U.S. patent application number 10/344724 was filed with the patent office on 2004-07-29 for use of haploid genomes for genetic diagnosis, modification and multiplication.
Invention is credited to Moreira, Pedro Nuno, Robl, James M..
Application Number | 20040146865 10/344724 |
Document ID | / |
Family ID | 22588424 |
Filed Date | 2004-07-29 |
United States Patent
Application |
20040146865 |
Kind Code |
A1 |
Robl, James M. ; et
al. |
July 29, 2004 |
Use of haploid genomes for genetic diagnosis, modification and
multiplication
Abstract
Methods for propagating haploid genomes of male or female
origina and genetic screening and modification thereof are
provided. These haploid genomes may be used to produce haploid
embryos, and embryonic stem-like cells and differentiated cells.
Also, these haploid genomes and cells containing, may be used as
nuclear transfer donors to produce diploid nuclear transfer units.
These diploid NT units e.g., human NT units, may be used to obtain
pluripotent cells and differentiated cells and tissues.
Inventors: |
Robl, James M.; (Brandon,
SD) ; Moreira, Pedro Nuno; (Madrid, ES) |
Correspondence
Address: |
MERCHANT & GOULD PC,
P.O. BOX 2903
MINNEAPOLIS
MN
55402-0903
US
|
Family ID: |
22588424 |
Appl. No.: |
10/344724 |
Filed: |
February 14, 2003 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10344724 |
Feb 14, 2003 |
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PCT/US00/30202 |
Nov 2, 2000 |
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60163086 |
Nov 2, 1999 |
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Current U.S.
Class: |
435/6.11 ;
435/325; 435/455; 435/6.17 |
Current CPC
Class: |
C12N 2517/04 20130101;
C12N 2510/00 20130101; C12N 15/873 20130101 |
Class at
Publication: |
435/006 ;
435/455; 435/325 |
International
Class: |
C12Q 001/68; C12N
015/85; C12N 005/06 |
Goverment Interests
[0001] The invention was developed as a result of the expenditure
of funds received from the United States Department of Agriculture
and accordingly the government has rights to this invention.
Claims
What is claimed is:
1. A method for selecting a haploid genome containing cell
comprising the following steps: (i) obtaining and amplifying the
number of cells containing a haploid genome of male or female
origin; (ii) subjecting the genome of said haploid cells to genetic
screening or analysis to determine whether said haploid genome
comprises a desired genetic makeup; and (iii) selecting cells that
contain said desired genetic makeup.
2. The method of claim 1, wherein said female-derived haploid cells
are produced by activation of an oocyte in which half of the
chromosomes are extruded in the polar body.
3. The method of claim 1, wherein said female-derived haploid cells
are produced by fertilization of an egg and removal of a male
pronucleus therefrom.
4. The method of claim 1, wherein said female-derived haploid cells
are produced by activation of an egg to provide an egg containing
two female pronuclei and removal of one of said pronuclei.
5. The method of claim 1, wherein said female-derived haploid cells
are produced by insertion of a diploid cell nucleus into an
immature oocyte followed by separation of said chromosomes in to
two haploid nuclei.
6. The method of claim 1, wherein said female-derived haploid cells
are produced by transfer of the nucleus of a parthenogenetic embryo
(contains half the chromosomes) but propagated with the full DNA
content (four chromatids) into an oocyte, and subsequent extrusion
of half the chromosomes therefrom.
7. The method of claim 1, wherein the male-derived haploid cell is
derived from a fertilized egg from which the female pronucleus is
removed.
8. The method of claim 1, wherein the male-derived haploid cell is
derived by fertilization of an enucleated egg.
9. The method of claim 1, wherein the male-derived haploid cell is
produced by artificial de-condensation of a sperm nucleus which is
then injected into a non-egg derived cytoplast.
10. The method of claim 1, wherein said female- or male-derived
haploid cells are amplified by a method selected from the group
consisting of (i) allowing a haploid egg cytoplast to undergo cell
division; (ii) allowing a haploid cell to produce a haploid embryo
which is then cultured to produce "propagating haploid" cells;
(iii) culturing a haploid embryo to produce propagating haploid
cells and allowing such embryonic stem-like cells to differentiate;
and (iv) culturing a haploid somatic cell cytoplast under
conditions that allow cell division.
11. The method of claim 1, wherein said selected haploid genome is
genetically modified.
12. The method of claim 11, wherein a selected male- and
female-haploid genome are both genetically modified.
13. The method of claim 1, which further comprises using said
selected male or female haploid genome or a cell containing said
selected haploid genome is used to produce a diploid embryo.
14. The method of claim 1 which further comprises using a selected
male and female haploid genome to produce a diploid embryo.
15. The method of claim 1, wherein said selected male or female
haploid genome or a cell containing said male or female genome is
used as a nuclear transfer donor.
16. The method of claim 15, wherein both a selected male or female
haploid genome are used as nuclear transfer donors to produce a
diploid nuclear transfer unit that contains said selected male and
female haploid genome.
17. The method of claim 15, wherein said haploid genome is
human.
18. The method of claim 15, wherein said haploid cell or genome
comprises a differentiated cell embryonic stem-like cell or inner
cell mass cell derived from a propagated haploid embryo.
19. The method of claim 18, wherein said differentiated cells or
embryonic stem-like cells are produced by in vitro culturing of a
haploid embryo.
20. The method of claim 13, wherein said selected haploid genome is
genetically modified.
21. The method of claim 19, wherein both said selected haploid
genomes are genetically modified.
22. The method of claim 20, wherein said selected haploid genome is
genetically modified by homologous recombination to eliminate,
insert, or substitute a particular DNA.
23. The method of claim 21, wherein both of said selected haploid
genomes are genetically modified by homologous recombination to
eliminate, insert or substitute a particular DNA.
24. The method of claim 1, wherein said genetic testing comprises
screening said amplified haploid genome for a genetic defect, a DNA
methylation defect associated with aberrant genomic imprinting, or
to select cells that contain a desired DNA, allelic trait, or which
lack a functional gene.
