tissue such injecting
is considered to be the same as injecting into veins 13 and/or 14
of FIG. 1 and is also the same as injecting into the veins "5" of
FIG. 1.
[0014] The present invention relates to methods of producing
transgenic avians and to transgenic avians and their eggs. Provided
for are methods of introducing large nucleic acid segments, for
example, large DNA segments present on plasmids, into avians, for
example, avians of egg bearing age. Furthermore, the present
invention provides for the targeting of certain tissue within
avians, for example, specific targeting of the oviduct tissue.
[0015] In one aspect of the present invention, there are provided
methods of producing transgenic avians which include locally
delivering nucleic acid (e.g., heterologous nucleic acids) to
certain tissue of avians, for example, to the oviduct tissue of
avians. In this aspect of the invention, the nucleic acid enters a
cell of the oviduct tissue (e.g., tubular gland cell) and is
expressed. In a particularly useful embodiment, the nucleic acid
integrates into the genome of the cell to produce a transgenic
animal.
[0016] In one aspect of the invention, the nucleic acid is
delivered under pressure to oviduct tissue (e.g., oviduct tissue
cells) of the avian. In one embodiment, the pressure is hydrostatic
pressure. The pressure is provided by injection of a solution
comprising the nucleic acid into a blood vessel. For example, the
injection may be an intravenous injection or may be an
intraarterial injection. In one embodiment, the nucleic acid is
delivered to the lumen of the oviduct of the avian.
[0017] In one aspect of the invention, nucleic acid is delivered to
oviduct tissue (e.g., oviduct tissue cells) by electroporation, for
example, in vivo electroporation. Procedures for electroporation
are well known in the art and may be accomplished by standard
techniques known to practitioners of ordinary skill.
[0018] The present invention may be employed for the transfection
of any egg laying animal. The invention is particularly useful for
the transfection of avians including, but not limited to, chicken,
quail, turkey, duck, goose, pheasants, parrots, finches, hawks,
crows and ratites including ostrich, emu or cassowary. In one
useful embodiment, the avians are prepubertal hens or mature
hens.
[0019] In accordance with the present invention, the nucleic acid
to be transfected into the avian may be in a solution, for example,
a physiologically acceptable solution. In one embodiment, the
nucleic acid is DNA. For example, the nucleic acid may be circular
DNA molecules. The nucleic acid may include a vector. In one
embodiment, the vector is an expression construct.
[0020] In one embodiment of the invention, the nucleic acid
comprises an attB site. Typically, in the embodiment of the
invention where the nucleic acid comprises an attB site, integrase
is delivered to the cell of the oviduct tissue before, during or
after the transfection. In one embodiment, the integrase is encoded
on a nucleic acid which is cotransfected with the nucleic acid of
interest.
[0021] In a particularly useful aspect of the present invention, a
polypeptide encoded by a coding sequence of the nucleic acid is
present in egg white of eggs laid by transgenic avians produced
according to methods disclosed herein. In one useful embodiment,
the polypeptide is a pharmaceutical protein or therapeutic protein.
In one embodiment, the pharmaceutical protein is a light chain or a
heavy chain of an antibody, for example a human antibody. In
another embodiment, the pharmaceutical protein is a cytokine, for
example, interferon.
[0022] Any combination of features described herein are included
within the scope of the present invention provided that the
features included in any such combination are not mutually
inconsistent. Such combinations will be apparent based on this
specification and on the knowledge of one of ordinary skill in the
art.
BRIEF DESCRIPTION OF THE DRAWINGS
[0023] FIG. 1 shows a diagrammatic representation of blood vessels
associated with the chicken oviduct. The structures are as follows:
1 represents the ovary; 2 represents the oviduct; 3 represents the
cloaca; 4 represents the anterior oviductal vein; 5 represents the
middle oviductal veins; 6 represents the hypogastric vein; 7
represents the anterior oviductal artery; 8 represents the middle
oviductal artery; 9 represents the superior uterine artery; 10
represents the inferior oviductal artery; 11 represents the middle
uterine artery; 12 represents the inferior uterine artery; 13
represents the renal portal vein; 14 represents the renal rehevens
vein and 15 represents the dorsal arota artery.
DEFINITIONS
[0024] For convenience, definitions of certain terms employed in
the specification, examples, and appended claims are collected
here.
[0025] As used in this specification and the appended claims, the
singular forms "a," "an" and "the" include plural references unless
the content clearly dictates otherwise. Thus, for example,
reference to "a nucleic acid" includes a mixture of two or more
nucleic acids.
[0026] The term "avian" as used herein refers to any species,
subspecies or race of organism of the taxonomic class ava, such as,
but not limited to chicken, turkey, duck, goose, quail, pheasants,
parrots, finches, hawks, crows and ratites including ostrich, emu
and cassowary. The term includes the various known strains of
Gallus gallus, or chickens, (for example, White Leghorn, Brown
Leghorn, Barred-Rock, Sussex, New Hampshire, Rhode Island,
Australorp, Minorca, Amrox, California Gray), as well as strains of
turkeys, pheasants, quails, duck, ostriches and other poultry
commonly bred in commercial quantities. It also includes an
individual avian organism in all stages of development, including
embryonic and fetal stages. The term "avian" also may denote
"pertaining to a bird", such as "an avian (bird) cell."
[0027] A "blood vessel" is an artery or a vein.
[0028] "Distended" means enlarged as a result of internal
pressure.
[0029] The term "inject" means to force a fluid into an object such
as a blood vessel. The term "nucleic acid" as used herein includes
any natural or synthetic linear and sequential array (e.g.,
polymer) of nucleotides or nucleosides, for example cDNA, genomic
DNA, mRNA, tRNA, oligonucleotides, oligonucleosides and derivatives
thereof. For ease of discussion, such nucleic acids may be
collectively referred to herein as "constructs," "plasmids," or
"vectors." The term "nucleic acid" further includes modified or
derivatized nucleotides and nucleosides such as, but not limited
to, halogenated nucleotides such as, but not only, 5-bromouracil,
and derivatized nucleotides such as biotin-labeled nucleotides.
[0030] The terms "polynucleotide," "oligonucleotide," and "nucleic
acid sequence" may be used interchangeably herein and include, but
are not limited to, coding sequences (polynucleotide(s) or nucleic
acid sequence(s) which are transcribed and translated into
polypeptide in vitro or in vivo when placed under the control of
appropriate regulatory or control sequences); control sequences
(e.g., translational start and stop codons, promoter sequences,
ribosome binding sites, polyadenylation signals, transcription
factor binding sites, transcription termination sequences, upstream
and downstream regulatory domains, enhancers, silencers, and the
like); and regulatory sequences (DNA sequences to which a
transcription factor(s) binds and alters the activity of a gene's
promoter either positively (induction) or negatively (repression)).
No limitation as to length or to synthetic origin are suggested by
the terms described above.
[0031] As used herein the terms "peptide," "polypeptide" and
"protein" refer to a polymer of amino acids in a serial array,
linked through peptide bonds. A "peptide" typically is a polymer of
at least two to about 30 amino acids linked in a serial array by
peptide bonds. The term "polypeptide" includes proteins, protein
fragments, protein analogues, oligopeptides and the like. The term
"polypeptides" contemplates polypeptides as defined above that are
encoded by nucleic acids, produced through recombinant technology
(isolated from an appropriate source such as a bird), or
synthesized. The term "polypeptides" further contemplates
polypeptides as defined above that include chemically modified
amino acids or amino acids covalently or noncovalently linked to
labeling moieties.
[0032] A "pharmaceutical" or a "pharmaceutical protein" is any
substance which may be useful as a therapeutic, alone or in
combination with one or more other substances.
[0033] The terms "recombinant nucleic acid" and "recombinant DNA"
as used herein refer to combinations of at least two nucleic acid
sequences that are not naturally found in a eukaryotic or
prokaryotic cell. The nucleic acid sequences may include, but are
not limited to, nucleic acid vectors, gene expression regulatory
elements, origins of replication, suitable gene sequences that when
expressed confer antibiotic resistance, protein-encoding sequences
and the like. The term "recombinant polypeptide" is meant to
include a polypeptide produced by recombinant DNA techniques. A
recombinant polypeptide may be distinct from a naturally occurring
polypeptide either in its location, purity or structure.
