Articles
The
Biology
History and
of
Cloning:
Rationale
ROBERTG. McKINNELL
AND MARIEA. DI BERARDINO
shouldbe
Althoughthe methodof nucleartransplantation
valuableprincipally
forthe studyof nucleardifferentiation,
it
mayalsohaveotheruses.(BriggsandKing1952,p. 462)
1997 was the year of the clone (Figure1).
The cover illustrationof
Clearly,
Nature (27 February1997)
announcedthe birth of Dolly,the ewe cloned from an adult
sheep in Scotland, and Science (19 December 1997)
proclaimed Dolly to be the "breakthrough"of the year.
Newspapers,news magazines,radio, and television were
even more fervent in their reports of Dolly. Even now,
severalyearslater,Dolly (Wilmutet al. 1997) and a number
of mice (Wakayamaet al. 1998, Wakayamaand Yanagimachi 1999) and calves(Katoet al. 1998,Wellset al. 1999),
all cloned from adult cells,continueto evokefascination.
Was Dolly the first animal to be cloned?Of course not.
Why,then, the sudden, almost unprecedentedattention to
the announcement of a cloned sheep? We believe that
because Dolly was the first animal cloned from an adult
cell, she stimulated scientists, theologians, ethicists, journalists, and politicians to contemplate the application of
cloning to humans.
The point of this articleis not to reconsiderthe extension of cloning to humans, a subjectthat has alreadybeen
covered (e.g., Silver 1997, Kolata 1998, Nussbaum and
Sunstein 1998), but to considerthe genuine rationalesthat
stimulated the original and continuing efforts in cloning
research.Cloning was never intended as a procedure for
the simple multiplication of animals. Frogs are cheap in
the United States,as are sheep in Scotland.The reasonsfor
cloning are more complex than simply producing identical animals, and in this article we consider those reasons
and the results that have been obtained with the procedure. We place cloning in the historical context of developmental biology and review results obtained with the
procedure.Some readersmay wonder how two scientists
with cloning experienceview the ethics of human cloning.
We offer our views in the epilogue.
Cloning'sroots in nineteenthcentury biology
How do cells become specialized during development?
One cell, the zygote,gives rise to a multiplicityof cells that
in time become increasinglyspecialized,or differentiated.
In the latter part of the nineteenth century,August Weis-
CLONING HAS PROVIDED INSIGHTS INTO
NUCLEAR DIFFERENTIATION,NUCLEAR
REPROGRAMMING,CELLULARAGING, AND
GENOMIC IMPRINTING
mann believed that differentiationresultsfrom the differential and sequential partitioning of the genome as the
cells divide (reviewed by Wilson 1928, Spemann 1938).
The attractiveness of the now-discarded Weismann
hypothesiswas that it could be tested.WilhelmRoux,in an
1888 experiment (Spemann 1938), killed one cell (blastomere) of a two-cell amphibianembryo and found that a
half-embryo developed, suggesting that some genes are
lost during cell replication.However,in 1892, Hans Driesch found that if the blastomeresof two-cell sea urchin
embryoswere physicallyseparated,entireembryosformed
from each blastomere (Spemann 1938); similar results
were obtainedwhen amphibianblastomereswereisolated,
provided that they contained a portion of cytoplasm
known as gray crescent material.Thus, the genome was
not diminished, but rather reproduced during cell division. (Roux'shalf-embryoswere later interpretedto result
from the inhibitory effect of the dead blastomere.)
Blastomereseparationof embryosbeyond the 2- to 16cell stage (depending on species) was noninformativefor
testing genomic potential because the cells had too little
RobertG. McKinnell(e-mail:[email protected])
is a professor of Geneticsand Cell Biologyat the Universityof Minnesota,
Saint Paul,MN55108-1095. He is the authorof Cloning:Nuclear
in Amphibia(1978) and Cloningof Frogs,Miceand
Transplantation
OtherAnimals(1985) and the 1998 recipientof the PrinceHitachi
Prize in Comparative Oncology, awarded by the Japanese
Foundationfor Cancer Research. MarieA. Di Berardino(e-mail:
is a professor emerita of
[email protected])
Biochemistryat MCP HahnemannUniversity,Philadelphia,PA
19129. She is the authorof GenomicPotentialof Differentiated
Cells(1997) and the 1996 recipientof the Jean BrachetMemorial
Inc. ?