25. The method of claim 24, wherein the genetic defect results in
one of the following genetic diseases .alpha.-1 antitrypsin
deficiency; .alpha.-Thallasemia; Adenomatous polyposis coli; adult
polycystic kidney disease; breast cancer susceptibility due to
defects in BRCA1 or BRCA2; .beta.-Thallasemia, Charcot-Marie Tooth
disease; colon cancer susceptibility due to defects in MSH2, MLH1,
PMS1 or PMS2; congenital adrenal hyperplasia; cystic fibrosis;
Duchenne/Becker muscular dystrophy; Fragile X syndrome; Hemophilia
types A and B; Gaucher's disease; Huntington's disease; Kennedy's
disease; Lesch-Nyhan's syndrome; Marfan's syndrome; medium chain
acyl-coenzyme A dehydrogenase deficiency; melanoma susceptibility;
multiple endocrine neoplasia types 1 or 2A; myotonic dystrophy;
neurofibromatosis type 1; ornithine transcarbamylase deficiency;
retinoblastoma; sickle cell anemia; steroid sulfatase; Tay-Sachs
disease; or Werdnig-Hoffman disease.
26. The method of claim 24, wherein the DNA methylation defect
tested is selected from the group consisting of: Prader-Willi
syndrome, Angelman syndrome, uniparental isodisomy,
Beckwith-Wiedermann syndrome, Wilm's tumor carcinogenesis and von
Hippel-Lindau syndrome.
27. The method of claim 24, wherein said genetic testing includes
the use of a nucleic acid sequence that is directly or indirectly
attached to a label that specifically binds to a nucleic acid
sequence correlating to a particular genetic defect or selecting
cells that contain a desired DNA or allelic trait or lack a
functional gene.
28. The method of claim 1, wherein said genetic testing includes
the use of mass spectroscopic, fluorometric or radionuclear
detection of a particular DNA sequence, allelic trait, or genetic
defect.
29. The method of claim 1, wherein such genetic screening comprises
RFLP analysis, SSCP analysis, STR analysis, VNTR analysis, or
denaturing gradient gel electrophoresis.
30. The method of claim 13, wherein said embryo is implanted into a
suitable female surrogate and allowed to develop into a viable
offspring.
31. The method of claim 1, wherein the haploid genome is that of a
bovine.
32. The method of claim 1, wherein the haploid genome is that of a
primate.
33. The method of claim 1, wherein the haploid genome is selected
from the group consisting of goat, sheep, bovine, porcine, caprine,
equine, ovine, canine, feline, murine, rabbit, primate, human,
elephant, guinea pig, mouse, rat.
34 A propagating haploid cell line developed using the method of
claim 10.
35. The propagating haploid cell line of claim 34, wherein the cell
line is a female cell- or male cell-derived haploid cell line.
36. A diploid embryo that is produced by nuclear transfer wherein
said nuclear transfer process comprises using as the nuclear
transfer donor a haploid genome or cell containing which is
produced according to claim 1.
37. The diploid embryo of claim 36 wherein said nuclear transfer
process comprises transplantation of both a male and female haploid
genome or cell containing produced according to claim 1.
38. Embryonic stem-like cells or differentiated cells produced from
a haploid embryo.
39. An improved nuclear transfer process that is used to produce a
clone embryo, fetus or animal wherein the improvement comprises
using as the nuclear transfer donor embryonic stem-like cells or
differentiated cells produced from a haploid embryo.
Description
FIELD OF THE INVENTION
[0002] This invention relates to the propagation and use of haploid
genomes for purposes of (1) genetic diagnosis, (2) genetic
selection and (3) genetic modification. The selected haploid
genomes are useful for the production of embryos and embryonic stem
cells when combined with another haploid genome, preferably one
having a desired genetic makeup.
BACKGROUND OF THE INVENTION
[0003] Gametes are specialized haploid cells (e.g., spermatozoa and
oocytes) produced by meiosis and involved in sexual reproduction.
By contrast, diploid cell has its chromosomes in homologous pairs,
and has two copies of each autosomal genetic locus.
[0004] The diploid number (2n) equals twice the haploid number and
is the characteristic number for most cells other than gametes. A
zygote is the diploid cell resulting from the fusion of male and
female gametes during fertilization. THE DICTIONARY OF CELL BIOLOGY
103, 139, 388 (J. M. Lackie et al., eds. 1995). Only a (diploid)
zygote is capable of giving rise to a viable offspring. By
contrast, while haploid gametes conditions may give rise to embryos
being parthenogenetic development of female-derived haploid cells
(oocytes) these embryos typically stop developing before
embroyogenesis is completed. Such embryos may be produced
spontaneously but more typically are produced by artificial
activation of an oocyte. Such gynogenetic embryos are useful for
the study of embryogenesis.
[0005] The production of properly haploid-derived pluripotent cell
lines has previously been reported. For example, purported
pluripotent haploid cells were allegedly created by obtaining eggs
from 129 SvE or C57BL x CBA hybrid mice and activating them
parthenogenetically following exposure to a 7% solution of ethanol
in phosphate buffered saline (PBS). However upon examining the
chromosomes of these early passage "haploid" cell lines, all the
cells were diploid with a modal number of 40 chromosomes (Kaufman
et al., J. Embryol. Exp. Morphol. 73: 249-61 (1983)).
[0006] While it has been well reported that mammalian embryos may
result from haploid genomes, such mammalian embryos have not been
used for genetic analysis. Rather, to the best of the inventors'
knowledge, prenatal genetic diagnosis is conventionally performed
in utero or ex utero using apparent normal (diploid) embryos.
However, in utero genetic diagnosis is invasive and can be
dangerous to the developing fetus (e.g., amniocentesis and
chorionic villi sampling). Fetuses diagnosed with disease can
either be aborted or gestated to term, as in utero surgery and gene
therapy are still highly risky and experimental.
[0007] In humans, ex utero genetic diagnosis is typically performed
on embryos produced by in vitro fertilization (IVF) technologies.
Typically one or two cells are taken from a recent embryo and
tested for such diseases as cystic fibrosis (CF), sex-linked
diseases, chromosomal abnormalities, fragile X syndrome, spinal
muscular atrophy and myotonic dystrophy (de Die-Smulders et al.,
Ned. Tijdschr. Geneeskd. 142: 2441-4 (1998)). Preimplantation
genetic diagnosis (PGD) can be performed using direct polymerase
chain reaction (PCR) or nested PCR to diagnose the common
.DELTA.F508 mutation of CF (Cui et al., Mol. Hum. Reprod. 2: 63-1
(1996); and Ao et al., Prenat. Diagn. 16: 13742 (1996)), as well as
other diseases (Ben-Ezra, Clin. Lab. Med. 15: 95-815 (1995)).