[0034] The term "gene" or "genes" as used herein refers to nucleic
acid sequences that encode genetic information for the synthesis of
a whole RNA, a whole protein, or any portion of such whole RNA or
whole protein. Genes that are not naturally part of a particular
organism's genome are referred to as "foreign genes," "heterologous
genes" or "exogenous genes" and genes that are naturally a part of
a particular organism's genome are referred to as "endogenous
genes". The term "gene product" refers to an RNA or protein that is
encoded by the gene. "Endogenous gene products" are RNAs or
proteins encoded by endogenous genes. "Heterologous gene products"
are RNAs or proteins encoded by "foreign, heterologous or exogenous
genes" and are, therefore, not naturally expressed in the cell.
[0035] The term "expressed" or "expression" as used herein refers
to the transcription from a gene to give an RNA nucleic acid
molecule at least complementary in part to a region of one of the
two nucleic acid strands of the gene which is expressed. The term
"expressed" or "expression" as used herein may also include the
translation from an RNA molecule to give a protein, a polypeptide
or a portion thereof.
[0036] The term "coding region" as used herein refers to a
continuous linear arrangement of nucleotides which may be
translated into a polypeptide. A full length coding region is
translated into a full length protein; that is, a complete protein
as would be translated in its natural state absent any
post-translational modifications. A full length coding region may
also include any leader protein sequence or any other region of the
protein that may be excised naturally from the translated
protein.
[0037] The terms "vector" or "nucleic acid vector" as used herein
refer to a natural or synthetic single or double stranded plasmid
or viral nucleic acid molecule (RNA or DNA) that can be transfected
or transformed into cells and replicate independently of, or
within, the host cell genome. The term "expression vector" as used
herein refers to a nucleic acid vector that comprises a
transcription regulatory region operably linked to a site wherein
is, or can be, inserted, a nucleotide sequence to be transcribed
and, optionally, to be expressed, for instance, but not limited to,
a sequence coding at least one polypeptide.
[0038] The term "transfection" as used herein refers to the process
of inserting a nucleic acid into a host cell.
[0039] The terms "recombinant cell" and "genetically transformed
cell" refer to a cell comprising a combination of nucleic acid
segments not found in a single cell with each other in nature. A
new combination of nucleic acid segments can be introduced into an
organism using a wide array of nucleic acid manipulation techniques
available to those skilled in the art. The recombinant cell may
harbor a vector that is extragenomic, i.e, that does not covalently
insert into the cellular genome, including a non-nuclear (e.g.,
mitochondrial) genome(s). A recombinant cell may further harbor a
vector or a portion thereof that is intragenomic, i.e., covalently
incorporated within the genome of the recombinant cell.
[0040] As used herein, a "transgenic avian" is any avian, as
defined above, including the chicken and quail, in which one or
more of the cells of the avian contain heterologous nucleic acid
introduced by manipulation, such as by transgenic techniques. A
recombinant DNA molecule may be integrated within a chromosome, or
it may be extrachromosomally replicating DNA.
[0041] The terms "chimeric animal" or "mosaic animal" are used
herein to refer to animals in which the recombinant gene is found,
or in which the recombinant is expressed, in some but not all cells
of the animal. The term "tissue-specific chimeric animal" indicates
that the recombinant gene is present or expressed in some tissues
but not others.
[0042] As used herein, the term "transgene" means a nucleic acid
sequence that is partly or entirely heterologous, i.e., foreign, to
the transgenic animal or cell into which it is introduced, or, is
homologous to an endogenous gene of the transgenic animal or cell
into which it is introduced, but which is designed to be inserted,
or is inserted, into the animal's genome in such a way as to alter
the genome of the cell into which it is inserted (e.g., it is
inserted at a location which differs from that of the natural gene
or its insertion results in a knockout).
[0043] The term "cytokine" as used herein refers to any secreted
polypeptide that affects a function of cells and modulates an
interaction between cells in the immune, inflammatory or
hematopoietic response. A cytokine includes, but is not limited to,
monokines and lymphokines. Examples of cytokines include, but are
not limited to, interferons, Interleukin-1 (IL-1), Interleukin-6
(IL-6), Interleukin-8 (IL-8), EPO, G-CSF, GM-CSF, Tumor Necrosis
Factor-.alpha. (TNF-.alpha.) and Tumor Necrosis Factor .beta.
(TNF-.beta.).
[0044] The term "antibody" as used herein refers to polyclonal and
monoclonal antibodies and fragments thereof, and immunologic
binding equivalents thereof. Antibodies may include, but are not
limited to polyclonal antibodies, monoclonal antibodies (MAbs),
humanized or chimeric antibodies, single chain antibodies, FAb
fragments, F(Ab').sub.2 fragments, fragments produced by a FAb
expression library, anti-idiotypic (anti-Id) antibodies, and
epitope-binding fragments of any of the above.
[0045] The term "immunoglobulin polypeptide" as used herein refers
to a constituent polypeptide of an antibody or a polypeptide
derived therefrom. An "immunological polypeptide" may be, but is
not limited to, an immunological heavy or light chain and may
include a variable region, a diversity region, joining region and a
constant region or any combination, variant or truncated form
thereof. The term "immunological polypeptides" further includes
single-chain antibodies comprised of, but not limited to, an
immunoglobulin heavy chain variable region, an immunoglobulin light
chain variable region and optionally a peptide linker.
[0046] A nucleic acid or nucleic acid fragment of interest may
additionally be a "marker nucleic acid" or expressed as a "marker
polypeptide". Marker genes encode proteins that can be easily
detected in transformed cells and are, therefore, useful in the
study of those cells. Examples of suitable marker genes include
.beta.-galactosidase, green or yellow fluorescent proteins,
enhanced green fluorescent protein, chloramphenicol acetyl
transferase, luciferase, and the like. Such regions may also
include those 5' noncoding sequences involved with initiation of
transcription and translation, such as the enhancer, TATA box,
capping sequence, CAAT sequence, and the like.
[0047] "Therapeutic proteins" or "pharmaceutical proteins" include
an amino acid sequence which is a drug or is a component of a
drug.
[0048] The term "transformed" as used herein refers to a heritable
alteration in a cell resulting from the uptake of nucleic acid, for
example, heterologous DNA.
[0049] Techniques useful for isolating and characterizing the
nucleic acids and proteins of the present invention are well known
to those of skill in the art and standard molecular biology and
biochemical manuals may be consulted to select suitable protocols
without undue experimentation. See, for example, Sambrook et al,
1989, "Molecular Cloning: A Laboratory Manual", 2nd ed., Cold
Spring Harbor, the content of which is herein incorporated by
reference in its entirety.
ABBREVIATIONS
[0050] Abbreviations which may be used in the present specification
include the following: aa which means amino acid(s); bp which means
base pair(s); cm which means centimeter(s); G which means gauge, h
which means hours; kb which means kilobase; IU which means
infectious units; mg which means milligram; ug which means
microgram; psi which means pound per square inch; sec which means
seconds; V which means volts; and msec which means
milliseconds.
DETAILED DESCRIPTION
[0051] The present invention is based, in part, on the discovery
that transgenic avians may be produced by in vivo transfection. In
particular, the present invention provides methods for producing
transgenic avians by delivering nucleic acid (e.g., heterologous
nucleic acid) to tissue, such as the oviduct tissue, of a live
avian. For example, transgenic avians may be produced by in vivo
electroporation of avian tissue. In another example, in vivo
transfection may be accomplished by applying nucleic acid to avian
tissue under pressure thereby transfecting the tissue. Though the
present invention is particularly useful for accomplishing in vivo
transfection, methods disclosed herein are also useful to effect or
facilitate virus transduction of avian tissue.
[0052] Avian tissue which may be transfected according to the
present invention includes, without limitation, oviduct tissue. For
example, tissue that may be transfected according to the present
methods include, without limitation, the magnum, the ovaries, the
infundibulum, and the isthmus.