Award,given by the International
Society of Differentiation,
1999 AmericanInstituteof BiologicalSciences.
November1999 / Vol.49 No. 11 * BioScience 875
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Figure 1. Dolly, the sheepproducedfrom the
transferof a nucleusof an adult mammarygland
cell at the RoslinInstitute in Scotland.
cytoplasm. But in 1894, Jacques Loeb fortuitously
observedthat fertilizedsea urchineggs sometimesruptured when exposedto hypotonicsolutions(Spemann
1938). The extrudedportion of the egg was usuallybereft
of a nucleusandremaineduncleaved.Occasionally,
nuclei
traversedthe isthmusbetweenthe cleavingegg and the
extrudedcytoplasmicmaterial.In such cases,the extruded
cytoplasm cleaved along with the main mass, resultingin
the formation of two entire embryos. In 1914, Hans Spemann performed a conceptuallyidentical experiment on
an amphibian egg (Spemann 1938). He constricted a
zygotewitha noosemadeof babyhair,causingthe eggto
assume the shape of a dumbbell. When the cleaving
(nucleated) portion reached the 8- or 16-cell stage, he
loosened the constriction and permitted a nucleus to
move to the non-nucleatedcytoplasmicportion.Here,
too, the non-nucleatedportion cleavedand formed a
clone of its nucleardonor.
These primitive nuclear transplantation (cloning)
experimentsaffirmedthe view that the complete genome
is replicatedduring cell division,at least during early
cleavage.The stagewas now set for modern cloning experiments to examine the genomic potential of older embryonic cells.However,Spemann (1938) "couldsee no way for
the moment" to manually insert a nucleus from older
embryos into enucleated cytoplasm. Robert Briggs and
ThomasJ.Kingfounda way.
Successin Philadelphia
Around1943,Briggs(1911-1983),an embryologist
workat
is
now
what
the
Fox
Chase
in
Cancer
Center
ing
wanted
to
determine
whether
the
Philadelphia,
genomeof
older somatic nuclei remainsequivalentto the zygote
nucleusthroughoutdevelopment(Pattersonn.d.). One
a somaticnucleusinto
possibleapproachwasto transplant
an oocytewhoseownnucleushadbeenremovedandthen
observewhat type of developmentoccurred.Briggswas
awarethatComandonandde Fonbrune(1939)hadtransamoeba,buthe recplantedsinglenucleiin theunicellular
876 BioScience * November1999 / Vol.49 No. 11
ognized that nuclear transfer in the oocytes of
metazoans would be more arduous because of
their complex physiological and biochemical
requirements for embryonic development. Over
the next 7 years,while analyzingthe development
of haploid and triploid frog embryos, he became
experienced with various microsurgical techniques that would contribute to the procedureof
nucleartransfer.In 1948,one of us (Di Berardino)
joined Briggs'slaboratoryand was thereto witness
the first cloning of metazoan animals in 1952.
In 1949, Briggs began searching for a research
fellow to develop a nucleartransplantationprocedure for the North American leopard frog, Rana pipiens.
He also applied to the National Institutes of Health for
funds to support the project.His first attempt was rejected because the reviewersconsideredhis proposed research
a harebrainedscheme with little chance of success. However, a second application was successful, and Briggs
broughtKingin as his researchfellow in 1950.The two scientists first determined that eggs lacking a functional
nucleus but containing a cell division center developed at
best into partialblastulae(Briggset al. 1951).This baseline
study establishedthe total developmentalpotential of the
recipient host; any further development could, therefore,
be creditedto an introducednucleus.
Briggs and King's nuclear transfer procedure involved
first activating an oocyte at metaphase II of meiosis by
pricking it with a glass needle, which initiated the metabolic changes normally induced by the sperm. Approximately 15 minutes later,a pit (blackdot) appearingon the
surface of the oocyte was microsurgicallyremoved with
another glass needle; this surgery removed the oocyte's
chromosomes, resulting in an enucleated egg. Finally, a
blastulacell was aspiratedinto a glass micropipettewhose
lumen was slightlysmallerthan the diameterof the cell to
gently breakthe cell membrane.The broken cell was then
transferredto the animal hemisphere of the enucleated
egg, which permittedthe nucleus to interactwith the cytoplasmic molecular milieu of the oocyte and to undergo
nuclearreprogramming(Figures2 and 3).