Genetic screening can also be done by single blastomere biopsy for
rhesus (RhD) blood group typing of early cleavage stage embryos
(Avner et al., Mol. Hum. Reprod. 2: 60-2 (1996)) or by blastocyst
biopsy (Verlinsky et al., Bailieres Clin. Obstet. Gynaecol. 8:
177-96 (1994)). Primed in-situ labeling (PRINS) and in-situ
hybridization can be used for detecting human chromosomal
abnormalities for PGD (Pellestor et al., Mol. Hum. Reprod. 2: 135-8
(1996)). PGD has also been performed using fluorescence in situ
hybridization (FISH) to prevent development of moles resulting from
a fertilization of an inactive oocyte by a haploid X-bearing
spermatozoon, which subsequently duplicates (Reubinoff et al., Hum.
Reprod. 12: 805-8 (1997)). PGD can be performed on oocytes to
diagnose single gene disorders by first polar body analysis and to
identify oocytes that contain maternal unaffected genes (Verlinsky
et al., Biochem. Mol. Med. 62: 182-7 (1997); Verlinsky et al.,
Curr. Opin. Obstet. Gynecol. 4: 720-5 (1992); and Verlinsky et al.,
Hum. Reprod. 5: 826-9 (1990)). In one case, individual spermatoza
of a father with two affected infants with osteogenesis imperfecta,
were separated by dilution and micromanipulation. A segment of the
type I collagen gene containing the mutation was amplified using
nested PCR and sequencing to detect the wild-type gene as well as
genes with a single point mutation (Iida et al., Mol. Hum. Reprod.
2:131-4 (1996)). Methods of selecting sperm have been developed in
response to use of intracytoplasmic sperm injection techniques
(ICSI) (Meschede et al., Hum. Reprod. 10: 2880-6 (1995)).
Sequential analysis of first and second polar body and multiplex
PCR can lead accurate genetic diagnosis in comparison to the
pitfalls encountered by single-cell DNA analysis (Richitsky et al.,
J. Assist. Reprod. Genet. 16: 192-8 (1999)).
[0008] Additional methods of genetic screening includes the
detection or change in restriction fragment length polymorphisms
(RFLPs), variable number of tandem repeat (VNTR) sequences and
dinucleotide or other short tandem repeat (STR) sequences.
Alternatively, allele specific amplification and allele specific
ligation, utilizing primers complimentary to either the wild type
or the mutant sequence, provide two alternative means for detection
of specific mutations. Other methods are available to screen for
the presence of mutations without identifying the specific mutation
itself. These methods include single-strand conformational
polymorphism (SSCP) analysis, denaturing gradient gel
electrophoresis (DGGE), and mismatch cleavage analysis by enzymatic
(RNAse A) or chemical (piperidine) means. See Fujimura, "Genetic
Testing," IN MOLECULAR BIOLOGY AND BIOTECHNOLOGY: A COMPREHENSIVE
DESK REFERENCE 374-379 (Robert A. Meyers, ed., 1995).
[0009] Thus, based on the foregoing, it is evident that although
research is ongoing in perfecting preimplantation genetic
screening, as well as manipulation of embryos created in vitro,
little progress has been achieved in the genetic screening of
gametes or the genetic manipulation of gametes to be used to make
transgenic animals.
[0010] Therefore, notwithstanding what has previously been reported
in the literature, there exists a need for improved methods of
genetic screening of gametes and genetically engineering haploid
cells for preparing transgenic animals.
SUMMARY AND OBJECTS OF THE INVENTION
[0011] It is an object of the present invention to provide a method
for selecting genomes for the production of embryos, embryonic stem
cells or embryonic germ cells comprising the steps of: (i)
culturing cells containing either a male or female-derived haploid
genetic content; (ii) genetically testing the genetic content of
said cultured cells to identify whether said haploid genome
comprises a genetic defect, a desired gene or lacks a functional
gene; and (iii) selecting cells that do not comprise a genetic
defect, or selecting cells that contain the desired gene or lack a
functional gene.
[0012] Specifically, in the case of female-derived haploid cells,
the cells can be obtained by one of five methods: (1) by activation
of an oocyte in which half of the chromosomes are extruded in the
polar body; (2) by fertilization of an egg and removal of a male
pronucleus therefrom; (3) by activation of an egg to provide an egg
containing two female pronuclei and removal of one of said
pronuclei; (4) by insertion of a diploid cell nucleus into an
immature oocyte followed by separation of said chromosomes in to
two haploid nuclei; and (5) by transfer of the nucleus of a
parthenogenetic embryo (contains half the chromosomes) but
propagated with the full DNA content (four chromatids) into an
oocyte, and subsequent extrusion of half the chromosomes
therefrom.
[0013] Another object of the invention is directed towards the
screening of male-derived haploid cells, which can be obtained by
one of the following methods: (1) obtaining the male-derived
haploid cell from a fertilized egg from which the female pronucleus
is removed; (2) obtaining the male-derived haploid cell by
fertilizing an enucleated egg; and (3) obtaining the male-derived
haploid cell by artificial decondensation of a sperm nucleus which
is then injected into a non-egg derived cytoplast.
[0014] Another object of the invention is a method of propagating
male- or female-derived haploid cells by a method selected from the
group consisting of (i) allowing a haploid egg cytoplast to undergo
cell division; (ii) allowing a haploid cell to produce a haploid
embryo which is then cultured to produce "propagating haploid"
cells; (iii) culturing a haploid embryo to produce embryonic
stem-like cells which are haploid and allowing such embryonic
stem-like cells to differentiate; and (iv) culturing a haploid
somatic cell cytoplast under conditions that allow cell
division.
[0015] Another object of the invention is to provide a propagated
haploid genome cell line of male or female origin, i.e., one which
comprises a desired genetic make-up or comprises a desired genetic
modification.
[0016] Still another object of the invention is to provide
pluripotent or embryonic-like stem cells produced from a haploid
cell line and differentiated cells derived therefrom, which
comprise a desired genetic make-up, e.g., comprise a desired
genetic modification.
[0017] Yet another object of the invention is to provide diploid
mammalian embryos produced from a genetically modified or selected
haploid male and/or female genome, as well as pluripotent cell
lines and differentiated cells derived therefrom.
[0018] Definitions
[0019] The invention relates to the production and multiplication,
by any method, of cells containing either a male or female-derived
haploid chromosome content, the use of these cells for genetic
evaluation, genetic modification or multiplication of a specific
haploid genome, and the use of these cells in producing an embryo
with a diploid content of DNA. The haploid genomes to be
propagated, screened and/or modified include ungulates, such as
bovine, ovine, porcine, equine, caprine; canine, feline, murine,
rabbit, and rodents (e.g., guinea pigs, hamsters and rats), human,
non-human primates, such as cynomolgus monkey, chimpanzees, baboon
and gorilla.
