U.S. patent application number 12/008399 was filed with the patent office on 2009-02-12 for vectors and methods for tissue specific synthesis of proteins in eggs of transgenic hens.
This patent application is currently assigned to Michigan State University. Invention is credited to William C. MacArthur.
Application Number | 20090042299 12/008399 |
Document ID | / |
Family ID | 26692444 |
Filed Date | 2009-02-12 |
United States Patent
Application |
20090042299 |
Kind Code |
A1 |
MacArthur; William C. |
February 12, 2009 |
Vectors and methods for tissue specific synthesis of proteins in
eggs of transgenic hens
Abstract
Vectors and methods are provided for introducing genetic
material into cells of a chicken or other avian species. More
particularly, vectors and methods are provided for transferring a
transgene to an embryonic chicken cell, so as to create a
transgenic hen wherein the transgene is expressed in the hen's
oviduct and the transgene product is secreted in the hen's eggs
and/or those of her offspring. In a preferred embodiment, the
transgene product is secreted in the egg white.
Inventors: |
MacArthur; William C.; (Ann
Arbor, MI) |
Correspondence
Address: |
WILMERHALE/BOSTON
60 STATE STREET
BOSTON
MA
02109
US
|
Assignee: |
Michigan State University
East Lansing
MI
|
Family ID: |
26692444 |
Appl. No.: |
12/008399 |
Filed: |
January 9, 2008 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10935905 |
Sep 8, 2004 |
7378086 |
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12008399 |
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08844175 |
Apr 18, 1997 |
6825396 |
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10935905 |
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60019641 |
Jun 12, 1996 |
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Current U.S.
Class: |
435/456 ;
435/320.1 |
Current CPC
Class: |
A01K 2267/01 20130101;
A01K 67/0275 20130101; C12N 2740/10043 20130101; C12N 15/86
20130101; A01K 2217/20 20130101; A01K 2227/30 20130101; A01K
2217/05 20130101; C12N 15/8509 20130101 |
Class at
Publication: |
435/456 ;
435/320.1 |
International
Class: |
C12N 15/63 20060101
C12N015/63; C12N 15/79 20060101 C12N015/79 |
Claims
1. A replication-defective retroviral vector comprising: a) a
transgene; and b) control elements operatively-linked to the
transgene and capable of directing synthesis of the transgene
product in avian egg white, wherein the control elements comprise a
promoter, a 5' untranslated region and a signal sequence.
2. The vector of claim 1, wherein the replication-defective
retroviral vector is derived from REV-A.
3. The vector of claim 1, wherein the promoter is chosen from the
group consisting of the ovalbumin, lysozyme, conalbumin and
ovomucoid promoters, and combinations thereof.
4. The vector of claim 1, wherein the avian is a chicken.
5. The vector of claim 1, wherein the control elements further
comprise an enhancer.
6. The vector of claim 3, wherein the promoter comprises an
ovalbumin promoter.
7. The vector of claim 5, wherein the enhancer comprises a steroid
hormone response element.
8. The vector of claim 5, wherein the enhancer is a viral
enhancer.
9. The vector of claim 8, wherein the enhancer is a portion of
SV40.
10. A replication-defective retroviral vector comprising: a) a
transgene; and b) control elements operatively-linked to the
transgene and capable of directing synthesis of the transgene
product in avian egg yolk, wherein the control elements comprise a
promoter, a 5' untranslated region, a signal sequence and an uptake
sequence.
11. The vector of claim 10, wherein the replication-defective
retroviral vector is derived from REV-A.
12. The vector of claim 10, wherein the promoter is chosen from the
group consisting of the vitellogenin and apolipoprotein A
promoters, and combinations thereof.
13. The vector of claim 10, wherein the avian is a chicken.
14. The vector of claim 10, wherein the control elements further
comprise an enhancer.
15. The vector of claim 14, wherein the enhancer comprises a
steroid hormone response element.
16. The vector of claim 14, wherein the enhancer is a viral
enhancer.
17. The vector of claim 16, wherein the enhancer is a portion of
SV40.
18. A method for transferring a transgene to an embryonic chicken
cell, comprising the step of introducing a replication-defective
retroviral vector into the cell, wherein the vector comprises the
transgene and control elements operatively-linked thereto and
capable of directing the synthesis of the transgene product in egg
white, wherein the control elements comprise a promoter, a 5'
untranslated region and a signal sequence.
19. The method of claim 18, wherein the replication-defective
retroviral vector is derived from REV-A.
20. The method of claim 18, wherein the control elements further
comprise an enhancer.
Description
RELATED APPLICATIONS
[0001] This application is a continuation-in-part of U.S.
Application Ser. No. 60/019,641, entitled "Vector For Expression Of
Proteins Into Eggs Of Transgenic Hens," filed Jun. 12, 1996.
FIELD OF THE INVENTION
[0002] The present invention relates generally to vectors and
methods for introducing genetic material into an embryo of a
chicken and other avian species and, more particularly, to vectors
and methods for transferring a gene of interest to an embryonic
chicken cell, so as to create a transgenic hen having the gene of
interest expressed in the hen's oviduct and the gene product
secreted in the hen's eggs and/or those of her offspring.
BACKGROUND OF THE INVENTION
[0003] Since the development of recombinant DNA technology some
twenty-five years ago, the prospect of producing proteins on a
large scale, rather than extracting them from tissue where they are
naturally expressed, has become a reality. In particular, over the
last two decades, progress in the development of expression vectors
has led to the production of thousands of recombinant proteins on a
laboratory scale. Production of commercial quantities of
recombinant proteins requires often difficult and expensive scaling
up procedures, but has nonetheless also been successful. In
addition, transgenic animals including mice, rabbits, pigs, sheep,
goats and cows have been engineered to produce human
pharmaceuticals in their tissues or secretions. Houdebine, L. M.,
J. Biotechnology 34:269-287 (1994).
[0004] Although egg white is thought to be an excellent host for
recombinant protein production, preparing transgenic avians has
proven to be technically difficult due in large part to problems
involved in manipulating the chicken embryo. When oviposition
occurs, the embryo has already reached a stage corresponding to a
mammalian late blastula or early gastrula. Genetic manipulation of
the embryo during earlier development requires reintroduction to
the female or in vitro culture, both difficult procedures.
Houdebine, L. M., J. Biotechnology 34:269-287 (1994). Despite these
difficulties, transgenic chickens have been produced that are
resistant to infection by avian leukosis (Crittenden and Salter,
"Transgenic Livestock Models In Medicine And Agriculture" pp. 73-87
(Wiley-Liss (1990))), or have high levels of circulating growth
hormone. Bosselman, R. A., et al., Science 243:533-535 (1989).
[0005] Four general methods for generating transgenic avians have
been reported. One method involves excision of a developing egg
from the oviduct, microinjection of DNA near the blastoderm, and in
vitro culture of the manipulated embryo in solution and surrogate
shells. Love, J., et al., Biotechnology 12:60-63 (1994). A second
method requires the culture and transfection of primordial germ
cells, with subsequent transplantation into an irradiated recipient
near the same stage of development as the donor. Carsience et al.,
Development 117:669-675 (1993); Etches et al., Poultry Science
72:882-889 (1993). Although technically very demanding, these two
approaches are attractive because large pieces of DNA can be
transferred.
[0006] A third method involves blind injection of replication
competent retrovirus with a needle near the blastoderm of a newly
laid egg. Petropoulos, C. J., et al., J. Virol. 65:3728-3737
(1991). Although this method is the simplest, it is also limited in
that the DNA to be transferred must be approximately 2 kb or less
in size and, the method results in viremic hens which shed
infective recombinant retrovirus. Petropoulos, C. J., et al., J.
Virol. 66(6):3391-3397 (1992).
[0007] The fourth method involves a replication-defective
retroviral vector system (see, e.g., U.S. Pat. Nos. 5,162,215 and
4,650,764, hereby incorporated by reference). One of these systems
(Watanabe and Temin, Mol. Cell. Biol. 3(12):2241-2249 (1983)) has
been derived from the reticuloendotheliosis virus type A (REV-A).
Sevoian et al., Avian Dis. 8:336-347 (1964). Replication-defective
retroviral vectors derived from the REV-A virus are based on the
helper cell line C3 (Watanabe and Temin, Mol. Cell. Biol.