In their classic paper reporting the outcome of the
experiment,Briggsand King (1952) showed that tadpoles
developed from some enucleatedeggs injected singly with
blastula nuclei. At this stage, the pioneers terminatedthe
experiment because they were interested not in cloning
animals per se but rather in investigatingnuclear potential. Later,however,when they tested early gastrulanuclei
(Briggsand King 1960), they did reartadpoles and showed
that the majority metamorphosed into normal juvenile
frogs.Briggsand Kingchose donor cells from blastulaand
earlygastrulastagesbecause previousstudies (reviewedin
Articles
in amphibians
Figure2. Nucleartransplantation
usingembryoniccellsas nucleardonors.Adult
and
leopardfrogs(A)arematedin thelaboratory,
theresultantfertilizedeggs(B)arepermittedto
developto theblastulastage(C),withapproximately
3000cells.Theblastulais dissociatedtoyieldsingle
cells(D) thatwill beusedas nucleardonors.A gravid
female(E)providesa recipientovum(F),whichis
artificiallyactivatedbyprickingwitha sharpglass
needle.Theactivatedovumis enucleated,eitherwith
anotherglassneedle,or,as in thiscase,witha pulse
of a rubylaser(G).A previouslydissociatedcell(D)
is drawnintoa micropipette
and insertedintothe
enucleatedovum(H).Whenall stagesof this
operationareperformedproperly,oneor more
clonedfrogs(I) areproduced.Reprinted
from
McKinnell(1985)andusedwithpermission.
Donor Preparation
A
Spemann 1938) had shown that when regions of
embryos at these stages were graftedto other areasof
autologous embryos, the grafts developed according
to the new site; hence, the cells of the transplanted
regions were still undetermined.If the procedurehad
not producednormal animalsfrom nuclei of undetermined regions, it could not have been applied to
nuclei from determined and differentiatedregions to
study nucleardifferentiation.The totipotency of blastula nuclei was eventuallydemonstratedwhen blastula nuclei were found to direct the development of
adult frogs that produced normal progeny (Xenopus
laevis, Gurdon 1961; R. pipiens, McKinnell 1962). In
both studies,the donor nuclei carrieda genetic marker (single nucleolus in Xenopusand pigment pattern in
Rana). The resulting frogs expressed the donor genetic
markers,thus proving that the frogs were derived from
donor nuclei and not oocyte nuclei left behind from faulty
enucleation.
Many other investigatorsin the United States,England,
France,China, and Japansoon enteredthe field. They too
confirmed the totipotency of blastulanuclei in other frog
species as well as in some salamanderspecies (reviewedin
Di Berardino1997a).The pioneersof nucleartransplantation concluded in a classicunderstatementthat "although
the method of nucleartransplantationshould be valuable
principallyfor the study of nucleardifferentiation,it may
also have other uses" (Briggs and King 1952, p. 462). We
discuss this research and other applications of cloning
below. In retrospect,it is clearthat the amphibiannuclear
transplantation procedure became the prototype for
cloning multicellularanimals.
Amphibian cloning
Earlygastrulacells that arefatedto give rise to neuralplate
if left in place will, if graftedto a site destined to give rise
to epidermis, develop as epidermis. On the other hand,
late gastrulacells that are destined to give rise to neural
Recipient Preparation
E
41
1
BO
Q
FertilizedEgg
F
Activation of
unfertilized ovum
C/
Blastula
Laser radiation
G
Laser enucleated
unfertilized ovum
Dissociation of
donor cells
H
Enucleated egg + nucleus from dissociated cell =
4nuclear
1
transplantation
One or more cloned frogs
plate differentiate into neural cells when placed in
anatomicallyinappropriatesites. Thus, late gastrulacells,
although not yet differentiatedas neural cells, are determined to follow a neural pathway(Spemann 1938).
The transferof embryonicnuclei. Kingand Briggs
(1956) found that with increasing embryonic age, more
and more transplantednuclei displayeda loss of differentiation potential. This observationwas confirmed by other investigatorsfor several frog and salamanderspecies
(reviewed by McKinnell 1978, Di Berardino 1997a,
1997b). What made this observation particularlycompelling was the fact that similar results were obtained by
King and Briggs (1956) with R. pipiens and by Gurdon
(1974) with the South Africanclawedtoad, X. laevis--two
very different anuran species. X. laevis is primitive and
aquatic,and it develops rapidly,whereasR.pipiensis more
evolutionarilyadvancedand terrestrialmuch of the year,
and it develops more slowly.Yet despite the evolutionary
differences between the animals, similar loss of nuclear
potential was observed in the transplantednuclei from
both species (reviewedby McKinnell1972).
