WO2001019181A1 - Cloning pigs using donor cells or nuclei from differentiated cells and production of pluripotent porcine. - Google Patents

Abstract

An improved method of nuclear transfer involving the transplantation of donor differentiated pig cell nuclei into enucleated pig oocytes is provided. The resultant nuclear transfer units are useful for multiplication of genotypes and transgenic genotypes by the production of fetuses and offspring. Production of genetically engineered or transgenic pig embryos, fetuses and offspring is facilitated by the present method since the differentiated cell source of the donor nuclei can be genetically modified and clonally propagated.

Description

CLONING PIGS USING DONOR CELLS OR NUCLEI FROM DEFFERENΗATED CELLS AND PRODUCΗON OF

PLURIPOTENT PORCINE.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation-in-part of U.S. Serial No. 08/888,057, filed July

3, 1997, which is a continuation-in-part of U.S. Serial No. 08/781,752, filed January 10,

1997, the contents of which are hereby incorporated by reference.

FIELD OF THE INVENTION

The present invention relates to cloning procedures in which cell nuclei derived

from differentiated pig cells are transplanted into enucleated porcine oocytes or

blastomeres. The nuclei are reprogrammed to direct the development of cloned embryos,

which can then be transferred into recipient females to produce fetuses and offspring, or

used to produce pluripotent cultured inner cell mass cells (CICM). The cloned embryos

can also be combined with fertilized embryos to produce chimeric embryos, fetuses

and/or offspring.

BACKGROUND OF THE INVENTION

The use of ungulate inner cell mass (ICM) cells for nuclear transplantation has also

been reported. For example, Collas et al., Mol. Reprod. Dev., 38:264-267 (1994)

discloses nuclear transplantation of bovine ICMs by microinjection of the lysed donor

cells into enucleated mature oocytes. Collas et al. disclosed culturing of embryos in vitro

for seven days to produce fifteen blastocysts which, upon transferral into bovine

recipients, resulted in four pregnancies and two births. Also, Keefer et al., Biol. Reprod.,

50:935-939 (1994), disclosed the use of bovine ICM cells as donor nuclei in nuclear

transfer procedures, to produce blastocysts which, upon transplantation into bovine recipients, resulted in several live offspring. Further, Sims et al., Proc. Natl. Acad. Sci.,

USA, 90:6143-6147 (1993), disclosed the production of calves by transfer of nuclei from

short-term in vitro cultured bovine ICM cells into enucleated mature oocytes.

The production of live lambs following nuclear transfer of cultured embryonic disc

cells has also been reported (Campbell et al., Nature, 380:64-68 (1996)). Still further, the

use of bovine pluripotent embryonic cells in nuclear transfer and the production of

chimeric fetuses has been reported (Stice et al., Biol. Reprod., 54:100-110 (1996); Collas

et al, Mol. Reprod. Dev., 38:264-267 (1994)). Collas et al demonstrated that granulosa

cells (adult cells) could be used in a bovine cloning procedure to produce embryos.

However, there was no demonstration of development past early embryonic stages

(blastocyst stage). Also, granulosa cells are not easily cultured and are only obtainable

from females. Collas et al did not attempt to propagate the granulosa cells in culture or

try to genetically modify those cells. Wilmut et al (Nature, 365:810-813 (1997))

produced nuclear transfer sheep offspring derived from fetal fibroblast cells, and one

offspring from a cell derived from an adult sheep.

Cloning pig cells is more difficult in comparison with cells of other species. This

phenomenon is illustrated by the following table:

There also exist problems in the area of producing transgenic pigs. By current

methods, heterologous DNA is introduced into either early embryos or embryonic cell

lines that differentiate into various cell types in the fetus and eventually develop into a

transgenic animal. However, many early embryos are required to produce one transgenic

animal and, thus, this procedure is very inefficient. Also, there is no simple and efficient

method of selecting for a transgenic embryo before going through the time and expense

of putting the embryos into surrogate females. In addition, gene targeting techniques

cannot be easily accomplished with early embryo transgenic procedures. Embryonic stem cells in mice have enabled researchers to select for transgenic

cells and perform gene targeting. This allows more genetic engineering than is possible

with other transgenic techniques. However, embryonic stem cell lines and other

embryonic cell lines must be maintained in an undifferentiated state that requires feeder

layers and or the addition of cytokines to media. Even if these precautions are followed,

these cells often undergo spontaneous differentiation and cannot be used to produce

transgenic offspring by currently available methods. Also, some embryonic cell lines

have to be propagated in a way that is not conducive to gene targeting procedures.

Methods for deriving embryonic stem (ES) cell lines in vitro from early

preimplantation mouse embryos are well known. (See, e.g., Evans et al., Nature, 29: 154-

156 (1981); Martin, Proc. Natl. Acad. Sci., USA, 78:7634-7638 (1981)). ES cells can be

passaged in an undifferentiated state, provided that a feeder layer of fibroblast cells

(Evans et al., Id.) or a differentiation inhibiting source (Smith et al., Dev. Biol., 121:1-9

(1987)) is present.

ES cells have been previously reported to possess numerous applications. For

example, it has been reported that ES cells can be used as an in vitro model for differen¬

tiation, especially for the study of genes which are involved in the regulation of early

development. Mouse ES cells can give rise to germline chimeras when introduced into

preimplantation mouse embryos, thus demonstrating their pluripotency (Bradley et al.,

Nature, 309:255-256 (1984)). In view of their ability to transfer their genome to the next generation, ES cells

have potential utility for germline manipulation of livestock animals by using ES cells

with or without a desired genetic modification. Moreover, in the case of livestock

animals, e.g., ungulates, nuclei from like preimplantation livestock embryos support the

development of enucleated oocytes to term (Smith et al., Biol. Reprod., 40:1027-1035

(1989); and Keefer et al, Biol. Reprod., 50:935-939 (1994)). This is in contrast to nuclei

from mouse embryos which beyond the eight-cell stage after transfer reportedly do not

support the development of enucleated oocytes (Cheong et al, Biol. Reprod., 48:958

(1993)). Therefore, ES cells from livestock animals are highly desirable because they

may provide a potential source of totipotent donor nuclei, genetically manipulated or

otherwise, for nuclear transfer procedures.

Some research groups have reported the isolation of purportedly pluripotent

embryonic cell lines. For example, Notarianni et al., J. Reprod. Fert. Suppl, 43:255-260

(1991), reports the establishment of purportedly stable, pluripotent cell lines from pig and

sheep blastocysts which exhibit some morphological and growth characteristics similar

to that of cells in primary cultures of inner cell masses isolated immunosurgically from

sheep blastocysts. Also, Notarianni et al., J. Reprod. Fert. Suppl, 41:51-56 (1990)

discloses maintenance and differentiation in culture of putative pluripotential embryonic

cell lines from pig blastocysts. Gerfen et al., Anim. Biotech, 6(1): 1-14 (1995) discloses

the isolation of embryonic cell lines from porcine blastocysts. These cells are stably maintained in mouse embryonic fibroblast feeder layers without the use of conditioned

medium, and reportedly differentiate into several different cell types during culture.

Further, Saito et al., Roux' Arch. Dev. Biol., 201:134-141 (1992) reports cultured,

bovine embryonic stem cell-like cell lines which survived three passages, but were lost

after the fourth passage. Handyside et al., Roux's Arch. Dev. Biol, 196:185-190 (1987)

discloses culturing of immunosurgically isolated inner cell masses of sheep embryos

under conditions which allow for the isolation of mouse ES cell lines derived from mouse

ICMs. Handyside et al. reports that under such conditions, the sheep ICMs attach, spread,

and develop areas of both ES cell-like and endoderm-like cells, but that after prolonged

culture only endoderm-like cells are evident.

Recently, Chemy et al., Theriogenology, 41:175 (1994) reported purportedly

pluripotent bovine primordial germ cell-derived cell lines maintained in long-term

culture. These cells, after approximately seven days in culture, produced ES-like colonies

which stained positive for alkaline phosphatase (AP), exhibited the ability to form

embryoid bodies, and spontaneously differentiated into at least two different cell types.

These cells also reportedly expressed mRNA for the transcription factors OCT4, OCT6

and HES1, a pattern of homeobox genes which is believed to be expressed by ES cells

exclusively.

Also recently, Campbell et al., Nature, 380:64-68 (1996) reported the production

of live lambs following nuclear transfer of cultured embryonic disc (ED) cells from day

nine ovine embryos cultured under conditions which promote the isolation of ES cell lines in the mouse. The authors concluded that ED cells from day nine ovine embryos are

totipotent by nuclear transfer and that totipotency is maintained in culture.

Van Stekelenburg-Hamers et al., Mol. Reprod. Dev., 40:444-454 (1995), reported

the isolation and characterization of purportedly permanent cell lines from inner cell mass

cells of bovine blastocysts. The authors isolated and cultured ICMs from 8 or 9 day

bovine blastocysts under different conditions to determine which feeder cells and culture

media are most efficient in supporting the attachment and outgrowth of bovine ICM cells.

They concluded that the attachment and outgrowth of cultured ICM cells is enhanced by

the use of STO (mouse fibroblast) feeder cells (instead of bovine uterus epithelial cells)

and by the use of charcoal-stripped serum (rather than normal serum) to supplement the

culture medium. Van Stekelenburg et al reported, however, that their cell lines resembled

epithelial cells more than pluripotent ICM cells.

Smith et al., WO 94/24274, published October 27, 1994, Evans et al, WO

90/03432, published April 5, 1990, and Wheeler et al, WO 94/26889, published

November 24, 1994, report the isolation, selection and propagation of animal stem cells

which purportedly may be used to obtain transgenic animals. Evans et al. also reported

the derivation of purportedly pluripotent embryonic stem cells from porcine and bovine

species which assertedly are useful for the production of transgenic animals. Further,

Wheeler et al, WO 94/26884, published November 24, 1994, disclosed purported

embryonic stem cells which are assertedly useful for the manufacture of chimeric and

transgenic ungulates. Thus, based on the foregoing, it is evident that many groups have attempted to

produce ES cell lines, e.g., because of their potential application in the production of

cloned or transgenic embryos and in nuclear transplantation.

Therefore, notwithstanding what has previously been reported in the literature,

there exists a need for improved methods of cloning pigs using cultured differentiated

cells as donor nuclei.

OBJECTS AND SUMMARY OF THE INVENTION

It is an object of the invention to provide novel and improved methods for

producing cloned pigs by nuclear transfer using differentiated cells or nuclei derived

therefrom as the donor cell or nucleus. Preferably, such differentiated cells will comprise

actively dividing, i.e., non-quiescent (proliferating) cells, in G G₂ or M cell phase, which

optionally may be genetically modified.

It is a more specific object of the invention to provide a novel method for cloning

pigs which involves transplantation of a differentiated pig cell or nucleus thereof into an

enucleated pig oocyte or blastomere.

