Visualization of avian eggs

Abstract

The invention includes methods of visualizing and manipulating avian eggs employing TPLSM.

Description

RELATED APPLICATION INFORMATION

This application is a continuation-in-part of U.S. patent application Ser. No. 09/654,293, filed Sep. 1, 2000, the disclosure of which is incorporated in its entirety herein by reference.

BACKGROUND OF THE INVENTION

The avian reproductive system, including that of the chicken, is well described. The production of an avian egg begins with formation of a large yolk in the ovary of the hen upon which an unfertilized oocyte or ovum is positioned. After ovulation or release from the ovary, the yolk and ovum pass into the infundibulum of the oviduct, where the ovum is fertilized if sperm are present, and then moves into the magnum of the oviduct which is lined with tubular gland cells. The tubular gland cells secrete egg-white proteins, including ovalbumin, lysozyme, ovomucoid, conalbumin and ovomucin, into the lumen of the magnum which is deposited onto the ovum and yolk.

The hen oviduct offers outstanding potential as a protein bioreactor because of high levels of protein production and ease of recovery. As a result, efforts have been made to manipulate avian eggs in order to produce transgenic chickens expressing exogenous proteins in the oviduct. For example, microinjection of exogenous nucleic acid sequences has been performed in order to produce transgenic avians which produce heterologous proteins in the magnum which are subsequently packaged into eggs laid by the avian. Successful manipulations of avian eggs can be limited by factors including the inability to clearly visualize the egg by standard magnification procedures (e.g., using a light microscope).

Therefore, it is an object of this invention to provide improved methods for the visualization of avian eggs and their nuclear structures. It is also an object of this invention to provide an improved method for visualization of the nuclear structures in a recipient cell to facilitate the process of enucleation and subsequent nuclear transfer. It is also an object of this invention to provide an improved method for ablation of the nucleus in a recipient cell to facilitate subsequent nuclear transfer.

SUMMARY OF THE INVENTION

In one aspect, the invention is directed to methods for visualizing an avian egg which include visualizing the egg by Two-Photon Laser Scanning Microscopy (TPLSM). The methods also include visualizing the nucleus of the avian egg by TPLSM. The avian egg can be unfertilized (e.g., oocyte) or fertilized (e.g., embryo or zygote). In the case of an embryo, the embryo may be, for example, a stage I, stage II, stage III, stage IV, stage V, stage VI, stage VII, stage VIII, stage IX, stage X, stage XI or stage XII embryo. In one useful embodiment, the embryo is an early stage embryo such as a stage I, stage II or stage III embryo. In one embodiment, the embryo contains more than one cell. For example, the embryo may contain between 1 and 100,000 cells.

Methods of the invention are also directed to visualizing an avian egg in order to facilitate manipulation of the egg. The invention contemplates any useful manipulation of avian eggs in conjunction with viewing the egg by TPLSM. For example, manipulation of the egg may include injection a substance, such as nucleic acid, into the egg and/or into a nucleus of the egg. In one particular embodiment, the methods provide for injection of an artificial chromosome into the avian egg, for example, injection of an artificial chromosome into the nucleus of the egg. Nucleic acid injected into the egg typically comprises a transgene. Ovum transfer or ex ovo culture may be used for production of a transgenic avian after the introduction, e.g., injection, of nucleic acid into an avian egg as disclosed herein.

The avian egg which is visualized and or manipulated as disclosed herein can be the egg of, for example, and without limitation, a chicken, turkey, duck, goose, quail, pheasant, parrot, finche, hawk, crow, ratite, ostrich, emu or cassowary.

The present invention contemplates cloned cells, cell lines, embryos, and animals and methods for their production, employing two-photon visualization, ablation or both. Cloned and transgenic avians, including knock-outs and knock-ins, are also contemplated.

In one embodiment of the invention, two-photon laser scanning microscopy (TPLSM) is used to visualize nuclear structures in a recipient cell. In one embodiment, following visualization, the cell can be enucleated by any useful method, for example, by two-photon laser-mediated ablation, to provide a recipient cytoplast. It is also contemplated that the recipient cell can be enucleated by cell splitting, aspiration of its nuclear structure(s), irradiation, or other enucleating procedure. The recipient cell may be removed from an animal, the nucleus visualized and ablated by two-photon laser-mediated ablation. It is contemplated that a donor nucleus can then be inserted into the recipient cell by cell fusion, microinjection, or other useful renucleation procedures. The replacement of the recipient cell's nucleus with the donor cell's nucleus yields a reconstructed zygote. It is contemplated that the reconstructed zygote may be activated and allowed to develop to term in vivo or in vitro.

Another aspect of the present invention contemplates methods of producing a cloned avian comprising nuclear transfer in combination with ovum transfer. TPLSM and two-photon laser-mediated ablation is used to perform nuclear transfer wherein the donor nucleus may be a normal karyotype or may be genetically modified. Accordingly, the replacement of the recipient cell's nucleus with the donor cell's nucleus results in a reconstructed zygote. The ovum may be cultured by ovum transfer, wherein the ovum containing the reconstructed zygote is transferred to a recipient avian or may be cultured in vitro. Once transferred, the embryo develops inside the recipient hen and travels through the oviduct of the hen where it is encapsulated by natural egg white proteins and a natural egg shell. The egg which contains endogenous yolk and a reconstructed embryo, is laid and can then be incubated and hatched to produce a chick. The resulting chick may be genetically modified. In one embodiment, the genetically modified cloned avian carries a transgene in all or some of its cells. After maturation, the transgenic avian may lay eggs that contain one or more exogenous protein(s). The combination of nuclear transfer and ovum transfer allows for the preparation of a cloned avian. In another embodiment, ex ovo culture is used instead of ovum transfer to produce the cloned avian.

Another aspect of the present invention contemplates methods of producing a transgenic avian by, for example, preparing a transgenic avian, carrying a gene encoding an exogenous protein, using nuclear transfer by two-photon visualization and/or ablation, and allowing the immature transgenic avian to grow to maturity, wherein the exogenous protein is secreted into the oviduct lumen of the mature avian and deposited into eggs laid by the avian. Preferably, the exogenous DNA comprises a stable transgene and the transgenic avians may be bred and propagated. In one embodiment, transgenic avians possess the ability to lay eggs that contain one or more desired, exogenous protein(s).

Yet, another aspect of the present invention contemplates methods of producing a knock-out or knock-in avian by (i) preparing a knock-out or knock-in avian according to nuclear transfer by two-photon visualization and/or ablation, and (ii) allowing the immature knock-out or knock-in egg-laying animal to grow to maturity. The knock-out avians are able to lay eggs that contain less than all endogenous proteins normally present in the egg. This allows for the elimination of potential undesired substances found in the egg (e.g., allergens) or suppression of a specific agronomic trait. The knock-in sequence may replace all or part of an endogenous gene of the animal by a functional homologous gene or gene segment of another animal.