3(12):2241-2249 (1983)) which contains the components of a
packaging defective helper provirus. The derivation of the C3
helping line and several replication-defective retroviral vectors
have been described in detail in U.S. Pat. No. 4,650,764 and
Watanabe and Temin, Mol. Cell. Biol. 3(12):2241-2249 (1983). This
method is more technically demanding than the replication competent
technique in that the blastoderm must be exposed, and
microinjection equipment must be used. Bosselman, R. A., et al.,
Science 243:533-535 (1989). Nonetheless, it results in transgenic
hens free of replication competent retrovirus, and can transfer DNA
as large as 8 kb in size.
[0008] Tissue specific expression of a foreign gene in a transgenic
chicken was achieved using the replication competent retrovirus
technique. Petropoulos, C. J., et al, J. Virol. 66(6):3391-3397
(1992). A replication competent retrovirus was used to deliver the
reporter gene chloramphenicol acetyl transferase (CAT), driven by a
muscle specific promoter, a action, to skeletal muscle. Tissue
specific expression of a recombinant protein in the egg of a
transgenic avian has not yet been successful.
[0009] It would thus be desirable to provide a vehicle and method
for transferring a gene to an embryonic chicken cell (or other
avian species) so as to create a transgenic hen wherein the gene is
expressed in a tissue specific manner. It would also be desirable
to provide a vehicle and method for transferring a gene to an
embryonic chicken cell, wherein the gene is expressed in the hen's
oviduct and secretion of the gene product is in the hen's eggs. It
would also be desirable to provide a vehicle and method for
transferring a gene to an embryonic chicken cell, wherein the gene
is expressed in the hen's oviduct and secretion of the gene product
is in the hen's eggs without compromise to the hen's health and the
health of other birds in contact with her.
SUMMARY OF THE INVENTION
[0010] Vectors and methods are provided for introducing genetic
material into cells of a chicken or other avian species. More
particularly, vectors and methods are provided for transferring a
transgene to an embryonic chicken cell, so as to create a
transgenic hen wherein the transgene is expressed in the hen's
oviduct and the transgene product is secreted in the hen's eggs
and/or those of her offspring. In a preferred embodiment, the
transgene product is secreted in the egg white.
[0011] In one embodiment, the vector comprises a portion of a
retroviral genome, capable of transfecting a cell and incapable of
replication, i.e., a replication-defective retroviral vector. The
vector further comprises a transgene, operatively-linked to
appropriate control elements such that the transgene may be
expressed in a tissue specific manner. In one embodiment, the
control elements include an enhanced promoter directing the
expression of the transgene in the oviduct, an untranslated region
5' to the structural gene (coding region) of appropriate length and
sequence to promote efficient translation, and a signal sequence
directing the secretion of the transgene product in the egg white.
In this embodiment, the promoter may be chosen, without limitation,
from the group consisting of ovalbumin, lysozyme, conalbumin and
ovomucoid promoters, and combinations thereof. In another
embodiment, the control sequences include a promoter directing the
expression of the transgene in the liver and a signal sequence
directing the uptake and secretion of the transgene product into
the egg yolk. In this embodiment, the promoter may be chosen,
without limitation, from the group consisting of vitellogenin and
apolipoprotein A promoters, and combinations thereof.
[0012] The vectors of the present invention may be used in
producing transgenic avians, particularly chickens, by methods
known to those skilled in the art, such as the four methods
described above (see, Background Of The Invention). For example, as
described in U.S. Pat. No. 5,162,215, herein incorporated by
reference, the vectors may be used to introduce a nucleic acid
sequence, e.g., a gene, into germ cells and stem cells of an embryo
of a chicken. In one embodiment, the vector is microinjected in a
newly laid chicken egg, in close proximity to (e.g, directly
beneath) the blastoderm. The egg is then sealed and incubated until
the chicken is hatched from the egg. The transgenic chicken is then
tested for expression of the transgene and if positive and the
chicken is female (hen), the eggs of the chicken are harvested and
the protein is isolated by methods known to those skilled in the
art. If the chicken is male (rooster), it can be bred to produce a
female transgenic chicken whose eggs may then be harvested.
Transgenic avians and eggs, as well as methods of making transgenic
avians and eggs, are thus provided.
[0013] Other features and advantages of the present invention will
become apparent from the following description and appended claims,
taken in conjunction with the accompanying drawings.
BRIEF DESCRIPTION OF THE DRAWINGS
[0014] The various advantages of the present invention will become
apparent to one skilled in the art by reading the following
specification and subjoined claims and by referencing the following
drawings in which:
[0015] FIG. 1 is a schematic illustrating the production of the
vectors of the present invention and methods of using same to
produce transgenic chickens;
[0016] FIG. 2 is a schematic illustrating a preferred vector of the
present invention;
[0017] FIG. 3 is a schematic illustrating construction of the
retroviral and expression vectors of the present invention;
[0018] FIG. 4 is a schematic illustrating the construction of
intermediate #1, pOVSV;
[0019] FIG. 5 is a schematic showing the construction of
intermediate #2, pSigI;
[0020] FIG. 6 is a schematic illustrating the construction of
intermediate #3, pSigPCR;
[0021] FIG. 7 is a schematic showing the construction of
intermediate #4, pUTR;
[0022] FIG. 8 is a schematic illustrating the construction of
intermediate #5, pUTRAN;
[0023] FIG. 9 is a schematic showing the construction of
intermediate #6, pERE 1;
[0024] FIG. 10 is a schematic showing the construction of
intermediate #7, pERE (note in FIG. 10 that arrows are for
orientation of the ERE sequence within the oligonucleotides, not
the oligonucleotide itself);
[0025] FIG. 11 is a schematic illustrating the modified proviral
vector;
[0026] FIG. 12 is a schematic illustrating the modification of the
3' end of the hygromycin B phosphotransferase gene; and
[0027] FIG. 13 is a schematic showing the modification of the
N-terminus of hygromycin B phosphotransferase gene.
DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS
[0028] Vectors and methods are provided for introducing genetic
material into cells of a chicken or other avian species. More
particularly, vectors and methods are provided for transferring a
transgene to embryonic chicken cells, so as to create a transgenic
hen wherein the transgene is expressed in the hen's oviduct and the
transgene product is secreted in the hen's eggs and/or those of her
offspring. FIG. 1 is a schematic illustrating the methods of the
present invention, including vector production and use to produce a
transgenic chicken.
[0029] In one embodiment, the vector comprises a portion of a
retroviral genome, capable of transfecting a cell and incapable of
replication, i.e., a replication-defective retroviral vector.
Replication-defective retroviral vectors derived from the REV-A
virus are preferred. The vector further comprises a gene of
interest also referred to herein as a transgene, operatively-linked
to appropriate control elements such that the transgene product may
be synthesized in a tissue specific manner.
[0030] A schematic of a preferred expression vector of the present
invention is set forth in FIG. 2. It will be appreciated that the 3
kb .beta.-galactosidase gene shown in FIG. 2 is merely a reporter
gene and is replaced with any transgene(s) or fragment thereof. For
example a gene which encodes a blood clotting protein such as
fvIII, may be employed. The transgene product or protein, is
secreted in the egg and then isolated. Once purified, the protein
may be used in pharmaceutical applications such as in the treatment
of hemophilia. Other preferred genes include, without limitation,
the genes encoding blood proteins including human serum albumin and
.alpha. 1-antitrypsin, hematopoietic growth factors including
erythropoietin, and lymphopoietic growth factors such as
granulocyte colony stimulating factors. Genes encoding industrial
proteins such as .alpha.-amylase and glucose isomerase may also be
employed. Moreover, genes encoding antibodies and immunoreactive
portions thereof, may also be included in the vectors of the
present invention (see, e.g., Lilley, et al., J. Immunol. Meth.
171:211-226 (1994) and Davis et al., Biotechnol. 9:165-169 (1991),
herein incorporated by reference).
[0031] The gene, or a fragment of the gene, to be transferred may
be produced and purified by any of several methods well known in
the art. Thus, a gene can be produced synthetically, or by treating
mRNA derived from the transcription of the gene with a reverse
transcriptase so as to produce a cDNA version of the gene, or by
the direct isolation of the gene from a genomic bank or from other
sources.