It is another object of the invention to provide a method for multiplying adult pigs

having proven genetic superiority or other desirable traits.

It is another object of the invention to provide an improved method for producing

genetically engineered or transgenic pigs (i.e., NT units, fetuses, offspring). The

invention also provides genetically engineered or transgenic pigs, including those made

by such a method. It is a more specific object of the invention to provide a method for producing

genetically engineered or transgenic pigs by which a desired DNA sequence is inserted,

removed or modified in a differentiated pig cell or cell nucleus prior to use of that

differentiated cell or cell nucleus for formation of a NT unit. The invention also provides

genetically engineered or transgenic pigs made by such a method.

It is another object of the invention to provide a novel method for producing pig

CICM cells which involves transplantation of a nucleus of a differentiated pig cell or such

differentiated pig cell into an enucleated pig oocyte or blastomere, and then using the

resulting NT unit to produce pluripotent CICM cells. The invention also provides

pluripotent pig CICM cells and cell lines produced by such a method.

It is another object of the invention to use such pig CICM cells for therapy or

diagnosis.

It is a specific object of the invention to use such pig CICM cells for treatment or

diagnosis of any disease wherein cell, tissue or organ transplantation is therapeutically

or diagnostically beneficial. The CICM cells may be used within the same species or

across species, e.g., for human therapy.

It is another object of the invention to use cells~or tissues derived from pig NT

units, fetuses or offspring for treatment or diagnosis of any disease wherein cell, tissue

or organ transplantation is therapeutically or diagnostically beneficial. Such diseases and

injuries include Parkinson's, Huntington's, Alzheimer's, ALS, spinal cord injuries,

multiple sclerosis, muscular dystrophy, diabetes, liver diseases, heart disease, cartilage replacement, burns, vascular diseases, urinary tract diseases, as well as for the treatment

of immune defects, bone marrow transplantation, cancer, among other diseases. The

tissues may be used within the same species or across species.

It is another specific object of the invention to use cells or tissues derived from pig

NT units, fetuses or offspring, or pig CICM cells produced according to the invention for

the production of differentiated cells, tissues or organs.

It is another specific object of the invention to use cells or tissues derived from pig

NT units, fetuses or offspring, or pig CICM cells produced according to the invention in

vitro, e.g. for study of cell differentiation and for assay purposes, e.g. for drug studies. For

example, pig CICMs can be introduced into SCID mice.

It is another object of the invention to use cells, tissues or organs produced from

such tissues derived from pig NT units, fetuses or offspring, or pig CICM cells to provide

improved methods of transplantation therapy. Such therapies include by way of example

treatment of diseases and injuries including Parkinson's, Huntington's, Alzheimer's, ALS,

spinal cord injuries, multiple sclerosis, muscular dystrophy, diabetes, liver diseases, heart

disease, cartilage replacement, bums, vascular diseases, urinary tract diseases, as well as

for the treatment of immune defects, bone marrow transplantation, cancer, among other

diseases.

It is another object of the invention to provide genetically engineered or transgenic

tissues derived from pig NT units, fetuses or offspring, or pig CICM cells produced by

inserting, removing or modifying a desired DNA sequence in a differentiated pig cell or cell nucleus prior to use of that differentiated cell or cell nucleus for formation of a NT

unit.

It is another object of the invention to use the transgenic or genetically engineered

tissues derived from pig NT units, fetuses or offspring, or pig CICM cells produced

according to the invention for gene therapy, in particular for the treatment and/or

prevention of the diseases and injuries identified, supra.

It is another object of the invention to use the tissues derived from pig NT units,

fetuses or offspring, or pig CICM cells produced according to the invention, or transgenic

or genetically engineered tissues derived from pig NT units, fetuses or offspring, or pig

CICM cells produced according to the invention as nuclear donors for nuclear

transplantation.

It is another object of the invention to use transgenic or genetically engineered pig

offspring produced according to the invention in order to produce pharmacologically

important proteins.

Thus, in one aspect, the present invention provides a method for cloning a pig

(e.g., embryos, fetuses, offspring). The method comprises:

(i) inserting a desired differentiated pig cell or cell nucleus into a pig oocyte,

or blastomere, which is optionally enucleated under conditions suitable for the formation

of a nuclear transfer (NT) unit;

(ii) removing the endogenous nucleus from said pig oocyte or blastomere if

recipient pig oocyte or blastomere was not previously enucleated; (iii) activating the resultant nuclear transfer unit; and

(iv) transferring said cultured NT unit to a host pig such that the NT unit develops

into a fetus.

Optionally, the activated nuclear transfer unit is cultured until greater than the 2-

cell developmental stage. The culture medium will comprise known substituents, e.g.,

hormones, salts, that promote NT embryo development and may further optionally be

cultured in the presence of compounds that inhibit apoptosis, e.g., caspase inhibitors.

However, this is not required as one cell NT embryos can be transferred with resultant

fetal development virtually immediately after NT activation.

Further, the host pig will optionally comprise "helper embryos", e.g., normal pig

embryos, parthenogenetic embryos or tetraploid embryos to facilitate development of

cloned embryo. The number of helper embryos will preferably number from two to one

about one hundred, preferably two to fifty, more preferably two to ten embryos.

The cells, tissues and/or organs of the fetus are advantageously used in the area of

cell, tissue and/or organ transplantation, or production of desirable genotypes.

The present invention also includes a method of cloning a genetically engineered

or transgenic pig, by which a desired DNA sequence is inserted, removed or modified in

the differentiated pig cell or cell nucleus prior to insertion of the differentiated pig cell

or cell nucleus into the optionally enucleated oocyte or blastomere. Genetically

engineered or transgenic pigs produced by such a method are advantageously used in the area of cell, tissue and/or organ transplantation, production of desirable genotypes, and

production of pharmaceutical proteins.

If the cloned fetus, embryo, or offspring is to be used to produce cells, tissues or

organs for transplantation, it is also desirable to introduce one or more genetic

modifications to inhibit the risk of rejection. For example, it is known that specific

carbohydrate epitopes are involved in rejection responses, i.e., Galαl-Gal on the vascular

endothelium. Therefore, it may be advantageous to knock out genes that encode these

epitopes or replace such epitopes (e.g., mask) with other carbohydrate epitopes

("competitive glycosylation") that are present in human proteins. In particular,

introduction of the gene for the human histo-blood 0(H) antigen, for which 90% of

humans do not elicit antibodies against, in order to delete 90% of αGal expression is one

option. Alternatively, the αGal epitopes may be removed enzymatically in vivo by

introducing the cDNA for αgalactosidase, preferably expressed under the control of a

strong regulatable or constitutive promoter. Still another option is to eliminate expression

of the galactosyl transferase enzyme, e.g., by homologous recombination. Also, as this

will introduce new carbohydrate epitopes, it may be desirable to eliminate these epitopes

also by knockout, or enzymatically.

Also, because it is known that major histo-compatibility complex (MHC) class I

antigens elicit an immune response, it may be desirable to eliminate expression of genes

involved in such expression, e.g., beta 2-microglobulin (a peptide that forms part of the

class I molecule which is necessary for assembly and expression ), the proteasomal subunits LMP-2 and LMP-7, and or the peptide transporters TAP-1 and/or TAP-2 (TAP-

1 and TAP-2 transport the peptide fragments across the membrane of the endoplasmic

reticulum at the start of their journey to the cell surface.)

Still further, supra-physiologic down-regulation of local expression in the donor

tissues of inhibitory cytokines, such as LL-4, soluble CTLA-4, CTLA4-Ig, anti-CD40,

anti-CD40-L (CD 154), other inhibitors of receptor-ligand pairs or Fas ligand may inhibit

rejection, e.g., by inducing tolerance to the transplanted xenograft. Also, rejection may

be prevented or inhibited by enhancing expression of protective genes to suppress pro-

inflammatory medications associated with endothelial cell activation, and to protect the

donor cells, tissue or organ from apoptosis. For example, expression of the stress

responsiveness gene, hemeoxygenase, (HO-1) can potentiate xenograft survival. Also,

anti-apoptotic genes, e.g, which inhibit transcriptional activation can be over-expressed

to enhance xenograft survival. Many genes which inhibit apoptosis have been cloned and

sequenced.

Still further, endogenous porcine retroviruses may be eliminated to prevent the risk

of such sequences inserting into the host genome.

Also, because complement activation is a critical mediator of hyper-acute

rejection, this pathway may be inhibited by genetic modification. Specifically, the

complement cascade is known to be closely regulated by a group of endothelial proteins

including Decay Accelerating Factor (DAF, CD55), Membrane Cofactor Protein (MCP,

CD46), and CD59, which ordinarily act as inhibitors at various points in the complement cascade. These proteins have restricted activity, i.e., they only act on homologous (same

species) target molecules. Therefore, it may be beneficial to introduce genes that encode

human complement-inhibiting proteins (e.g., DAF, MCP) on the vascular endothelium

of porcine tissues. Also, it may be advantageous to combine these genetic approaches in

order to obtain optimal results, i.e., to produce cells, tissues or organs having very low

capability to elicit a rejection response.

Still further, the cells, tissues or organs may be cultured in vitro in the presence of

donor cells and other agents, e.g., CTLA-4 Ig, immunotoxins, anti-CD40-L, prior to

implantation into recipients in order to induce tolerance prior to implantation.

Of course, it may still be necessary to administer anti-rejection agents after

transplantation, which include by way of example cyclosporine, glucocorticoids, FK-506,

rapamycin, imuran, and derivatives thereof.

Also provided by the present invention are pigs obtained according to the above

method, and offspring of those pigs.

In another aspect, the present invention provides a method for producing pig

CICM (pluripotent) cells. The method comprises:

(i) inserting a desired differentiated pig cell or cell nucleus into a pig oocyte

or blastomere, optionally enucleated, under conditions suitable for the formation of a

nuclear transfer (NT) unit;

(ii) optionally removing the endogenous nucleus of the oocyte or blastomere

if not previously enucleated; (iii) activating the resultant nuclear transfer unit; and

(iv) culturing cells obtained from said cultured NT unit to obtain pig CICM cells.

Optionally, the activated nuclear transfer unit is cultured until greater than the 2-

cell developmental stage. The resultant pig CICM cells are advantageously used in the

area of cell, tissue and organ transplantation.

With the foregoing and other objects, advantages and features of the invention that

will become hereinafter apparent, the nature of the invention may be more clearly

understood by reference to the following detailed description of the preferred

embodiments of the invention and to the appended claims.

DETAILED DESCRIPTION OF THE INVENTION

The present invention provides improved procedures for cloning pigs by nuclear

transfer or nuclear transplantation. In the subject application, nuclear transfer or nuclear

transplantation or NT are used interchangeably.