Cloned non-human cells, cell lines, embryos, and animals, optionally genetically modified, are encompassed by the instant invention. Transgenic, knock-out, and knock-in animals are also provided. In one embodiment, reconstituted avian embryos, particularly chick embryos, prepared by transferring the nucleus of a donor cell into a suitable recipient cell, are provided. The donor cell may be quiescent or non-quiescent.

Intact avian eggs containing protein(s) exogenous to naturally occurring avian eggs are included in the present invention.

Any useful combination of features described herein is included within the scope of the present invention provided that the features included in any such combination are not mutually inconsistent as will be apparent from the context, this specification, and the knowledge of one of ordinary skill in the art.

Additional objects and aspects of the present invention will become more apparent upon review of the detailed description set forth below.

Definitions

The following definitions are set forth to illustrate and define the meaning and scope of the certain terms used to describe the invention herein.

The term “TPLSM” refers to two-photon laser scanning microscopy. TPLSM relies upon the phenomenon of two-photon excited fluorescence in which two photons collide simultaneously with a fluorescent molecule. Their combined energy is absorbed by the fluorophore inducing a fluorescent emission, which is detected by a photomultiplier and converted into a digital image. The major advantage of TPLSM lies in its ability to generate images of living and optically dense structures for prolonged periods of time, while not affecting their viability. This is the case because TPLSM utilizes biologically innocuous pulsed infrared light that is able to penetrate much deeper into scattering specimens. Hence this method provides the capability for producing noninvasive, three-dimensional, real-time images of the optically dense oocyte (e.g., avian egg). In addition to visualization, TPLSM may also be used for enucleation.

The terms “ovum” and “oocyte” are used interchangeably herein. Although only one ovum matures at a time, an animal is born with a finite number of ova. During fertilization, sperm penetrate the blastodisc or germinal disc, which contains the nuclear material. When sperm enters the germinal disc an embryo begins to form.

“Egg” or “avian egg” refers to an unfertilized ovum or a fertilized ovum. In particular, “egg” can refer to the germinal disc, which may be fertilized or unfertilized, present on the yolk.

A “donor cell” is used herein to describe the source of the nuclear structure that is transplanted to the recipient cytoplast. All cells of normal karyotype, including embryonic, fetal, and adult somatic cells, preferably in a quiescent state, may be nuclear donors. The use of non-quiescent cells as nuclear donors has been described as well by Cibelli, et al. Science 280:1256-8, 1998, the disclosure of which is incorporated in its entirety herein by reference.

A “recipient cell” is used herein to describe the enucleated recipient cell, for example, an enucleated metaphase I or II oocyte or an enucleated unactivated oocyte, or an enucleated preactivated oocyte. Enucleation may be accomplished by splitting the cell into halves, aspirating the metaphase plate, pronulceus or pronuclei, or even by irradiation. In one embodiment, enucleation is done through two-photon laser-mediated ablation. Alternatively, TPLSM could be used to guide mechanical enucleation.

A “nucleic acid sequence” or “polynucleotide” includes, but is not limited to, eucaryotic mRNA, cDNA, genomic DNA, and synthetic DNA and RNA sequences, comprising the natural nucleotide bases adenine, guanine, cytosine, thymidine, and uracil. The term also encompasses sequences having one or more modified nucleotide(s). The terms “polynucleotide”, “oligonucleotide”, and “nucleic acid sequence” are 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 herein.

The terms “endogenous nucleic acid sequence” and “endogenous DNA” are used interchangeably herein. The term “endogenous” as it relates to nucleic acid sequences such as coding sequences, control sequences, and regulatory sequences denotes sequences that are normally associated with a particular cell or tissue. Hence, endogenous sequences are found in nature. Endogenous proteins are the expression products of endogenous DNA, such as endogenous coding sequences.

The terms “exogenous nucleic acid sequence” and “exogenous DNA” are used interchangeably herein. The term “exogenous” as it relates to nucleic acid sequences such as coding sequences, control sequences, and regulatory sequences denotes sequences that are “not” normally associated with a particular cell or tissue. Thus, an “exogenous” region of a nucleic acid is an identifiable segment of nucleic acid within or attached to another nucleic acid molecule that is not found in association with the other molecule in nature. Exogenous DNA may be integrated into the genome of the donor cell or may exist independently of the genome of the donor cell. For example, exogenous DNA may not be integrated into the genome of the donor cell but may exist as part of a non-integrated vector in the donor cell.

The term “transgene” refers to exogenous polynucleotide sequence(s) which can include a desired coding sequence and control sequences in operable linkage, so that cells transformed with these sequences are capable of producing the encoded product. In order to effect transformation, the transgene may be included on a vector. For example, the vector may be an integrative vector which has or can become integrated into the host chromosome. A “transgenic animal” is an animal that contains one or more exogenous nucleotide sequences in its genome.

The term “knock-out animal” refers to an animal that lacks all or a portion of a specific gene that is normally present in its genome.

The term “knock-in animal” refers to an animal that carries a specific nucleic acid sequence such as a “knock-in sequence” in a predetermined coding or noncoding region, wherein the knock-in sequence may be introduced through methods of recombination, such as homologous recombination. The recombination event comprises replacing all or part of a gene of the animal by a functional homologous gene or gene segment of another animal, where the respective knock-in sequence is placed in the genomic sequence.

“Vector” means a nucleotide sequence useful to introduce DNA into a cell. In one embodiment, the vector is comprised of DNA or RNA which may be single strand, double strand, circular and/or supercoiled. In one embodiment, the vector contains a coding sequence and a regulatory sequence. The positioning and orientation of the coding sequence with respect to the regulatory sequence may be such that the coding sequence is transcribed being at least partially under the “control” of the regulatory sequence.

A “plasmid” is a small, circular DNA vector capable of independent replication within a bacterial or yeast host cell.

The terms “transformation”, “transduction”, and “transfection” all denote the introduction of a polynucleotide into a cell, such as an embryonic or somatic cell.

The term “exogenous protein” means a protein or polypeptide not naturally present in a particular composition, tissue or cell, a protein that is the expression product of an exogenous gene, an exogenous expression construct or a transgene, or a protein not naturally present in a given quantity in a particular tissue or cell.

The term “avian” means a bird of any known species or type. The term includes the various know strains of Gallus gallus, or chickens, (for example, White Leghorn, Brown Leghorn, Barred-Rock, Sussex, New Hampshire, Rhode Island, Ausstralorp, Minorca, Amrox, California Gray, Italian Partridge-colored), as well as turkeys, pheasants, quails, duck, ostriches and other poultry commonly bred for commercial purposes.

DETAILED DESCRIPTION OF THE INVENTION

The invention is drawn to the use of TPLSM in visualizing avian eggs and the nuclei of avian eggs. For example, the invention includes visualizing fertilized or unfertilized avian ovum (e.g., fertilized or unfertilized germinal discs) and their nuclear structures. TPLSM is particularly useful for visualizing these structures because, for example, the opaque yolk of an egg can substantially obscure visualization using techniques such as standard light microscopy.