[0032] Control elements which flank the transgene include promoters
and enhancers, UTRs and signal sequence(s), that allow tissue
specific expression of the transgene. In one embodiment, the
promoter directs expression of the transgene in the oviduct of the
transgenic avian. A preferred promoter of the present invention is
chosen from the group consisting of ovalbumin, lysozyme, conalbumin
and ovomucoid promoters, and combinations thereof. Signal sequences
included in the vector direct secretion of the transgene product
into the egg white. In an alternative embodiment, the promoter
drives expression of the transgene in the liver and signal
sequences included in the vector direct the secretion and uptake of
the transgene product into the egg yolk. In this embodiment, the
promoter is chosen from the group consisting of vitellogenin and
apolipoprotein A promoters, and combinations thereof. Preferred
enhancers are viral enhancers including, but not limited to, the
SV40 enhancer, or portion thereof. Lysozyme enhancers may also be
employed in addition to synthetic DNAs thought to bind
transcription factors, such as a steroid hormone response element,
e.g., the tandem EREs described herein.
[0033] In one embodiment of the present invention, shown in FIG. 2,
control elements which flank the gene of interest include the SV40
enhancer, three tandem estrogen response elements (ERE), 1.3 kb of
the ovalbumin promoter (5' flank), 77 bp of 5' untranslated region
(UTR), the N-terminal signal peptide sequence from the chicken
lysozyme gene, and the polyadenylation and termination signals from
the SV40 small T antigen. Sequence Listing 1 sets forth the
nucleotide sequence of the preferred construct. In a preferred
embodiment of the present invention, this construct is contained on
a 5 kb Xba I fragment which is inserted into a
replication-defective retroviral vector for transgenesis. The
preferred proviral vector is a derivative of plasmid pSW272.
Emerman, M., et al., Cell 39:459-467 (1984); U.S. Pat. No.
4,650,764. As described in U.S. Pat. No. 4,650,764, herein
incorporated by reference, cell lines have been constructed to
complement these vectors and produce the viral proteins necessary
to package replication-defective retroviral vectors. The packaged
vector may infect a cell once, but is incapable itself of
subsequent rounds of infection.
[0034] The vectors of the present invention are particularly useful
in producing transgenic avians, particularly chickens, by methods
known to those skilled in the art. For example, as described in
U.S. Pat. No. 5,162,215, herein incorporated by reference, the
vectors may be used to introduce a nucleic acid sequence, e.g., a
gene, into cells of an embryo of a chicken. In one embodiment, the
vector is microinjected in a newly laid chicken egg arrested at
stage X (not generally more than seven days old, unincubated), in
close proximity to, e.g., directly underneath, the blastoderm. More
specifically, an opening about 5 mm in diameter is made in the side
of the egg, normally by the use of a drilling tool fitted with an
abrasive rotating tip which can drill a hole in the eggshell
without damaging the underlying shell membrane. The membrane is
then cut out by use of a scalpel or 18 gauge needle and thumb
forceps, so that a portion of the shell and membrane is removed
thereby exposing the embryo. The embryo is visualized by eye or
with an optical dissecting microscope with a 6.times.-50.times.
magnification. A solution, usually tissue culture medium,
containing the vector of the present invention, is microinjected
into an area beneath and around the blastoderm, using a
micro-manipulator and a very small diameter needle, preferably
glass, 40-60 .mu.M outer diameter at the tip, 1 mm outer diameter
along it's length. The volume of solution for microinjection is
preferably 5-20 .mu.l. After microinjection, the egg is sealed with
shell membrane and a sealing material, preferably glue or paraffin.
The sealed egg is then incubated at approximately 38.degree. C.
(99.5.degree. F.) for various time periods up to and including the
time of hatching to allow normal embryo growth and development. DNA
from embryos and from newly hatched chicks is tested for the
presence of sequences from the microinjected vector. The presence
of the inserted sequences is detected by means known in the art and
appropriate to the detection of the specific gene or if desirable,
gene product if the gene or gene product, i.e., protein, is
present, eggs from the transgenic chicken are collected and the
protein isolated.
[0035] In another embodiment, the vector or transfected cells
producing the virus containing the transgene is injected into
developing oocytes in vivo, for example, as described in Shuman and
Shoffner, Poultry Science 65:437-1444 (1986), herein incorporated
by reference. The same steps of incubation, hatching, etc. are
followed.
[0036] As referred to herein, by the term "gene" or "transgene" is
meant a nucleic acid, either naturally occurring or synthetic,
which encodes a protein product. The term "nucleic acid" is
intended to mean natural and/or synthetic linear, circular and
sequential arrays of nucleotides and nucleosides, e.g., cDNA,
genomic DNA (gDNA), mRNA, and RNA, oligonucleotides,
oligonucleosides, and derivatives thereof. The phrase
"operatively-linked" is intended to mean attached in a manner which
allows for transgene transcription. The term "encoding" is intended
to mean that the subject nucleic acid may be transcribed and
translated into either the desired polypeptide or the subject
protein in an appropriate expression system, e.g., when the subject
nucleic acid is linked to appropriate control sequences such as
promoter and enhancer elements in a suitable vector (e.g., an
expression vector) and when the vector is introduced into an
appropriate system or cell. As used herein, "polypeptide" refers to
an amino acid sequence which comprises both full-length protein and
fragments thereof.
[0037] The term "replication-defective retroviral vector" refers to
a vector comprising a portion of a retroviral genome capable of
infecting a cell but incapable of unrestricted replication, i.e.,
multiple rounds of infection, usually due to mutations or deletions
in the virus genome. The term "REV-derived replication-defective
vector" refers to a reticuloendotheliosis viral vector that is
incapable of unrestricted replication.
[0038] The term "avian species" includes, without limitation,
chicken, quail, turkey, duck and other fowl. The term "hen"
includes all females of the avian species. A "transgenic avian"
generally refers to an avian that has had a heterologous DNA
sequence, or one or more additional DNA sequences normally
endogenous to the avian (collectively referred to herein as
"transgenes") chromosomally integrated into the germ cells of the
avian. As a result of such transfer and integration, the
transferred sequence may be transmitted through germ cells to the
offspring of a transgenic avian. The transgenic avian (including
its progeny) will also have the transgene fortuitously integrated
into the chromosomes of somatic cells.
[0039] In order to more fully demonstrate the advantages arising
from the present invention, the following examples are set forth.
It is to be understood that the following is by way of example only
and is not intended as a limitation on the scope of the
invention.
SPECIFIC EXAMPLE 1
Vector Construction
Discussion
[0040] Promoter. The protein ovalbumin is the most abundant protein
in egg white. Ovalbumin is synthesized in the tubular gland cells
of the oviduct magnum and secreted directly into the lumen, where
it joins the forming egg. The ovalbumin promoter is a well
characterized and complex promoter. Houdebine, L. M., J. Biotech
34:269-287 (1994). The ovalbumin promoter is regulated by all known
classes of steroid hormones (Gaub, M. P., et al., Cell 63:1267-1276
(1990)), and at least eight different regulatory proteins or groups
of proteins are thought to bind to a region spanning 1.1 kb 5' to
the cap site. These proteins include the TATA binding protein
complex (TFIID), the estrogen receptor, activator protein 1 (AP-1),
which includes the fos and jun gene products and related peptides
(Curran, T., et al., Cell 55:395-397 (1988)), the chicken ovalbumin
upstream promoter transcription factor (COUP-TF) (Wang, L., et al.,
Nature 340:163-166 (1989)) and an associated protein S300-II
(Sagami, I., et al., Mol. Cell. Biol. 6(12):4259-4267 (1986)), a
NF-.nu.B-like nuclear protein (Schweers, L., et al., J. Biol. Chem.
266(16):10490-10497 (1991)), and a nuclear factor I (NF-I) homolog.
Bradshaw, M. S., et al., J. Biol. Chem. 263(17):8485-8490 (1988).