[0020] By "genetic screening," "genetic diagnosis," "genetic
analysis" and "genetic testing" is meant the analysis of the
haploid genome by conventional methods to detect the presence or
absence of a specific DNA associated with a phenotype, disease or
condition. Such methods include in situ hybridization, polymerase
chain reaction, nested polymerase chain reaction, fluorometric
detection methods, RFLP analysis VNTR or STR detection methods
(which screen for usage in a number of tandem repeat dinucleotide
or other short tandem repeat (STR) sequences, single-strand
conformational polymorphism (SSCP) analysis, denoting gradient gel
electrophoresis (DGGE) and mismatch cleavage analysis i.e., by
enzymatic (RNAse A) or chemical (piperidine) means. Such methods
are reviewed in Fujimura "Genetic Testing", IN MOLECULAR BIOLOGY
AND BIOTECHNOLOGY: A COMPREHENSIVE DESK REFERENCE 374-379 (Robert
A. Meyers, ed., 1995).
[0021] By "genetic selection" is meant the directed choice of a
genotype using genetic testing.
[0022] By "genetic modification" or "genetic manipulation" is meant
the modification of the genome of a cell, typically a haploid cell.
This includes insertion, deletion and substitute modifications.
Preferably the modification will be effected at a target site in
the genome. In a preferred embodiment, the modified haploid cell
will eventually be used in nuclear transplantation for production
of an animal which expresses the modified/manipulated gene.
[0023] By "multiplication" is meant increasing the number of cells
comprising the desired haploid genome of male or female origin.
[0024] By "haploid cell" is meant a cell with a haploid number (n)
of chromosomes. "Gametes" are specialized haploid cells (e.g.,
spermatozoa and oocytes) produced by meiosis and involved in sexual
reproduction. A "diploid cell" has its chromosomes in homologous
pairs, and has two copies (2n) of each autosomal genetic locus. A
"zygote" is the diploid cell resulting from the fusion of a male
and a female gamete during fertilization.
[0025] The term "nuclear transfer" or "nuclear transplantation"
refers to a method of cloning wherein the nucleus from a donor cell
is transplanted into an enucleated oocyte. Nuclear transfer
techniques or nuclear transplantation techniques are known in the
literature (Campbell et al., Theriogenology 43: 181 (1995); Collas
et al., Mol. Reprod. Dev. 38: 264-267 (1994); Keefer et al., Biol.
Reprod. 50: 935-939 (1994); Sims et al., Proc. Natl. Acad. Sci. USA
90: 6143-6147 (1993); Evans et al., WO 90/03432 (Apr. 5, 1990);
Smith et al., WO 94/24274 (Oct. 27, 1994); Wheeler et al., WO
94/26884 (Nov. 24, 1994)). Also, U.S. Pat. Nos. 4,994,384 and
5,057,420 describe procedures for bovine nuclear transplantation.
See also U.S. Pat. No. 5,945,577; WO 97/06668 and WO 97/06669,
which respectively name The University of Massachusetts and Roslin
Institute as the Assignee or Applicant. This patent and
applications are incorporated by reference herein. In the subject
application, nuclear transfer or nuclear transplantation or NT are
used interchangeably. The present definition also embraces the
implantation of one or two selected haploid genomes to produce an
embryo.
[0026] By "lack a functional gene" is meant either the entire gene
is missing from the subjects genome, or the gene is mutated to an
extent that it can no longer function (e.g., produce a wild-type
protein).
[0027] By "genetic defect" is meant a nucleic acid deletion or
insertion which corresponds to an alteration in transcription of
the gene, translation of the gene's mRNA into a protein, alteration
of the half-life of the protein or the gene's mRNA or other change
from wild-type expression of the gene. Different forms of a given
gene are called "alleles." The "wild-type alleles" of a gene are
those that exist at relatively high frequencies in natural
populations and yield wild-type or normal phenotypes. Alleles of a
gene that result in abnormal or non-wild-type phenotypes are
"mutant alleles."
[0028] By "propagating haploid cell line" is meant a cell line of
proliferating haploid cells produced artificially outside of the
haploid cell's host organism. Typically such haploid cell line will
be comprised in an in vitro culture. Alternatively, a haploid cell
may be propagated in vivo, e.g. by injection into a SKID mouse to
produce differentiated cell types.
DETAILED DESCRIPTION OF THE INVENTION
[0029] As discussed, the present invention is directed toward the
production and propogation of haploid genomes, the selection of
desirable haploid genomes from said propogated haploid genomes by
genetic analysis, and the use of said selected haploid genomes to
produce diploid embryos. As noted in the background of this
application, it is known to conduct genetic evaluation of
preimplantation embryos as a means of selecting embryos suitable
for implantation and the production of offspring. Such methods
involve genetic evaluation of the genome of one or more cells of
the embryo prior to implantation.
[0030] However, such methods may pose ethical issues in that an
embryo is manipulated, and potentially may be destroyed if it
exhibits undesirable genetic characteristics. Most especially, such
methods may pose ethical issues in the context of human
preimplantation embryos, especially those produced by nuclear
transfer or conventional in vitro fertilization.
[0031] By contrast, the present invention selects haploid DNA for
use in the production of diploid embryos by genetic testing of a
haploid cell genome. Such methods should not pose the same ethical
concerns as haploid cells cannot give rise to viable offspring.
Thus, the disposal of non-desirable haploid genomes or manipulation
of haploid genomes should obviate ethical issues associated with
manipulation and destruction of diploid embryos, e.g. human diploid
embryos.
[0032] Because the present invention involves genetic testing of
haploid genomes, it requires a propagated source of such haploid
genome. This-initially entails constructing or obtaining a cell
containing a haploid genome, and providing for proliferation
thereof.
[0033] Various methods for producing cells containing either male
or female haploid genomes may be utilized. For example, methods of
producing haploid cells containing haploid genomes of female origin
include by way of example:
[0034] (i) activating in vitro an oocyte in which half the
chromosomes are extruded in the polar body;
[0035] (ii) fertilizing an egg and removal of the male
pronucleus;
[0036] (iii) activating in vitro an egg which comprises two female
pronuclear and removal of one of said pronuclear therefrom;
[0037] (iv) insertion of a diploid cell nucleus into an immature
oocyte and separation of the chromosomes into two haploid nuclei
and
[0038] (v) transfer of a parthenogenetic nucleus (which contains
half the number of chromosomes) but is propagated with the full DNA
content (four chromotides)-into an oocyte and half the chromatides
are extruded therefrom.