TPLSM is based on two-photon excited fluorescence in which two photons collide simultaneously with a fluorescent molecule. Their combined energy is absorbed by the fluorophore, inducing fluorescent emission, which is detected by a photomultiplier tube and converted into a digital image. See Squirrell et al., Nat. Biotechnol. 17:763-7, 1999 and Piston et al., Trends Cell Biol., 9:66-9, 1999, the disclosure of which is incorporated in its entirety herein by reference. TPLSM allows for the generation of images of living, optically dense structures for prolonged periods of time, while not necessarily affecting their viability. This is possible because TPLSM can use biologically innocuous pulsed near infrared light, usually at a wavelength of about 700 nm to about 1000 nm, which is able to penetrate much deeper into scattering specimens. TPLSM may employ different lasers, such as a mode-locked laser, where the wavelength is fixed, or a tunable laser that can be tuned between about 700 nm and about 1000 nm., depending upon the range of emission of the dye used. For DAPI and Hoescht 33342 dyes, 720-770 nm is preferred. New fluorophores are being produced with different ranges of emission and the invention is not limited to the presently available dyes and their respective emission ranges.

Lasers used in TPLSM can be grouped into femtosecond and picosecond lasers. These lasers are distinguished by their pulse duration. Femtosecond lasers can be particularly suitable for visualization without harming the specimen, however, the invention is not limited thereto.

In one embodiment, TPLSM is useful for producing noninvasive, three-dimensional, real-time images of the optically dense avian egg. In one embodiment, the albumen capsule is removed and the ovum placed in a dish with the germinal disc facing the top. Any remnants of the albumen capsule can be removed from the top of the germinal disc. An aqueous solution, e.g. phosphate-buffered saline (PBS), can be added to prevent drying of the ovum. In one embodiment, a cloning cylinder is placed around the germinal disc and DAPI in PBS is added to the cylinder. In another embodiment, a DAPI-PBS solution is injected into the germinal disc by using a glass pipette, after which the dye moves into the nuclear structures. In the case of injecting the dye, removal of the albumen capsule is not necessary. However, injection of nuclei into the disk can be facilitated in the absence of the capsule. In one embodiment, following exposure to DNA-specific dyes, images of the inside of the early avian embryo can be generated through the use of TPLSM. In certain embodiments, visualization is performed after about 10 to 15 minutes after administration of the dye. During visualization, the germinal disc is placed under the microscope objective and the egg is viewed using relatively low laser powers of about 3-6 milliwatts. The invention also contemplates the viewing or visualization of avian eggs using TPLSM without the use of DNA specific dyes.

The invention contemplates the delivery of any useful substance to an egg such as an avian egg (e.g., delivery to the nucleus of the avian egg) in conjunction with viewing by TPLSM. In a particularly useful embodiment, the invention provides for the delivery of an aqueous solution to the egg and/or the nucleus, by injection. In one embodiment, the aqueous solution includes a biomolecule such as nucleic acid (e.g., DNA or RNA). Any useful type of nucleic acid may be employed in the present invention. For example, the nucleic acid may be linear or circular (e.g., open circular or closed circular). In one embodiment, the nucleic acid is associated with protein, for example, a chromosome (e.g., an artificial chromosome) may be delivered to an avian egg or nucleus. The invention also contemplates the delivery of a nucleus to the egg. In one embodiment, “delivery” means introducing into, for example, inside of the egg (e.g., into the nucleus of the egg) by injection.

In one embodiment, the present invention is useful to create a transgenic (e.g., transchromosomic) avian by injecting a nucleic acid component (e.g., an artificial chromosome, see, for example, U.S. patent application Ser. No. 11/068,115, filed Feb. 28, 2005, the disclosure of which is incorporated in its entirety herein by reference) into an avian reproductive cell such as a cell of a germinal disc which is atop a yolk and is viewed by TPLSM. In one embodiment, the invention provides for a minimally invasive delivery of DNA or other substance to a germinal disc thereby providing for a germinal disc which remains viable after injection. In one particularly useful embodiment, an artificial chromosome is delivered into the nucleus of the avian egg.

To produce a transgenic avian, a fertilized ova (stage I embryo) can be isolated from a euthanized hen (female bird), for example, 45 min to 4 h after oviposition of the previous egg. Alternatively, the eggs can be isolated from hens whose oviducts have been fistulated according to the techniques of Gilbert & Wood-Gush, J. Reprod. Fertil., 5: 451-453 (1963) and Pancer et al, Br. Poult. Sci., 30: 953-7 (1989), each incorporated by reference herein in their entireties. In one embodiment, the yolk having the fertilized ova is placed in a dish with the germinal disc upwards. Ringer's buffer medium can be added to the dish to prevent drying. In one embodiment, nucleic acid is injected into the germinal disc by visualizing the germinal disc by TPLSM and guiding the injection needle of the device into the germinal disc. In one embodiment, a piezo unit operably linked to the injection needle can be activated for a period of time sufficient for the needle to penetrate the oolemma and or nuclear membrane following which the nucleic acid is injected, e.g., into the nucleus. Certain aspects of the delivery of nucleic acid into the avian egg by microinjection are disclosed in U.S. patent application Ser. No. 11/159,973, filed Jun. 23, 2005, the disclosure of which is incorporated in its entirety herein by reference.

Injected embryos can be surgically transferred to a recipient hen as described, for example, in Olsen & Neher, J. Exp. Zool., 109: 355-66 (1948) and Tanaka et al, J. Reprod. Fertil., 100: 447-449 (1994), the disclosure of which is incorporated herein in its entirety by reference. In one embodiment, the injected embryos are surgically transferred to recipient hens by the ovum transfer method of Christmann et al in PCT/US01/26723, published Aug. 27, 2001, the disclosure of which is incorporated herein by reference in its entirety, and hard shell eggs are incubated and hatched. The embryo is allowed to proceed through the natural in vivo cycle of albumin deposition and hard-shell formation. The transgenic embryo is then laid as a hard-shell egg which is incubated until hatching of the chick.

In accordance with the present invention, the germinal disc may be a germinal disc of any animal which produces a germinal disc, in particular avians including, but not limited to, chickens, ducks, turkeys, quails, pheasants and ratites.

In one embodiment, the invention is directed to devices useful for the delivery of an object or a substance such as an isolated cell nucleus, a spermatozoon or a fluid containing biomolecules such as nucleic acid by microinjection into an avian oocyte or embryo. The present invention is also directed to providing methods of microinjecting an isolated cell nucleus, a spermatozoon or a fluid having a nucleic acid therein, into an avian embryo or embryonic cell. In one useful embodiment, the invention provides devices and methods useful for delivering a nucleic acid to an avian embryo or avian embryonic cell. For example, the invention provides for devices and methods useful for delivering a nucleic acid to an avian germinal disc. In one useful embodiment, the invention provides for the delivery of one or more chromosomes to a germ cell or an embryo, for example, delivery into the nucleus of a germinal disc.