According to the invention, cell nuclei derived from differentiated pig cells are

transplanted into enucleated pig oocytes or blastomeres. The nuclei are reprogrammed

to direct the development of cloned embryos, which can then be transferred into recipient

females to produce fetuses and offspring, or used to produce CICM cells. The cloned

embryos can also be combined with fertilized embryos to produce chimeric embryos,

fetuses and/or offspring.

Prior art methods have used embryonic cell types in cloning procedures. This

includes work by Campbell et al (Nature, 380:64-68, 1996) and Stice et al (Biol. Reprod., 54:100-110, 1996). In both of those studies, embryonic cell lines were derived from

embryos of less than 10 days of gestation. In both studies, the cells were maintained on

a feeder layer to prevent overt differentiation of the donor cell to be used in the cloning

procedure. The present invention uses differentiated cells.

Adult cells and fetal fibroblast cells from a sheep have purportedly been used to

produce sheep offspring (Wilmut et al, 1997). Studies have shown, however, that the

cloning of pigs is more difficult than cloning sheep. In fact, of the mammalian species

studied, cloning of sheep appears to be the easiest, and pig cloning appears to be the most

difficult. Therefore, the successful cloning of pigs using differentiated cell types,

preferably actively dividing non-quiescent cells, i.e., in the G_l5 G₂ or M cell phase,

according to the present invention is an unexpected outcome.

Thus, according to the present invention, multiplication of superior genotypes of

pigs is possible. This will allow the multiplication of adult pigs with proven genetic

superiority or other desirable traits. Genetic progress will be accelerated in the pig. By

the present invention, potentially billions of fetal or adult pig cells can be harvested and

used for the cloning procedure. This will result in many identical offspring in a short

period.

The present invention also allows simplification of transgenic procedures by

working with a cell source that can be clonally propagated. This eliminates the need to

maintain the cells in an undifferentiated state. Thus, genetic modifications, both random

integration and gene targeting, are more easily accomplished. Also by combining nuclear transfer with the ability to modify and select for these cells in vitro, this procedure is more

efficient than previous transgenic embryo techniques. According to the present invention,

these cells can be clonally propagated without cytokines, conditioned media and/or feeder

layers, further simplifying and facilitating the transgenic procedure. When transfected

cells are used in cloning procedures according to the invention, transgenic pig embryos

are produced which can develop into fetuses and offspring. Also, these transgenic cloned

embryos can be used to produce CICM cell lines or other embryonic cell lines.

Therefore, the present invention eliminates the need to derive and maintain in vitro an

undifferentiated cell line that is conducive to genetic engineering techniques.

In a preferred embodiment, which is particularly applicable for complex genetic

modifications, desired differentiated cells will be genetically modified, preferably in

tissue culture, these genetically modified cells used to produce a cloned fetus or animal,

and differentiated cells are then derived from the cloned fetus or animal, subjected to an

additional genetic modification, and the resultant twice genetically modified cells used

as a cell or nuclear donor for cloning. This process, which the inventors refer to as

"recloning", is useful for producing complex genetic modifications which require

substantial time. Essentially, by effecting such genetic modifications in different steps,

followed by cloning, it is possible to produce the desired genetic modifications without

the problem of the cells potentially becoming senescent prior to effecting all the desired

genetic modifications. Theoretically, this process can be repeated as many times as

necessary. Transgenic CICM cells produced according to the invention can be maintained

indefinitely in vitro, thereby providing a limitless supply of undifferentiated pluripotent

cells for the later production of desired differentiated cell types. In a preferred

embodiment, these CICM's will be maintained in an undifferentiated state according to

the method disclosed in commonly assigned U.S. Patent No. 5,905,042, which is

incorporated by reference in its entirety herein.

The present invention can also be used to produce cloned pig fetuses, offspring or

CICM cells which can be used, for example, in cell, tissue and organ transplantation. By

taking a fetal or adult cell from a pig and using it in the cloning procedure a variety of

cells, tissues and possibly organs can be obtained from cloned fetuses as they develop

through organogenesis. Cells, tissues, and organs can be isolated from cloned offspring

as well. This process can provide a source of "materials" for many medical and

veterinary therapies including cell and gene therapy. If the cells are transferred back into

the animal from which the cells were derived, then immunological rejection is averted.

Also, because many cell types can be isolated from these clones, other methodologies

such as hematopoietic chimerism can be used to avoid immunological rejection among

animals of the same species as well as between species.

Thus, in one aspect, the present invention provides a method for cloning a pig. In

general, the pig will be produced by a nuclear transfer process comprising the following

steps: (i) obtaining desired differentiated pig cells to be used as a source of donor

nuclei or donor cells;

(ii) obtaining pig oocytes or blastomeres;

(iii) optionally enucleating said oocytes or blastomeres;

(iv) transferring the desired differentiated cell or cell nucleus into the optionally

enucleated oocyte or blastomere, e.g., by fusion or injection, to form NT units;

(v) enucleating the NT unit to remove endogenous oocyte or blastomere

nucleus if not previously enucleated;

(vi) activating the resultant NT unit; and

(vii) transferring said cultured NT unit to a host pig such that the NT unit

develops into a fetus.

Optionally, the activated nuclear transfer unit is cultured until greater than the 2-

cell developmental stage. Also, optionally the host pig will contain one or more "helper"

embryos, e.g., normal pig embryos, tetraploid embryos, or parthenogenetic embryos to

promote development of cloned embryos.

The present invention also includes a method of cloning a genetically engineered

or transgenic pig, by which a desired DNA sequence is inserted, removed or modified in

the differentiated pig cell or cell nucleus prior to insertion of the differentiated pig cell

or cell nucleus into the optionally enucleated oocyte or blastomere (enucleation can be

effected after insertion of donor cell or nucleus). Also provided by the present invention are cloned pigs obtained according to the

above method, and offspring of those pigs. In contrast to previous transgenic and bred

pigs, these clones will comprise the identical genotype as a previously existing

differentiated cell or nucleus used as the nuclear transfer donor.

In addition to the uses described above, the genetically engineered or transgenic

pigs according to the invention can be used to produced a desired protein, such as a

pharmacologically important protein. That desired protein can then be isolated from the

milk or other fluids or tissues of the transgenic pig. Alternatively, the exogenous DNA

sequence may confer an agriculturally useful trait to the transgenic pig, such as disease

resistance, decreased body fat, increased lean meat product, improved feed conversion,

or altered sex ratios in progeny. Also, the exogenous DNA may encode one or more

DNAs that inhibit rejection of such cells in a heterologous host, e.g., human. In a

particularly preferred embodiment, the pig will express one or more human genes, e.g.,

those encoding structural proteins such as collagens, immune proteins, hormones,

enzymes, clotting factors, preferably inserted in favor of the porcine counterpart. This

will facilitate later recovery of the human protein as it will eliminate the need to remove

homologous porcine protein, e.g., porcine factor VIII, if human factor VIII is expressed

therein.

The present invention further provides for the use of NT fetuses and NT and

chimeric offspring in the area of cell, tissue and organ transplantation. In another aspect, the present invention provides a method for producing pig

CICM cells. The method comprises:

(i) inserting a desired differentiated pig cell or cell nucleus into an optionally

enucleated pig oocyte or blastomere, under conditions suitable for the formation of a

nuclear transfer (NT) unit;

(ii) removing endogenous oocyte or blastomere nucleus if not previously

enucleated;

(iii) activating the resultant nuclear transfer unit; and

(iv) culturing cells obtained from said cultured NT unit to obtain pig CICM

cells.

As noted above, a preferred culturing procedure is disclosed in U.S. Patent No.

5,905,042, incoφorated by reference in its entirety herein. Optionally, the activated

nuclear transfer unit is cultured until greater than the 2-cell developmental stage.

The pig CICM cells are advantageously used in the area of cell, tissue and organ

transplantation, or in the production of fetuses or offspring, including transgenic fetuses

or offspring.

As used herein, a fetus is the unborn young of a viviparous animal after it has

taken form in the uterus. In pigs, the fetal stage occurs from 30 days after conception

until birth. A mammal is an adult from birth until death.

Optionally, the NT units will be cultured to a size of at least 2 to 400 cells,

preferably 4 to 128 cells, and most preferably to a size of at least about 50 cells. Nuclear transfer techniques or nuclear transplantation techniques are known in the

literature and are described in many of the references cited in the Background of the

Invention. See, in particular, Campbell et al, Theriogenology, 43:181 (1995); Collas et

al, Mol. Report Dev., 38:264-267 (1994); Keefer et al, Biol. Reprod., 50:935-939 (1994);

Sims et al, Proc. Natl. Acad. Set, USA, 90:6143-6147 (1993); WO 94/26884; WO

94/24274, and WO 90/03432, which are incoφorated by reference in their entirety herein.

Also, U.S. Patent Nos. 4,944,384 and 5,057,420 describe procedures for bovine nuclear

transplantation.

Differentiated refers to cells having a different character or function from the

surrounding structures or from the cell of origin. Differentiated pig cells are those cells

which are past the early embryonic stage. More particularly, the differentiated cells are

those from at least past the embryonic disc stage (day 10 of bovine embryogenesis). The

differentiated cells may be derived from ectoderm, mesoderm or endoderm.

In a preferred embodiment, the differentiated cell will be an active proliferating

(non-quiescent) cell, i.e., in G G₂ or M cell phase. Such cells may be obtained directly

from an adult or fetal porcine, or may be isolated from an in vitro culture. Still further,

such differentiating cells may be derived from a non-porcine animal, e.g., a SCID mouse,

e.g., implanted with porcine immune cells. Suitable differentiated cells useful as the

donor cell or nuclei include somatic and germ cells, and nuclei derived therefrom.

Pig cells may be obtained by well known methods. Pig cells useful in the present

invention include, by way of example, epithelial cells, cumulus cells, neural cells, epidermal cells, keratinocytes, hematopoietic cells, melanocytes, chondrocytes,

lymphocytes (B and T lymphocytes), erythrocytes, dendritic cells, macrophages,

monocytes, mononuclear cells, and other immune cells, fibroblasts, cardiac muscle cells,

and other muscle cells, etc. Moreover, the pig cells used for nuclear transfer may be

obtained from different organs, e.g., skin, lung, pancreas, liver, stomach, intestine, heart,

reproductive organs, bladder, kidney, urethra and other urinary organs, etc. Also, stem

cells for specific differentiated cell types may be useful donor cells, e.g., hematopoietic

stem cells. These are just examples of suitable donor cells. Suitable donor cells, i.e.,

cells useful in the subject invention, may be obtained from any cell or organ of the body.

As noted, donor cells are intended to include both somatic and germ cells.

For example, in the case of germ cells this will include in particular primordial

germ cells.