The cis acting sequences responsible for these interactions are
included in the 1.3 kb fragment used as the preferred promoter in
the present invention. Although the natural system of ovalbumin
expression was mimicked as closely as possible in the vectors and
methods of the present invention, the ovalbumin 5' regulatory
region spans some 8 kb (Gaub, M. P., et al., Cell 63:1267-1276
(1990)), which, together with the other downstream elements
(LeMeur, M. A., et al., EMBO Journal 3(12):2779-2786 (1994)), is
too large for a replication-defective retroviral vector. Emerman,
M., et al., Cell 39:459-467 (1984). Thus, the 1.3 kb fragment was
used. However, it will be appreciated by those skilled in the art,
that the ovalbumin promoter may include any portion of the
ovalbumin transcription unit capable of driving expression of a
transgene in the oviduct. Moreover, although the ovalbumin promoter
is discussed in detail herein, it will be appreciated that other
promoters that drive expression in cells generating the egg white
may be employed, including but not limited to, lysozyme, conalbumin
and ovomucoid promoters, and combinations thereof.
[0041] In an alternative embodiment, a promoter which drives
expression of the transgene in the liver is employed, such as the
vitellogenin or apolipoprotein A promoter, and combinations
thereof. Although vitellogenin and apolipoprotein A are very
abundant proteins in the yolk, they are synthesized in the liver
and are then transported to the yolk through the blood. It is
deposited in the yolk via a specific receptor which recognizes an
N-proximal fragment of the vitellogenin precursor. Thus, the
vectors of the present invention, when containing the vitellogenin
or apolipoprotein A promoters (or combinations thereof), they also
contain a signal sequence or separate sequences directing the
secretion and uptake of the protein in the yolk. Although a
blood-borne intermediate step is required, this type of vector is
useful particularly for antibody production or compounds found in
blood of other species.
[0042] Enhancer. The SV40 enhancer has been previously used to
increase expression from the ovalbumin promoter. Dierich, A., et
al., EMBO Journal 6(8):2305-2312 (1987). AP-1 has been shown to act
on the proximal portion of the ovalbumin promoter, and the SV40
enhancer may increase the local concentration of the AP-1 complex
or some of its components. Curran, T., et al., Cell 55:395-397
(1988). There are other control elements found in the ovalbumin 5'
flank which are not included in the 1.3 kb ovalbumin promoter. Kaye
et al., EMBO Journal 5(2):277-285 (1986), discovered four hormone
dependent DNAase I hypersensitive sites in the 5' flank of
ovalbumin chromatin which are correlated with expression of the
ovalbumin gene. Two sites are contained within the preferred
promoter used herein, and the other two lie 3.3 kb and 6 kb 5' to
the cap site (sites III and IV respectively). Site III, at -3.3 kb
is contained on a 675 bp Pst I-Xba I fragment from approximately
3.7 kb to 3.1 kb 5' to the cap site. Within this fragment are four
half palindromic estrogen response elements (EREs) which enhance
expression from the ovalbumin promoter in a synergistic fashion.
Kato, S., et al., Cell 68:731-742 (1992). The half EREs are spaced
more than 100 base pairs apart from each other. Nonetheless, fusion
and deletion studies have shown both the functionality and
necessity of these elements in conferring estrogen responsiveness
to a truncated ovalbumin promoter. Kato, S., et al., Cell
68:731-742 (1992). It is thought that several weakly bound estrogen
receptors interact synergistically at this locus to result in more
stable receptor-DNA complexes, which then either destabilize the
helix, or increase the local concentration of transcription factors
in the vicinity of the promoter.
[0043] This region III fragment is not included in the preferred
vector of the present invention, but instead is replaced by a
synthetic oligonucleotide containing a full palindromic ERE
adjacent and 5' to a single ERE. The estrogen receptor binds
palindromic EREs as a dimer with much greater affinity than to a
single half site. The tandem arrangement of palindromic ERE and a
single ERE spaced seven base pairs away adds even further
stability. Klein-Hitapa.beta., L., et al., J. Mol. Biol.
201:537-544 (1988). It is thought that this oligonucleotide
functionally replaces the -3.3 kb hypersensitive site in vivo.
[0044] It is likely that the tandem EREs have a positive effect on
gene expression. EREs have been shown to enhance expression in
estrogen responsive cells, and with promoters containing imperfect
EREs. Tsai, S. Y., et al., Cell 57:443-448 (1989);
Ponglikitmongkol, M., et al., EMBO Journal 9(7):2221-2231 (1990).
There are imperfect EREs in the ovalbumin promoter, and it is
likely that a synergism occurs between the synthetic perfect
consensus EREs and the natural ones.
[0045] The hormone dependent DNAase I hypersensitive site at -6 kb
is contained within 1.2 kb. Fusion studies with this DNA fragment
show no evidence of estrogen responsive enhancement of the
ovalbumin promoter. Kato, S., et al., Cell 68:731-742 (1992). For
this reason, no part or analog was included in the vector shown in
FIG. 2.
[0046] Previous investigators have demonstrated an absolute
requirement for an intracellular phosphorylation cascade; via
somatomedin, insulin, or cAMP for induction of the ovalbumin gene
in response to estrogen. Evans, M. I., et al., Cell 25:187-193
(1981); Evans, M. I., et al., Endocrinology 115(1):368-377 (1984).
Although these studies are more than ten years old and the
intracellular second messenger cascade mechanisms are now
understood in greater detail, the exact mechanism with respect to
specific cis acting sequences in the ovalbumin promoter has not
been demonstrated definitely. It is not unreasonable to suggest,
however, that the mechanism involves AP-1 binding, the cis acting
sequence of which is included in both the preferred ovalbumin
promoter and the SV40 enhancer. Curran, T., et al., Cell 55:395-397
(1988).
[0047] 5' Untranslated Region. The 5' untranslated region (UTR) is
that of ovalbumin RNA. The ovalbumin gene contains a 5' leader exon
that is spliced to the first coding exon to generate an
untranslated region 65 bases in length. O'Hare, K., et al., Nucleic
Acids Research 7(2):321-334 (1979). The vector UTR sequence is
copied almost exactly off the cDNA to yield a 5'. UTR that very
closely resembles that of ovalbumin RNA. The only difference is a
one base mutation near the 5' end which was necessary for
construction, and an additional 3' linker, resulting in a UTR 77 bp
in length. A 77 base leader is more consistent with Kozak's study
which suggest that a minimum of 77 bases is required for maximum
translational efficiency (Kozak, M., et al., J. Cell Biol.
115(4):887-903 (1991)), however, any UTR with a functional sequence
around the start codon may be used.
[0048] Signal Sequence. The signal peptide is responsible for
transport of the protein out of the cell, and signal peptide
sequence theory is well developed. von Heijne, G., Eur. J. Biochem.
133:17-21 (1983); von Heijne, G., J. Mol. Bio. 173:243-251 (1984);
and von Heijne, G., J. Mol. Biol. 184:99-105 (1985). In the
majority of secreted proteins, the sequence is at the N-terminus of
the nascent protein and is cleaved during synthesis and
translocation into the endoplasmic reticulum. In the case of
ovalbumin, however, the sequence is internal to the protein and is
not cleaved (Robinson, A., et al., FEBS 203(2):243-246 (1986)),
thus rendering it inappropriate for use in an expression vector.
The signal sequence of egg white lysozyme was used in the vectors
of the present invention as a translocation signal because it is a
cleaved N-terminal sequence, it functions in vivo in the chicken
oviduct, and will release a protein with a native N-terminus in
Saccharomyces. Jigami, Y., et al., Gene 43:273-279 (1986). However,
it will be appreciated by those skilled in the art that any signal
sequence(s) may be used.
[0049] Gene. The .beta.-galactosidase gene was utilized in the
vector set forth in FIG. 2 for two reasons. First, at 3 kb, it is
the largest of the available reporter genes; many genes encoding
commercially valuable proteins are much smaller than this. Thus, if
this system can express .beta.-galactosidase into the egg, then
other genes will likewise be expressed. Second,
.beta.-galactosidase expression can be easily assayed, which
facilitates screening of eggs produced from the transgenic hens of
the present invention. It will be appreciated that any transgene(s)
or fragment thereof, may be employed.
[0050] 3' Control. Since the transgenic vector of the present
invention is a retrovirus, the genome is RNA and, a transcription
termination signal in the orientation of genome synthesis could
prematurely stop synthesis and result in low titers of retrovirus.