[0039] Of the above methods (i), (iii), (iv) and (v) are preferred,
as the methods at no time result in a diploid embryo wherein half
its DNA content is of male and the other half is of female origin.
Thus, even if implanted, they would be incapable of developing into
a full-term offspring.
[0040] Methods for providing haploid genomes of male origin
include:
[0041] (i) fertilization of an egg and removal of the female
pronucleus;
[0042] (ii) fertilization of a enucleated oocyte; and
[0043] (iii) artificial decondensation of a sperm nucleus and
injection into a non egg-derived cytoplast.
[0044] The above-described haploid cells and other haploid cells
may be propagated by various methods. For example, haploid genomes
may be propagated by inducing division of egg cytoplasts.
Alternatively, haploid embryos may be used for the product of
embryonic stem-like cells. This may be effected by culturing the
embryo using known media and methods for maintaining embryos in
culture and culturing the inner cell mass or cells derived
therefrom to produce embryonic stem-like cells. For example, this
may be effected by placing the inner cell mass or cells of the
inner cell mass of a haploid-genome derived embryo on a feeder
layer, e.g. murine fetal fibroblasts, to produce a culture
containing embryonic stem-like cells which give rise to different
differentiated cell types, e.g., when removed from the feeder
layer.
[0045] Still alternatively, embryonic stem-like cells derived from
haploid embryos may be used to produce differentiated cells which
have the genome of the parent haploid genome. Yet another means of
propagating haploid genomes comprises inducing division of haploid
somatic cell cytoplasts produced by introduction of a haploid
genome into a cytoplast.
[0046] As noted, in its preferred embodiment the haploid genome
will be of human origin, e.g. that of human sperm, or oocyte.
However, the present invention embraces the construction of haploid
genomes of any mammalian species origin, e.g. non-human primate,
dog, cat, mouse, rat, rabbit, bear, cow, horse, pig, sheep, guinea
pig, buffalo, goat, antelope, etc. Essentially, the invention is
applicable for the selection of any animal that is desirably
propagated, e.g. by nuclear transfer, that contains a desired
genetic makeup of particular importance are agricultural animals,
especially animals having a long gestation period. The present
invention should enable rapid screening for haploid genomes that
will give rise to diploid embryos having desired genetic
characteristics. For example, the presence or absence of sex-linked
genetic diseases can be the basis of the genetic screen.
[0047] Also, the invention allows for haploid cell line produced
according to the invention to be genetically modified, by
homologous recombination.
[0048] This is an advantageous aspect of the invention because
allelic differences at a locus will not interfere with the desired
recombination events. Also, the present invention allows for the
same locus to be targeted in both the male and female haploid cell
lines, and the resultant modified male and female haploid genomes
to be combined to produce a diploid embryo that is homozygous for
the particular modification, e.g. deletion of a particular
gene.
[0049] As discussed, the invention described herein improves upon
prior methods of preimplantation genetic diagnosis (PGD), because
these methods do not involve the manipulation of an embryo.
Generally, few embryos are available for screening. Moreover,
removal of the cells from an embryo for testing can be harmful for
further development of the embryo. Often only one or very few cells
are available for genetic testing, which can lead to inaccurate
results due to DNA loss or DNA contamination. Finally, there are
ethical considerations regarding embryo disposal. Genetic screening
of haploid DNA offers the advantage that if male and/or female
gametes are screened then, even with few gametes, the total
possible combination becomes large.
[0050] In the case of sex-linked genetic diseases, screening can be
done on sperm only, which is typically easy to obtain in large
quantities. If the sperm is not available in large quantities, then
multiplication of the sperm genome can also be useful. The
technique makes many identical copies of the genome available for
screening to minimize the likelihood of misdiagnosis, and permits
additional samples to be analyzed for verification of results. The
ethical concerns about working with and manipulating sperm are
minimal in comparison with those for working with embryos.
[0051] Screening of haploid cells can also be performed e.g., to
determine whether genetic or DNA methylation defects in the haploid
cell may cause any adult animal developed therefrom to contract
cancer or other disease. Screening for genetic conditions and
predispositions would be useful in eliminating defective haploid
cells containing such defects. The present invention can be used to
screen for chromosomal aberrations and DNA sequences that are
correlated to disease or other undesirable traits. These haploid
genomes will typically be disposed of. However, in some instances
such haploid genomes may be retained. For example, the production
of haploid genomes that encode genes that are involved in disease
may be useful in producing animals for research purposes, e.g. for
evaluating the efficacy of putative therapeutics or prophylactics.
Also, the present invention can be used to select haploid genomes
that contain a desired genetic makeup, e.g., comprise DNA sequences
that are involved in enhanced growth, disease resistance, milk
production, or other desirable traits. For example, genetic
analysis of haploid cells using DNA probes and linkage (L) or
mutation (M) detection can be made on the following human diseases
listed in Table 1:
1TABLE 1 Condition Chromosome L/M Cloned .alpha.-1 antitrypsin
deficiency 14 M Yes .alpha.-Thallasemia 16 M Yes Adenomatous
polyposis coli 5 L, M Yes Adult polycystic kidney disease 16 L No
Breast cancer susceptibility (BRCA1) 17 L, M Yes Breast cancer
susceptibility (BRCA2) 13 L No .beta.-Thallasemia 11 M Yes
Charcot-Marie-Tooth disease 1 M Yes Colon cancer susceptibility
(MSH2) 2 M Yes Colon cancer susceptibility (MLH1) 3 M Yes Colon
cancer susceptibility (PMS1) 2 M Yes Colon cancer susceptibility
(PMS2) 7 M Yes Congenital adrenal hyperplasia 6 M, L Yes Cystic
Fibrosis (CF) 7 M Yes Duchenne/Becker muscular dystrophy X M, L Yes
Fragile X syndrome X M, L Yes Hemophilia A X M, L Yes Gaucher's
disease 1 M Yes Hemophilia B X M, L Yes Huntington's disease 4 M, L
Yes Kennedy's disease X M Yes Lesch-Nyhan syndrome X L, M Yes
Marfan's syndrome 15 M Yes Medium chain acyl-coenzyme A 1 M Yes
dehydrogenase deficiency Melanoma susceptibility 9 M Yes Multiple
endocrine neoplasia 1 11 L No Multiple endocrine neoplasia 2A 10 L,
M Yes Myotonic dystrophy 19 M, L Yes Neurofibromatosis type 1 17 L,
M Yes Ornithine transcarbamylase deficiency X M, L Yes
Retinoblastoma 13 M, L Yes Sickle cell anemia 11 M Yes Steroid
sulfatase deficiency X L, M Yes Tay-Sachs disease 15 M Yes
Werdnig-Hoffman disease 5 L No
[0052] Frank K. Fujimura, "Genetic Testing," IN MOLECULAR BIOLOGY
AND BIOTECHNOLOGY: A COMPREHENSIVE DESK REFERENCE (Robert A.