In one useful embodiment, the injection needle can be positioned (i.e., moved to a certain location) by employing a micromanipulator operably attached to the needle. The invention contemplates the movement of the needle provided by the micromanipulator to be in one, two or three axes. That is, the needle can be placed at any useful position on the egg and at any useful angle to the egg and can be moved to pierce the membrane of the egg and the nucleus.

In one embodiment, the microinjection device includes a piezo unit. Typically, the piezo unit is operably attached to the needle to impart oscillations to the needle. However, any configuration of the piezo unit which can impart oscillations to the needle is included within the scope of the invention. Typically, the “piezo drill” provides for a tunable frequency and amplitude which provides for optimization of the piezo's performance (e.g., passage of the needle into the egg, i.e., into a germinal disc).

The oscillations of the needle imparted by the piezo may be in any useful direction. For example, and without limitation, the oscillations may be side to side, back and forth, up and down, in circular, oval, square, rectangular motions or other patterns or combinations thereof. In one useful embodiment, the oscillations are side to side.

In one embodiment, the piezo unit is operably attached to the needle meaning the piezo unit is able to impart oscillations to the needle. In one embodiment, the piezo unit is activated during the penetration of the oolemma by the needle. For example, the needle may be a piezo electrically-driven needle, i.e., the needle punctures the surface of the egg (e.g., oolemma) in a manner facilitated (e.g., substantially facilitated) by the action of the piezo unit.

The invention contemplates the employment of any useful frequency of oscillations imparted to the needle by the piezo. In one embodiment, a frequency of greater than 100 Hz is used. The invention contemplates the upper limit for frequency as being limited by the mechanics of the piezo. For example, and without limitation, a frequency of between about 100 Hz and about 100,000 kHz is within the scope of the invention. In one useful embodiment, the frequency is between about 100 Hz and about 100 kHz, for example, about 500 Hz to about 50 kHz. In one embodiment, the frequency is between about 500 Hz and about 10 kHz, for example, about 500 to about 5 kHz. In one certain embodiment, the frequency is about 3100 Hz.

The invention contemplates the employment of any useful amplitude of oscillations imparted to the needle by the piezo. For example, the travel distance of the needle is contemplated as being between about 0.001 nm and about 100 μm. In one embodiment, the travel distance of the needle is between about 0.11 nm and about 50 μm. In one embodiment, the travel distance of the needle is about 1 nm to about 20 μm or about 1 nm to about 10 μm. In one useful embodiment, the travel distance of the needle is about 0.01 μm to about 20 μm. In one particularly useful embodiment, the travel distance of the needle is about 0.1 μm to about 20 μm, for example, about 1.0 μm to about 10 m (e.g., 5 μm or 7.5 μm).

In one particular embodiment, the piezo unit includes one or more of, for example, all of: a Signal Generator (BK Precision Model # 4011A) set to operate at a frequency of 5 KHz; an Amplifier (Physik Instrumente GmbH, Amplifier: PI-Polytec E-505 PZT-Power Amplifier, Average power 30 W, output voltage −20 to +120 V and optimized for 100V PiezoDrive); and a Piezo actuator (Physik Instrumente GmbH, catalog # P-840.10, 5 μm travel for latitudinal vibration).

The needle may approach the egg from any useful angle. In one useful embodiment, the longitudinal axis of the needle is visible when viewing the egg. That is, the TPLSM viewing is not directly above the needle (i.e., the viewing axis is not parallel to the longitudinal axis of the needle). However, the invention also contemplates the TPLSM viewing being directly above the egg (i.e., the viewing axis being parallel to the longitudinal axis of the needle). Other aspects of needle angles useful in the present invention are disclosed in U.S. patent application Ser. No. 11/159,973, filed Jun. 23, 2005. In one embodiment of the invention, the needle is coated with a fluorescent dye which facilitates visualization of the needle by TPLSM. In another embodiment, the needle is impregnated with a fluorescent dye which facilitates visualization of the needle by TPLSM.

The present invention also contemplates methods for producing cloned animals by nuclear transfer and by combinations of nuclear transfer and embryo transfer. Nuclear transfer allows the cloning of animal species, wherein individual steps are common to the procedures of embryonic, fetal and adult cell cloning. These steps can include, but are not limited to, preparation of a chromosome-free recipient cell called a cytoplast (which involves chromosome removal often referred to as enucleation); donor cell nucleus (nuclear donor) isolation and transfer to the cytoplast to produce a reconstructed embryo; optional reconstructed embryo culture; and embryo transfer to a synchronized host animal.

A novel approach to nuclear transfer in animals, employing two-photon visualization, is described in the instant invention. In one embodiment, the fertilized or unfertilized egg is removed from an animal and manipulated in vitro, wherein the nucleus or genetic material of the egg is visualized and removed or ablated and a donor nucleus is inserted. In one embodiment, the donor nucleus is genetically modified. Two-photon laser scanning microscopy (TPLSM) is used to visualize the nuclear structures.

The use of light microscopy to visualize the metaphase plate or pronucleus in avian eggs during nuclear transfer can be hindered by the presence of the yolk, which makes visualization of these nuclear structures difficult or impossible. But two-photon imaging with femtosecond lasers operating in the near infrared allows visualization of nuclear structures without damaging cellular constituents, despite the unfavorable optical properties of the egg yolk. Prior to visualization, specimens may be incubated or injected with DNA-specific dyes such as DAPI (4′,6′-diamidino-2-phenylindole hydrochloride) or Hoescht 33342 (bis-benzimide). Typically visualization may be performed after about 10 to 15 minutes of incubation or about 10 minutes after injection. During visualization, the germinal disc can be placed under the microscope objective and the pronuclear structures searched within the disk using relatively low laser powers of about 3-6 milliwatts. In one embodiment, once the structures are found they may be ablated by using higher laser power or mechanically removed, guided by TPLSM.

Nuclear transfer typically requires the destruction or enucleation of the pronucleus before a nuclear donor can be introduced into the oocyte. In order to enucleate the oocyte to produce a cytoplast donor, it is essential to visualize the pronucleus which resides about 25 μm beneath the ovum's vitelline membrane within the germinal disc. Microsurgery is one method to accomplish pronuclear removal or enucleation. However, two-photon laser-mediated ablation of nuclear structures provides an alternative to microsurgery. Higher laser powers than those used for imaging are used for enucleation, with minimal collateral damage to the cell. As during visualization, the wavelength for ablation generally ranges from about 700 nm to 1000 nm, normally about 750 nm. TPLSM and two-photon laser-mediated ablation are more efficient than other certain methods because they can be less operator dependent and less invasive, which results in higher viability of the recipient cell. Following visualization, pronuclear structures may be ablated using higher laser powers of about 30 to about 70 milliwatts.