Fibroblast cells are an ideal cell type because they can be obtained from

developing fetuses and adult pigs in large quantities. Fibroblast cells are differentiated

somewhat and, thus, were previously considered a poor cell type to use in cloning

procedures. Importantly, these cells can be easily propagated in vitro with a rapid

doubling time and can be clonally propagated for use in gene targeting procedures. Again

the present invention is novel because differentiated cell types are used. The present

invention is advantageous because these cells can be easily propagated, genetically

modified and selected in vitro. Methods for isolation of oocytes are well known in the art. Essentially, this will

comprise isolating oocytes from the ovaries or reproductive tract of a pig. A readily

available source of pig oocytes is slaughterhouse materials.

For the successful use of techniques such as genetic engineering, nuclear transfer

and cloning, oocytes can be matured in vitro before these cells are used as recipient cells

for nuclear transfer. This process generally requires collecting immature (prophase I)

oocytes from pig ovaries, e.g., pig ovaries obtained at a slaughterhouse, and maturing the

oocytes in a maturation medium prior to fertilization or enucleation until the oocyte

attains the metaphase II stage, which in the case of pig oocytes generally occurs about 35-

45 hours post-aspiration. For puφoses of the present invention, this period of time is

known as the "maturation period." As used herein for calculation of time periods,

"aspiration" refers to aspiration of the immature oocyte from ovarian follicles. A current

preferred protocol for aspiration of porcine ovarian follicles is disclosed in the Examples

which follow.

Additionally, metaphase II stage oocytes, which have been matured in vivo have

been successfully used in nuclear transfer techniques. For example, mature metaphase

II oocytes have been collected surgically from either non-superovulated or superovulated

cows or heifers 35 to 48 hours past the onset of estrus or past the injection of human

chorionic gonadotropin (hCG) or similar hormone. Similar procedures can be used in

pigs. A suitable procedure is described in the Examples which follow. The stage of maturation of the oocyte at enucleation and nuclear transfer has been

reported to be significant to the success of NT methods. (See e.g., Prather et al.,

Differentiation, 48, 1-8, 1991). However, it is anticipated that non-mature oocytes can

also be fused with differentiated cells or nucleus and used to produce nuclear transfer

embryos. For example, the oocyte may be matured in vitro after fusion. However, in

general, successful mammalian embryo cloning practices use the metaphase II stage

oocyte as the recipient oocyte because at this stage it is believed that the oocyte can be

or is sufficiently "activated" to treat the introduced nucleus as it does a fertilizing sperm.

In domestic animals, the oocyte activation period generally ranges from about 16-52

hours, preferably about 35-45 hours post-aspiration.

For example, immature oocytes can be matured in vitro in suitable maturation

medium. Preferably, but not necessarily, the porcine oocytes are matured in vitro, e.g.,

by placing such oocytes for about 22 hours in NCSU 37 medium (substituents identified

infra), supplemented with pFF, β-mercaptoethanol, cysteine, EGF (epidermal growth

factor), HCG/PMSG and cAMP, which are then washed with HECM/HEPES and

sucrose, preferably three times, and then placed for about 20 hours in same NCSU 37

medium, except that the hormones are eliminated. This is preferably effected in a four

well nunc plate.

Matured oocytes, wherein maturation may be effected in vitro (e.g., as described

above) or in vivo, are also preferably processed prior to enucleation. This is effected in order to remove cumulus cells. This can preferably be effected by treatment with

hyaluronidase followed by vortexing.

As noted, oocytes may be matured in vivo, followed by vortexing by inducing the

formation of oocyte maturation in vivo and collecting such mature oocytes. For example,

female porcines can be injected with PG600 and mature oocytes collected, typically,

about 5 to 6 days later, i.e., 24 to 36 hours after estrus. This may be effected by removal

of uterine traits from animals sent to slaughter, from which the oviduct is then dissected,

preferably flushed with suitable media, and the oocytes then stripped of cumulus cells.

This will be effected by the same methods as for in vitro matured oocytes, e.g., by

treatment with hyaluronidase followed by vortexing.

After maturation, which if effected in vitro typically takes about 30 to 50 hours,

and preferably about 40 hours, the oocytes are preferably then enucleated. However, this

is not necessary as enucleation can alternatively be effected after transplantation of the

donor cell or nucleus. The stripped oocytes produced by the above-described or

alternative procedures are preferably screened for polar bodies, and the selected

metaphase II oocytes, as determined by the presence of polar bodies, are preferably used

for nuclear transfer. Enucleation may be effected before or after introduction of donor

the differentiated cell or nucleus. Enucleation may be effected by known methods, such

as described in U.S. Patent No. 4,994,384, which is incoφorated by reference herein. In

a preferred embodiment, oocytes will be exposed to NCSU 23 medium (containing .2567

mg/10 ml of sucrose) and HXT for 20 minutes or longer. Enucleation is then conducted preferably in HECM/HEPES and sucrose (.2567 mg/10 ml) media, also containing

cytochalasin B. After enucleation, the oocytes are placed in a suitable medium, e.g.,

NCSU 23 containing sucrose (.2567 mg/10 ml). Alternatively, metaphase II oocytes can

be placed in HECM, optionally containing 7.5 micrograms per milliliter cytochalasin B

(CB) and 0.15 M sucrose, for immediate enucleation. Optionally, these oocytes may be

maintained in a suitable medium, for example an embryo culture medium such as NCSU

23 (see Table in the Examples) at 39°C and 5% C0₂, and then enucleated later, preferably

not more than 24 hours later.

Enucleation may be accomplished microsurgically using a micropipette to remove

the polar body and the adjacent cytoplasm. The oocytes are screened to identify those of

which have been successfully enucleated. This screening is preferably effected by

staining the oocytes with a suitable dye, e.g., 1 microgram per milliliter 33342 Hoechst

dye, for 20 minutes in a suitable medium, e.g., NCSU 23, and then determining visually

whether enucleation has been accomplished, e.g., by viewing the oocytes under ultraviolet

irradiation for less than 10 seconds. The oocytes that have been successfully enucleated

can then be placed in a suitable culture medium, e.g., NCSU 23 and sucrose (.2567 mg/10

ml), HECM and 0.15 M sucrose.

In the present invention, the recipient oocytes will preferably be enucleated at a

time ranging from about 30 hours to about 50 hours after the initiation of maturation,

more preferably from about 38 hours to about 46 hours after initiation of maturation, and

most preferably about 42 hours after initiation of maturation. A single porcine differentiated cell, e.g., a somatic or germ cell, pig cell or nucleus

will then be transferred into the perivitelline space of a preferably enucleated oocyte or

blastomere used to produce the NT unit. However, as has been noted, enucleation can

be effected after fusion if so desired. The pig cell and the enucleated oocyte will be used

to produce NT units according to methods known in the art. For example, the cells may

be fused by electrofusion. Electrofusion is accomplished by providing a pulse of

electricity that is sufficient to cause a transient breakdown of the plasma membrane. This

breakdown of the plasma membrane is very short because the membrane reforms rapidly.

Thus, if two adjacent membranes are induced to breakdown and upon reformation the

lipid bilayers intermingle, small channels will open between the two cells. Due to the

thermodynamic instability of such a small opening, it enlarges until the two cells become

one. Reference is made to U.S. Patent 4,997,384 by Prather et al., (incoφorated by

reference in its entirety herein) for a further discussion of this process. A variety of

electrofusion media can be used including e.g., sucrose, mannitol, sorbitol and phosphate

buffered solution. Fusion can also be accomplished using Sendai virus as a fusogenic

agent (Graham, Wister lnot. Symp. Monogr., 9, 19, 1969). A preferred fusion medium

used in the Examples which follow comprises 0.28 M mannitol, 10 μM CaCl₂, 100 μM

MgS0₄ and 10 mM histidine, pH 7.0.

In some cases (e.g. with small donor nuclei) it may be preferable to inject the

nucleus directly into the oocyte rather than using electroporation fusion. Such techniques are disclosed in Collas and Barnes, Mol. Reprod. Dev., 38:264-267 (1994), incoφorated

by reference in its entirety herein.

Alternatively, prior to introduction into the fusion chamber, the NT units can be

gradually exposed to the fusion medium via 3 incubations containing HECM to fusion

medium in ratios of 2:1, 1:2 and 0:1. The pig cell and oocyte can be electrofused by

various methods, e.g., by treatment in a 500 μm chamber by application of an electrical

pulse of 90- 120V for about 30 μsec, about 44 hours after initiation of oocyte maturation.

After fusion, the resultant fused NT units are maintained in fusion medium for 5 min, then

placed in HECM for 10 min, and then in NCSU 23 plus 7.5 mg/ml CB until activation.

Typically activation will be effected shortly thereafter, typically less than 24 hours later,

and preferably about 1-9 hours later, and most preferably about 2 hours later.

Currently, a preferred protocol is to transfer optionally enucleated oocytes which

have been treated with pronase, preferably about 400 μl/1 well, and then diluted in

suitable media, e.g., HECM/HEPES, and then centrifuged, preferably at about 6 kRPM

for four minutes, and then resuspended in suitable media, e.g., HECM/HEPES.

Alternatively, the oocytes can be treated with TE using the same dissociation conditions

as above.

In the current preferred protocol, transfer of the donor nucleus or cell is effected

in a suitable medium, e.g., HECM/HEPES + sucrose (.2567 mg/10 ml). After transfer is

complete, the resultant NT embryos are then transferred to a suitable medium, e.g., NCSU

23 containing sucrose (.2567 mg/10 ml). The NT embryos are then fused, preferably by applying 110V current for about thirty μ seconds in a suitable fusion media. As noted,

the current preferred fusion medium comprises 500 ml of Sigma water, .28 mannitol

(25.51 g), 100 μM MgS0₄ (.0123 g), and 100 mM Histidine (.776 g). However, other

known fusion media can be substituted therefor. Preferably, the fused NT units will then

be placed in a suitable medium, e.g., HECM/HEPES and then placed in NCSU 23 and

cytochalasin B (3 μl/2 ml) for about two hours prior to activation.

Optionally one or more caspase inhibitors may be used during maturation,

manipulation (stripping of cumulus cells) and/or activation to enhance blastocyst

development and the production of live offspring. Examples thereof include caspase 3,

caspase 8, and caspase 9.

The NT unit may be activated by known methods. Activation may be effected

before, simultaneous, or after. Such methods include, e.g., culturing the NT unit at sub-

physiological temperature, in essence by applying a cold, or actually cool temperature

shock to the NT unit. This may be most conveniently done by culturing the NT unit at

room temperature, which is cold relative to the physiological temperature conditions to

which embryos are normally exposed.

Suitable activation protocols include the following:

1. Activation by Ionomycin and DMAP

1- Place oocytes in Ionomycin (5 μM) with 2 mM of DMAP for 4

minutes; 2- Move the oocytes into culture media with 2 mM of DMAP for 4

hours;

3- Rinse four times and place in culture.

2. Activation by Ionomycin DMAP and Roscovitin

1- Place oocytes in Ionomycin (5 μM) with 2 mM of DMAP for four

minutes;

2- Move the oocytes into culture media with 2 mM of DMAP and 200

microM of Roscovitin for three hours;

3- Rinse four times and place in culture.