Other investigators have used termination and polyadenylation
signals and found relatively little effect. Bradyopadhy, P. K., et
al., Mol. Cell. Biol. 4(4):749-754 (1984). A transcription
termination signal should not disrupt genome synthesis if placed in
the opposite orientation, however, but may not benefit from the
enhancing effect of a more proximal LTR in the retroviral vector.
Therefore, both orientations of the expression vector with respect
to the retroviral vector were constructed. Standard stop codons and
the proven polyadenylation signal from the SV40 small T antigen are
included 3' to the structural gene.
Materials and Methods
[0051] Introduction. The .beta.-galactosidase gene together with
transcription termination signals and the polyadenylation signal
from the SV40 small T antigen are contained on a 3.5 kb Cla I-Xba I
fragment of the expression vector pSV.beta.-galactosidase,
purchased from Promega Inc. The ovalbumin promoter is contained on
a 1.7 kb Pst I Eco RI fragment of the plasmid pOV1.7 (sequence in
Helig, R., et al., J. Mol. Biol. 156:1-19 (1982), Genback accession
# J00895 M24999). The SV40 enhancer is contained on a 247 bp Nco
I-Eco RI fragment of the plasmid pCAT-enhancer, purchased from
Promega Inc. All other DNAs in the construct were synthesized de
novo. FIG. 3 is a schematic illustrating the construction of the
retroviral and expression vectors.
[0052] Construction of intermediate #1; pOVSV. The plasmid pOV1.7
contains a Hind III site in the first intron of the ovalbumin gene,
and a Pst I site 1.37 kb 5' to the cap site (see FIG. 4). This 1.6
kb Pst I Hind III fragment of pOV1.7 was joined to the Hind III and
Nsi I sites of pSV.beta.-galactosidase, (Nsi I has compatible ends
with Pst I), resulting in a plasmid called pOVSV, shown in FIG. 4.
pOVSV is the first of 8 intermediates generated to construct the
most complex version of the vector.
[0053] Construction of intermediate #2; pSigI. A synthetic linker
containing the nucleotide sequence encoding the signal peptide from
chicken lysozyme was inserted into the Ssp BI and Cla I sites of
pOVSV as shown in FIG. 5. The resulting plasmid is called pSigI.
The nucleotide sequence is included in FIG. 5, along with the amino
acid sequence of the signal peptide and the start codon.
[0054] Construction of intermediate #3; pSigPCR. The plasmid pSig I
contains undesirable deletions in the 3' end of the ovalbumin
promoter and in the 5' end of the .beta.-galactosidase gene. The
.beta.-galactosidase gene was restored using PCR. A 3' primer was
used that hybridizes 35 bp 3' to a unique Sac I site within the
gene. Its sequence and the design of the PCR are shown in FIG. 6.
The 5' primer hybridizes to the 5' end of the .beta.-galactosidase
gene and contains a 17 base 5' overhang containing a unique Csp 45
I site and eight 5' nucleotides. Csp 45 I digestion generates end
compatible with Cla I digestion. PCR was performed for 30 cycles,
and the products were digested with Sac I and Csp 45 and then
purified on a low-melt gel. This 1.9 kb fragment was ligated into
the unique Cla I and Sac I sites of pSig 1, restoring the
.beta.-galactosidase gene and putting it directly 3' to and in
frame with the signal sequence codons (see FIG. 6). This plasmid is
called pSigPCR, and was verified by Pvu I digestion, and subsequent
sequence analysis.
[0055] Construction of intermediate #4; pUTR. A synthetic
oligonucleotide encoding the 5' UTR of ovalbumin was ligated into
the Bgl II Ssp BI sites of pSigPCR. This oligonucleotide also
contains an Acc 65 I site near its 5' end (centered around the cap
site) to allow restoration of the promoter in the following steps
(see FIG. 7). Proper constructs were verified by Kpn I digestion.
This plasmid is called pUTR, and contains all necessary elements 3'
to the cap site.
[0056] Construction of intermediate #5; pUTRAN. The promoter was
restored by ligating a 1.4 kb Ssp BI partial-Nco I restriction
fragment containing the entire intact promoter from pOVSV into the
Nco I and Acc 65 I sites of pUTR, shown in FIG. 8. Proper
recombinants were verified by Bgl II Ssp B double digestion. This
plasmid, pUTRAN, has a 1.3 kb ovalbumin promoter driving all
necessary downstream elements of the construct.
[0057] Construction of intermediate #6; pERE 1. The tandem estrogen
response elements (ERE) are contained on a synthetic
oligonucleotide. Because the invested repeats contained within the
EREs form stem loop structures which prevent annealing into a
double stranded structure, the oligonucleotide was inserted in two
steps. The first oligonucleotide contains two EREs in the same
orientation, separated by unique Hind III and Spe I sites. This
oligonucleotide was ligated into the unique Nsi I and Nco I sites
of pUTRAN, forming the plasmid pERE 1. pERE 1 also contains Acc III
and Nco I sites useful for insertion of the SV40 enhancer, and a
terminal Xba I site to allow insertion of subsequent constructs
into the unique Xba I site of the retroviral vector. Proper
recombinants were verified by Xba I digestion and sequence
analysis
[0058] Construction of intermediate #7; pERE. A full palindromic
ERE was created by ligation of a synthetic oligonucleotide
containing the 3' half site into the unique Hind III and Spe I
sites of pERE. The resulting plasmid, pERE, contains a full
palindromic ERE and a single ERE half site spaced 7 base pairs away
(see FIGS. 9 and 10). Proper recombinants were verified by Hind III
Bgl II double digestion, since ligation of the second
oligonucleotide obliterates the unique Hind III site.
[0059] Construction of intermediate #8; pUCERE. The plasmid pERE
contains all elements of the expression vector except the SV40
enhancer. The SV40 enhancer is contained on a 247 bp Eco RI Nco I
fragment of the plasmid pCAT-enhancer, available from Promega. pERE
contains 3 Eco RI sites and 2 Nco I sites, necessitating its
subcloning into a vector which lacks these sites.
[0060] The plasmid pUC18 contains only one Eco RI sites and lacks
an Nco I site altogether. pUC18 was digested with Eco RI and Bam HI
(both in the multiple cloning site), blunted with Klenow
polymerase, and autoligated. Proper deletions were verified by Eco
RI-Sca I double digestion. The modified vector is called
pUC.DELTA.BE, and contains a unique Xba I site useful in subcloning
the construct. Subsequently, the 5 kb Xba I fragment of pERE,
containing the construct, was ligated into the modified vector at
that site. This plasmid is called pUCERE.
[0061] Construction of pWM0. pUCERE contains Nco I and Eco RI sites
5' to the EREs, and 3' to the Xba I site necessary for subcloning
into the retroviral vector. It also contains an extra Eco RI site
within the .beta.-galactosidase gene, which necessitates a partial
digestion strategy. pUCERE was partially digested with Eco RI, and
the linear band isolated. This DNA was digested with Nco I, and the
8 kb fragment recovered from a low-melt gel. The 247 bp Eco RI-Nco
I fragment of pCAT-enhancer was isolated by standard means, and
ligated to the pUCERE preparation. Proper recombinants were
verified by Xba I-Bgl II double digestion. This plasmid is called
pWV0, and contains all elements of the transgene on a 5 kb Xba I
fragment.
[0062] Construction of pBCWM. pWM0 and pSW272 both confer
ampicillin resistance to their hosts. To reduce the level of
background plasmid when subcloning into the ampicillin resistant
REV vectors, the 5 kb insert of pWM0 was cloned into the unique Xba
I site of pBCSK+, purchased from Stratagene (La Jolla, Calif.),
which confers chloramphenicol resistance to its host. pWM0 was
digested with Xba I, and the 5 kb fragment isolated from a low melt
gel. pBCSK+ was digested with Xba I, dephosphorylated, and purified
on agarose. The vector and insert fragments were ligated together,
and proper recombinants were verified by Xba I digest on cultures
grown from colonies recovered from LB chloramphenicol (34 .mu.g/ml)
plates. This plasmid contains the entire expression vector on a
background of chloramphenicol resistance, ready for insertion into
the replication defective retroviral vector.