In one embodiment, enucleation is followed by renucleation, where a cultured somatic cell nucleus (nuclear donor) is injected into the enucleated recipient cytoplast. Renucleation may be performed using TPLSM and a micromanipulation unit comprising a microinjector and a micromanipulator. Following ablation, the nuclear donor may be introduced into the germinal disc though guided injection using episcopic illumination (i.e., light coming through the objective onto the sample), for example. The reconstructed zygote may then be surgically transferred to the oviduct of a recipient hen to produce a hard shell egg. Alternatively, the reconstructed embryo may be cultured for 24 hours and screened for development prior to surgical transfer. The egg can be further incubated to generate a cloned chick, optionally genetically modified. The cloned chick may carry a transgene in all or most of its cells. After maturation, the transgenic avian may lay eggs that contain one or more desired, exogenous protein(s). Alternatively, the resulting chick may be a knock-out animal capable of laying eggs that contain less than all endogenous proteins normally present in the egg. The cloned chick may also be a knock-in chick expressing an alternative phenotype or capable of laying eggs having an exogenous protein therein. The reconstructed egg may be cultured to term using the ex ovo method described by Perry (U.S. Pat. No. 5,011,780, issued Apr. 30, 1991).

The replacement of the recipient cell's nucleus with the donor cell's nucleus results in a reconstructed zygote. In one embodiment, the cytoplasmic membrane of the cell used as nuclear donor is disrupted to expose its nucleus to the ooplasm of the recipient cytoplast. The nuclear donor may be injected into the germinal disc, where it undergoes reprogramming and becomes the nucleus of the reconstructed embryo. Alternatively, a donor cell may be fused to the recipient cell using methods well known in the art, for example, by use of fusion-promoting chemicals, such as polyethylene glycol, inactivated viruses, such as Sendai virus, or electrical stimulation.

The methodologies of TPLSM visualization and two-photon laser-mediated ablation described herein, can also be used for selective visualization and destruction of specific structures within germ and/or somatic cells, for example, as used in nuclear transfer in mammalian species and other vertebrate species. The skilled artisan will be able to readily adapt the methods established for avians described herein to other types of animals including, but not limited to, mammals, fish, reptile(s), amphibian(s), and insect(s).

Another aspect of the present invention contemplates methods of producing a cloned animal comprising nuclear transfer in combination with ovum transfer. Two-photon visualization and ablation may be used to perform nuclear transfer, as described herein. Accordingly, the replacement of the recipient cell's nucleus with the donor cell's nucleus results in a reconstructed zygote. In one embodiment, pronuclear stage eggs are used as recipient cytoplasts already activated by fertilization. Alternatively, unactivated metaphase II eggs may serve as recipient cytoplast and activation may thereafter be induced after renucleation. The ovum may be cultured by ovum transfer, wherein the ovum containing the reconstructed zygote is transferred to a recipient hen. The ovum is surgically transferred into the oviduct of the recipient hen shortly after oviposition. This is accomplished according to normal husbandry procedures oviposition, incubation, and hatching; see Tanaka, et al. J Reprod Fertil 100: 447-449, 1994, the disclosure of which is incorporated herein in its entirety by reference.

In another embodiment, the ovum may be cultured to stage X prior to transfer into a recipient hen. More specifically, reconstructed stage I embryos are cultured for 24-48 hours to stage X. This allows for developmental screening of the reconstructed embryo prior to surgical transfer. Stage I embryos are enclosed within a thick albumen capsule. In this procedure, the albumen capsule is removed, after which the nuclear donor is injected into the germinal disc. Subsequently, the capsule and germinal disc are recombined by placing the thick capsule in contact with the germinal disc on top of the yolk. Embryos develop to stage X at similar rates as those cultured with their capsules intact. At stage X, the embryo is transferred to the oviduct of a recipient hen.

Once transferred, the embryo develops inside the recipient hen and travels through the oviduct of the hen where it is encapsulated by natural egg white proteins and a natural egg shell. The egg which contains endogenous yolk and an embryo from another hen, is laid and can then be incubated and hatched to produce a chick. The resulting chick may carry a transgene in all or most of its cells. Following maturation, the cloned avian may express a desired phenotype or may be able to lay eggs that contain one or more desired, exogenous protein(s).

Ovum transfer can facilitate the production of a genetically modified avian. Genetically modified avians encompassed by the instant invention include transgenic avians, for example, transgenic avians produced by injection into an egg (e.g., injection into a nucleus) as disclosed herein, cloned avians and knock-in and knock-out avians.

In another embodiment, ex ovo culture may be used instead of ovum transfer to produce genetically modified avians. See, for example, Perry (U.S. Pat. No. 5,011,780, issued Apr. 30, 1991). Avians include, but are not limited to, chickens, ducks, quails, turkeys, pheasants and ostriches. The invention disclosed herein also relates to mammals, fish, reptiles, amphibians, and insects.

The present invention provides for cloned egg-laying animals produced by nuclear transfer and by combinations of nuclear transfer and ovum transfer as described herein. A novel approach of nuclear transfer, employing two-photon visualization and ablation is used to produce the cloned animals. In addition, the present invention encompasses cloned animals that are genetically modified including, but not limited to, transgenic, knock-out, and knock-in animals. The instant invention satisfies the need for a rapid route to the expression and deposition of exogenous proteins in eggs. Eggs containing protein(s) exogenous to an egg are also provided by the present invention.

The present invention contemplates methods of producing cloned, transgenic animals through nuclear transfer by two-photon visualization and ablation, and ovum transfer. Transgenic animals may have their hereditary properties permanently modified by the introduction of recombinant DNA into their germ cells. The combination of zygote reconstruction followed by ovum transfer, as disclosed herein, promises a more efficient and flexible route to accomplish the cloning of animals and production of transgenics. One use of this technology is the modification of poultry and livestock genomes to improve agronomic traits. Candidate genes, whose introduction or deletion would enhance agronomic traits, can be targeted through use of the instant invention. Most importantly, the possibility of cloning avian species or producing transgenic avians promises tremendous gains for the market place. The new technology disclosed herein may be used in selective poultry breeding, leading to enhanced traits in chickens and their eggs. Further, nuclear transfer techniques developed herein (e.g., laser-mediated selective ablation of nuclear structures of oocytes) also have a wide range of applications in fields such as mammalian transgenesis, genetics, cell therapies, and transplantation.

One aspect of the present invention provides for a method of producing a transgenic animal, comprising the steps of (i) preparing the transgenic animal according to nuclear transfer by two-photon visualization and optionally, laser-mediated ablation, and ovum transfer which contains exogenous DNA in its cells, and (ii) allowing the immature transgenic animal to grow to maturity. In the case of an avian, an exogenous protein may be secreted into the oviduct lumen of the mature animal and deposited into eggs laid by the animal. In one embodiment of the instant invention, the exogenous DNA comprises a transgene and the resulting transgenic animals can be bred and propagated. Transgenic avians produced by the instant invention also possess the ability to lay eggs that contain one or more desired, exogenous protein(s).