Although endoderm cells of tailbud-stage R. pipiens
embryos have lost nuclear potential, Hennen (1970) was
November
1999/ Vol.49 No. 11 * BioScience877
Articles
Figure3. Normal
leopardfrog Rana
pipiens,producedby
nucleartransplantation
(RobertG.McKinnell,
data).
unpublished
able to reverse these
otherwisestablenuclear
changeswith technical
modifications to the
transplantationprocedure.Sheaddedthepolycationic amine, spermine, to complexchromatinproteinsand loweredthe
environmental
temperatureto lengthenthe cell cycleof
the oocytehost.Of the completeblastulaethatdeveloped
fromenucleatedegg cellsinto whichtailbud-stage
nuclei
had been transplantedusing these modifications,62%
developedintonormallarvae.Bycontrast,whenthemodificationswerenot used,only25%of the blastulaedeveloped into normallarvae.Almost30 yearsago, Hennen
involves
(1970)concludedthat"if normaldifferentiation
the selective repressionof genetic information,then
howeverstableit mightbe undernormalconrepression,
ditions,is reversibleas far as nuclei from tailbudpreTheimplicationof Hensumptivemidgutareconcerned."
nen'swork is that modificationsin the nucleartransfer
procedurecan enhancethe developmental
expressionof
nucleifromadvanceddevelopmental
stages.
eggs to develop into fertilefrogs:one gut nucleus from Pleurodelesprehatchinglarva (Aimar
1972) and, from Xenopusswimming larvae,20
gut nuclei (Gurdon 1962), 2 intestinal nuclei
(Gurdonand Uehlinger 1966), and 2 epidermal
nuclei (Brun and Kobel 1972, Kobel et al.
1973). However, it is not known whether the
few totipotent nuclei originatedfrom differentiated cells or from contaminatingstem cells.In
contrastto the few totipotent amphibiannuclei
from larval stages, no adult nuclei were found
to be totipotent (reviewed by Di Berardino
1997a, 1997b).
Extensivenuclear transfer studies of differentiated larval and adult cells from R. pipiens
andX. laevisrevealedthe nucleiof thesecellsto be multipotentbut not totipotent:Xenopusnucleifrommelanoskin,andlymphocytesinjectedinto
phores,erythroblasts,
enucleatedeggsdirectedthe developmentof pre-or posthatchingtadpoles (reviewedby Di Berardino1997a,
1997b).ThemostadvancedtadpolesensuedfromR.pipienserythrocytenucleifromjuvenilefrogs:7.8%directed
the formationof feedingtadpolesthatsurvivedforup to a
month(Di Berardinoet al. 1986).
Thesestudiesareimportantforseveralreasons.One is
thatthe terminallydifferentiated
statewasobviousby the
and
color
of
the
donor
red
bloodcell.Thecelltype
shape
of donor nuclei in a cloningexperimentis not always
knownbecauseof the complexityof biologicaltissuesthat
arecomposedof both differentiated
and stemcells.With
matureerythrocytes,
therecan be no doubtas to the cell
typebecauseof theirovalmorphologyandredcolor.Furlarval
and
adult
nuclei.
a
small
the uniquemorphologyof the matureamphibthermore,
Only
Cloningof
of
larval
nuclei
directed
enucleated
ian
nucleated
bloodcellis alwaysassociatedwith a particminority
amphibian
ularphaseof the cellcycle(G0,in thisinstance)and
the virtualabsenceof transcriptional
activity.
Figure4. Tadpoleensuingfromthetransplantation
of a terminally
Anothernotable aspect of the study was that
nucleus.
The
nucleus
was
differentiated
erythrocyte
initially
nucleiwerefirstinjectedintometaphase
incubatedin oocytecytoplasmandsubsequently
intoan erythrocyte
transplanted
I
and
"conditioned"
for one daywhile the
oocytes
enucleatedegg.Thetadpolehadhindlimbbuds(arrow)anda
matured
into
oocytes
metaphaseII oocytes(theconfunctionaldigestivesystem(noterectumwithfecesbelowthelimb
ventional
The
maturedoocytes were then
host).
bud),and it wasabletofeed.Reprinted
fromDi Berardinoet al.
activated
parthenogenetically
by insertionof a glass
(1986).
needle, and the maternal (oocyte) nucleus was
nucleusin the
removed,leavingonlythe erythrocyte
On
the
blastulae
had
cytoplasm.
following day,
that
then
became
nuclear
donors
for
enudeveloped
cleatedmetaphaseII oocytes.Theseclonederythrocyte embryosdevelopedinto feedinglarvaewith
hind limb buds (Figure4). The use of metaphaseI
oocytes for conditioningthe erythrocytegenome
wasbasedon previousstudies,in whicherythrocyte
nucleitransferred
to metaphaseII oocytesfailedto
promotedevelopmentof the host beyondthe early
gastrulastage;however,thosenucleiexposedfirstto
metaphaseI and then metaphaseII oocyte cytoplasmdirectedthe hoststo developinto larvae(Di
878 BioScience* November
1999/ Vol.49 No. 11
Articles
Berardino and Hoffner 1983). These experiments were
based on the hypothesisthat molecularcomponents of the
oocyte cytoplasm that prepare oocyte chromosomes to
participate in fertilization would similarly condition the
genetic material of erythrocytes.Obviously,the developmental potential of these terminally differentiatederythrocyte nuclei was enhanced by this conditioning; however,the actualmolecularmechanismsresponsiblefor this
nuclearreprogrammingrequireinvestigation.