3. Activation by exposure to Ionomycin followed by cytochalasin and

cycloheximide.

1- Place oocytes in Ionomycin (5 microM) for four minutes;

2- Move oocytes to culture media containing 5 μg/ml of cytochalasin

B and 5 μg/ml of cycloheximide for five hours;

3- Rinse four times and place in culture.

4. Activation by electrical pulses

1- Place eggs in mannitol media containing 100 μM CaCL₂;

2- Deliver three pulses of 1.0 kVcm^"1 for 20 μsec, each pulse 22

minutes apart;

3- Move oocytes to culture media containing 5 μg/ml of cytochalasin

B for three hours. 5. Activation by exposure with ethanol followed by cytochalasin and

cycloheximide

2- Place oocytes in 7% ethanol for one minute;

3- Move oocytes to culture media containing 5 μg/ml of cytochalasin

B and 5 μg/ml of cycloheximide for five hours;

4- Rinse four times and place in culture.

6. Activation by microinjection of adenophostin

1- Inject oocytes with 10 to 12 picoliters of a solution containing 10

μM of adenophostin;

2- Put oocytes in culture.

7. Activation by microinjection of sperm factor

1 - Inj ect oocytes with 10 to 12 picoliters of sperm factor isolated either

from primates, pigs, bovine, sheep, goats, horses, mice, rats, rabbits

or hamsters;

2- Put eggs in culture.

8. Activation by microinjection of recombinant sperm factor.

9. Alternative DMAP/Ionomycin Protocol

Place oocytes or NT units, typically about 22 to 28 hours post maturation in about

2 mM DMAP for about one hour, followed by incubation for about two to twelve hours,

preferably about eight hours, in 5 μg/ml of cytochalasin B and 20 μg/ml cycloheximide. A current preferred method for effecting activation is in HECM/HEPES (H/H)

medium containing 1 mg/ml BSA, by a three step activation protocol. In the first step,

the NT fusions are placed in said H H BSA medium also containing 10 μm ionomycin

(4 μl 2 ml) for about four minutes, followed by rinsing in said H/H medium, and then

placing the NT fusions in NCSU 23 medium containing DMAP (1 μl ml) for about thirty

minutes. In the second step, the NT fusions are again placed in H/H medium containing

1 mg/ml BSA and also containing 5 μM ionomycin for about four minutes, followed by

rinsing in H/H, and placing the rinsed oocytes in NCSU 23 and DMAP (1 μl/ml) for

about thirty minutes. In the third step, the NT fusions are again placed in H/H containing

1 mg/ml BSA and further comprising 5 μM ionomycin and DMAP (1 μl/ml) for two

hours. After such activation protocol, the activated NT units are then preferably

transferred to a suitable medium, e.g., NCSU 23 medium. This medium, or other suitable

medium, is changed as necessary, typically about the third day. On day five, about 5%

FBS is preferably added and the NT units cultured to enable blastocyst formation.

However, as discussed above, the NT embryos can be transferred essentially immediately

after production, i.e., there may be successfully transferred at day one, while in the one

cell stage.

Alternatively, the pig NT units can be activated in a 500 μm chamber by

application of an electrical pulse of 30V for 30 μsec in an activation medium containing

0.28 M mannitol, 100 μM CaCl₂, 100 μM MgS0₄ and 10 mM histidine, pH 7.0. One hour later a second pulse of 15V is applied for 30 μsec. Between pulses the NT units are

maintained in NCSU 23 with CB at 39°C and 5% C0₂.

Still alternatively, activation may be achieved by application of known activation

agents. For example, penetration of oocytes by sperm during fertilization or the

activation factor contained in sperm cells can activated NT units. Also, treatments such

as electrical or chemical shock, calcium ionophores, and protein kinase inhibitors may be

used to activate NT embryos after fusion.

Yet alternatively, chemical activation can be effected about one to two hours after

fusion by placing NT units in HECM/HEPES containing 5 μM ionomycin for four

minutes, followed by washing three times in HECM/HEPES, and then placing in

HECM/HEPES plus cytochalasin B of ionomycin for about four minutes, washed three

times in HECM/HEPES and then placed in NCSU 23 containing 2mM of DMAP (6-

dimethylaminopurine) for about three hours. Afterward, the NT units are preferably

washed about three to four times in HECM/HEPES and then placed in NCSU 23 prior to

embryo transfer.

Further alternatively, after activation the NT units can be cultured for 3 to 4 hours

in NCSU 23 plus CB, and thereafter in NCSU 23 without CB. As noted, the NT units can

be transferred into the recipient female anytime after activation.

Alternatively, the activated NT units may then be cultured in a suitable in vitro

culture medium to produce CICM cells and cell colonies. Culture media suitable for

culturing and maturation of embryos are well known in the art. Examples of known media include Ham's F-10 + 10% fetal calf serum (FCS), Tissue Culture Medium- 199

(TCM-199) + 10% fetal calf serum, Tyrodes-Albumin-Lactate-Pyruvate (TALP),

Dulbecco's Phosphate Buffered Saline (PBS), Eagle's and Whitten's media. One of the

most common media used for the collection and maturation of oocytes is TCM-199, and

1 to 20% serum supplement including fetal calf serum, newborn serum, estrual cow

serum, lamb, pig, or steer serum. A preferred maintenance medium includes TCM-199

with Earl salts, 10% fetal calf serum, 0.2 mM Na pyruvate and 50 μg/ml gentamycin

sulphate. More preferably, the medium used is NCSU 23, and 2 to 5 days after activation

the NT units are cultured in fresh NCSU 23 and 5 to 10% fetal calf serum. Any of the

above may also involve co-culture with a variety of cell types such as granulosa cells,

oviduct cells, BRL cells, uterine cells, and STO cells.

Another maintenance medium is described in U.S. Patent 5,096,822, to

Rosenkrans, Jr. et al., which is incoφorated herein by reference. This embryo medium,

named CR1, contains the nutritional substances necessary to support an embryo.

The NT units can be cultured in NCSU 23 plus 5 to 10% FCS until the NT units

reach a desired size, whereupon they are transferred to a recipient female, or are used to

produce CICM cells or cell colonies. For example, these NT units can be cultured until

at least about 2 to 400 cells, more preferably about 4 to 128 cells, and most preferably at

least about 50 cells. Alternatively, NT embryos which are 1 cell can be introduced into

recipient females, i.e., those produced on the first day of activation. The culturing will

be effected under suitable conditions, i.e., about 38.5 °C and 5% C0₂, with the culture medium changed in order to optimize growth typically about every 2-5 days, preferably

about every 3 days.

The methods for embryo transfer and recipient animal management in the present

invention can be effected using standard procedures used in the embryo transfer industry.

Synchronous transfers are desirable, i.e., the stage of the NT embryo is in synchrony with

the estrus cycle of the recipient female. This advantage and how to maintain recipients

are discussed in Wall et al ("Development of porcine ova that were centrifuged to permit

visualization of pronuclei and nuclei," Biol. Reprod., 32:645-651 (1985)), the contents of

which are hereby incoφorated by reference.

As discussed previously, it is desirable but not necessary that the recipient female

also comprise "helper" embryos. Such helper embryos include normal porcine embryos,

e.g., produced by natural or artificial methods, parthenogenetic embryos (activated, non-

fertilized embryos which will not give rise to live offspring), and tetraploid embryos.

This has been found to enhance the development and maintenance of cloned porcines.

The number of such helper embryos can vary significantly, i.e., from about one

to two to as many as one hundred. It is hypothesized that these helper embryos may

produce materials, e.g., hormones and growth factors, that enhance embryonic

development and/or implantation of cloned embryos. An advantage of parthenogenetic

helper embryo or tetraploid helper embryo is that the only offspring which will develop

into viable offspring will be the clones. However, the production of cloned offspring can

be confirmed by genetic analysis, e.g., by PCR or by detecting expression or presence of cloned DNA sequence, e.g., by in situ hybridization, or by detecting expression of cloned

DNA, e.g., by use of radiolabeled antibody or other probe.

As noted, the present invention is particularly useful for producing cloned

genetically engineered or transgenic pigs. The present invention is advantageous in that

transgenic procedures can be simplified by working with a differentiated cell source that

can be clonally propagated. In particular, the differentiated cells used for donor nuclei

have a desired DNA sequence inserted, removed or modified. Those genetically altered,

differentiated cells are then used for nuclear transplantation with enucleated oocytes.

Any known method for inserting, deleting or modifying a desired DNA sequence

from a mammalian cell may be used for altering the differentiated cell to be used as the

nuclear donor. These procedures may remove all or part of a DNA sequence, and the

DNA sequence may be heterologous. Included is the technique of homologous

recombination, which allows the insertion, deletion or modification of a DNA sequence

or sequences at a specific site or sites in the cell genome. In a preferred embodiment,

endogenous porcine genes will be "knocked out" and the human homolog, e.g., a DNA

encoding an immunological protein, hormone, structural protein, clotting factor, enzyme,

receptor, or other cloned gene, knocked in.

The present invention can thus be used to provide adult pigs with desired

genotypes. Multiplication of adult pigs with proven genetic superiority or other desirable

traits is particularly useful, including transgenic or genetically engineered animals, and

chimeric animals. Thus, the present invention will allow production of single sex offspring, and production of pigs having improved meat production, reproductive traits

and disease resistance. Furthermore, cell and tissues from the NT fetus, including

transgenic and/or chimeric fetuses, can be used in cell, tissue and organ transplantation

for the treatment of numerous diseases as described below in connection with the use of

CICM cells. Hence, transgenic pigs have uses including models for diseases,

xenotransplantation of cells and organs, and production of pharmaceutical proteins.

As discussed, in a prefened embodiment, endogenous structural genes, e.g.,

collagens, will be replaced by human collagen genes. In another preferred embodiment,

porcine serum albumin gene will be replaced by HSA gene.

For production of CICM cells and cell lines, after NT units of the desired size are

obtained, the cells are mechanically removed from the zone and are then used. This is

preferably effected by taking the clump of cells which comprise the NT unit, which

typically will contain at least about 50 cells, washing such cells, and plating the cells onto

a feeder layer, e.g., uradiated fibroblast cells. Typically, the cells used to obtain the stem

cells or cell colonies will be obtained from the inner most portion of the cultured NT unit

which is preferably at least 50 cells in size. However, NT units of smaller or greater cell

numbers as well as cells from other portions of the NT unit may also be used to obtain ES

cells and cell colonies. The cells are maintained in the feeder layer in a suitable growth

medium, e.g., alpha MEM supplemented with 10% FCS and 0.1 mM β-mercaptoethanol

(Sigma) and L-glutamine. The growth medium is changed as often as necessary to

optimize growth, e.g., about every 2-3 days. This culturing process results in the formation of CICM cells or cell lines. One

skilled in the art can vary the culturing conditions as desired to optimize growth of the

particular CICM cells. Also, genetically engineered or transgenic pig CICM cells may

be produced according to the present invention. That is, the methods described above can

be used to produce NT units in which a desired DNA sequence or sequences have been

introduced, or from which all or part of an endogenous DNA sequence or sequences have

been removed or modified. Those genetically engineered or transgenic NT units can then

be used to produce genetically engineered or transgenic CICM cells. As noted

previously, a prefened means for maintaining such CICM's in culture in an

undifferentiated state is discussed in U.S. Patent No. 5,905,042, incoφorated by reference

herein.