In one embodiment, it is contemplated that transgenes can be introduced into the ovum of an animal through nuclear transfer by two-photon visualization and ablation, wherein the nuclear donor contains a desired exogenous DNA sequence in its genome. One of ordinary skill in the art will be able to readily adapt conventional methods to insert the desired transgene into the genome of the nuclear donor prior to injection of the nuclear donor into the recipient cytoplast, or prior to fusion of the nuclear donor cell with the recipient cell. For example, a vector that contains one or more transgene(s) may be delivered into the nuclear donor cell through the use of a delivery vehicle. The transgene is then transferred along with the nuclear donor into the recipient ovum. Following zygote reconstruction, the ovum can be transferred into the reproductive tract of a recipient hen. In one embodiment, the ovum is transferred into the infundibulum of the recipient hen. After reconstruction, the embryo containing the transgene develops inside the recipient hen and travels through the oviduct of the hen where it is encapsulated by natural egg white proteins and a natural egg shell. The egg is laid and can be incubated and hatched to produce a transgenic chick. The resulting transgenic chick will carry one or more desired transgene(s) in its germ line. Following maturation, the transgenic avian may lay eggs that contain one or more desired, exogenous protein(s) which can be easily harvested.

In one embodiment of the invention, a nuclear donor cell is transfected with a vector construct that contains a transgene. Methods for transfection of somatic cell nuclei are well known in the art and include, by way of example, the use of retroviral vectors, retrotransposons, adenoviruses, adeno-associated viruses, naked DNA, lipid-mediated transfection, electroporation and direct injection into the nucleus. Such techniques, particularly as applied to avians, are disclosed in Bosselman (U.S. Pat. No. 5,162,215), Etches (PCT Publication No. WO99/10505), Hodgson (U.S. Pat. No. 6,027,722), Hughes (U.S. Pat. No. 4,997,763), Ivarie (PCT Publication No. WO99/19472), MacArthur (PCT Publication No. WO97/47739), Perry (U.S. Pat. No. 5,011,780, issued Apr. 30, 1991), Petitte (U.S. Pat. Nos. 5,340,740 and 5,656,749), and Simkiss (PCT Publication No. WO90/11355), the disclosures of which are incorporated herein in their entirety by reference.

Another aspect of the present invention provides for producing a knock-out or knock-in animal, comprising the steps of (i) preparing the knock-out or knock-in animal according to nuclear transfer by two-photon visualization and/or ablation, and (ii) allowing the immature knock-out or knock-in animal to grow to maturity.

In one embodiment of the instant invention, the knock-out animal has been manipulated such that an endogenous gene has been removed from the genome of the donor nucleus. This may be accomplished using described protocols for the production of knock-out mice, including the transformation of the nuclear donors with a targeting vector comprising genomic DNA containing the desired modification, flanked by positive (neomycin resistance gene for instance) and/or negative (herpes simplex virus thymidine kinase) or other applicable selectable marker genes, using a number of described strategies such as the so-called “hit and run” (Hasty, et al., Nature 350:243-6, 1991 and Valancius and Smithies, Mol. Cell Biol. 11:1402-8, 1991), tag and exchange (Askew, et al., Mol. Cell Biol. 13:4115-24, 1993) and double replacement (Stacey, et al., Mol. Cell Biol. 14:1009-16, 1994).

The resulting knock-out animal can be bred and propagated. Animals produced in this fashion are suitable for research purposes, for example, to study the effects of specific drugs on the breeding of poultry and certain agronomic traits. These knock-out animals also possess the ability to lay eggs that contain less than all endogenous proteins normally present in the egg, which allows for the elimination of potential undesired substances found in the egg (e.g., allergens).

In another embodiment of the instant invention, a knock-in animal is manipulated such that it carries a specific nucleic acid sequence such as a “knock-in sequence” in a predetermined coding or noncoding region of its genome. The knock-in sequence may replace all or part of an endogenous gene of the animal by a functional homologous gene or gene segment of another animal. Knock-in animals can be prepared according to a variation of the standard knock-out method, comprising the introduction of a foreign gene into the targeting vector, in such a way that the introduced gene would be under the control of the regulatory elements that normally control the expression of the endogenous gene. See, for example, Le, et al., Proc. Natl. Acad. Sci. USA 87:4712-6, 1990 and McCreath, et al., Nature 405:1066-1069, 2000, the disclosures of which are incorporated in their entirety herein by reference.

Another embodiment of the invention provides methods of producing a protein derived from cloned or otherwise genetically modified egg-laying animals, e.g., transgenic avians, produced as disclosed herein. The methods typically include producing a hard-shell egg that contains exogenous protein and then isolating the exogenous protein from the egg. An intact avian egg containing protein exogenous to an avian egg is contemplated by the present invention. The transgenic animals of the instant invention include avians that have a transgene encoding an exogenous protein in their oviducts, wherein the avians secrete into their eggs the protein expressed by the transgene. A transgenic avian that makes a human protein (e.g., human interferon) will recognize this substance as its own and will therefore not produce an immune response against it. This makes the egg-laying transgenic avian ideally suited for the production of large quantities of human protein. In this respect, the avian egg provides an ideal container for the production of recombinant proteins because its interior may be sterile and contains antibacterial compounds, and it is easily accessible. As a consequence, the purity of the protein products can be improved and their efficacy tested more efficiently. Several proteins that may be produced in this fashion are contemplated by the present invention. These proteins include, but are not limited to, human growth hormone, interferon, β-casein, α−1 antitrypsin, antithrombin III, collagen, factor VIII, factor IX, factor X, fibrinogen, hyaluronic acid, insulin, lactoferrin, protein C, erythropoietin (EPO), granulocyte colony-stimulating factor (G-CSF), granulocyte macrophage colony-stimulating factor (GM-CSF), tissue-type plasminogen activator (tPA), feed additive enzymes, somatotropin, and chymotrypsin. Other proteins contemplated for production as disclosed herein are disclosed in, for example, U.S. patent application Ser. No. 11/193,750, filed Jul. 29, 2005, the disclosure of which is incorporated in its entirety herein by reference.

The disclosures of publications, patents, and patent applications referenced in this application are hereby incorporated by reference into the present disclosure to more fully describe this invention.

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 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.

The following specific Examples are intended to illustrate the invention and should not be construed as limiting the scope of the claims.

EXAMPLE 1

Preparation of the Recipient Cytoplast

Incubation

Ova were isolated from euthanized hens between 2 and 4 hours after oviposition of the previous egg. Alternatively, eggs were isolated from hens whose oviducts have been fistulated. See, for example, Gilbert and Woodgush, Journal of Reproduction and Fertility 5:451-453, 1963 and Pander, et al. Br Poult Sci 30:953-7, 1989, the disclosures of which are incorporated in their entirety herein by reference.