Finally,the experiments resulted in the most developmentally advanced cloned animals produced with adult
nuclei before the advent of Dolly, the sheep cloned from
an adult mammarygland cell by Wilmut et al. (1997). Like
the larvaein the erythrocytestudy,Dolly developed from
a "quiescent"nucleus, one in the Go part of the cell cycle.
One cannot but ponder the significanceof the quiescent
phase associatedwith the successfulresultsof these donor
nuclei. Perhapsthe successfulresultsof these donor nuclei
in the quiescent phase suggest that Go nuclei integrate
more normallyinto the cell cycle of the host than those in
the other cell cycle phases (Campbell 1999).
Cloning a cancer genome. Until recently,the traditional dogma of pathology assertedthat cancer cells can
only give rise to more cancer cells. This view that mitotic
progeny of cancer cells must alwaysbe malignant mandates that cancercure can only follow the death of all cancer cells in a patient. Killing cancer cells in a patient with
cytotoxic drugs is hazardous because a wide range of
chemotherapeuticagents do not discriminatein their toxicity between normal cells and cancercells (Lipp 1999).
When one of us (McKinnell)joined the laboratoryof
King in 1958, it was decided that the cloning procedure
could be used to determine whether the molecular environment of the oocyte and that of the subsequent developing embryo could reversethe malignant phenotype. If
normal cell differentiationwould ensue in the embryo or
larva produced from the transplantationof a cancer cell
nucleus into an enucleatedegg, it would demonstratethat
mitotic progeny of a cancer genome could, under certain
circumstances, give rise to normal differentiation. This
finding in turn would provide support for the notion that
nontoxic differentiationtherapymight somedaybe developed as a mode of treatmentfor cancer.This then was the
rationalefor the cloning of a cancergenome.
The malignancy chosen for cloning studies (King and
McKinnell 1960) was the herpesvirus-induced(Tweedell
1967, Davison et al. 1999) Luck6 renal carcinoma of R.
pipiens.Although little was known about the competence
of cancercells to give rise to normal cells by mitosis at the
time these studies began, significantlymore is known now
(Pierceand Speers 1988, McKinnellet al. 1998).
Because normal, or near-normal, chromosomes are
required for successful nuclear transplantation experiments, it was gratifyingto learn that most Luck6tumor
cells have a normal karyotype(Di Berardinoet al. 1963,Di
Berardinoand Hoffner 1969). The cancer cell nuclei were
shown to induce both partial and complete cleavage in
enucleatedova; some of the complete blastulaedeveloped
into abnormal embryos (King and McKinnell 1960) and
larvae (King and Di Berardino 1965). These studies
revealed that the neoplastic nucleus retains the genetic
program for limited embryonic and larval development.
However, because development of nuclear transplants
from normal cells diminished as the age of the nuclear
donor increased (in these experiments,the donor cancer
nuclei were derived from adult animals), it became critically important to provide evidence that the tumor
nuclear transplants developed from the inserted cancer
nucleus and not from an inadvertentlyretainedegg nucleus. Also, it was necessaryto show that developmentensued
from a nucleus from a neoplastic cell and not from one of
the few stromal cells present in the tumor. The evidence
was obtainedwith severalprocedures,including the use of
chromosomallytaggeddonor tumor nuclei, the identification of the malignant origin of the donor cells by their
fluorescencein ultravioletlight after treatmentwith acridine orange,and the mode of tumor cell dissociation (discussed in McKinnell et al. 1998). The results led to the
conclusion that the Luckecancergenome could direct the
development of earlylarvae.
To determine if more advanced tissue differentiation
could be directedby the Lucketumor genome, tissue fragments of tumor nuclear transplant embryos were allograftedto normal hosts of a differentploidy, and the hosts
were maintainedfor 40 days,until shortlybeforethe onset
of metamorphosis (Lust et al. 1991). Well-differentiated
tissues of all three germ layersthat were equivalentto the
tissues of controls of the same age were observedin histological sections. Therewas no evidence of neoplasiain any
of the grafts.
The reprogrammingof Lucke cancer cells is especially
important in the context of the emerging field of cancer
researchknown as differentiationtherapy (Warrell1997).
The induction of differentiationin mitotic progenyof the
Luckecancergenome, and induced differentiationof other malignant cell types, supports the view that the new
mode of cancertreatmentknown as differentiationtherapy may supplant,in selected malignancies,older forms of
cytotoxic chemotherapy.