SPECIFIC EXAMPLE 2
Retroviral Vector Design and Construction
Retroviral Vector Design
[0063] The plasmid pSW272 (Emerman and Temin, Cell 39:459-467
(1984)) contains a deletion mutant of spleen necrosis virus (SNV),
now the reticuloendotheliosis virus (REV). The provirus within the
plasmid comprises the LTRs, the packaging sequence and the
thymidine kinase gene and its promoter as a selectable marker for
determination of viral titer. There is a unique Xba I site 5' to
the thymidine kinase promoter. In a previous study, the neomycin
phosphotransferase gene had been inserted in this vicinity (in a
Hind III site) resulting in a second construct pME III (Emerman and
Temin, Cell 39:459-467 (1984)) which was used successfully to
generate a transgenic chicken (Bosselman, et al., Science
243:533-535 (1989)). In the same study, the gene encoding chicken
growth hormone was cloned into pSW272, and resulting transgenic
chickens had significantly higher levels of circulating growth
hormone than nontransgenic controls. pSW272 was modified to better
serve as a vehicle for the expression vector.
[0064] The goals in modifying the retroviral architecture include:
replacement of the thymidine kinase gene with the gene conferring
resistance to hygromycin B (this eliminates the need for
co-transfection, however, it also requires remodeling the ends of
the hygromycin gene); elimination of the 5' promoter driving the
hygromycin gene, and its polyadenylation signal (this will provide
a more stable architecture); provide an Xba I site 3' to the
hygromycin gene for insertion of the expression vector.
[0065] pSW272 was reconstructed in three phases. The first phase
was the deletion of the herpes virus thymidine kinase promoter and
structural gene, and replacement with a synthetic linker. This
linker contains sites necessary for subsequent manipulations,
including a unique Xba I site at the 3' end which allows insertion
of the 5 kb expression construct contained in pBCWM. The second
phase was the introduction of linkers at the 5' and 3' ends of the
hygromycin resistance gene that allow for more specific
construction of control sequences at the ends of the gene. The
third phase tested 4 different arrangements of control sequences
for their ability to stably transfect the C3 cell line. The
arrangement with the fewest control sequences that can stably
transfect the C3 cells was chosen as a preferred proviral
vector.
[0066] The retroviral vector pSW272 contains the
reticuloendotheliosis virus (REV) long terminal repeats (LTR)s, and
also the selectable marker thymidine kinase (TK) driven by its own
promoter. The LTRs lie at the ends of the provirus, and can
function as promoters.
[0067] By itself, pSW272 is a stable architecture, stable in this
case referring to the ability to generate full length retrovirus
with no internal deletions from its genome. The selectable marker
is useful for titering retrovirus on TK.sup.- cells, but not
helpful for transfecting the helper cells necessary to generate the
retrovirus.
[0068] Additionally, problems may arise when the expression vector
is inserted into pSW272, and the entire construct is then
transfected into the C3 cell line. The structure of the construct
then includes two internal promoters. The 5' or left promoter (in
this case the ovalbumin promoter) may be unstable in this
environment, meaning retrovirus produced from cells transfected
with such a construct experience frequent deletions in this region
(Emerman and Temin, J. Virology 50(1):42-49 (1984)).
[0069] The same study provided evidence that a structural gene
alone in that location is stable and can be expressed by the LTR,
eliminating the need for a promoter. Since that gene can be
virtually any structural gene, it can be the selectable marker. The
C3 packaging cell line contains endogenous Tk activity and
consequently it must be co-transfected with a plasmid conferring
hygromycin B resistance. The gene encoding hygromycin B
phosphotransferase was cloned into the retroviral vector to
generate an improved architecture.
[0070] The expression vector was then inserted at the unique Xba I
site, resulting in a stable architecture, elimination of the need
for co-transfection and still enabling the titration of virus on
CEF cells.
Retroviral Vector Construction
[0071] Construction of pREV.DELTA. schematic illustrating the
construction of the retroviral vector is set forth in FIG. 3. The
promoter and structural gene of thymidine kinase are carried on a 2
kb Xba I-Xma I fragment, both of which are unique in pSW272. pSW272
was digested with Xba I and Xma I, and the larger fragment
(approximately 7 kb) recovered from a low melt get. This fragment
was ligated to a synthetic oligonucleotide containing 5 restriction
sites. The resulting construction, pREV.DELTA., was confirmed by
Cla I digestion, as Cla I recognizes a site in the synthetic
linker, and a second site outside of the proviral DNA.
Modification of Hygromycin B Phosphotransferase Gene
[0072] Modification to pREP4. The hygromycin B phosphotransferase
gene is contained on the plasmid pREP-4, purchased from Invitrogen.
However, there are problems with the hygromycin B
phosphotransferase gene at both the 5' and 3' ends. At the 5' end,
there is an unfavorable sequence surrounding the start codon,
specifically a second out-of-frame start codon 4 bp upstream. The
3' end contains both a polyadenylation signal, which may interfere
with retroviral titer, and the LTR from Raus Sarcoma Virus (RSV),
which must be removed. The 3' end also lacks convenient restriction
sites necessary to generate the desired constructs, and with the
remodeling, these sites are included.
[0073] The plasmids are named after the nature of their control
signals. For example, the construct containing both a promoter and
a polyadenylation signal is designated p++. Similarly, the plasmid
containing a promoter, but no polyadenylation signal is called
p+-.
[0074] Construction of p++. The 3' end of the hygromycin B
phosphotransferase gene was modified using synthetic double
stranded oligos. FIG. 12 shows the modification of the 3' end of
the Hyg gene. A unique Sca I site located 60 bp from the stop
codon, within the gene (see FIG. 12). A synthetic oligonucleotide
containing a Sca I site, the C-terminal codons and stop codon of
the hygromycin marker gene, a Hind III site and flanking Nsi I and
Sal I ends was cloned into the Nsi I and Sal I sites on pREP4.
Proper recombinants were verified by HindIII-Nru I double
digestion, and are called p++.
[0075] Construction of p+-. p++ was partially digested with Sca I
and the 6.3 kb fragment recovered from a low melt gel and
autoligated, resulting in a hygromycin gene construct lacking 3'
control elements except the stop codons (see FIG. 12). Proper
recombinants were verified by Sca 1-Cla I double digestion, and
called p+-.
[0076] Construction of p-+ and p--. The N-terminal codons were
modified in a similar manner. Afl III is unique in p++, and lies
just 5' to the start codon of the hygromycin B phosphotransferase
gene. An Aat II site lies 25 bases into the hygromycin gene, making
an Afl III-Aat II double digestion convenient for removal of the
promoter. Aat II is not unique to p++, and thus a two enzyme
strategy was used. p++was digested with Cla I and Aat II and in a
separate reaction Cla I and Afl Ill. The digestion products were
run on a low melt gel, and the 5.5 kb Cla I Aat II product
recovered, and the 2.4 kb Cla I Afl III product recovered. These
two DNAs were ligated with a synthetic oligonucleotide (see FIG.
13) resulting in p-+. Proper recombinants were verified by Bcl I
Alw NI double digestion and sequence analysis. The plasmid p+- was
treated the same way, resulting in the plasmid p--.
[0077] The manipulation to the N-terminal portion of the gene was
done independently for p++, and p+-. The resulting four constructs
contain all the permutations for the control signals as: 1) with
promoter, with poly A signal--contained on an Nru 1-Hind III
fragment of p++; 2) with promoter, without poly signal--contained
on an Nru I-Hind III fragment of p+-; 3) without promoter, with
poly A signal--contained on a Bcl I-Hind III fragment of p-+; and
4) without promoter, without poly A signal--contained on an Bcl
I-Hind III fragment of p--.
[0078] The fragments with promoters (1 and 2 above) were cloned
into pREV.DELTA. at the Sma I (Xma I) and Hind III sites in the
multiple cloning site. Since recircularization secondary to
incomplete digestion is always a concern, the plasmid pREV.DELTA.
was digested at three sites Sma I, Hind III, and Acc Ill. Recovery
of the 2 fragments of the appropriate size from low melt gels
ensured digestion at both sites within the MCS, and when ligated to
the Nru I-Hind III fragments in a 3-molecule ligation, resulted in
desired plasmid readily. These plasmids are called p++R and
p+-R.