[0053] The present methods may be used for the in vivo introduction
of nucleic acid (e.g., heterologous nucleic acid) in any avian or
any animal capable of laying an egg including, but not limited to,
chicken, quail, duck, turkey, goose, pheasants, parrots, finches,
hawks, crows and ratites including ostrich, emu and cassowary. In
one aspect of the invention, the present methods are believed to be
very useful for the in vivo introduction of nucleic acid into
chicken, quail and duck. In one embodiment, the present methods are
particularly useful for the in vivo introduction of nucleic acid
into chicken.
[0054] In one embodiment of the present invention, nucleic acid
enters cells of the avian, for example, cells comprising oviduct
tissue. Typically, the nucleic acid is expressed in the recipient
cells to produce a heterologous polypeptide. In one embodiment, an
expressed heterologous polypeptide is deposited in egg white
produced in the avians. In this case, without wishing to limit the
invention to any theory or mechanism of operation, it is believed
that upon transfection, the nucleic acid enters cells of the
oviduct (e.g., tubular gland cells) and may incorporate into the
genome of the cells. Subsequently, a coding region of the nucleic
acid is expressed to produce a recombinant protein which is
excreted into the lumen of the oviduct along with egg white
proteins excreted from oviduct cells (e.g., tubular gland
cells).
[0055] In vivo transfection of avian tissue may be performed on
adult avians or on immature avians. In one embodiment, an immature
avian is subject to steroid hormone treatment prior to in vivo
transfection. An immature avian can be classified as an avian from
hatchling to seventeen weeks of age. The steroid treatment may
include administration of a hormone, or combination of hormones,
which provide for a transiently increased proliferation of cells in
the magnum of the immature avian. Hormones that may be administered
include, without limitation, estrogen, progesterone, testosterone
and related hormones. In one embodiment, estrogen is administered
to the immature avian. In another embodiment, estrogen is
administered to the immature avian in conjunction with progesterone
or testosterone. Estrogen can be in any useful form. For example,
estrogen may be in the form of estrone, estradiol or
diethylstilbestrol (DES). Doses can be any useful amount. For
example, a useful does range for DES is about 0.1 mg per kilogram
of body weight to about 10 mg per kilogram of body weight once per
day. The hormone may be administered, for example, injected, with
any useful adjuvant such as vegetable oil. Further useful aspects
of hormone induction in immature avians are readily apparent and
well known to those skilled in the art.
[0056] In one useful embodiment, transfection is accomplished by
the delivery of nucleic acid to tissue of an avian wherein the
nucleic acid is under pressure. Typically, although not
exclusively, the delivery of the nucleic acid to tissue by pressure
is accomplished with the nucleic acid in a solution. In one
embodiment, the solution is applied to the tissue with pressure
exerted between the solution and the tissue. That is, an
application of force is present between the solution and the
tissue. The pressure may be any useful amount of positive pressure.
The pressure may be in a range of about 0.001 psi to 200 psi, for
example, in a range of about 0.05 psi to about 40 psi (e.g., about
0.1 psi to about 15 psi).
[0057] In one embodiment, the nucleic acid solution is applied to
the tissue under hydrostatic or hydrodynamic pressure. For example,
nucleic acid in solution may be applied to avian tissue, for
example oviduct tissue (e.g, tissue of the magnum), wherein
pressure is exerted from the weight of the solution. In another
embodiment, the pressure may be provided by an instrument such as a
syringe or any other such useful device. In any case, the pressure
results from injecting.
[0058] In one embodiment of the present invention, transfection of
an avian is accomplished by delivering a nucleic acid (e.g.,
heterologous nucleic acid) solution to avian tissue through a blood
vessel. For example, nucleic acid may be delivered through an
artery or a vein supplying blood to avian tissue, for example,
oviduct tissue. In a particularly useful embodiment, the nucleic
acid solution is provided to the tissue through the blood vessel
under pressure, i.e., injected.
[0059] For the methods of in vivo avian transfection disclosed
herein, the tissue to be transfected may be surgically exposed, for
example, removed wholly or in part from the avian, prior to the
transfection. However, exposing or removing the tissue prior to
transfection is not required. In a case where exposure of the
tissue to be transfected is desired, the avian may be prepared for
surgery by methods known in the field avian surgery. After
preparation, an incision may be made in the bird in the vicinity of
the tissue to be transfected. In the case of transfecting oviduct
tissue, a vertical incision may be made in the skin of the avian on
the left side of the abdominal cavity beginning at the junction of
the sternal rib to the breastbone and running parallel to the
breastbone. In this case, the muscle layer directly below the skin
incision may be cut following the incision line made in the skin.
The fat layer may be pulled away from the site of the incision, for
example, pulled away toward the bird's left side. The peritoneal
membrane may be incised exposing the reproductive tract. At this
point, the reproductive tract may be eternalized. Preferably, if
externalized, the reproductive tract is handled in a gentle manner.
In one useful embodiment, the first or second loop of the magnum is
stabilized by techniques known in the field of avian surgery and a
catheter is slowly introduced into one or more oviductal arteries
such as the anterior oviductal, inferior, superior and middle
uterine artery and the artery is gently occluded. In one embodiment
the artery is occluded above and/or below the catheter site. In one
embodiment, the artery is occluded on the side of the catheter
distal to the oviduct tissue.
[0060] Typically, a nucleic acid solution is injected into the
blood vessel in a manner which results in an application of
positive pressure between the nucleic acid solution and the tissue
to be transfected. The present invention contemplates the
employment of any useful amount of pressure. For example, the
pressure may range from an amount which slightly distends the
tissue exposed to the pressure to an amount of pressure less than
that required to rupture the tissue. Any mechanical device useful
for such injection may be employed in the present invention. For
example a syringe, may be used. Other methods for introduction of
the nucleic acid solution into a blood vessel, for example, an
artery in a manner which applies positive pressure to the tissue
which the nucleic acid solution comes in contact with are
contemplated, such as the use of hydrostatic or hydrodynamic
pressure. In one embodiment, where the blood vessel is occluded
above and below the site of catheter insertion, the occlusion
proximal to the tissue to be transfected (e.g., oviduct tissue) may
be removed resulting in a large and rapid flow of nucleic acid
solution to the tissue to be transfected.
[0061] A period of time after exposure of the tissue to be
transfected to the nucleic acid solution, the blood vessel is
unblocked. The period of time between injection and unblocking of
the blood vessel may range from about one second to about 24 hours.
For example, the period of time may be in a range of about one
minute to about one hour. In one embodiment, the period of time
between injection and unblocking of the blood vessel is about two
minutes to about 6 minutes, for example, about three minutes.
[0062] The oviduct may be returned to the abdominal cavity and the
fat layer positioned to cover the reproductive tract. Both the
muscle and skin incisions may be closed utilizing simple continuous
interlocking stitches. After surgery, the hen may be allowed to
recover with free access to both feed and water.
[0063] Transfection may be performed by applying the nucleic acid
to oviduct tissue inside the lumen of the oviduct. This may be
achieved by any useful technique. In one embodiment, oviduct tissue
is exposed surgically as described above. After exposure of the
oviduct, a loop of the magnum, for example, the first loop of the
magnum, is stabilized and the oviduct is occluded above and below
the site where a hollow needle, such as a 20 gauge needle, is
introduced into the loop of the magnum. A nucleic acid solution is
injected into the lumen through the needle until expansion of the
lumen is detected. A period of time after the injection the
occlusions are removed. The period of time between injection and
removal of the occlusions may range from about one second to about
24 hours. For example, the period of time may be in a range of
about one minute to about one hour. In one embodiment, the period
of time between removal of the occlusions is about two minutes to
about 6 minutes, for example, about three minutes.
[0064] The application of the nucleic acid solution to avian tissue
by force or pressure may be accomplished by any useful technique.
For example, the nucleic acid solution may be propelled onto the
tissue in the form of a mist, or a stream or in a spray.
[0065] It is not required that the nucleic acid be in solution when
being delivered to avian tissue in accordance with the present
invention. For example, the nucleic acid may be in a substantially
dry form such as a precipitate which is delivered to the avian
tissue (e.g., oviduct tissue) by force. For example, the nucleic
acid may be delivered to avian tissue by a gene gun. In such a case
the nucleic acid may be associated with a carrier, for example
microspheres.
