Animal Cloning
1
(Proc. Natl. Acad. Sci. USA, 38:4550-463, 1952)
2
(animalscience.ucdavis.edu/.../images/Image5.gif)
3
Dolly and her first-born lamb, Bonnie
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Cloning of Sheep
Megan and Morag –
The first animals cloned from
cultured cells
Dolly and her 3 lambs born
in April 1998
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供卵
Cloning of Sheep
供核
電融合
1/277
= 0.4%
Dolly and her mother
Mammary cells
Nuclear transplantation
體外培養
移置至代理孕母
(Nature 385:810-813, 1997)
6
Cloning of Mouse
31/1385
= 2.2%
No. Oocyte
(Nature 394:369-374, 1998)
- 2468
7
Cloning of Transgenic Calves
ACT3, ACT4, ACT5; 3 weeks of age
N
C
A
D
A,B. Transgenic fibroblast
C,D. Nontransgenic fibroblast
Normal
PCR amplified segment of the pCMV/GEO
construct of DNA from cell line and calf
5/276 =1.8%
Southern blot of genomic DNA
(Science 280:1256-1258, 1998)
8
Cloning of Pig
Single NT
0/185 = 0% - 3 Recipients
No. oocytes - 283
Double NT
5/401 = 1.3% - 7 Recipients
No. oocytes - 1869
(Nature 407:86-90, 2000)
9
Cloned female mouflon
with the sheep foster
mother
Nuclear source:
Granulosa cells from dead ones
1/22 = 4.5%
(Nature Biotechnology 19:962-964, 2002)
a. Nuclear-donor (cumulus cells) cat;
b. cloned kitten with its surrogate
mother. 1/87 = 1.2%
(Nature 415:859, 2002)
10
Rabbits born from
somatic nuclear
transfer
Nuclear source:
Cumulus cells
6/755 = 0.8% (2 died)
(Nat. Biotechnol. 20:366369, 2002)
11
Mouse created without father
2/371
= 0.6 % (survived)
No. Oocytes
- 457
Kaguya
輝夜姬
Parthenogenesis
Imprinted genes
(Nature 428:860-864, 2004)
12
Meiosis
produces
haploid
cells
Meiosis reduces
the number of
chromosomes
from the diploid
to the haploid
number.
13
Oogenesis
in mammals
After the germ cells
that form oocytes
enter the
embryonic ovary,
they divide
mitotically a few
times and then
enter the prophase
of the first meiotic
division. No further
cell multiplication
occurs, but the
oocyte increases
100-fold mass.
14
Spermatogenesis in
mammals
Germ cells that
develop into sperm
enter the embryonic
testis and become
arrested at the G1
stage of the cell
cycle. After birth,
they begin to divide
mitotically again,
forming a population
of stem cell
(spermatogonia).
15
The importance of parental genomes
male pronucleus
Pronuclear-stage
mouse embryos
female pronucleus
Exchange of pronuclei
Isoparental
mouse embryos
Gynogenetic embryos
Androgenetic embryos
(Cell 37:179-183, 1984; Nature 311:374-376, 1984; Cell 45:127-136, 1986)
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Gynogenotes /
Parthenotes
Grew to the early
somatic stages before
involution
The conceptuses were relatively
normal in size and appearance
but showed inadequate, severely
stunted placental tissues
Androgenotes
Developed to the late
preimplantation stage, very
small percentage which
were able to implant
The implanted embryos gave rise
to predominantly extraembryonic
plancetal tissues with severely
stunted development of the embryo
proper.
17
Paternal and maternal genomes are both required
for normal mouse development
(Wolpert, L. Ch 12, 2002)
18
Chromosomal translocations
Meiotic or mitotic non-disjunctions and produce
uniparental disomies (UPDs) for particular
chromosomes or chromosomal regions
No net change in the coding information of the genomes
Fetus: contained two copies of a large portion of the
maternal chromosome 7
retarded development, small placentas, died in
utero at mid-gestation
Fetus: contained two copies of paternal and no
maternal chromosome 7
died at a much earlier stage
19
Human disorders with chromosome defect
Ex. Chromosomal deletions in bands 15q11-q13
Prader-Willi syndrome
 Mild to moderate in mental retardation, slow moving,
overweight due to hyperphagia
 Paternal deletions ; Maternal UPD (fairly frequent)
Angelman syndrome
 Severe in mental retardation, thin and hyperactive,
a“
happy puppet”appearance, inappropriate laughter
 Maternal deletions; Paternal UPD (rare cases)
20
Non-Mendelian phenomena
 X-inactivation
(Nat. Rev. Genet. 3:662-673 2002)
21
Non-Mendelian phenomena (cont.)