Insect and fish cloning
Cloning studies in Drosophilawere launched during the
late 1960s.Becauseenucleationof the fragilehost eggs was
not feasible, investigators injected several genetically
marked nuclei into the posterior region of each unfertilized (Illmensee 1973) or fertilized (Zalokar 1973) egg at
the site where pole cells, the progenitorsof the germ cells,
would later form. The assumption-which turned out to
be correct-was that in some nuclear transplants,host as
well as injected nuclei would populate the pole cells. In
many cases,the fertilizedegg hosts developed into normal
November1999 / Vol.49 No. 11 * BioScience 879
Articles
Donor Nuclei
RecipientCytoplasm
embryo from IVF
M IIoocyte
Grecovery
recovery
enucleation
remove zona
peliucida
disaggregation
of blastomeres
'~-'~Jnuclei
for recipient
cytoplasm
for donor
NuclearTransfer
culture
-+
electrofusion
embryo transfers
to foster mothers
I.
'00___rhesus
monkeys
with Identical
composition
9genetic
in therhesusmonkey.
Figure5. Nucleartransplantation
(upperleft)Dissociatedembryoniccellsfor donornuclei
areobtainedfroman in vitro-fertilized
egg.(upperright)
Recipientcytoplasmis preparedbyenucleationof an
oocyte.(lowerpanel)A donornucleusis insertedunderthe
zonapellucidaof an enucleatedoocyte.Theeggand its
cellarefusedbyelectricpulses,thuspermitting
transferred
thedonornucleusto interactwith therecipientcytoplasm.
Theeggcontainingthetransferred
nucleusis initially
to a foster
culturedin vitroandsubsequently
transferred
mother.Theblackshadingof thedonormonkeyindicates
thatit differsgenetically
fromthemonkeythatprovidesthe
recipientoocytecytoplasm(grayshading).Thethreecloned
monkeysexpressthegenotypeof thenucleardonor,not the
fromMeng
genotypeof therecipientcytoplasm.
Reprinted
et aL(1997)withpermission.
adults.The unfertilized(activated)egg hosts developed
into defectivenucleartransplantsthat were rescuedby
the pole cellsof the embryosor the gonads
transplanting
of the larvaeinto normalhosts.Manyof these rescued
embryosdevelopedinto normaladults.Theseadultswere
fertile and producednormal progenythat arose from
gametescontainingeither host or donor nuclei. Thus,
880 BioScience * November1999 / Vol 49 No. 11
these experiments demonstrated the totipotency of the
original transplantedpreblastoderm(Zalokar 1973) and
early gastrula(Illmensee 1973) nuclei.
Nuclear transfersin fish were initiated by the late T. C.
Tung (Tong Dizhou) in the early 1960s in China and
extendedby Yan(1998). One of their goals was to produce
fish clones for agriculturaland commercial purposes. To
this end, they produced nucleocytoplasmic hybrids by
transplanting a blastula nucleus of one species into an
enucleatedoocyte from a differentspecies. Combinations
of nuclei and cytoplasm from differentgenera and different subfamilies resulted in fertile adults, demonstrating
the totipotency of fish blastula nuclei. Compared to the
original species, the inter-genera constructs exhibited
higher growth rates,increasedprotein content, and lower
fat content, indicating the feasibility of producing commerciallyvaluablefish by this technique.
Mammalian cloning
Cloning of mammals via nuclear transfer was initially
reportedin mice during the early 1980s,approximately30
years after the first tadpole clones were produced.
Although there was considerable interest in extending
cloning to mammalian species, these efforts were delayed
until numerous technical parameterswere modified for
the small (approximately 100 Vm in diameter) mammalian oocyte, including enucleation and in vitro culture
of viviparous oocytes and embryos. Investigatorsusing
mammaliancells were interestedin the same fundamental
question of nuclear potency during embryogenesis that
was posed for cells of invertebrateand vertebratespecies.
In fact, a series of experimentsinvolvingblastomereseparation and fusion, bisection of blastocysts,and injection of
inner cell mass cells into the blastocyst had already
demonstratedthat totipotency is maintainedin mammals
until at least the blastocyststage (reviewedin Di Berardino 1997a, 1997b).In addition to these basic studies, investigatorsseekingto improvethe genetic content of livestock
species to benefit agriculturefocused on nuclear transfer
to oocytes of large domestic animals.
Nuclear transfersin mammals are performedprimarily
by cell fusion, using a proceduresimilar to the one originally developed for murine nucleartransplants(McGrath
and Solter 1983) but with some modifications (Figure5).