[0066] In vivo transfection of avian tissue (e.g., oviduct tissue)
may be accomplished by in vivo electroporation. In one embodiment,
the avian tissue (e.g., oviduct tissue) to be electroporated is
exposed surgically prior to electroporation. In one embodiment,
after exposure of the oviduct, a loop of the magnum, for example,
the first loop of the magnum, is stabilized and the oviduct is
occluded above and below the site where a 20 gauge needle is
introduced into the loop of the magnum. A nucleic acid solution may
be injected into the lumen through the needle. In one embodiment, a
volume of nucleic acid solution sufficient to distend the lumen is
injected into the lumen. In another embodiment, a volume of nucleic
acid solution sufficient to substantially fill the lumen is
injected into the lumen; however, filling of the lumen, or
substantial filling of the lumen, with the nucleic acid solution
may not be required for the present electroporation methods. An
electrical current can be applied to the occluded area of the
magnum which holds the injected nucleic acid. Any voltage useful
for electroporating avian tissue may be used. For example, the
voltage may range from about 10 V to about 10,000, for example,
about 20 V to about 600 V. In one particularly useful embodiment,
the voltage ranges from about 100 V to about 250 V. The current may
be applied to the tissue for any useful amount of time. For
example, the current may be applied for a period of time in a range
of about 0.001 msec to about 1 h. For example, the current may be
applied for about 0.01 msec to about 1 sec (e.g., about 0.1 msec to
about 0.5 sec). In one particularly useful embodiment the current
is applied for about 10 to about 20 msec. The current may be
administered any number of useful times. In one embodiment, the
current is administered between 1 and about 1,000,000 times per
site of administration. For example, the current may be
administered between 1 and about 100,000 times (e.g., 1 to about
1000 times) per site of administration. In one particularly useful
embodiment, the current is administered between 1 and about 100
times per site of administration, for example between 4 and 24
times per site of administration.
[0067] The nucleic acid may be, for example, and without
limitation, a plasmid or construct that incorporates a region
capable of expressing a protein or selected polypeptide in a
recipient cell. The nucleic acid may comprise elements that
regulate or are necessary for the transcription or translation of
nucleic acid (e.g., heterologous nucleic acid) in the transfected
tissue of the avian. The nucleic acid may be in a physiologically
acceptable solution and may further comprise agents to facilitate
the uptake of the nucleic acid by the recipient cells of the
oviduct such as, but not limited to, polyanionic substances such as
PEI, lipofectin, liposomes and the like.
[0068] Any useful concentration of nucleic acid may be used in the
present methods. For example, the nucleic acid may be present in
the solution in a range of about 1 ug per ml to an amount where the
solution is saturated with the nucleic acid. In one embodiment, the
nucleic acid is present in solution in a range of about 10 ug per
ml to about 10 mg per ml, for example, about 0.1 mg per ml to about
2 mg per ml.
[0069] The nucleic acid solution may include salts such as sodium
chloride at a useful concentration, buffers such as a phosphate
buffer at a useful concentration or sugars such as glucose at a
useful concentration. Any useful volume of nucleic acid solution
may be used in accordance with the present methods. In one
embodiment, a useful volume is about 0.05 ml to about 200 ml. For
example, a useful volume may be about 0.5 ml to about 50 ml (e.g.,
about 1 ml to about 20 ml).
[0070] Any tissue of an avian may be transfected using the present
methods as is obvious to a practitioner of ordinary skill in the
field. For example, testicular tissue or oviduct tissue may be
transfected by methods of the present invention. The nucleic acid
transfected into the avian tissue (e.g., oviduct tissue) may
include a nucleotide region which encodes a useful biological
product such as, a pharmaceutical protein.
[0071] The present invention can be useful for the production of
many biological molecules such as hormones including cytokines
(i.e., secreted polypeptides that affect a function of cells and
modulates an interaction between cells in an immune, inflammatory
or hematopoietic response), antibodies and other useful
pharmaceutical molecules which include polypeptides. Cytokines
includes, but are not limited to, monokines and lymphokines.
Examples of cytokines include, but are not limited to, interferon
.alpha.2b, Interleukin-1 (IL-1), Interleukin-6 (IL-6),
Interleukin-8 (IL-8), Tumor Necrosis Factor-.alpha. (TNF-.alpha..)
and Tumor Necrosis Factor .beta. (TNF-.beta.), antibodies such as
polyclonal and monoclonal antibodies and fragments thereof, and
immunologic binding equivalents thereof. Antibodies may include,
but are not limited to polyclonal antibodies, monoclonal antibodies
(MAbs), humanized or chimeric antibodies, single chain antibodies,
FAb fragments, F(Ab').sub.2 fragments, fragments produced by a FAb
expression library, anti-idiotypic (anti-Id) antibodies, and
epitope-binding fragments thereof. Also contemplated is the
production of antibody fusion proteins, for example, Fc fusion
proteins in accordance with the present methods. The methods of the
present invention can also be useful for producing immunoglobulin
polypeptides which are constituent polypeptides of an antibody or a
polypeptide derived therefrom. An "immunological polypeptide" may
be, but is not limited to, an immunological heavy or light chain
and may include a variable region, a diversity region, joining
region and a constant region or any combination, variant or
truncated form thereof. Immunological polypeptides also include
single-chain antibodies comprised of, but not limited to, an
immunoglobulin heavy chain variable region, an immunoglobulin light
chain variable region and optionally a peptide linker.
[0072] Certain specific examples of pharmaceutical proteins which
are contemplated for production as disclosed herein include, with
out limitation, Factor VIII (e.g., Recombinate.RTM., Bioclate.RTM.,
Kogenate.RTM., Helixate.RTM. (Centeon), B-domain deleted Factor
VIII (e.g., ReFacto.RTM.), Factor VIIa (e.g., NovoSeven.RTM.),
Factor IX (e.g., Benefix.RTM.), anticoagulant; recombinant hirudin
(e.g., Revasc.RTM., Refludan.RTM.) Alteplase, tPA (e.g.,
Activase.RTM.), Reteplase, tPA, tPA-3 of 5 domains deleted,
Ecokinase.RTM., Retavase.RTM., Rapilysin.RTM., insulin (e.g.,
Humulin.RTM., Novolin.RTM., Insuman.RTM.) insulin lispro (e.g.,
Humalog.RTM.), Bio Lysprol, Liprolog.RTM.), insulin Aspart,
iNovoRapid.RTM., insulin glargine, long-acting insulin analog
(e.g., Lantus.RTM.), rhGH (e.g., Protropin.RTM., Humatrope.RTM.,
Nutropin.RTM., BioTropin.RTM., Genotropin.RTM., Norditropin.RTM.,
Saizen.RTM., Serostim.RTM.), glucagons (e.g., Glucagen.RTM.), TSH
(e.g., Thyrogeng, Gonal F.RTM., Puregon.RTM.), follitropin-beta,
FSH (e.g., Follistim.RTM.), EPO (e.g., Epogen.RTM., Procrit.RTM.,
Neorecormon.RTM., GM-CSF (e.g., Leukine.RTM., Neupogen.RTM.), PDGH
(e.g., Regranex.RTM.), IFN alpa2a (e.g., Roferon A.RTM.), INF-apha
(e.g., Infergen.RTM.), IFN alpa2, IFN alpa2a, IFN alpa2b (e.g.,
Intron A.RTM., Alfatronol.RTM., Virtron.RTM.), ribavirin &
INF-alpha 2b (e.g., Robetron.RTM.), IFN-beta, INF-beta 1b, (e.g.,
Betaferon.RTM.), IFN-beta 1a (e.g., Avonex.RTM., Rebif.RTM.),
IFN-gamma, IFN-gammalb (e.g., Actimmune.RTM.), IL-2 (e.g.,
Proleukin.RTM.) rIL-11 (e.g., Neumega.RTM.), rHBsAg (e.g.,
Recombivax.RTM.), Combination vaccine containing HBsAgn as one