The resultant CICM cells and cell lines have numerous therapeutic and diagnostic

applications. Most especially, such CICM cells may be used for cell transplantation

therapies.

In this regard, it is known that mouse embryonic stem (ES) cells are capable of

differentiating into almost any cell type, e.g., hematopoietic stem cells. Therefore, pig

CICM cells produced according to the invention should possess similar differentiation

capacity. The CICM cells according to the invention will be induced to differentiate to

obtain the desired cell types according to known methods. For example, the subject pig

CICM cells may be induced to differentiate into hematopoietic stem cells, neural cells,

muscle cells, cardiac muscle cells, liver cells, cartilage cells, epithelial cells, urinary tract cells, neural cells, etc., by culturing such cells in differentiation medium and under

conditions which provide for cell differentiation. Medium and methods which result in

the differentiation of CICM cells are known in the art as are suitable culturing conditions.

For example, Palacios et al, Proc. Natl. Acad. Set, USA, 92:7530-7537 (1995)

teaches the production of hematopoietic stem cells from an embryonic cell line by

subjecting stem cells to an induction procedure comprising initially culturing aggregates

of such cells in a suspension culture medium lacking retinoic acid followed by culturing

in the same medium containing retinoic acid, followed by transfenal of cell aggregates

to a substrate which provides for cell attachment.

Moreover, Pedersen, J. Reprod. Fertil. Dev., 6:543-552 (1994) is a review article

which references numerous articles disclosing methods for in vitro differentiation of

embryonic stem cells to produce various differentiated cell types including hematopoietic

cells, muscle, cardiac muscle, nerve cells, among others.

Further, Bain et al, Dev. Biol, 168:342-357 (1995) teaches in vitro differentiation

of embryonic stem cells to produce neural cells which possess neuronal properties. These

references are exemplary of reported methods for obtaining differentiated cells from

embryonic or stem cells. These references and in particular the disclosures therein

relating to methods for differentiating embryonic stem cells are incoφorated by reference

in their entirety herein.

Thus, using known methods and culture medium, one skilled in the art may culture

the subject CICM cells, including genetically engineered or transgenic CICM cells, to obtain desired differentiated cell types, e.g., neural cells, muscle cells, hematopoietic

cells, etc. It has been demonstrated by the successful production of chimeric animals

(pigs and bovines) that the culturing method disclosed in U.S. Patent 5,905,042, gives rise

to pluripotent CICMs.

The subject CICM cells may be used to obtain any desired differentiated cell type.

Therapeutic usages of such differentiated cells are unparalleled. For example,

hematopoietic stem cells may be used in medical treatments requiring bone manow

transplantation. Such procedures are used to treat many diseases, e.g., late stage cancers

such as ovarian cancer and leukemia, as well as diseases that compromise the immune

system, such as AIDS. Hematopoietic stem cells can be obtained, e.g., by fusing adult

somatic cells of a cancer or AIDS patient, e.g., epithelial cells or lymphocytes with an

enucleated oocyte, obtaining CICM cells as described above, and culturing such cells

under conditions which favor differentiation, until hematopoietic stem cells are obtained.

Such hematopoietic cells may be used in the treatment of diseases including cancer and

AIDS.

The present invention can be used to replace defective genes, e.g., defective

immune system genes, or to introduce genes which result in the expression of

therapeutically beneficial proteins such as growth factors, lymphokines, cytokines,

clotting factors, receptors, enzymes, etc.

DNA sequences which may be introduced into the subject CICM cells include, by

way of example, those which encode epidermal growth factor, basic fibroblast growth factor, glial derived neurotrophic growth factor, insulin-like growth factor (I and II),

neurotrophin-3, neurotrophin-4/5, ciliary neurotrophic factor, AFT-1, cytokines

(interleukins, interferons, colony stimulating factors, tumor necrosis factors (alpha and

beta), etc.), therapeutic enzymes, etc.

The present invention includes the use of pig cells in the treatment of human

diseases. Thus, pig CICM cells, NT fetuses and NT and chimeric offspring (transgenic

or non-transgenic) may be used in the treatment of human disease conditions where cell,

tissue or organ transplantation is wananted. In general, CICM cell, fetuses and offspring

according to the present invention can be used within the same species (autologous,

syngenic or allografts) or across species (xenografts). For example, brain cells from pig

NT fetuses may be used to treat Parkinson's disease.

Also, the subject CICM cells, may be used as an in vitro model of differentiation,

in particular for the study of genes which are involved in the regulation of early

development. Also, differentiated cell tissues and organs using the subject CICM cells

may be used in drug studies.

Further, the subject CICM cells may be used as nuclear donors for the production

of other CICM cells and cell colonies.

In order to more clearly describe the subject invention, the following examples are

provided. EXAMPLES

MATERIALS AND METHODS FOR PIG CLONING

Modified NCSU 37 Medium (mNCSU 37)

Use 18 mohm, RO, DI water. pH should be 7.4, Check osmolarity and record.

Sterilize by vacuum filtration (0.22 μm), date and initial bottle.

Store at 4°C and use within 10 days.

Modified TL-Hepes-PVA Medium (Hepes-PVA)

**60% syrup

* Add CaCl₂2H₂0 last, slowly to prevent precipitation

Use 18 mohm, RO, DI water.

Adjust pH to 7.4, Check osmolarity and record.

Sterilize by vacuum filtration (0.22 μm), date and initial bottle.

Store at 4°C and use within 10 days.

NCSU 23 Medium

Use 18 mohm, RO, DI water. pH should be 7.4, Check osmolarity and record.

Sterilize by vacuum filtration (0.22 μm) using red Nalgene filters, date and initial bottle.

Store at 4°C and use within 10 days.

NOTE: BSA type is important. Preferably use Sigma BSA catalog # A-7906. Also, Pen

G/Strept is optional.

Maturation Medium fMATt;

18.0 ml mNCSU 37

2.0 ml porcine follicular fluid (pFF)

7.0 μl of diluted β-Mercaptoethanol (dilute 10 μl β-Mercaptoethanol to 990 μl mNCSU

37; 50 μM final concentration)

0.002 g cysteine (0.6 mM final concentration)

20 μl EGF Stock (Epidermal Growth Factor from 10 ng/μl EGF stock)

Protocol for Aspiration of Porcine Ovarian Follicles:

Follicles are graded visually for size. Follicles that are 3 mm x 3 mm up to 7 mm

x 7 mm are considered to be good candidates for aspiration. By contrast, follicles that are

larger, especially those that are larger than 1 cm x 1 cm are poor candidates for aspiration. A 10 cc syringe with an 18 gauge needle is preferably used to draw up 1 ml of

heparin (concentration of 100 IU/ml) which is then held upright and the heparin solution

drawn down to the 10 cc line to coat the inside of the syringe. The heparin is then

discarded from the syringe. The ovary is then held in one hand and the syringe in the

other hand.

The needle is then positioned bevel down and pushed into the follicle with slight

drawback on the syringe until the follicle collapses because all the follicular fluid along

with the follicle have been extracted. A slight wiggling motion back and forth during the

aspiration process inside the follicle has been found to promote the collapse of the follicle

and removal of all the follicle contents.

Aspiration is continued to obtain as many follicles as possible until the 10 cc mark

is obtained. The needle is then removed from the syringe and the follicular fluid is

deposited in a collection tube.

Care should be taken to remove the needle when depositing the follicular fluid in

the tube in order to avoid stripping of the cumulus cells as well as damaging the oocytes.

Also, it is desirable to aspirate as many follicles of good size in a particular ovary before

discarding the ovary and moving on to the next one. For optimal results, it is also

desirable to change the heparin-coated syringe for each set of tracts. Porcine Follicular Fluid Preparation

Porcine follicular fluid (pFF), typically about 3-6 mm follicles, is collected from

prepubertal gilts. The oocytes and follicular cells are allowed to settle therefrom, e.g., by

waiting for about 5-10 minutes. The pFF is then aspirated and removed to 15 ml conical

tubes. This is then centrifuged on Sorvall at 4°C at 3000 φm for 30 minutes. The tubes

are removed and the pFF is collected from above the pellet, pooled, and filtered through

a 0.8 μm, followed by a 0.45 μm filter (Sterivex). The filtered material is then aliquoted

to 1.5 ml sterile microfuge tubes and then frozen at -20 °C until use.

Epidermal Growth Factor Stock (EGF^ and Preparation

100 μg EGF

10 ml mNCSU 37 with 0.1% BSA

Mix well. Aliquot to 25 μl, freeze at -20 °C.

Equine Chorionic Gonadotropin and human Chorionic Gonadotropin Stock for MAT (TMSG/hCG^ and Preparation

ECG (PMSG 6000; Intervet Inc., Millsboro; DE 19966)

This material is diluted from 6000 IU to 2000 IU/ml by the addition of 3 ml dH₂0.

hCG (Chorulon; Intervet Inc.)

The hCG material is diluted from 10,000 IU to 2000 IU/ml by the addition of 5 ml

dH,0. Afterward, 1 ml of diluted PMSG and 1 ml of diluted hCG are mixed to get 1000

IU/ml of each hormone. Fifty μl aliquots are then made up and frozen at -20°C. The

remaining PMSG and hCG stocks are also frozen.

db-cAMP 100 mM Stock and Preparation

25 mg db-cAMP (stored in dessicator at -20 ° C)

0.509 ml dH₂0

The above materials are well mixed and used to produce 50 μl aliquots which are

then frozen at -20°C.

Activation Medium

Same as HECM/HEPES except contains 1 mg/ml BSA.

Antibiotic/ Antimycotic (Ab/Am)

100 U/l Penicillin, 100 μg/1 streptomycin and 0.25 μg/1 amphotericin B, (Gibco

#15240-062)

Ten ml aliquot of the above materials are added per liter of saline. Ten μl of this

mixture is added per ml.

Oocyte-Cumulus Complex (OCO Collection

Ovaries are transported to the lab at 25 °C and immediately washed with 0.9%

saline with antibiotic/antimycotic (10 ml L; Gibco #600-5240g). Follicles between 3-6

mm are aspirated using 18g needles and 50 ml Falcon tubes connected to vacuum system

(GEML bovine system). After a tube is filled, the OCC's are allowed to settle for 5-10 minutes. Follicular fluid (pFF) is aspirated and saved for use in culture system if needed

(see pFF preparation protocol below).