A micropipetteis insertedthrough the zona pellucida (i.e.,
the noncellular envelope surrounding the mammalian
oocyte) and positioned over the oocyte's spindle; the
micropipette is then used to withdraw the spindle, chromosomes, and first polar body. Next, a micropipettecontaining an intact, geneticallymarkeddonor cell is inserted
through the zona pellucida,and the cell is gently injected
into the cavity between the oocyte's membrane and the
zona pellucida.The two cells are usually fused by electrofusion, whereby the electrical dischargecauses breaks in
the cell membranesof the oocyte and donor cell, permitting the contents of each to mingle beforemembraneheal-
Articles
ing. The electrical discharge can activate some oocytes,
although in most cases, additional treatment is required.
Alternatively,in some mouse experiments,nuclei may be
injected into oocytes that are subsequently activated
(Wakayama et al. 1998, Wakayama and Yanagimachi
1999). The nucleartransplantsare rearedin vitro through
various cleavagestagesand then transferredto the uteri of
surrogatemothers.
Totipotencyhas been demonstratedfor nuclei from various preimplantationstagesof mouse and cattle (reviewed
by Sun and Moor 1995, Di Berardino1997a, 1997b,Fulka
et al. 1998). In all cases, the clones developed into adults
and produced normal progeny.So far, the only primates
cloned by nucleartransferaretwo rhesusmonkeys (Figure
5) from 8-cell embryos (Meng et al. 1997). We await a
reporton tests of their fertility.
In some cases, multiple clones were produced from a
single donor animal; these will be valuable for testing
pharmaceuticals on animals with an identical genetic
(nuclear)background.For example, isogenic groups were
reportedin murine twins and triplets (Kono et al. 1991)
and in a group of over 30 animals produced by serial
cloning (i.e., the cloning of clones; Park et al. 1993,
Wakayamaet al. 1998). Similarly,multiple calves ensued
from one donor (Bondioli et al. 1990, Willadsen et al.
1991, Chesne et al. 1993), and 10 calveswere producedby
serial cloning (Stice and Keefer 1993). Finally, serial
cloning of goat nuclei from the 32-cell stage resultedin 45
kids (Zang and Li 1998). Three identical transgenicgoats
were cloned from fetal cells bearing the human gene for
antithrombinIII,and one goat is producingthe protein in
her milk (Baguisiet al. 1999).
Although cloning of preimplantation mammalian
nuclei has been relativelysuccessful,the cloning efficiency
from more advanced donor stages, as in amphibians,
decreases.Nevertheless,a cloning efficiency adequate for
commercialuse has been achievedwith fetal lamb and calf
fibroblastand muscle cells, as well as with adult sheep
mammarygland, mouse cumulus, and calf cumulus and
oviductalcells.The percentagesof newborns,based on the
number of embryo clones transferredto uteri, are 5-20%
for lambs (7 total) from fetal fibroblastcells (Schniekeet
al. 1997), 11%for calves (3 total) from fetal fibroblastcells
(Cibelliet al. 1998), and 7% for calves (2 total) from fetal
muscle cells (Vignon et al. 1998). With respect to the
cloning efficiency from adult cells, the percentages are 3%
for lambs (1 total) from mammary epithelium (Wilmut et
al. 1997), 3% for mice (33 total) from cumulus cells
(Wakayama et al. 1998), 1% for mice (3 total) from tail-tip
cells (Wakayama and Yanagimachi 1999), 80% for calves
(8 total) from cumulus and oviductal cells (Kato et al.
1998), and 10% for calves (10 total) from mural granulosa
cells (Wells et al. 1999). However, for those persons speculating on the application of cloning to humans, we emphasize the high mortality rate occurring throughout the
experimental procedure. The Dolly experiment, as we will
discuss subsequently,began with 434 attempts to fuse a
mammarygland cell to an oocyte; the developmentof this
one ewe representsa success rate of only 0.2%, and the
remainingattemptsresultedin death, either during fusion
or during various pre- and postnatal stages.Although the
success rates for mouse (1-2%) and calf (3.2%) cloning
from adult cells are higher than for humans, the resulting
mortality rate is still high.
Severalstudies in sheep and cattle deserve further discussion. Sims and First (1993) initially cloned four calves
from cultured cells of the inner cell mass of blastocysts.
This result establishedthe feasibilityof cloning from cultured cells, and it suggested that transgenic clones might
be produced by transfectingcultured cells with a foreign
gene and then using these cells as donor cells for nuclear
transfer. Indeed, two laboratories have now produced
transgenic cloned animals. Schnieke et al. (1997) cloned
three transgeniclambs (two of which survived)containing
the human gene for clotting factor IX. Fetal lamb cells in
culture grew as fibroblastsand were transfectedwith constructs composed of the coding sequences for neomycin
resistance and human clotting factor IX placed downstream of the promoter sequence for ovine B-lactoglobin.