component (e.g., Comvax.RTM., Tritarix.RTM., Twinrix.RTM.,
Primavax.RTM., Procomax.RTM.), OspA, a lipoprotein found on the
surface of B burgoeri (e.g., Lymerix.RTM.), murine MAb directed
against t-lymphocyte antigen CD3 (e.g., Orthoclone OKT3.RTM.g),
murine MAb directed against TAG-72, tumor-associated glycoprotein
(e.g., OncoScint CR/OV.RTM.), FAb fragments derived from chimeric
MAb, directed against platelet surface receptor GPII(b)/III(a)
(e.g., ReoPro.RTM.), murine MAb fragment directed against
tumor-associated antigen CA125 (e.g., Indimacis.RTM.), murine MAb
fragment directed against human carcinoembryonic antigen, CEA
(e.g., CEA-scan.RTM.), murine MAb fragment directed against human
cardiac myosin (e.g., MyoScint.RTM.), murine MAb fragment directed
against tumor surface antigen PSMA (e.g., ProstaScint.RTM.), murine
MAb fragments (FAb/FAb2 mix) directed against HMW-MAA (e.g.,
Tacnemab.RTM.), murine MAb fragment (FAb) directed against
carcinoma-associated antigen (e.g., Verluma.RTM.), MAb fragments
(FAb) directed against NCA 90, a surface granulocyte nonspecific
cross reacting antigen (e.g., LeukoScan.RTM.), chimeric MAb
directed against CD20 antigen found on surface of B lymphocytes
(e.g., Rituxan.RTM.), humanized MAb directed against the alpha
chain of the IL2 receptor (e.g., Zenapax.RTM.), chimeric MAb
directed against the alpha chain of the IL2 receptor (e.g.,
Simulect.RTM.), chimeric MAb directed against TNF-alpha (e.g.,
Remicade.RTM.), humanized MAb directed against an epitope on the
surface of respiratory synctial virus (e.g., Synagis.RTM.),
humanized MAb directed against HER 2, i.e., human epidermal growth
factor receptor 2 (e.g., Hercepting), human MAb directed against
cytokeratin tumor-associated antigen (e.g., Humaspect.RTM.),
anti-CTLA4, chimeric MAb directed against CD 20 surface antigen of
B lymphocytes (e.g., Mabthera.RTM.), domase-alpha DNAse (e.g.,
Pulmozyme.RTM.), beta glucocerebrosidase (e.g., Cerezyme.RTM.),
TNF-alpha (e.g., Beromun.RTM.), IL-2-diptheria toxin fusion protein
that targets cells displaying a surface IL-2 receptor (e.g.,
Ontak.RTM.), TNFR-lgG fragment fusion protein (e.g., Enbrelg),
Laronidase, Recombinant DNA enzyme, (e.g., Aldurazyme.RTM.),
Alefacept, Amevive.RTM.&, Darbepoetin alfa (Colony stimulating
factor) (e.g., Aranesp.RTM.), Tositumomab and iodine 1 131
tositumomab, murine MAb, Bexxar.RTM., Alemtuzumab, Campath.RTM.,
Rasburicase, Elitek.RTM.), Agalsidase beta, Fabrazyme.RTM.,
FluMist.RTM., Teriparatide, Parathyroid hormone derivative (e.g.,
Forteo.RTM.), Enfuvirtide Fuzeon.RTM., Adalimumab (IgG1) (e.g.,
Humira.RTM.), Anakinra, Biological modifier (e.g., Kineret.RTM.),
nesiritide, Human B-type natriuretic peptide (hBNP) (e.g.,
Natrecor.RTM.), Pegfilgrastim, Colony stimulating factor (e.g.,
Neulasta.RTM.), Pegvisomant, human growth hormone receptor
antagonist, (e.g., Somavert.RTM.), recombinant activated protein C
(e.g., Xigris.RTM.), Omalizumab, Immunoglobulin E (lgE) blocker
(e.g., Xolairg) and lbritumomab tiuxetan (murine MAb) (e.g.,
Zevalin.RTM.).
[0073] Other exemplary therapeutic proteins for production in
accordance with the invention include, without limitation,
erythropoietin, GM-CSF, interferon .beta., fusion protein, CTLA4-Fc
fusion protein, growth hormones, cytokines, structural proteins,
interferon, lysozyme, .beta.-casein, albumin, .alpha.-1
antitrypsin, antithrombin III, collagen, factors VIII, IX, X (and
the like), fibrinogen, lactoferrin, protein C, tissue-type
plasminogen activator (tPA), somatotropin, and chymotrypsin,
immunoglobulins, antibodies, immunotoxins, factor VIII, b-domain
deleted factor VIII, factor VIIa, factor IX, anticoagulants;
hirudin, alteplase, tpa, reteplase, tpa, tpa--3 of 5 domains
deleted, insulin, insulin lispro, insulin aspart, insulin glargine,
long-acting insulin analogs, glucagons, tsh, follitropin-beta, fsh,
pdgh, inf-beta, inf-alpha 1, ifn-alpha 2, inf-beta, inf-beta 1b,
ifn-beta 1a, ifn-gamma, ifn-gamma 1b, il-2, il-11, hbsag, ospa,
domase-alpha dnase, beta glucocerebrosidase, tnf-alpha,
il-2-diptheria toxin fusion protein, tnfr-lgg fragment fusion
protein laronidase, dnaases, alefacept, tositumomab, murine mab,
alemtuzumab, rasburicase, agalsidase beta, teriparatide,
parathyroid hormone derivatives, adalimumab (lggl), anakinra,
nesiritide, human b-type natriuretic peptide (hbnp), colony
stimulating factors, pegvisomant, human growth hormone receptor
antagonist, recombinant activated protein c, omalizumab,
immunoglobulin e (lge) blocker, lbritumomab tiuxetan, ACTH,
glucagon, somatostatin, somatotropin, thymosin, parathyroid
hormone, pigmentary hormones, somatomedin, luteinizing hormone,
chorionic gonadotropin, hypothalmic releasing factors, etanercept,
antidiuretic hormones, prolactin and thyroid stimulating hormone,
an immunoglobulin polypeptide, immunoglobulin polypeptide D region,
immunoglobulin polypeptide J region, immunoglobulin polypeptide C
region, immunoglobulin light chain, immunoglobulin heavy chain, an
immunoglobulin heavy chain variable region, an immunoglobulin light
chain variable region and a linker peptide.
[0074] The present invention contemplates the use of certain
compositions and methods disclosed in U.S. patent application Ser.
No. 10/790,455, now abandoned, and in U.S. patent application Ser.
No. 10/811,136, now abandoned, the disclosures of which are
incorporated in their entirety herein by reference, for use in
combination with the present methods. For example, and without
limitation, integrase or nucleic acid which encodes integrase may
be used in combination with the present invention. In addition,
other agents which facilitate transfection or chromosome
integration disclosed in the patents and patent applications
incorporated by reference herein may be used in combination with
the present methods.
[0075] Certain gene expression controlling regions, such as
promoters, may be included in nucleic acid which is transfected
into avian tissue in accordance with the present methods. Any
useful gene expression controlling region or coding region may be
used in accordance with the present invention. Examples include,
without limitation, those gene expression controlling regions or
coding regions disclosed in US patent publication No. 2004/0210954,
published Oct. 21, 2004; US patent publication No. 2004/0255345,
published Dec. 16, 2004; U.S. Pat. No. 6,730,822, issued May 4,
2004, U.S. Pat. No. 7,049,480, issued May 23, 2006, U.S. Pat. No.
6,875,588, issued, Apr. 5, 2005, U.S. Pat. No. 7,135,562, issued
Nov. 14, 2006, and US patent publication No. 2006/0130170,
published Jun. 15, 2006. The disclosures of each of these four
issued patents and three published patent applications are
incorporated herein in their entirety by reference.
[0076] The methods of the invention are useful for expressing
nucleic acid sequences that are optimized for expression in avian
cells and which encode desired polypeptides or derivatives and
fragments thereof. Derivatives include, for instance, polypeptides
with conservative amino acid replacements, that is, those within a
family of amino acids that are related in their side chains. These
families are commonly known as acidic, basic, nonpolar, and
uncharged polar amino acid families. Phenylalanine, tryptophan, and
tyrosine are sometimes classified jointly as aromatic amino acids
and other groupings are known in the art (see, for example,
"Biochemistry", 2nd ed, L. Stryer, ed., W.H. Freeman & Co.,
1981). Peptides in which more than one replacement is made can
readily be tested for activity in the same manner as derivatives
with a single replacement, using conventional polypeptide activity
assays (e.g., for enzymatic or ligand binding activities).