Prior to generating images of the early stage avian embryo, incubation of the DNA specific dye was performed according to the following protocol: The albumen capsule was removed and the ovum placed in a dish with the germinal disc facing the top. Remnants of the albumen capsule were removed from the top of the germinal disc. Phosphate buffered saline was added to the dish to prevent drying of the ovum. A cloning cylinder was placed around the germinal disc and 1.0 μg/ml of DAPI in PBS was added to the cylinder. Visualization was performed after approximately 15 minutes of incubation.

Injection

Preparation of the egg was done as described for incubation. Following removal of the capsule, 10-50 nanoliters of a 0.1 μg/ml solution of DAPI in PBS was injected into the germinal disc using a glass pipette. Visualization was performed approximately 15 minutes after injection.

Visualization

Following incubation, images of the inside of the avian early embryo were generated through the use of TPLSM. The germinal disc was placed under the microscope objective, and the pronuclear structures were searched within the central area of the disk, to a depth of 60 μm using low laser power of 3-6 milliwatts at a wavelength of 750 nm. Once the structures were found they were subsequently ablated.

Nuclear Ablation and Enucleation

Pronuclear structures were subjected to laser-mediated ablation. In these experiments, an Olympus 20x/0.5 NA (Numerical Aperture) water immersion lens was used. The x and y planes to be ablated were defined with the two photon software, while the z plane (depth) was just under 10 μm for this type of objective. Since the pronuclear structure was about 20 μm in diameter, the ablation comprised two steps (2 times 10 μm). The focal point was lowered to visualize the remaining of the pronucleus, which was subsequently ablated. The laser power used to ablate the pronuclei was between 30 and 70 milliwatts at a wavelength of 750 nanometers. For the ablation experiments described above, the image was zoomed by a factor of 4 to 5, giving an area compression of 16-25 fold. Then the power was increased 10-12 fold for a total intensity increase of 160-300 fold compared to the visualization intensity of 3-6 milliwatts. The ablation intensity (power density) is the functional parameter, i.e. the power increase of 10-12 fold results in ablation power of 30-70 milliwatts, but the zoom factor compressed this power into an area 16-25× smaller giving a power density increase of 160-300 fold.

EXAMPLE 2

Preparation of the Nuclear Donor Cell

Isolation of the Donor Nucleus

Fibroblast cells in cultured were trypsinized (0.25% Trypsin and 1 μM EDTA, Gibco catalog #25200-056), centrifuged twice in PBS containing 5% of Fetal Calf Serum and placed in a 60 mm plastic dish in PBS containing 5% of Fetal Calf Serum. Using the microscope/micromanipulation unit described below, under transmission light, the nuclear donors were then isolated by repeated pipetting of the cells, which disrupted the cytoplasmic membrane and released the nucleus from inside the cell.

EXAMPLE 3

Preparation of the Reconstructed Zygote

Injection

A micromanipulation unit, comprising a IM-16 microinjector and a MM-188NE micromanipulator, both from Nikon/Marishige, were adapted to an upright Nikon Eclipse E800. This microscope was adapted to operate under both transmission and reflective light conditions. This unique configuration has allowed us to morphologically examine and prepare (isolate the nuclei, as described above) somatic cells in suspension and to load the injection pipette using dry or water immersion lenses under diascopic illumination or transmitted light. This was followed by prompt localization and positioning of the germinal disc under the microscope and subsequent guided injection of the somatic cells, using dry and long distance lenses under fiber optic as well as episcopic illumination (light coming from the side and through the objectives onto the sample respectively).

EXAMPLE 4

Ovum Transfer

At the time of laying, recipient hens are anesthetized by wing vein injection with pentobarbital (0.7 ml of a 68 mg/ml solution). At this time, the infundibulum is receptive to receiving a donor ovum but has not yet ovulated. We have also established that pentobarbital is the anesthetic of choice. Feathers are removed from the abdominal area, and the area is scrubbed with betadine, and rinsed with 70% ethanol. The bird is placed in a supine position and a surgical drape is placed over the bird with the surgical area exposed. An incision is made beginning at the junction of the sternal rib to the breastbone and running parallel to the breastbone. The length of the incision is approximately two inches. After cutting through the smooth muscle layers and the peritoneum, the infundibulum is located. The infundibulum is externalized and opened using gloved hands and the donor ovum is gently applied to the open infundibulum. The ovum is allowed to move into the infundibulum and into the anterior magnum by gravity feed. The internalized ovum is placed into the body cavity and the incision closed using interlocking stitches both for the smooth muscle layer and the skin. The recipient hen is returned to her cage and allowed to recover with free access to both feed and water. Recovery time for the bird to be up, moving and feeding is usually within 45 min. of the operation's end. Eggs laid by the recipient hens are collected the next day, set, and incubated in a Jamesway incubator. They will hatch 21 days later.

Alternatively, a hen whose oviduct is fistulated allows the collection of eggs for enucleation (Gilbert and Woodgush, Journal of Reproduction and Fertility 5:451-453, 1963) and (Pancer, et al. Br Poult Sci 30: 953-7, 1989) as mentioned previously, but also the transfer of the reconstructed embryo to a recipient hen for the production of a hard shell egg (Wentworth, Poultry Science 39:782-784, 1960).

EXAMPLE 5

Production of Transgenic Hens by Microinjection of an Ovomucoid Promoter-Bacterial Artificial Chromosome Expression Vector

BAC clones OMC24-IRES-LC and OCM24-IRES-HC are used to produce transgenic chickens by microinjection. A detailed description of these BACs is disclosed in U.S. patent application Ser. No. 11/047,184, filed Jan. 31, 2005, the disclosure of which is incorporated in its entirety herein by reference. Briefly, each BAC includes a 70 kb chicken ovomucoid gene region with a coding sequence for either a heavy chain (HC) or light chain (LC) of a particular human IgG1 antibody. The HC and LC sequences are under the translational control of an internal ribosome entry site (IRES) which is inserted in the 5′ UTR of the ovomucoid gene region.

The BACs are linearized by enzymatic restriction digest. The digested DNA is phenol/CHCl₃ extracted, ethanol precipitated, suspended in 0.25 M KCl and is diluted to a working concentration of approximately 60 μg/ml (30 μg/ml OMC24-IRES-LC and 30 μg/ml OMC24-IRES-HC) with SV40 T antigen nuclear localization signal peptide (NLS) being added yielding a peptide:DNA molar ratio of 100:1 (Collas and Alestrom, 1996, Mol. Reprod. Develop. 45: 431-438, the disclosure of which is incorporated by reference in its entirety). The DNA samples are allowed to associate with the SV40 T antigen NLS peptide by incubation at room temperature for 15 minutes.

Introduction of the DNA-NLS complex into an avian egg is accomplished by microinjection employing a microinjection needle in conjunction with TPLSM. Briefly, TPLSM is used to visualize the germinal disc or zygote and the tip of the injection needle is inserted into the germinal disc such that the DNA-NLS complex can be injected directly into a nucleus contained in the germinal disc.