 Genomic imprinting
The process by which alleles are marked in
the two parental germlines for differential
expression in the offspring is called genomic
imprinting or, alternatively, parental or
gametic imprinting
22
Maternally imprinted
Expression of genes is inhibited after
passage through the mother’
s
germline
Paternally imprinted
Expression of genes is inhibited when
transmitted by father’
s germline
23
Theory of imprinting: selective pressure
The placental mode of reproduction
in mammals which mated promiscuously
Five genes (Igf2, Ins-2, Mas, Grf-1, Peg1, Peg3) with
growth-promoting activity are maternally imprinted in at least
some developing tissues of the mouse
 Three genes (Igf2R, H19, p57KIP2) with growth-restraining
activity are paternally imprinted
The maternal-paternal conflict theory of
imprinting is still controversial
24
(Reprod. 130:389-399, 2005)
25
Imprints are erased, then reprogrammed in the germline and
are heritable in somatic lineages.
seminar in Cell Dev Biol 14:43-49 2003
26
Table The imprinted genes in mice
Reproduction 122:185-193 2001
27
Table The imprinted genes in mice (cont.)
Reproduction 122:185-193 2001
28
Methylation levels of individual imprinted genes and nonimprinted regions of the genome were assessed over the period
of oocyte growth and development.
Reproduction 130:389-399, 2005
29
The modification of the DNA or chromatin
accounted for imprinting must satisfy the
following requirements:
 must be made prior to fertilization
 must be able to confer transcriptional silencing
 must be stably transmitted through mitosis in
somatic cells
 must be reversible on passage through the
opposite parental germline
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Epigenetic modifications
 These changes are faithfully propagated in
dividing somatic cells.
 These changes affect gene expression and
phenotype.
 These changes are completely reversible
under appropriate conditions, since no
change in the coding information of the DNA
31
 Chromatin modification
 Chromatin remodelling
 DNA methylation at CpG dinucleotides
 Modifications to core histones
32
Dogs cloned from adult somatic cells
Two Afghan pups could help to unravel the genetics
behind the assorted traits of other canine breeds
Afghan
hound
Nuclear
donor
Nuclear source: Ear fibroblasts
Snuppy
2/1095 = 0.2 % (one died on day 22)
No. recipient - 123
Snuppy
Labrador
Surrogate
Mother
Snuppy - Seoul National
University puppy
(Nature, 436:641, 2005)
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1
(Nature, 445-800-801, 2007)
34
(Nature, 445-802, 2007)
Cloned wolves by somatic cell nuclear transfer. (A) “
SNUWOLF,”the first cloned wolf
at 129 days after birth; (B) “
SNUWOLFFY,”second cloned wolf derived at 121 days
after birth.
(Cloning Stem Cells, 9-130-137, 2007)
35
Techniques of nuclear transfer in mammals
(Rev. Reprod. 1:40-46, 1996)
36
Schematic represtation of the major event during oocyte maturation and the
first cell cycle of fertilized zygote.
Cl: cleavage; PB: polar body; GV: germinal vesicle; MII: metaphase II; MPF:
maturation/meiosis/mitosis-promoting factor.
(Rev. Reprod. 1:40-46, 1996)
37
Effects of nuclear
transfer of karyoplasts
at defined cell cycle
stages into cytoplasts
with either high (group
A) or low (group B)
maturation-promoting
factor (MPF) activity
upon DNA synthesis
during the first cell
cycle and potential
effects upon the ploidy
of the reconstructed
embryo.
(Rev. Reprod. 1:40-46, 1996)
38
Somatic-cell nuclear transfer using abnormal embryos.
(Nature 447:649-650, 2007)
39
The first embryonic cell cycle.
(Nature 447:679-689, 2007)
40
Chromosome transfer into
zygotes arrested in mitosis.
HMC: Hoffman modulation contrast.
MB: microtubule birefringence.
(Nature 447:679-689, 2007)
41
Dox: doxycycline
Developmental potential in vitro and in vivo after chromosome
transfer from ES cells into mitotic zygotes.
(Nature 447: 679-689, 2007)
42
Derivation of ES cell lines from
somatic-cell chromosome
transfer blastocysts.
(Nature 447: 679-689, 2007)
43
Aneuploid zygotes with more than two pronuclei can be used as
recipients for chromosome transfer.
(Nature 447: 679-689, 2007)
44
Methods for nuclear transfer
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Enucleation
Nuclear Transplantation
PB1
Donor cell
A
C
PB1
Donor cell
B
D
(沈，2004)
46
Electrofusion
A
B
C
D
Procedures of nuclear transfer in cattle
50μm
(沈，2004)
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(Nature 407:86-90, 2000))
48
(Science 289:118-1190, 2000)
(Nature 394:369-374, 1998)
49
Diagrammatic representation of six mouse NT protocols.
(J. Reprod. Dev. 53:13-26, 2007) 50
Diagrammatic representation of six mouse NT protocols
(Cont.).
(J. Reprod. Dev. 53:13-26, 2007)
51