Preferred protocol for In Vivo Oocyte Recovery and Transfer of Nuclear Transfer Pig Embryos

Landrance x York or Landrance x Hampshire Gilts of 225 to 275 pounds are

injected with PG600 and 5 to 6 days later (24 to 36 hours after the onset of estrus) the

animals are then sent to slaughter. The uterine tracts are recovered and placed in an

insulated container, and taken to the laboratory.

The oviduct is dissected from the uterus and flushed with buffer media.

Thereafter, oocytes are stripped from cumulus cells as described supra for the in vitro

matured oocytes.

After activation, eggs are loaded into a 5 ^XA inch Tom Cat catheter (Sovereign Cat.

#8890-703021) and surgically transfened into the oviduct of a synchronous recipient gilt

(same breed and 200 to 300 pounds). Half of the total number of embryos is transfened

into each oviduct and typically no more than 100 NTs are transfened per animal. In some

instances, 10 to 20 naturally produced two to four-cell embryos are transfened along with

the NTs as "helper" embryos. Caspase inhibitors optionally may be included in the buffer

media to enhance embryo development and maintenance. The gilts are anesthetized using

1 ml per 50 pounds of a mix of the 250 mg of Xylazine, 250 mg of ketamine and 500 mg

of Telazol reconstituted in 5 ml. After implantation, recipient gilts are preferably moved

to a new isolated facility. Pregnancy check is preferably performed thirty days after surgery, typically by

ultrasound. The fetuses can be retrieved at that time by C-section, and analyzed by PCR

and Southern Blot in order to determine which one of them was produced by nuclear

transfer. Alternatively, cloned fetuses will be allowed to develop to full term and be bom

by natural methods or C-section.

OCC Washing

OCCs are resuspended in 20 ml Hepes-PVA and allowed to settle; repeat 2 times.

After last wash, OCCs are moved to grid dishes and selected for culture. Selected OCCs

are washed twice in 60 mm dishes of Hepes-PVA. All aspiration and oocyte recovery are

performed at room temperature (approx. 25 °C).

In Vitro Maturation fTVHVn

The washed OCCs (about 50) are placed in a four-well Nunc plate tje we;;s pf

which each contain 0.5 ml of Maturation medium (described above) for about 22 hours.

Afterward, the oocytes are then placed in the same medium except lacking hormones for

about twenty hours.

Isolation of primary cultures of porcine embryonic and adult fibroblast cells

Primary cultures of porcine fibroblasts are obtained from pig fetuses 30 to 114

days post-fertilization, preferably 35 days. The head, liver, heart and alimentary tract are

aseptically removed, the fetuses minced and incubated for 30 minutes at 37 °C in

prewarmed trypsin EDTA solution (0.05% trypsin/0.02% EDTA; GIBCO, Grand Island,

NY). Fibroblast cells are plated in tissue culture dishes and cultured in fibroblast growth medium (FGM) containing: alpha-MEM medium (BioWhittaker, Walkersville, MD)

supplemented with 10% fetal calf serum (FCS) (Hyclone, Logen, UT), penicillin (100

IU/ml) and streptomycin (50 μl/ml). The fibroblasts are grown and maintained in a

humidified atmosphere with 5% C0₂ in air at 37 °C.

Adult fibroblast cells are isolated from the lung and skin of a pig. Minced lung

tissue is incubated overnight at 10 °C in trypsin EDTA solution (0.05% trypsin/0.02%

EDTA; GIBCO, Grand Island, NY). The following day tissue and any disassociated cells

are incubated for one hour at 37°C in prewarmed trypsin EDTA solution (0.05%

trypsin/0.02% EDTA; GIBCO, Grand Island, NY) and processed through three

consecutive washes and trypsin incubations (one hr). Fibroblast cells are plated in tissue

culture dishes and cultured in alpha-MEM medium (BioWhittaker, Walkersville, MD)

supplemented with 10% fetal calf serum (FCS) (Hyclone, Logen, UT), penicillin (100

IU/ml) and streptomycin (50 μl/ml). The fibroblast cells can be isolated at virtually any

time in development, ranging from approximately post embryonic disc stage through

adult life of the animal (porcine day 9 to 10 after fertilization to 5 years of age or longer).

Preparation of fibroblast cells for nuclear transfer

Examples of fetal fibroblasts which may be used as donor nuclei are:

1. Proliferating fibroblast cells that are not synchronized in any one cell stage

or serum starved or quiescent can serve as nuclear donors. The cells from the above

culture are treated for 10 minutes with trypsin EDTA and are washed three times in 100%

fetal calf serum. Single cell fibroblast cells are then placed in micromanipulation drops of HbT medium (Bavister et al., 1983). This is done 10 to 30 min prior to transfer of the

fibroblast cells into the enucleated pig oocyte. Preferably, proliferating transgenic

fibroblast cells having the CMV promoter and green fluorescent protein gene (9th

passage) are used to produce NT units.

2. By a second method, fibroblast cells are synchronized in Gl or GO of the

cell cycle. The fibroblast cells are grown to confluence. Then the concentration of fetal

calf serum in the FGM is cut in half over four consecutive days (day 0 = 10%, day 1 =

5%, day 2 - 2.5%, day 3 = 1.25%, day 4 = 0.625%. On the fifth day the cells are treated

for 10 minutes with trypsin EDTA and washed three times in 100% fetal calf serum.

Single cell fibroblasts are then placed in micromanipulation drops of HbT medium. This

is done within 15 min prior to transfer of the fibroblast cells into the enucleated pig

oocyte.

Alternatively, donor cells, e.g., fibroblasts can be obtained directly from a live

animal, e.g., an adult porcine, e.g., from a tissue or fluid source.

Removal of cumulus cells

After a maturation period, which ranges from about 30 to 50 hours, and preferably

about 42 hours, the oocytes can preferably be enucleated. Removal of cumulus cells is

preferably effected prior to enucleation by contacting cells with H/H media containing

.68 mg/ml of hyaluronidase followed by vortexing for about three minutes. Alternatively,

prior to enucleation the oocytes can be removed and placed in HECM (Seshagiri and

Bavister, 1989) containing 1 milligram per milliliter of hyaluronidase prior to removal of cumulus cells. This may be effected by repeated pipetting through very fine bore pipettes

or by vortexing briefly (about 3 minutes). The stripped oocytes are then screened for

polar bodies, and the selected metaphase II oocytes, as determined by the presence of

polar bodies, are then used for nuclear transfer.

Enucleation:

A cunent prefened procedure comprises exposure of oocytes to NCSU 23 +

sucrose + HXT for at least 20 minutes, followed by enucleation effected in H/H medium

containing sucrose and cytochalasin B. After enucleation, the oocytes are preferably

placed in NCSU 23 containing sucrose before transfer.

Transfer:

Cells for transfer are preferably treated with pronase or TE. In the case of pronase,

the cells are treated with 400 μl/1 well, allowed to incubate and then diluted in H/H and

then spun at 6 kRPM for four minutes, and then resuspended in H/H for use.

In the case of TE, the same protocol is used. Transfer is then preferably

effected in H/H containing sucrose and when complete, the cells are placed in NCSU 23

containing sucrose for fusion.

Fusion Media:

Recipe: 500 mL of Sigma water

.28 mannitol 25.5 lg

100 μM MgSO₄ .0123g

lO mM histidine .776 g Fusion:

Nuclear transfer units are preferably put through a gradient of H/H and mannitol

1:2, 1:1, and 0:2, and then fused in a 600 μm chamber, and flooded with mannitol.

The cells are electrofused, by placing cells for 30 μsec @100 V. After fusion, the

NT units are placed in pure H/H and then into NCSU 23 + Cyto B (3 μl/2ml), preferably

for two hours before activation.

Activation

Examples of methods of activation which may be used, typically at about 47 to

49 hours post maturation, include the following procedures, identified previously in the

present application. As noted, activation may be effected prior, proximate, or after

fusion.

1. Ionomycin/DMAP procedure:

NT units are placed in H/H medium containing 1 mg/ml BSA + 10 μM ionomycin

(4 μl/2 ml) for four minutes, rinsed with H/H and then placed in NCSU 23 + DMAP (1

μl/ml) for thirty minutes. Afterward, the NT units are placed in the same H/H medium

containing BSA (1 μl/ml) BSA and 5 μM ionomycin, and treated for thirty minutes.

Afterward, the NT units are placed in the same H/H medium containing 1 mg/ml

BSA + 2.5 μM ionomycin and DMAP for two more hours.

The NT units are then rinsed to remove DMAP, and then cultured in NCSU 23.

The media is changed on day three and 5% FBS is added late on day five, and the NT

units cultured until blastocysts form. 2. Single activation pulse. NT units are removed from the NCSU 23 plus CB and

washed three times in activation medium. After equilibration, the NT units are placed

into the fusion chamber (500 μm gap) filled with activation medium as described in the

fusion procedure. A pulse of 30 V for 30 μsec is applied. Then the NT units are

immediately washed three times in HECM HEPES and cultured (39°C, 5% C0₂) in

NCSU 23 for 2 more hours until embryo transfer or in vitro culture (39°C, 5% C0₂ in

NCSU 23). If cultured, NT units are placed in fresh NCSU 23 plus 5% fetal calf serum

on day 2 of culture. The results in Table 1 indicate that oocytes can be activated using

this procedure and that they have developmental capabilities.

3. Two activation pulses. NT units are removed from the NCSU 23 plus CB and

washed three times in activation medium. After equilibration the NT units are placed

back into the fusion chamber (500 μm gap) filled with activation medium as described

in the fusion procedure. A pulse of 30 V for 30 μsec is applied. Then the NT units are

immediately washed three times in HECM HEPES, placed back in NCSU 23 plus CB,

and cultured in this at 39 °C, 5% C0₂, until the next electrical pulse 1 hr later. After 1 hr

this time the activation medium equilibration step is repeated and a pulse of 15 V for 30

μsec is applied. Then the NT units are immediately washed three times in HECM

HEPES, placed back in NCSU 23 plus CB, and cultured in this medium at 39°C, 5% C0₂,

for 2 to 6 more hours. The NT units are then cultured using the same procedure described

above in 1. The results in Table 1 indicate that oocytes can be activated using this

procedure and that they have developmental capabilities. The same is true for nuclear transfer embryos. Four blastocyst stage NT units were produced with the two pulse

activation procedure.

4. Sperm factor. First described in mammalian sperm by Stice and Robl (Mol.

Reprod. Dev., 25:272-280 (1990)) (the contents of which are hereby incoφorated by

reference), this factor causes activation in oocytes. The method of sperm factor isolation

from pig sperm cells and microinjection is described in Fissore et al. (Mol. Reprod. Dev.,

46:176-189 (1997)), the contents of which are hereby incoφorated by reference. NT

units are removed from the NCSU 23 plus CB and placed in micromanipulation plates

described above for enucleation and fibroblast transfer. Using a micro-injection needle

(1 μm opening) filled with sperm factor the oocytes undergo activation after the delivery

of the factor into the cytoplasm of the NT unit. After microinjection, the NT embryos are

washed in HECM HEPES and held in NCSU 23 plus CB for 2 to 6 hours, and thereafter

in NCSU 23 until embryo transfer.