An injection needle, mounted on a micromanipulator, comprising a drawn out glass capillary tube coated with a thin layer of DAPI is employed to inject the DNA-NLS complex into nuclei contained in avian embryos. Stage I White Leghorn chicken embryos are immersed in Ringer's buffer and approximately 10 to 50 nanoliters of a 0.1 μg/ml solution of DAPI in PBS is injected into each germinal disc. After incubation for approximately 15 minutes the nuclei within the germinal disc are visualized using TPLSM in conjunction with an Olympus 20x/0.5 NA (Numerical Aperture) water immersion lens. Low laser power is used, for example, about 1-6 milliwatts at a wavelength of 750 nm. Approximately 10 nanoliters of the DNA-NLS mixture is injected into each of one or more nuclei in the embryos with the positioning of the needle and progress of the injection being monitored by TPLSM.

Injected embryos are surgically transferred to recipient hens by ovum transfer according to the method of Christmann et al. (see, for example, U.S. patent application Ser. No. 10/679,034, filed Oct. 2, 2003, the disclosure of which is incorporated herein in its entirety by reference) and hard shell eggs are incubated and hatched. See, Olsen and Neher, 1948, J. Exp. Zoo. 109: 355-366, the disclosure of which is incorporated in its entirety herein by reference.

Genomic DNA samples from one-week old chicks are analyzed for the presence of OMC24-IRES-LC and OMC24-IRES-HC by PCR. Female chicks that test positive for the transgene are grown to maturity and lay eggs containing the antibody, present primarily in the egg white.

EXAMPLE 6

Production of Transchromosomic Chickens Using Satellite DNA-Based Artificial Chromosomes

Satellite DNA-based artificial chromosomes, as described in Lindenbaum et al Nucleic Acids Res (2004) vol 32 no. 21 el72, the disclosure of which is incorporated in its entirety herein by reference, are isolated by a dual laser high-speed flow cytometer as described previously. See, for example, de Jong, G, et al. Cytometry 35: 129-133, 1999, the disclosure of which is incorporated by reference herein in its entirety.

The flow-sorted chromosomes are pelleted by centrifugation of a 750 μl sample containing approximately 10⁵ chromosomes in injection buffer at 2500×g for 30 min at 4° C. The supernatant, except the bottom 75 microliters (μl) containing the chromosomes, is removed resulting in a concentration of about 1000 chromosomes per μl of injection buffer. See, Monteith, et al. Methods Mol Biol 240: 227-242, 2004, the disclosure of which is incorporated by reference herein in its entirety.

Embryos for this study are collected from 24-36 week-old hens from commercial White Leghorn variety of G. gallus. Embryo donor hens are inseminated weekly using pooled semen from roosters of the same breed to produce fertile eggs for injection.

On the day of egg collection, fertile hens are euthanized 2 h post oviposition by cervical dislocation. Typically, oviposition is followed by ovulation of the next egg after about 24 minutes (Morris, Poultry Science 52: 423-445, 1973). The recently ovulated and fertilized eggs are collected from the upper magnum region of the oviduct under sterile conditions and placed in a glass well and covered with Ringers' Medium (Tanaka, et al. J Reprod Fertil 100: 447-449, 1994) and maintained at 41° C. until microinjection.

Injection of artificial chromosomes into a nucleus of a stage I embryo is achieved using TPLSM in combination with microinjection essentially as disclosed in example 5. Chromosomes are injected into a nuclear structure of the stage I embryos. Each injected nucleus is injected with approximately 1-5 chromosomes.

Following microinjection, the embryos are transferred to the oviduct of recipient hens using an optimized ovum transfer (OT) procedure (Olsen, M and Neher, B. J Exp Zool 109: 355-66, 1948), with the exception that the hens are anesthetized by isofluorane gas. Typically, about 26 h after OT, the recipient hens lay a hard shell egg containing the manipulated ovum. Eggs are incubated for 21 days in an incubator until hatching of the birds. Transchromosomic birds are identified by PCR analysis of DNA recovered from blood samples.

To verify the chromosomes are intact in the transchromosomic avians, metaphase spreads from fibroblast cells derived from founders are made and analyzed as described previously (Garside and Hillman (1985) Experientia 41: 1183-1184).

All references cited herein are incorporated by reference herein in their entirety and for all purposes to the same extent as if each individual publication, patent or patent application is specifically and individually indicated to be incorporated by reference in its entirety for all purposes.

The citation of any publication is for its disclosure prior to the filing date and should not be construed as an admission that the present invention is not entitled to antedate such publication by virtue of prior invention.

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.

Claims

1. A method comprising:

visualizing a nucleus of an avian egg by TPLSM.

2. The method of claim 1 wherein a substance is injected into the nucleus.

3. The method of claim 1 wherein the nucleus is injected with nucleic acid.

4. The method of claim 1 wherein the egg is an oocyte.

5. The method of claim 1 wherein the avian egg is an egg of an avian selected from the group consisting of avians of chicken, turkey, duck, goose, quail, pheasant, parrot, finche, hawk, crow, ratite, ostrich, emu and cassowary.

6. A method comprising:

visualizing a nucleus of an avian embryo by TPLSM.

7. The method of claim 6 wherein the embryo comprises more than one cell.

8. The method of claim 6 wherein the embryo contains between 1 and 100,000 cells.

9. The method of claim 6 wherein a substance is injected into the nucleus.

10. The method of claim 6 wherein the nucleus is injected with nucleic acid.

11. The method of claim 10 wherein the nucleic acid comprises a transgene.

12. The method of claim 6 wherein the avian egg is an egg of an avian selected from the group consisting of avians of chicken, turkey, duck, goose, quail, pheasant, parrot, finche, hawk, crow, ratite, ostrich, emu and cassowary.

13. The method of claim 6 wherein the egg is an early stage embryo.

14. The method of claim 6 wherein the egg is selected from the group consisting of a stage I, stage II, stage III, stage IV, stage V, stage VI, stage VII, stage VIII, stage IX, stage X, stage XI and stage XII embryo.

15. A method comprising visualizing an avian egg using TPLSM and injecting an artificial chromosome into a nucleus.

16. The method of claim 15 wherein the egg is an oocyte.

17. The method of claim 15 wherein the egg is a zygote.

18. The method of claim 15 wherein the avian egg is an egg of an avian selected from the group consisting of avians of chicken, turkey, duck, goose, quail, pheasant, parrot, finche, hawk, crow, ratite, ostrich, emu and cassowary.

19. The method of claim 15 wherein the embryo is an early stage embryo.

20. The method of claim 15 wherein the embryo is selected from the group consisting of a stage I, stage II, stage III, stage IV, stage V, stage VI, stage VII, stage VIII, stage IX, stage X, stage XI and stage XII embryo.