Meyers, ed. 1995).
[0053] Methods for screening genomes for the presence of specific
DNA sequences or chromosomal aberrations are well known. Such
screening methods include by way of example polymerase chain
analysis (PCR) techniques including nested PCR and direct PCR
amplification, SSCP analysis, RFLP analysis, primed in situ
labeling (PRINS) methods (see Pellestor et al., 1996), fluorescence
in situ hybridization (FISH) analysis, and analysis of VNTRs or
STRs, denaturing gradient gel electrophoresis (DGGE), and mismatch
cleavage analysis using enzymatic (e.g., RNAse A) or chemical
(e.g., piperidine) methods.
[0054] Other screening methods include DNA methylation analysis
which is useful for identifying syndromes associated with genomic
imprinting. Syndromes and diseases in humans associated with
genomic imprinting include: Prader-Willi syndrome (PWS), Angelman
syndrome (AS), uniparental isodisomy, Beckwith-Wiedermann syndrome
(BWS), Wilm's tumor carcinogenesis and von Hippel-Lindau (VHL)
disease. For methods of performing DNA methylation analysis, see
Buchholz et al., Hum. Genet. 103: 535-9 (1998). PWS can be caused
by genetic mutations, such as deletions, as well as abnormal
genomic imprinting (Barabash et al., Med. Clin. (Barc) 108: 304-6
(1997)). In animals, genomic imprinting has also been linked to
coat color. For example, the mouse agouti gene confers wild-type
coat color, and differential expression of the Aiapy allele
correlates with the methylation status of the gene's upstream
regulatory sequences (Michaud et al., Genes Dev. 8: 1463-72).
Genetic screening in agriculture can be used for genetic selection
to produce optimal combinations that minimize recessive mutations,
increases heterozygosity or homozygosity or to accumulate
beneficial or otherwise desired alleles.
[0055] As noted above, many genetic screening and testing methods
are known in the art and may be used in the present invention.
Also, many sequences have been identified that correlate to desired
or undesired traits.
[0056] The methods of the present invention can be used for genetic
selection, both in animals, e.g., agricultural, laboratory or
domestic animals as well as in humans. Currently, the combination
of gamete genomes that constitute the embryo is random. However, by
performing genetic screening on gametes, the optimal combinations
could be made to minimize recessive mutations, increase
heterozygosity, increase homozygosity or accumulate beneficial
alleles. Haploid genomes that are selected to have desirable
genetic makeup would be used to provide diploid embryos and
offspring.
[0057] As further discussed, the methods of producing propagating
haploid cells can also be used to prepare genetically modified
haploid cells. In the cases of homologous recombination, allelic
differences at a locus will not interfere with the recombination
event. Furthermore, targeting both male and female cell lines can
result in the preparation of homozygous modifications.
[0058] Methods for effecting genomic modification are well known in
the art and include by way of example the use of retroviral
vectors, microinjection, and transformation with DNAs comprising
sequences that are to be inserted. Preferably, the genetic
modification will be made at a targeted site in the genome. Methods
for effecting targeted insertion, deletion and substitute
modifications of genomes, and particularly mammalian genomes have
been well reported and are the subject of numerous patents.
[0059] Essentially, in the present invention a particular haploid
genome contained in a propogated haploid cell line will be
genetically modified in order to remove, add or substitute a
particular DNA sequence with another. After such genetic
modification has been effected, e.g. by homologous recombination,
the haploid genome will be tested or screened to determine that it
indeed comprises the modification. For example, this can be
effected by one of the genetic screening methods identified supra,
or by expression of a particular marker contained in the inserted
DNA, e.g., enzyme, antibiotic resistance marker, fluorescent or
radiolabel, etc.
[0060] After the genetically modified haploid genome has been
produced, it preferably will be amplified by the methods discussed
previously.
[0061] The resultant selected haploid of male or female origin,
genomes which may be genetically modified, are especially useful
for nuclear transfer or transplantation. Essentially, such methods
will comprise the introduction of a selected male and female
haploid genome into an enucleated oocyte, or the introduction of a
selected male or female haploid genome into a haploid oocyte
wherein such haploid DNA is either of male or female origin.
Thereby, diploid nuclear transfer unit will be obtained, wherein
either or both the male or female DNA therein has been selected
based on its genetic makeup. Those diploid nuclear transit units
can be used to provide progeny that have a desired genetic makeup,
e.g., contain genes involved in disease resistance, growth, or a
heterologous DNA that encodes a desired product.
[0062] Nuclear transfer techniques or nuclear transplantation
techniques are well known in the literature. See, in particular,
Sims et al., Proc. Natl. Acad. Sci. USA 90: 6143-6147 (1993);
Collas et al., Mol. Report Dev. 38: 264-267 (1994); Keefer et al.,
Biol. Reprod. 50: 935-939 (1994); Campbell et al., Theriogenology,
43: 181 (1995); Campbell et al., Nature, 380: 64-66 (1996);
Schnieke et al., Science 278: 2130-3 (1997); Wells et al., Biol.
Reprod. 57: 385-393 (1997); Wilmut et al., Nature 386: 810-813
(1997); Cibelli et al., Science 280: 1256-8 (1998); Kato et al.,
Science 282: 2095-8 (1998); Wakayama et al., Nature 394: 369-74
(1998); Wolf et al., J. Biotechnol. 65: 99-110 (1998); Baguisi et
al., Nat. Biotechnol. 17: 456-61 (1999); Dominko et al., Biol.
Reprod. 60: 1496-1502 (1999); Wolf et al., Biol. Reprod. 60:199-204
(1999); PCT/US99/00045; WO 94/26884; WO 94/24274; and WO 90/03432,
which are herein incorporated by reference in their entirety. Also,
U.S. Pat. Nos. 4,944,384 and 5,057,420 describe procedures for
bovine nuclear transplantation. See also, U.S. Pat. No. 5,945,577,
incorporated by reference in its entirety.