[0077] If the nucleic acid molecules are transfected into a
recipient chicken cell, the sequence of a nucleic acid insert to be
expressed can be optimized for chicken codon usage. This may be
determined from the codon usage of at least one, and preferably
more than one, protein expressed in a chicken cell according to
well known principles. For example, in the chicken the codon usage
could be determined from the nucleic acid sequences encoding the
proteins such as lysozyme, ovalbumin, ovomucin and ovotransferrin
of chicken. Optimization of the sequence for codon usage can
elevate the level of translation in avian eggs.
[0078] The present invention further relates to methods for gene
expression by avian cells from nucleic acid vectors, and transgenes
derived therefrom, that include more than one polypeptide-encoding
region wherein, for example, a first polypeptide-encoding region
can be operatively linked to an avian promoter and a second
polypeptide-encoding region is operatively linked to an Internal
Ribosome Entry Sequence (IRES). It is contemplated that the first
polypeptide-encoding region, the IRES and the second
polypeptide-encoding region of a recombinant DNA of the present
invention may be arranged linearly, with the IRES operably
positioned immediately 5' of the second polypeptide-encoding
region. This nucleic acid construct, when inserted into the genome
of an avian cell or a bird and expressed therein, will generate
individual polypeptides that may be post-translationally modified
and combined in the white of a hard shell bird egg. Alternatively,
the expressed polypeptides may be isolated from an avian egg and
combined in vitro.
[0079] The invention, therefore, includes methods for producing
multimeric proteins including immunoglobulins, such as antibodies,
and antigen binding fragments thereof. Thus, in one embodiment of
the present invention, the multimeric protein is an immunoglobulin,
wherein the first and second heterologous polypeptides are
immunoglobulin heavy and light chains respectively. Illustrative
examples of this and other aspects of the present invention for the
production of heterologous multimeric polypeptides in avian cells
are fully disclosed in US patent publication No. 20020108132,
published Aug. 8, 2002, and U.S. Pat. No. 7,312,374, issued Dec.
25, 2007, the disclosures of each of which are incorporated herein
by reference in their entirety.
[0080] Accordingly, the invention further provides immunoglobulin
and other multimeric proteins that have been produced by transgenic
avians of the invention.
[0081] The present invention is further illustrated by the
following examples, which are provided by way of illustration and
should not be construed as limiting. The contents of all
references, published patents and patents cited throughout the
present application are hereby incorporated by reference in their
entireties.
[0082] It will be apparent to those skilled in the art that various
modifications, combinations, additions, deletions and variations
can be made in the present invention without departing from the
scope or spirit of the invention. For instance, features
illustrated or described as part of one embodiment can be used in
another embodiment to yield a still further embodiment. It is
intended that the present invention covers such modifications,
combinations, additions, deletions and variations as come within
the scope of the appended claims and their equivalents.
EXAMPLE 1
Expression of Luciferase and Green Fluorescent Protein in
Transgenic Chickens Produced by In Vivo Transfection by
Electroporation in the Presence of Integrase Encoding DNA
[0083] Plasmid DNA encoding luciferase, plasmid DNA encoding GFP,
each also encoding an attB site, and plasmid DNA encoding .PHI.
C-31 integrase, the coding sequence of each plasmid operably linked
to a CMV promoter, were prepared using a Qiagen endotoxin-free
plasmid purification kit. Each of the plasmid preparations was
suspended in 150 mM NaCl prior to use.
[0084] Five sexually mature hens (17-18 weeks) were prepared for
surgery. After oviposition of an egg, each hen was anesthetized
using isofluorane gas. The bird was placed in a supine position and
feathers removed from the left abdominal and leg area. The surgical
area was scrubbed with betadine and rinsed with 70% Alcohol. A
surgical drape was placed over the bird with the surgical field
exposed. A 5-7 cm vertical incision was made in the skin on the
left side of the abdominal cavity beginning at the junction of the
sternal rib to the breastbone and running parallel to the
breastbone. The muscle layer directly below the skin incision was
cut following the same lines as above. The fat layer was pulled
leftward and the thin peritoneal membrane was punctured. The
reproductive tract was located and gently externalized.
[0085] The first loop of the magnum was stabilized and the oviduct
was gently occluded about 2-3 cm above and below the site where a
20 gauge needle was introduced into the loop. A solution comprising
100 ug/ml of plasmid encoding GFP, 400 ug/ml of plasmid encoding
luciferase and 500 ug/ml of plasmid DNA encoding .PHI. C-31
integrase was injected into the lumen through the needle
substantially filling the lumen.
[0086] Electrical current was applied directly to, and to areas
surrounding, the injection site. The voltage ranged from 100 V to
250 V and was applied for 10 to 20 msec. The current was
administered 4 to 24 times per site.
[0087] The oviduct was returned to the abdominal cavity and the fat
layer was manipulated so that it covered the entire reproductive
tract. Both the muscle and skin incisions were closed utilizing
simple continuous interlocking stitches and the hens were allowed
to recover with free access to both feed and water. Two days later
the hens were sacrificed and the electroporated tissue was examined
for the presence of GFP or luciferase. Four of the five birds
tested positive for luciferase production in the tissue of the
magnum and three of the five birds tested positive for GFP in the
tissue of the magnum.
EXAMPLE 2
Expression of Human Interferon Alpha in Transgenic Chickens
Produced by In Vivo Transfection by Electroporation in the Presence
of Integrase Encoding DNA
[0088] Plasmid DNA which includes an attB site and encodes human
interferon operably linked to a CMV promoter and plasmid DNA
encoding .PHI. C-31 integrase operably linked to a CMV promoter
were prepared using a Qiagen endotoxin-free plasmid purification
kit. Each plasmid DNA was suspended in 150 mM NaCl prior to
use.
[0089] Two prepubertal hens, age approximately 14 weeks, were moved
from 12 hours of light exposure per day to 18 hours of light
exposure per day two days prior to the beginning of hormone
induction. Hens were injected with 1.0 mg of diethylstilbestrol and
0.8 mg of progesterone each day for three days prior to
electroporation, the day of electroporation and two days after the
electroporation for a total of six days.
[0090] For each bird, the reproductive tract was surgically
externalized as described in Example 1. The magnum was stabilized
and a 20 G needle was introduced into the lumen. A solution
comprising 200 ug/ml of plasmid encoding human interferon and 500
ug/ml of plasmid DNA encoding .PHI. C-31 integrase was injected
into the lumen through the needle until expansion of the lumen was
detected. Electrical current was applied to 8-10 sites per bird.
Voltage ranged from 150 V to 200 V for 10-20 msec with 16-24 pulses
per site. The oviduct was returned to the abdominal cavity and the
incisions were closed as described in Example 1. The hens were
allowed to recover with free access to both feed and water. After
hormone treatment was ceased, the birds were allowed to develop to
sexual maturity.
[0091] Eggs were collected from the birds and the egg white was
subjected to ELISA analysis for interferon. Both birds produced
eggs in which human interferon was detected.
EXAMPLE 3
Expression of Human Interferon Alpha in Transgenic Chickens
Produced by In Vivo Transfection by DNA Injection into an
Artery
[0092] DNA preparation: Plasmid DNA encoding human interferon alpha
operably linked to a CMV promoter was prepared using a Qiagen
endotoxin-free plasmid purification kit. 0.15 milligrams of plasmid
DNA was suspended in 2 ml of a 5% glucose solution.
[0093] Fifteen adult chickens were transfected with DNA encoding
human interferon alpha operably linked to a CMV promoter. For each
bird the reproductive tract was located and gently externalized as
described in Example 1. The first or second loop of the magnum was
stabilized and an oviductal artery such as the anterior oviductal,
inferior, superior and middle uterine artery was located. A
pediatric catheter was slowly introduced into the artery and the
artery was gently occluded about 2-3 cm both below and above the
catheter. The DNA solution was slowly injected into the artery. A
few minutes after injection the catheter was removed and the artery
was unblocked. The oviduct was returned to the abdominal cavity and
the incisions were closed as described in Example 1. The hens were
allowed to recover with free access to both feed and water.