Table 1. Development of activated oocytes and NT units using different activation procedures.

Embryo transfer

Methods of one cell embryo transfer in pigs are well known (see, for example,

Pinkert et al., 1993, the contents of which are hereby incoφorated by reference).

Typically, 20 to 30 NT and up to 100 NT units are synchronously transfened into the

oviduct of bred or unbred gilts. After and beyond 29 days of gestation, nuclear transfer

fetuses (transgenic or non-transgenic) can be recovered from the recipient gilt.

Alternatively, the fetuses are allowed to go to term (114 day gestation) and cloned pig

offspring are produced. As noted, such gilts will also preferably contain "helper"

embryos, e.g., normal porcine embryos, parthenogenetic porcine embryos, or tetraploid

embryos. The number of helper embryos may vary from 1 to about 50, typically 2 to 4. Use of Caspase Inhibitor During In Vitro Maturation. Stripping of Oocytes or Activation

As discussed above, it has been discovered that the addition of caspase inhibitors,

e.g., Caspase 3, 8 or 9, enhances the number of blastocysts that arise from NT procedures.

Some data in support of this discovery are provided in the Table below.

Also, for the reader's convenience, the cunent prefened protocol for transfer of

porcine oocytes being used by the inventors is summarized below. Summary of Preferred Cloning Methods and Materials

Protocol for Nuclear Transfer of Porcine Oocytes:

In Vitro Maturation:

22 hours in NCSU 37 + PFF + B-mercaptoethanol + cysteine + EGF + 1000 IU

of HCG/PMSG & cAMP during shipping, rinse with H/H three times

20 hours in above media without hormones in a four-well nunc plate.

Processing of Porcine Oocytes:

Stripping with .68 mg/ml of Hyal and vortex on three for three minutes.

Media needed:

HECM/HEPES + sucrose (.2567 mg/10 ml)

NCSU 23 + sucrose (.2567 ml/10 ml)

NCSU 23

Fusion Media:

10 μM Mannitol

Recipe: 500 mL of Sigma water

.28 Mannitol 25.51 g

100 μM MgSO₄ .0123 g

lO mM Histidine .776 k Fusion:

Nuclear Transfer Units are put through a gradient of H/H and Mannitol 1:2, 1 :1,

and 0:2 and then fused in a 600 μm chamber, and flooded with Mannitol.

Fusion is effected by applying 100V electricity for 30μ seconds. Fusions are

immediately placed in pure H/H and then into NCSU 23 + Cyto B (3 μl/2 ml) for two

hours before activation.

Enucleation:

Oocytes are exposed to NCSU 23 + sucrose + HXT for at least twenty minutes.

Enucleation is performed in H/H + sucrose + cyto B.

Oocytes are then put into NCSU 23 + sucrose before transfer.

Transfer:

Cells prepared with either pronase or TE

Pronase — 400 μl per well, let sit, dilute in H/H spin 6 kRPM for four

minutes, resuspend in H/H for use

TE — Same protocol for cells disassociation.

Transfer is done in H/H + sucrose and when complete back into NCSU 23 +

sucrose for fusion.

Activation Media:

H/H Zl (same as H/H but with 1 mg/ml BSA)

Three separate treatments separated by thirty minutes. 1. H/H Z 1 + 10 μM Ionomycin (4 μl/2 ml) for four minutes, rinse with

H/H and then put in NCSU 23 + DMAP (1 μl/ml) for thirty minutes.

2. H/H Zl + 5 1 + 2.5 μM Ionomycin and then DMAP for two more

hours.

Activated NT units are rinsed to remove DMAP and then cultured in NCSU 23.

Media is changed at day three and 5% FBS is added late on day five, and cultured

until blastocysts.

While the invention has been described with respect to certain specific

embodiments, it will be appreciated that many modifications and changes thereof may be

made by those skilled in the art without departing from the spirit of the invention. It is

intended, therefore, by the appended claims to cover all modifications and changes that

fall within the true spirit and scope of the invention.

Claims

WHAT IS CLAIMED IS:

1. A method of cloning a porcine fetus or live offspring, comprising:

(i) inserting a desired differentiated pig cell or cell nucleus into an optionally

enucleated pig oocyte or blastomere, under conditions suitable for the formation of a

nuclear transfer (NT) unit;

(ii) removing the endogenous nucleus from said oocyte or blastomere if not

previously removed;

(iii) activating the resultant nuclear transfer unit;

(iv) optionally culturing said NT unit; and

(v) transferring said optionally cultured NT unit to a host mammal such that the

NT unit develops into a porcine fetus or animal.

2. The method according to Claim 1 , which results in a live offspring.

3. The method according to Claim 1, wherein a desired DNA is inserted,

removed or modified in said differentiated pig cell or cell nucleus, thereby resulting in

the production of a genetically altered NT unit.

4. The method according to Claim 3, which further comprises developing the

fetus to an offspring.

5. The method according to Claim 1, which comprises culturing said activated

nuclear transfer unit until greater than the 2-cell developmental stage.

6. The method of Claim 1, wherein the transfened NT unit is one cell.

7. The method of Claim 6, wherein transfer is effected on the same day as

activation.

8. The method of Claim 1 , wherein the oocyte is enucleated after introduction

of said differentiated cell or nucleus.

9. The method of Claim 1, wherein the differentiated cell or nucleus is that of

a proliferating (non-quiescent) cell.

10. The method of Claim 9, wherein said differentiated cell is in G_l5 G₂ or M.

11. The method of Claim 1 , wherein the pig oocyte is matured in vitro.

12. The method of Claim 1, wherein the pig oocyte is matured in vivo.

13. The method of Claim 1, wherein said differentiated pig cell or nucleus is

obtained from a somatic cell.

14. The method of Claim 1, wherein said differentiated pig cell or nucleus is

obtained from a germ cell.

15. The method of Claim 1, wherein the host animal comprises one or more

helper embryos.

16. The method of Claim 1, wherein the differentiated cell comprises one or

more genetic modifications that inhibit rejection upon implantation of cells, tissues, or

organs of said cloned fetus or animal in a human.

17. The method according to Claim 1 , wherein the differentiated pig cell or cell

nucleus is derived from mesoderm.

18. The method according to Claim 1 , wherein the differentiated pig cell or cell

nucleus is derived from ectoderm.

19. The method according to Claim 1 , wherein the differentiated pig cell or cell

nucleus is derived from endoderm.

20. The method according to Claim 1 , wherein the differentiated pig cell or cell

nucleus is a fibroblast cell or cell nucleus.

21. The method according to Claim 1 , wherein the differentiated pig cell or cell

nucleus is an adult differentiated cell or nucleus derived therefrom.

22. The method according to Claim 1 , wherein the differentiated pig cell or cell

nucleus is an embryonic or fetal cell or cell nucleus.

23. The method according to Claim 1, wherein the differentiated pig cell or

nucleus is a proliferating cell obtained directly from a pig.

24. The method of Claim 1, wherein said differentiated cell or nucleus is

obtained from a proliferating cell that has been expanded in tissue culture.

25. The method of Claim 1, wherein said differentiated cell or nucleus is

proliferated in vivo, prior to nuclear transfer.

26. The method of Claim 25, wherein said proliferating pig cell is obtained

from a SCID mouse which has been injected with porcine cells.

27. The method of Claim 25, wherein said proliferating cell has been

genetically modified.

28. The method of Claim 27, wherein said genetic modification is effected by

use of a recombinant virus, viral vector, naked DNA, or plasmid vector.

29. The method according to Claim 1 , wherein the enucleated oocyte is matured

prior to enucleation.

30. The method according to Claim 1 , wherein the fused nuclear transfer unit

is activated by exposure to a single or multiple electrical pulses.

31. The method according to Claim 1 , wherein the fused nuclear transfer unit

is activated by exposure to ionomycin and DMAP.

32. The method according to Claim 1 , wherein the fused nuclear transfer unit

is activated by exposure to at least one activating factor derived from sperm cells.

33. The method according to Claim 3, wherein microinjection is used to insert

said heterologous DNA.

34. The method according to Claim 3, wherein electroporation is used to insert

a heterologous DNA.

35. A cloned, optionally transgenic porcine fetus or animal obtained according

to the method of Claim 1, wherein said fetus or animal has the same genotype as a

previously existing non-embryonic differentiated porcine cell which is optionally

genetically modified.

36. The method according to Claim 1, which further comprises combining the

cloned NT unit with a fertilized embryo to produce a chimeric embryo.

37. The method according to Claim 36, which includes developing the chimeric

embryo to an offspring.

38. A chimeric fetus obtained according to the method of Claim 36.

39. A chimeric offspring obtained according to the method of Claim 37.

40. Progeny of the chimeric offspring according to Claim 39.

41. A method of producing a porcine CICM (pluripotent) cell line, comprising: (i) inserting a desired differentiated pig cell or cell nucleus into an optionally

enucleated pig oocyte, under conditions suitable for the formation of a nuclear transfer

(NT) unit;

(ii) removing the endogenous oocyte nucleus if not already effected;

(iii) activating the resultant nuclear transfer unit; and

(iv) culturing cells obtained from said cultured NT unit to obtain a pig CICM cell

line, which is pluripotent and may be maintained indefinitely in tissue culture.

42. The method of Claim 41, which comprises culturing said activated nuclear

transfer unit until a discernible trophectoderm and inner cell mass is obtained.

43. A CICM cell line obtained according to the method of Claim 41 , wherein

said cell line is pluripotent and comprises the same genotype as a previously existing,

non-embryonic differentiated cell.

44. The method according to Claim 41, wherein a desired DNA is inserted,

removed or modified in said differentiated pig cell or cell nucleus, thereby resulting in

the production of a genetically altered NT unit.

45. A transgenic CICM cell line obtained according to Claim 44, wherein said cell

line has the identical genotype as a previously existing porcine differentiated cell that has

been genetically modified.

46. The method of Claim 41, wherein the resultant CICM cell line is induced

to differentiate.

47. The method of Claim 44, wherein the CICM cell is allowed to differentiate.

48. Differentiated cells obtained by the method of Claim 46.

49. A method for cloning a porcine fetus or live offspring comprising the

following steps:

(i) activating a porcine oocyte that optionally is enucleated;

(ii) transferring a desired differentiated pig cell or nucleus into said porcine

oocyte after or proximately simultaneous to said activating step (i) to produce a NT unit;

(iii) removing the endogenous oocyte nucleus if oocyte not previously

enucleated; and

(iv) transferring said NT unit, optionally after a culturing step, into a female

porcine to produce a porcine fetus or animal.