[0079] A similar procedure was used to clone the hygromycin B
phosphotransferase gene without a promoter into REV. The hygromycin
constructs (p-+ and p--) were digested with Bcl I and Hind III, and
cloned in a 3 molecule ligation to gel purified Bcl I, Hind III,
and Acc III fragments of pREV.DELTA., and called p-+R and p--R.
[0080] The insertion of the expression vector into these retroviral
vectors is as follows. Each retroviral vector contains a unique Xba
I site. The appropriate plasmid was opened with Xba I,
dephosphorylated, and gel purified. pBCWM contains the expression
vector on a chloramphicicol resistant plasmid as a 5 kb Xba I
fragment pBCWM was digested with Xba I and the 5 kb fragment
recovered from a low melt gel and ligated to the appropriate
retroviral vector. Proper recombinants were verified by Xba I
digestion, and orientation was checked by Eco RI digestion. These
plasmids were named as for their retroviral vectors, with the
addition of E and the clone number. For instance: p++RE1, with
promoter, with poly adenylation, in REV and with expression vector
in orientation 1.
SPECIFIC EXAMPLE 3
Transgenesis
Method 1
[0081] Each of the two orientation constructs for a given
retroviral vector, was transfected into the C3 cell line, and
stable clones selected. DNA is isolated from the clones and
analyzed for integrated intact proviral DNA by Southern blot.
Appropriate clones are propagated and assayed for retrovirus on CEF
cells selected for hygromycin resistance. Clones producing high
titers are used to generate retrovirus, which is further
concentrated by filtration and or centrifugation.
[0082] When a clone is found producing high titers of intact viral
DNA, eggs are injected as described in U.S. Pat. No. 5,162,215 and
Bosselman, R. A. et al., Science 243:533-535 (1989), herein
incorporated by reference. Newly laid line 0 SPF eggs are obtained
from SPAFAS (Preston, Conn.), and maintained at 20.degree. C. on
one side for at least 5 hours. The top of the egg is prepped with
70% ethanol, and air dried. The shell is then opened with a dremmel
mototool fitted with a steel burr. 15-25 microliters of a solution
containing retrovirus is microinjected beneath the blastoderm. The
eggs are sealed and incubated in a Humidaire incubator until
hatching.
[0083] Ten days after hatching, blood is collected from the chicks,
and assayed for the presence of viral DNA in their genomes by
Southern blot and PCR. All chickens are grown to maturity, at which
time, the eggs of these chimeric chickens are tested for the
presence of .beta.-galactosidase, and the rooster semen is tested
for viral DNA by Southern blot. Semen positive roosters are used to
sire G2 chickens which are true heterozygous transgenic
chickens.
Method 2
[0084] The 5 kb insert (the expression vector) of pBCWM was ligated
to pREV.DELTA. or p+-, cut with Xba I, dephosphorylated and
purified on a low melt gel Clones were screened for the insert by
Xba I digestion, and orientation was checked by digestion with Eco
RI.
[0085] C3 cells were seeded at 2-3.times.10.sup.5 cells per well in
a 6 well plate (35 mm well diameter) and grown overnight in DMEM
with high glucose supplemented with L-glutamine, 10 mM HEPES, 7%
calf serum, 400 .mu.g/ml G418, 100 .mu.g/ml gentamicin, 5 .mu.g/ml
fungizone (amphotericin B), 100 units/ml penicillin G, 100 .mu.g/ml
streptomycin sulfate, at 37.degree. C. in 10% CO.sub.2. The cells
were transfected using lipofectamine (Gibco Life Technologies) at a
ratio of 1.5 .mu.g DNA to 8 ul lipofectamine, according to the
manufacturers specifications. After 5 hours the transfection medium
was aspirated and replaced with 0.5 ml of DMEM with 7% calf serum,
and HEPES. The medium was removed after 48 hours of incubation and
used for microinjection or concentrated by ultrafiltration 20-fold
with a filter with a 50 kd cut-off and used for microinjection.
[0086] Newly laid fertilized SPF white leghorn eggs were obtained
from SPAFAS and maintained on their side for at least 5 hours. A
pentagonal shaped piece of shell approximately 0.5 cm.sup.2 was
removed intact from the top-most portion of the egg using a Dremmel
mototool fitted with a steel cutter (part 113). The shell membrane
was removed with an 18 gauge needle. Micropipettes were pulled on a
Sufter puller, trimmed with a razor blade, and checked for diameter
and tip angle under a microscope. 15 to 20 .mu.l of medium was
injected into the subgerminal space using a Narishige
micromanipulator (model MN-151) and microinjector (model IM-6). The
hole was patched using donor membranes harvested from eggs in the
same lot held briefly in PBS with penicillin G and streptomycin
sulfate used at the concentrations stated above. The shell fragment
was replaced on top of the donor membrane, and air dried for 10
minutes. Duco cement was used to seal the edges, and air dried for
at least 30 minutes. The eggs were then set in a Humidaire
incubator (model 21) and hatched according to manufacturers
specifications.
[0087] Those skilled in the art can now appreciate from the
foregoing description that the broad teachings of the present
invention can be implemented in a variety of forms. Therefore,
while this invention has been described in connection with
particular examples thereof, the true scope of the invention should
not be so limited since other modifications will become apparent to
the skilled practitioner upon a study of the drawings,
specification and following claims.
[0088] All patents and other publications cited herein are
expressly incorporated by reference.
TABLE-US-00001 Sequence ID No. 1 Xba I Nco I 1 TCTAGACCAT
GGAGCGGAGA ATGGGCGGAA CTGGGCGGAG TTAGGGGCGG 51 GATGGGCGGA
GTTAGGGGCG GGACTATGGT TGCTGACTAA TTGAGATGCA 101 TGCTTTGCAT
ACTTCTGCCT GCTGGGGAGC CTGGGGACTT TCCACACCTG 151 GTTGCTGACT
AATTGAGATG CATGCTTTGC ATACTTCTGC CTGCTGGGGA 201 GCCTGGGGAC
TTTCCACACC CTAACTGACA CACATTCCAC AGCAGATCCC 251 CCGGAATTCG
GTCAAGCTGA CCTACTAGTG GTCATCATGC A TTTCATA GGTAGAGATA ACATTTACTG
GGAAGCACAT CTATCATCAT AAAAAGCAGG CAAGATTTTC AGACTTTCTT AGTGGCTGAA
ATAGAAGCAA AAGACGTGAT TAAAAACAAA ATGAAACAAA AAAAATCAGT TGATACCTGT
AAGACGTGAT TAAAAACAAA ATGAAACAAA AAAAATCAGT TGATACCTGT GGTGTAGACA
TCCAGCAAAA AAATATTATT TGCACTACCA TCTTGTCTTA AGTCCTCAGA CTTGGCAAGG
AGAATGTAGA TTTCTACAGT ATATATGTTT TCACAAAAGG AAGGAGAGAA ACAAAAGAAA
ATGGCACTGA CTAAACTTCA GCTAGTGGTA TAGGAAAGTA ATTCTGCTTA ACAGAGATTG
CAGTGATCTC TATGTATGTC CTGAAGAATT ATGTTGTACT TTTTTCCCCC ATTTTTAAAT
CAAACAGTGC TTTACAGAGG TCAGAATGGT TTCTTTACTG TTTGTCAATT CTATTATTTC
AATACAGAAC AATAGCTTCT ATAACTGAAA TATATTTGCT ATTGTATATT ATGATTGTCC
CTCGAACCAT GAACACTCCT CCAGCTGAAT TTCACAATTC CTCTGTCATC TGCCAGGCCA
TTAAGTTATT CATGGAAGAT CTTTGAGGAA CACTGCAAGT TCATATCATA AACACATTTG
AAATTGAGTA TTGTTTTGCA TTGTATGGAG CTATGTTTTG CTGTATCCTC AGAAAAAAAG