[0094] Egg white from eggs laid after oviduct injection were
analyzed for the presence of interferon by ELISA. Four of the
fifteen adult chickens transfected with plasmid DNA encoding human
interferon alpha operably linked to a CMV promoter laid eggs with
detectable levels of human interferon present in the egg white. Six
of the fourteen adult chickens transfected with plasmid DNA
encoding human interferon alpha operably linked to a 10 kb chicken
ovomucoid promoter laid eggs with interferon present in the egg
white.
EXAMPLE 4
Expression of a Human Monoclonal Antibody in Transgenic Chickens
Produced by In Vivo Transfection by Electroporation in the Presence
of Integrase Encoding DNA
[0095] Plasmid DNA which includes an attB site and encoding a human
monoclonal antibody operably linked to an ovomucoid promoter and
plasmid DNA encoding .PHI. C-31 integrase operably linked to a CMV
promoter were prepared using a Qiagen endotoxin-free plasmid
purification kit. Each plasmid DNA was suspended in 150 mM NaCl
prior to use.
[0096] Four prepubertal hens, age approximately 14 weeks, were
moved from 12 hours of light exposure per day to 18 hours of light
exposure per day 2 days prior to the beginning of hormone
induction. Hens were injected with 1 mg of diethylstilbestrol and
0.8 mg of progesterone each day for three days prior to
electroporation, the day of electroporation and two days after the
electroporation treatment for a total of six days.
[0097] For each bird, the reproductive tract was surgically
externalized as described in Example 1. The magnum was stabilized
and a 20 G needle was introduced into the lumen. A solution
comprising 200 ug/ml of plasmid encoding a human antibody and 500
ug/ml of plasmid DNA encoding .PHI. C-31 integrase was injected
into the lumen through the needle until expansion of the lumen was
detected. Electrical current was applied to 8-10 sites per bird.
Voltage ranged from 150 V to 200 V for 10-20 msec with 16-24 pulses
per site. The oviduct was returned to the abdominal cavity and the
incisions were closed as described in Example 1. The hens were
allowed to recover with free access to both feed and water. After
hormone treatment was ceased, the birds were allowed to develop to
sexual maturity.
[0098] One of the four hens did not lay eggs. Eggs were collected
from the remaining birds and the egg white was subjected to ELISA
analysis for the human antibody. Antibody was detected in the egg
white of two of the three hens analyzed.
EXAMPLE 5
Expression of Human Interferon Alpha in Transgenic Chickens
Produced by Mature Lumen Injection
[0099] Plasmid DNA encoding human interferon alpha operably linked
to a CMV promoter was prepared using a Qiagen endotoxin-free
plasmid purification kit. The plasmid DNA was suspended in 150 mM
NaCl prior to use.
[0100] The reproductive tract of two adult hens was surgically
externalized as described in Example 1. The magnum was stabilized
and a 20 G needle was introduced into the lumen. A solution
comprising 200 ug/ml of plasmid encoding human interferon alpha and
in 5% PEI was injected into the lumen through the needle until
expansion of the lumen was detected. The oviduct was returned to
the abdominal cavity and the incisions were closed as described in
Example 1. The hens were allowed to recover with free access to
both feed and water
[0101] Eggs were collected from the birds and the egg white was
subjected to ELISA analysis for human interferon. Interferon was
detected in the egg white of one of the two hens.
EXAMPLE 6
Expression of Human Interferon Alpha in Transgenic Chickens
Produced by In Vivo Transfection by Electroporation
[0102] Plasmid DNA encoding human interferon alpha operably linked
to a CMV promoter is prepared using a Qiagen endotoxin-free plasmid
purification kit. The DNA is suspended in 150 mM NaCl prior to
use.
[0103] Twelve prepubertal hens, age approximately 14 weeks, are
moved from 12 hours of light exposure per day to 18 hours of light
exposure per day 2 days prior to the beginning of hormone
induction. The hens are injected with 1 mg of diethylstilbestrol
and 0.8 mg of progesterone each day for three days prior to
electroporation, the day of electroporation and two days after the
electroporation treatment for a total of six days.
[0104] For each bird, the reproductive tract is surgically
externalized as described in Example 1. The magnum is stabilized
and a 20 G needle is introduced into the lumen. 1 ml of a solution
comprising 200 ug/ml of plasmid encoding human interferon is
injected into the lumen through the needle. Electrical current is
applied to 8-10 sites per bird. Voltage ranges from 150 V to 200 V
for 10-20 msec with 16-24 pulses per site. The oviduct is returned
to the abdominal cavity and the incisions are closed as described
in Example 1. The hens are allowed to recover with free access to
both feed and water. After hormone treatment is ceased, the birds
are allowed to develop to sexual maturity.
[0105] Eggs are collected from the birds and the egg white is
subjected to ELISA analysis for interferon. Eggs are produced in
which interferon is detected by three of the twelve birds in the
study.
EXAMPLE 7
Expression of a Monoclonal Antibody Directed Against the Alpha
Chain of an IL-2 Receptor in Transgenic Chickens Produced by In
Vivo Electroporation
[0106] Plasmid DNA encoding a monoclonal antibody directed against
the alpha chain of the IL-2 receptor operably linked to an avian
lysozyme promoter is prepared using a Qiagen endotoxin-free plasmid
purification kit. The DNA is suspended in 150 mM NaCl prior to
use.
[0107] Eight prepubertal hens, age approximately 14 weeks, are
moved from 12 hours of light exposure per day to 18 hours of light
exposure per day 2 days prior to the beginning of hormone
induction. The hens are injected with 1 mg of diethylstilbestrol
and 0.8 mg of progesterone each day for three days prior to
electroporation, the day of electroporation and 2 days after the
electroporation treatment for a total of six days.
[0108] For each bird, the reproductive tract is surgically
externalized as described in Example 1. The magnum is stabilized
and a 20 G needle is introduced into the lumen. A solution
comprising 200 ug/ml of plasmid encoding a monoclonal antibody
directed against the alpha chain of the IL-2 receptor is injected
into the lumen through the needle until expansion of the lumen is
detected. Electrical current is applied to 8-10 sites per bird.
Voltage ranges from 150 V to 200 V for 10-20 msec with 16-24 pulses
per site. The oviduct is returned to the abdominal cavity and the
incisions are closed as described in Example 1. The hens are
allowed to recover with free access to both feed and water. After
hormone treatment is ceased, the birds are allowed to develop to
sexual maturity.
[0109] Eggs are collected from the birds and the egg white is
subjected to ELISA analysis for monoclonal antibody directed
against the alpha chain of the IL-2 receptor. Three of the eight
birds produce eggs in which the monoclonal antibody is
detected.
EXAMPLE 8
Expression of Human Erythropoietin in Transgenic Chickens Produced
by Mature Lumen Injection
[0110] Plasmid DNA encoding human EPO operably linked to an
ovomucoid promoter is prepared using a Qiagen endotoxin-free
plasmid purification kit. Each plasmid DNA is suspended in 150 mM
NaCl prior to use.
[0111] The reproductive tract of seven adult hens is surgically
externalized as described in Example 1. The magnum is stabilized
and a 20 G needle is introduced into the lumen. A solution
comprising 200 ug/ml of plasmid encoding a human EPO is injected
into the lumen through the needle until expansion of the lumen is
detected. The oviduct is returned to the abdominal cavity and the
incisions are closed as described in Example 1. The hens are
allowed to recover with free access to both feed and water
[0112] Eggs are collected from the birds and the egg white is
subjected to ELISA analysis for human EPO. Eggs are produced by two
of the seven birds in which EPO is detected.
EXAMPLE 9
Expression of a MAb Directed Against CD 20 Surface Antigen of B
Lymphocytes in Transgenic Chickens Produced by In Vivo
Electroporation
[0113] Plasmid DNA encoding a MAb directed against CD 20 surface
antigen of B lymphocytes operably linked to an avian lysozyme
promoter is prepared using a Qiagen endotoxin-free plasmid
purification kit. The DNA is suspended in 150 mM NaCl prior to
use.