[0063] Oocytes used for nuclear transfer may be obtained from
animals including mammals and amphibians. Suitable mammalian
sources for oocytes include sheep, bovines, ovines, pigs, horses,
rabbits, guinea pigs, mice, hamsters, rats, primates, human and
non-human etc. In the preferred embodiments, the oocytes will be
obtained from primates, e.g., human oocytes, or ungulates.
[0064] 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 mammal, e.g., a bovine. A readily
available source of bovine oocytes is from slaughterhouse
materials.
[0065] For the successful use of techniques such as genetic
engineering, nuclear transfer and cloning, oocytes must typically
are matured in vitro before these cells may be used as recipient
cells for nuclear transfer, and before they can be fertilized by
the sperm cell to develop into an embryo. This process generally
requires collecting immature (prophase I) oocytes from ovaries
(e.g., bovine ovaries obtained at a slaughterhouse) and maturing
the oocytes in a maturation medium prior to fertilization or
enucleation until the oocyte attains the metaphase 1] stage, which
in the case of bovine oocytes generally occurs about 18-24 hours
post-aspiration. For purposes 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. Also, the invention
includes the isolation of human oocytes by aspiration from
consenting donors.
[0066] Alternatively, metaphase II stage oocytes, which have been
matured in vivo can be used in nuclear transfer techniques. For
example, mature metaphase II oocytes are 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 hormones.
[0067] 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); Tanaka et al., Anim. Reprod. Sci. 49: 113-23 (1997)). In
general, successful mammalian embryo cloning practices use
metaphase II stage oocytes as recipient oocytes, because at this
stage it is believed that the oocyte can be or is sufficiently
"activated" to treat the introduced nucleus as it would a
fertilizing sperm. In domestic animals, and especially cattle, the
oocyte activation period generally ranges from about 16-52 hours,
preferably about 28-42 hours post-aspiration.
[0068] For example, immature oocytes may be washed in HEPES
buffered hamster embryo culture medium (HECM) as described in
Seshagine et al., Biol. Reprod. 40: 544-606 (1989), and then placed
into drops of maturation medium consisting of 50 .mu.l of tissue
culture medium (TCM) 199 containing 10% fetal calf serum (FCS),
which contains appropriate gonadotropins such as luteinizing
hormone (LH) and follicle stimulating hormone (FSH), and estradiol
under a layer of lightweight paraffin or silicon at 39.degree.
C.
[0069] After a fixed maturation period, which ranges from about 10
to 40 hours, and preferably about 16-18 hours, oocytes can be
enucleated. Prior to enucleation the oocytes are preferably removed
and placed in HECM containing 1 mg/ml of hyaluronidase prior to
removal of cumulus cells. This may be effected by either repeated
pipetting through very fine bore pipettes or by vortexing briefly.
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
follows.
[0070] Enucleation may be effected by known methods, such as
described in U.S. Pat. No. 4,994,384, which is herein incorporated
by reference. For example, metaphase 11 oocytes are either placed
in HECM, optionally containing 7.5 .mu.g/ml cytochalasin B, for
immediate enucleation, or may be placed in a suitable medium, for
example CR1 aa, plus 10% estrus cow serum, and then enucleated
later, preferably not more than 24 hours later, and more preferably
16 to 18 hours later.
[0071] Enucleation may be accomplished microsurgically using a
micropipette to remove the polar body and the adjacent cytoplasm.
The oocytes may then be screened to identify those of which have
been successfully enucleated. This screening may be effected by
staining the oocytes with 1 .mu.g/ml 33342 Hoechst dye in HECM, and
then viewing the oocytes under ultraviolet irradiation for less
than 10 seconds. The oocytes that have been successfully enucleated
then can be placed in a suitable culture medium, e.g., CR1aa plus
10% serum.
[0072] In the present invention, one or two selected, potentially
genetically modified haploid genomes will be transplanted into a
perivitelline space of an optionally enucleated oocyte or other
cytoplast. The resultant haploid genome containing oocyte or
cytoplast which is diploid is 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. Essentially, 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. Pat. No. 4,997,384 by Prather et al., 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 Inst. Symp. Monogr. 9:
19 (1969)
[0073] Also, in some cases (e.g., with small donor nuclei) it may
be preferable to inject the haploid cell or nucleus directly into
the oocyte rather than using electroporation fusion. Such
techniques are disclosed in Collas et al., Mol. Reprod. Dev. 38:
264-267 (1994).
[0074] Human or animal cells and oocytes or cytoplasts can be
electrofused by known methods, e.g., in a 500 .mu.m chamber by
application of an electrical pulse of 90-120 V for about 15
.mu.sec, about 24 hours after initiation of oocyte maturation.
After fusion, the resultant fused NT units are then placed in a
suitable medium until activation. Activation can be effected
shortly before or after fusion, typically less than 24 hours later,
and preferably about 4-9 hours later.
[0075] The NT unit may be activated by known methods. 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.
[0076] Alternatively, activation may be achieved by application of
known activation agents. For example, penetration of oocytes by
sperm during fertilization has been shown to activate perfusion
oocytes to yield greater numbers of viable pregnancies and multiple
genetically identical calves after nuclear transfer. Also,
treatments such as electrical and chemical shock may be used to
activate NT embryos after fusion. Oocyte activation methods are the
subject of U.S. Pat. No. 5,496,720, to Susko-Parrish et al.
[0077] Additionally, activation may be affected by simultaneously
or sequentially:
[0078] (i) increasing levels of divalent cations in the oocyte,
and
[0079] (ii) reducing phosphorylation of cellular proteins in the
oocyte.
[0080] This will generally be affected by introducing divalent
cations into the oocyte cytoplasm, e.g., magnesium, strontium,
barium or calcium, e.g., in the form of an ionophore. Other methods
of increasing divalent cation levels include the use of electric
shock, treatment with ethanol and treatment with caged
chelators.
[0081] Phosphorylation may be reduced by known methods, e.g., by
the addition of kinase inhibitors, such as serine-threonine kinase
inhibitors (e.g., 6-dimethylamino-purine, staurosporine,
2-aminopurine, and sphingosine). Alternatively, phosphorylation of
cellular proteins may be inhibited by introduction of a phosphatase
into the oocyte (e.g., phosphatase 2A and phosphatase 2B).