TTTGTTATAA AGCATTCACA CCCATAAAAA GATAGATTTA AATATTCCAG CTATAGGAAA
GAAAGTGCGT CTGCTCTTCA CTCTAGTCTC AGTTGGCTCC TTCACATGCA TGCTTCTTTA
TTTCTCCTAT TTTGTCAAGA AAATAATAGG TCACGTCTTG TTCTCACTTA TGTCCTGCCT
AGCATGGCTC AGATGCACGT TGTAGATACA AGAAGGATCA AATGAAACAG ACTTCTGGTC
TGTTACTACA ACCATAGTAA TAAGCACACT AACTAATAAT TGCTAATTAT GTTTTCCATC
TCTAAGGTTC CCACATTTTT CTGTTTTCTT AAAGATCCCA TTATCTGGTT GTAACTGAAG
CTCAATGGAA CATGAGCAAT ATTTCCCAGT CTTCTCTCCC ATCCAACAGT CCTGATGGAT
TAGCAGAACA GGCAGAAAAC ACATTGTTAC CCAGAATTAA AAACTAATAT TTGCTCTCCA
TTCAATCCAA AATGGACCTA TTGAAACTAA AATCTAACCC AATCCCATTA AATGATTTCT
ATGGCGTCAA AGGTCAAACT TCTGAAGGGA ACCTGTGGGT GGGTCACAAT TCAGGCTATA
TATTCCCCAG GGCTCAGCCA GTGTCTGTAC CTACAGCTAG AAAGCTGTAT TGCCTTTAGC
ACTCAAGCTC AAAAGACAAC TCAGAGTTCA CCTGTACATA CAGCTATGAG GTCTTTGCTA
ATCTTGGTGC TTTGCTTCCT GCCCCTGGCT GCTCTGGGGA ATAT
Sequence CWU 1
1
2911753DNAGallus gallus 1tctagaccat ggagcggaga atgggcggaa
ctgggcggag ttaggggcgg gatgggcgga 60gttaggggcg ggactatggt tgctgactaa
ttgagatgca tgctttgcat acttctgcct 120gctggggagc ctggggactt
tccacacctg gttgctgact aattgagatg catgctttgc 180atacttctgc
ctgctgggga gcctggggac tttccacacc ctaactgaca cacattccac
240agcagatccc ccgaattcgg tcaagctgac ctactagtgg tcatcatgca
tttcataggt 300agagataaca tttactggga agcacatcta tcatcatcaa
aaagcaggca agattttcag 360actttcttag tggctgaaat agaagcaaaa
gacgtgatta aaaacaaaat gaaacaaaaa 420aaatcagttg atacctgtgg
tgtagacatc cagcaaaaaa atattatttg cactaccatc 480ttgtcttaag
tcctcagact tggcaaggag aatgtagatt tctacagtat atatgttttc
540acaaaaggaa ggagagaaac aaaagaaaat ggcactgact aaacttcagc
tagtggtata 600ggaaagtaat tctgcttaac agagattgca gtgatctcta
tgtatgtcct gaagaattat 660gttgtacttt tttcccccat ttttaaatca
aacagtgctt tacagaggtc agaatggttt 720ctttactgtt tgtcaattct
attatttcaa tacagaacaa tagcttctat aactgaaata 780tatttgctat
tgtatattat gattgtccct cgaaccatga acactcctcc agctgaattt
840cacaattcct ctgtcatctg ccaggccatt aagttattca tggaagatct
ttgaggaaca 900ctgcaagttc atatcataaa cacatttgaa attgagtatt
gttttgcatt gtatggagct 960atgttttgct gtatcctcag aataaaagtt
tgttataaag cattcacacc cataaaaaga 1020tagatttaaa tattccacac
tataggaaag aaagtgtgtc tgctcttcac tctagtctca 1080gttggctcct
tcacatgcac gcttctttat ttctcctatt ttgtcaagaa aataataggt
1140cacgtcttgt tctcacttat gtcctgccta gcatggctca gatgcacgtt
gcacatacaa 1200gaaggatcaa atgaaacaga cttctggtct gttactacaa
ccatagtaat aagcacacta 1260actaataatt gctaattatg ttttccatct
ccaaggttcc cacatttttc tgttttctta 1320aagatcccat tatctggttg
taactgaagc tcaatggaac atgagcaata tttcccagtc 1380ttctctccca
tccaacagtc ctgatggatt agcagaacag gcagaaaaca cattgttacc
1440cagaattaaa aactaatatt tgctctccat tcaatccaaa atggacctat
tgaaactaaa 1500atctaaccca atcccattaa atgatttcta tggcgtcaaa
ggtcaaactt ctgaagggaa 1560cctgtgggtg ggtcacaatt caggctatat
attccccagg gctcagccag tgtctgtacc 1620tacagctaga aagctgtatt
gcctttagca ctcaagctca aaagacaact cagagttcac 1680ctgtacatac
agctatgagg tctttgctaa tcttggtgct ttgcttcctg cccctggctg
1740ctctggggaa tat 1753214PRTArtificial SequenceDescription of
Artificial Sequence Synthetic peptide 2Met Arg Ser Leu Leu Ile Leu
Val Leu Cys Phe Leu Pro Leu 1 5 10354DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide construct 3gtacatacag ctatgaggtc tttgctaatc
ttggtgcttt gcttcctgcc cctg 54450DNAArtificial SequenceDescription
of Artificial Sequence Synthetic oligonucleotide construct
4caggggcagg aagcaaagca ccaagattag caaagacctc atagctgtat
5056PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 5Ala Ala Leu Gly Asn Ile 1 5617DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide construct 6gctgctctgg ggaatat 17719DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide construct 7cgatattccc cagagcagc 19838DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide construct 8cgttatcttt cgaaggtgtc gttttacaac
gtcgtgac 38924DNAArtificial SequenceDescription of Artificial
Sequence Synthetic oligonucleotide construct 9accaccgcga cctaccattc
ggcg 241082DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide construct 10gatctaccgc ggacggtacc
tacagctaga aagctgtatt gcctttagca ctcaagctca 60aaagacaact cagagttcac
ct 821182DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide construct 11gtacaggtga actctgagtt
gtcttttgag cttgagtgct aaaggcaata cagctttcta 60gctgtaggta ccgtccgcgg
ta 821277DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide construct 12tcgacagtac tcgccgatag
tggaaaccga agaccatcta cacgaccgaa gtcaaaggaa 60tagtagaagc ttatgca
771369DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide construct 13taagcttcta ctattccttt
gacttcggtc gtgtagatgg tcttcggttt ccactatcgg 60cgagtactg
691479DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide construct 14gtacatctag accatggtcc
ggacagcgaa atggagaatt cggtcaagct tcactgacct 60gactagtggt catcatgca
791571DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide construct 15tgatgaccac tagtcaggtc
agtgaagctt gaccgaattc tccatttcgc tgtccggacc 60atggtctaga t
711679DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide construct 16gtacctctag accatggtcc
ggacagctca atggagaatt cggtcaagct tcactggcct 60gactagtggt catcatgca
791771DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide construct 17tgatgaccac tagtcaggcc
agtgaagctt gaccgaattc tccattgagc tgtccggacc 60atggtctaga g
711846DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide construct 18gtacctctag accatggtcc
ggacagctca atggagaatt cggtca 461950DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide construct 19agcttgaccg aattcaccat tgagctgtcc
ggaccatggt ctagaggtac 502017DNAArtificial SequenceDescription of
Artificial Sequence Synthetic oligonucleotide construct
20ctagtggtca tcatgca 172113DNAArtificial SequenceDescription of
Artificial Sequence Synthetic oligonucleotide construct
21agcatgatga cca 132210DNAArtificial SequenceDescription of
Artificial Sequence Synthetic oligonucleotide construct
22agctgaccta 102310DNAArtificial SequenceDescription of Artificial
Sequence Synthetic oligonucleotide construct 23ctagtaggtc
102438DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide construct 24gaattcggtc aagctgacct
actagtggtc atcatgca 382538DNAArtificial SequenceDescription of
Artificial Sequence Synthetic oligonucleotide construct
25tgcatgatga ccactagtag gtcagcttga ccgaattc 382637DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide construct 26ctagtcccgg gtgatcatcg attgaagctt
tctagaa 372737DNAArtificial SequenceDescription of Artificial
Sequence Synthetic oligonucleotide construct 27ccggttctag
aaagcttcaa tcgatgatca cccggga 372845DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide construct 28cgcgatgatc agtcaccatg aaaaagcctg
aactcaccgc gacgt 452937DNAArtificial SequenceDescription of
Artificial Sequence Synthetic oligonucleotide construct
29cgcggtgagt tcaggctttt tcatggtgac tgatcat 37
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