[0114] Eight prepubertal hens, age approximately 14 weeks, are
moved from 12 hours of light exposure per day to 18 hours of light
exposure per day 2 days prior to the beginning of hormone
induction. The hens are injected with 1 mg of diethylstilbestrol
and 0.8 mg of progesterone each day for three days prior to
electroporation, the day of electroporation and 2 days after the
electroporation treatment for a total of six days.
[0115] For each bird, the reproductive tract is surgically
externalized as described in Example 1. The magnum is stabilized
and a 20 G needle is introduced into the lumen. A solution
comprising 200 ug/ml of plasmid encoding a MAb directed against CD
20 surface antigen of B lymphocytes is injected into the lumen
through the needle until expansion of the lumen is detected.
Electrical current is applied to 8-10 sites per bird. Voltage
ranges from 150 V to 200 V for 10-20 msec with 16-24 pulses per
site. The oviduct is returned to the abdominal cavity and the
incisions are closed as described in Example 1. The hens are
allowed to recover with free access to both feed and water. After
hormone treatment is ceased, the birds are allowed to develop to
sexual maturity.
[0116] Eggs are collected from the birds and the egg white is
subjected to ELISA analysis for a MAb directed against CD 20
surface antigen of B lymphocytes. Three of the eight birds produce
eggs in which the monoclonal antibody is detected.
EXAMPLE 10
Expression of a Monoclonal Antibody Directed Against TNF-Alpha in
Transgenic Chickens Produced by In Vivo Electroporation
[0117] Plasmid DNA encoding a monoclonal antibody directed against
TNF-alpha operably linked to a CMV promoter is prepared using a
Qiagen endotoxin-free plasmid purification kit. The DNA is
suspended in 150 mM NaCl prior to use.
[0118] Eight prepubertal hens, age approximately 14 weeks, are
moved from 12 hours of light exposure per day to 18 hours of light
exposure per day 2 days prior to the beginning of hormone
induction. The hens are injected with 1 mg of diethylstilbestrol
and 0.8 mg of progesterone each day for three days prior to
electroporation, the day of electroporation and 2 days after the
electroporation treatment.
[0119] For each bird, the reproductive tract is surgically
externalized as described in Example 1. The magnum is stabilized
and a 20 G needle is introduced into the lumen. A solution
comprising 200 ug/ml of plasmid encoding a monoclonal antibody
directed against TNF-alpha is injected into the lumen through the
needle until expansion of the lumen is detected. Electrical current
is applied to 8-10 sites per bird. Voltage ranges from 150 V to 200
V for 10-20 msec with 16-24 pulses per site. The oviduct is
returned to the abdominal cavity and the incisions are closed as
described in Example 1. The hens are allowed to recover with free
access to both feed and water. After hormone treatment is ceased,
the birds are allowed to develop to sexual maturity.
[0120] Eggs are collected from the birds and the egg white is
subjected to ELISA analysis for antibodies directed against
TNF-alpha. Eggs in which the human monoclonal antibody is detected
are produced by two of the eight birds.
EXAMPLE 11
Expression of a Human CTLA4 Monoclonal Antibody in Transgenic Quail
Produced by In Vivo Electroporation
[0121] Plasmid DNA encoding a human CTLA4 monoclonal antibody
operably linked to a CMV promoter is prepared using a Qiagen
endotoxin-free plasmid purification kit. The DNA is suspended in
150 mM NaCl prior to use.
[0122] Ten adult quail are prepared for surgery and the
reproductive tract is surgically externalized essentially as
described in Example 1. The magnum is stabilized and a 20 G needle
is introduced into the lumen. A solution comprising 200 ug/ml of
plasmid encoding a human CTLA4 antibody is injected into the lumen
through the needle until expansion of the lumen is detected.
Electrical current is applied to 8-10 sites per bird. Voltage
ranges from 150 V to 200 V for 10-20 msec with 16-24 pulses per
site. The oviduct is returned to the abdominal cavity and the
incisions are closed essentially as described in Example 1. The
birds are allowed to recover with free access to both feed and
water. After hormone treatment is ceased, the birds are allowed to
develop to sexual maturity.
[0123] Eggs are collected from each of the birds and the egg white
is subjected to ELISA analysis for human CTLA4 antibody. Four of
the ten birds produce eggs in which the monoclonal antibody is
detected.
EXAMPLE 12
Expression of Human Erythropoietin in Transgenic Quail Produced by
Mature Lumen Injection
[0124] Plasmid DNA encoding human EPO operably linked to an
ovomucoid promoter is prepared using a Qiagen endotoxin-free
plasmid purification kit. Each plasmid DNA is suspended in 150 mM
NaCl prior to use.
[0125] The reproductive tract of 14 adult quail is surgically
externalized essentially as described in Example 1. The magnum is
stabilized and a 20 G needle is introduced into the lumen. A
solution comprising 200 ug/ml of plasmid encoding a human EPO is
injected into the lumen through the needle until expansion of the
lumen is detected. The oviduct is returned to the abdominal cavity
and the incisions are closed as described in Example 1. The quail
are allowed to recover with free access to both feed and water
[0126] Eggs are collected from the quail and the egg white of each
is subjected to ELISA analysis for human EPO. Eggs are produced by
three of the fourteen birds in which EPO is detected.
EXAMPLE 13
Expression of Human Interferon Alpha in Transgenic Ducks Produced
by In Vivo Transfection by Electroporation
[0127] Plasmid DNA encoding human interferon alpha is prepared
using a Qiagen endotoxin-free plasmid purification kit. The DNA is
suspended in 150 mM NaCl prior to use.
[0128] Sixteen prepubertal ducks are moved from 12 hours of light
exposure per day to 18 hours of light exposure per day 2 days prior
to the beginning of hormone induction. The ducks are injected with
1 mg of diethylstilbestrol and 0.8 mg of progesterone each day for
three days prior to electroporation, the day of electroporation and
two days after the electroporation treatment for a total of six
days.
[0129] For each bird, the reproductive tract is surgically
externalized essentially as described in Example 1. The magnum is
stabilized and a 20 G needle is introduced into the lumen. 1.0 ml
of a solution comprising 200 ug/ml of plasmid encoding human
interferon is injected into the lumen through the needle.
Electrical current is applied to 8-10 sites per bird. Voltage
ranges from 150 V to 200 V for 10-20 msec with 16-24 pulses per
site. The oviduct is returned to the abdominal cavity and the
incisions are closed as described in Example 1. The ducks are
allowed to recover with free access to both feed and water. After
hormone treatment is ceased, the birds are allowed to develop to
sexual maturity.
[0130] Eggs are collected from the birds and the egg white is
subjected to ELISA analysis for Interferon. Eggs are produced in
which the interferon is detected by four of the sixteen birds in
the study.
EXAMPLE 14
Expression of Antibody Directed Against TNF-alpha in Transgenic
Ducks Produced by Mature Lumen Injection
[0131] Plasmid DNA encoding an antibody directed against TNF-alpha
operably linked to an ovomucoid promoter is prepared using a Qiagen
endotoxin-free plasmid purification kit. Each plasmid DNA is
suspended in 150 mM NaCl prior to use.
[0132] The reproductive tract of eighteen adult ducks is surgically
externalized essentially as described in Example 1. The magnum is
stabilized and a 20 G needle is introduced into the lumen. A
solution comprising 200 ug/ml of plasmid encoding an antibody
directed against TNF-alpha is injected into the lumen through the
needle until expansion of the lumen is detected. The oviduct is
returned to the abdominal cavity and the incisions are closed as
described in Example 1. The ducks are allowed to recover with free
access to both feed and water.
[0133] Eggs are collected from the ducks and the egg white of each
is subjected to ELISA analysis for antibody directed against
TNF-alpha. Eggs are produced by three of the eighteen birds in
which antibody is detected.
[0134] While this invention has been described with respect to
various specific examples and embodiments, it is to be understood
that the invention is not limited thereto and that it can be
variously practiced with the scope of the following claims.
* * * * *