[0082] One means of effecting NT activation is by briefly exposing
the fused NT unit to a TL-HEPES medium containing 5 .mu.M ionomycin
and 1 mg/ml BSA, followed by washing in TL-HEPES containing 30
mg/ml BSA within about 24 hours after fusion, and preferably about
4 to 9 hours after fusion. Alternatively, activation can be
effected by use of ethanol or repeated electrical pulse.
[0083] The activated NT units produced from one or two selected
haploid genomes may then be cultured in a suitable in vitro culture
medium until the generation of embryonic or stem-like cells and
cell colonies. Culture media suitable for culturing and maturation
of embryos are well known in the art. Examples of known media,
which may be used for bovine embryo culture and maintenance,
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 plus an 1 to 20% serum supplement, including fetal calf
serum, newborn serum, estrual cow serum, lamb serum or steer serum.
A preferred maintenance medium includes TCM-199 with Earl salts,
10% fetal calf serum, 0.2 mM Na pyruvate and 50 .mu.g/ml gentamicin
sulphate. 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.
[0084] Afterward, activation of the cultured NT unit or units are
preferably washed and then placed in a suitable media, e.g., CRIaa
medium containing 10% FCS and 6 mg/ml contained in well plates
which preferably contain a suitable confluent feeder layer.
Suitable feeder layers include, by way of example, fibroblasts and
epithelial cells, e.g., fibroblasts and uterine epithelial cells
derived from ungulates, chicken fibroblasts, murine (e.g., mouse or
rat) fibroblasts, STO and SI-m220 feeder cell lines, and BRL
cells.
[0085] The NT units are cultured on the feeder layer until the NT
units reach a size suitable for obtaining cells which may be used
to produce embryonic stem-like cells or cell colonies. Preferably,
these NT units will 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 The culturing will be effected under suitable
conditions, e.g., about 38.5.degree. C. and 5% CO.sub.2, with the
culture medium changed in order to optimize growth typically about
every 2-5 days, preferably about every 3 days.
[0086] After NT units of the desired size are obtained, the cells
are mechanically removed from the zone and are then used to produce
embryonic or stem-like cells and cell lines. 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., irradiated
fibroblast cells. Typically, the cells used to obtain the stem-like
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-like 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 .beta.-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 embryonic or stem-like cells or
cell lines. The culture time before such cells are produced may
vary dependent upon the particular nuclear donor cell, specific
oocyte and culturing conditions. One skilled in the art can vary
the culturing conditions as desired to optimize growth of the
particular embryonic or stem-like cells.
[0087] The embryonic or stem-like cells and cell colonies produced
from said haploid genome generated embryos should exhibit an
appearance similar to native embryonic or stem-like cells of the
species used as the nuclear cell donor.
[0088] The present invention has been described with reference to a
preferred embodiment. However, it will be readily apparent to those
skilled in the art that it is possible to embody the invention in
specific forms other than as described above without departing from
the spirit of the invention. The preferred embodiments described in
the examples below are illustrative and should not be considered
restrictive in any way. The scope of the invention is given by the
appended claims, rather than the preceding description, and all
variations and equivalents which fall within the range of the
claims are intended to be embraced therein.
EXAMPLE
Production of a Haploid Cell Line
[0089] Production of Large Murine A9 Cells
[0090] Murine A9 cells (HPRT-) are cultured in 3.75 .mu.g/ml
cytochalasin B (Sigma, location) in alphamem (Biowhittaker,
location) supplemented with 10% fetal bovine serum for 96 hrs.
Cytochalasin B is an inhibitor of microfilaments and will prevent
the cells from undergoing cytokinesis while allowing the cell to
synthesize DNA and increase in size. After 24 hrs recovery from the
drug, cells can be removed from the culture surface and
manipulated. Resulting cells are approximately 30 .mu.m in
diameter.
[0091] Education
[0092] Round glass discs, approximately 2.5 cm in diameter are
coated with poly-D-lysine. Cytochalasin B treated A9 cells are
plated at 60-80% confluency on the discs and allowed to adhere for
24 hrs. Discs are placed cell-side down in centrifuge tubes
containing 5 ml enucleation medium (phosphate buffered saline, 10%
fetal bovine serum, 10 .mu.g/ml cytochalasin B). Cells are
incubated for 20 min at 37.degree. C. Centrifuge tubes are placed
in 37.degree. C. ultracentrifuge and spun at 23,000 g for an
additional 20 min. Resulting cytoplasts are viable for 24-48
hrs.
[0093] Cytoplasts are removed from the glass surface by
trypsinization. HAT supplement is added to culture medium at 1X
concentration to kill remaining nucleated cells. An alternative to
this is to add the HAT supplement following introduction of the
donor nucleus. This will eliminate any nucleated A9 cells while any
unfused cytoplasts will lyse within 48 hrs.
[0094] Introduction of Donor Nucleus
[0095] Sperm collected from transgenic mice, carrying the neomycin
resistance gene (Neo), are prepared for fusion by either
capacitation or treatment with protease. These treatments are used
to ensure that the sperm will stick to the cytoplasts. Transgenic
markers are useful for verifying the source of the sperm but are
not necessary for the procedure. Alternative haploid donors are the
male and female pronuclei (haploid karyoplasts) removed from newly
fertilized embryos by micromanipulation.
[0096] Fusion
[0097] Both the A9 cytoplasts and sperm are treated with protease
or with PHA to increase the likelihood of cytoplast to sperm
adhesion and fusion. The appropriate concentration of sperm or
donor nuclei and cytoplasts should be used to enhance the number of
resulting cells with a single nucleus. An AC pulse can be used to
orient nuclear/cytoplast couplets so that the membranes to be fused
are perpendicular to the flow of current. A DC pulse will be
administered to induce fusion between the nuclear donor cell and
the cytoplast. Other methods of cell fusion could also be used in
the procedure such as polyethylene glycol, fusion-inducing viruses
or liposomes.
[0098] Selection
[0099] Several days following fusion, selection for A9-haploid
nuclear hybrids will be started. HAT sensitive A9 cells will be
used as a source of cytoplasts, therefore, any colonies that form
in the HAT medium will be from haploid-cytoplast hybrids. Non
enucleated A9 cells will not survive selection. Resulting hybrids
will be clonally propagated until there are sufficient numbers to
analyze. We will determine whether hybrids are haploid or diploid
by fluorescent in situ hybridization or karyotyping.
[0100] Fertilization
[0101] Haploid cells can be used as donor nuclei in the
fertilization of oocytes. Nuclear transfer is effected using
standard procedures. Embryos will be activated using a method that
results in second polar body extrusion and haploidization of the
female chromatin.
* * * * *