The cultures were then exposed to G418, which kills all
cells except those expressing neomycin resistance. Only
those cells that had integrated the genes for neomycin
resistanceand, therefore,clotting factor IX survived and
were used for nuclear transfer.The promoter sequence of
the ovine fi-lactoglobin gene directs the gene for human
clotting factor IX to be expressedin a tissue-specificmanner,that is, in the mammarygland.Afterappropriateclinical trials,the protein harvestedfrom the milk will be used
to treat hemophiliacs (Wilmut 1998).
Recently,three transgeniccalveswere cloned from cells
of a fetal fibroblasticcell line that contained a fi-galactosidase-neomycin resistancefusion gene driven by a constitutive promoter (Cibelli et al. 1998). In the future, transgenic clones can be designed to produce a variety of
complex human proteinsfor human use. It should be noted that the production of transgenicanimalsby cloning is
considerablymore efficientthan by gene injection into the
pronuclei of fertilized eggs. There are several reasons for
this increasedefficiency,including the fact that transgenic
cells can be selected in culturebefore nucleartransfer.
Other biologicalproblems examined
by cloning
Cloning experiments have provided valuable insight into a
number of important cellular processes, such as nuclear
reprogramming, cellular aging, and genomic imprinting.
Nuclear reprogramming. The phrase"nuclearreprogramming" was used in frog cloning to designate the morphological and molecular changes occurring in nuclei
transplanted into oocyte cytoplasm. The cytoplasm of
activated eggs induces transplanted nuclei to cease RNA
November1999 / Vol.49 No. 11 * BioScience 881
Articles
synthesis and synthesize DNA. The transplanted nuclei
resume RNA synthesis at later embryonic stages, at the
same time as embryonic nuclei from fertilizedeggs begin
to synthesizeRNAs.This reversibilityof nuclear function
also applies to specific genes (reviewedin Gurdon 1974).
Moreover,during the first cell cycle of frog nucleartransplants,non-histone proteinsmove bidirectionallybetween
the transplantednucleus and the egg cytoplasm,whereas
histone proteins primarilymove from the cytoplasm into
the nucleus (Di Berardinoand Hoffner 1975, Hoffner and
Di Berardino 1977). This result suggests that the chromatin proteins are being modified.
Today,techniques are availableto analyzethe remodeling of chromatinproteinsdirectly.When transcriptionally
inactivespermnuclei wereincubatedin extractsfrom activated amphibian eggs, sperm-specific histone proteins
were replacedby somatic histones H2A and H2B via the
molecularchaperonnucleoplasmin(Katagiriand Ohsumi
1994). Similarly, erythrocyte chromatin was remodeled
when the nuclei were incubated in extracts of activated
amphibian eggs: somatic histones H1 and H10 were
releasedfrom chromatin into the egg cytoplasm, oocytespecific linker histone B4 and HMG1 were incorporated
into remodeledchromatin,and somatic histones H2A and
H4 were phosphorylated (Dimitrov and Wolffe 1996).
With respect to mammalian nuclei, recent studies in
nucleartransplantembryosof mice, rabbit,pig, and cattle
confirmed that changes similar to those observed in
amphibian nuclear transplantsalso occur during nuclear
reprogramming (Fulka et al. 1998). The importance of
nuclear reprogrammingis emphasized by the fact that
incomplete nuclear reprogramming or its failure in
amphibian and mammalian nuclear transplants causes
abnormal and arrested development (reviewed in Di
Berardino1997a).
Scientistsare only beginning to understandthe molecular changes involved in nuclear reprogramming,yet this
line of basic researchmay resultin some of the most beneficial applicationsof cloning to humans. For example, if
scientistscould explainin moleculartermshow a differentiated nucleus is de-differentiated,it might be possible to
repaircertaindiseasedtissues-a small amount of normal
tissue could be removed from a patient and de-differentiated in culture. After the cell population is expanded,
appropriateinducerscould be added to promote a desired
type of cell differentiation(e.g., bone, cartilage,or muscle). Then, the tissue could be graftedto the patient'sdiseased areas,wherethe cells would be recognizedas self and
not rejected.
Cellular aging. Cloning experiments have examined
the replication potential of a genome during cellular
aging. Normal cells culturedin vitro have a finite replication limit (Hayflickand Moorhead 1961). However,even
after serial cloning through 145 cell cycles,nuclei of blastula cells were still able to direct tadpole development
882 BioScience * November1999 / Vol.49 No. 11
(Robert G. McKinnell, unpublished study). After serial
transplantationto oocytes, even terminally differentiated
erythrocytenuclei that had gone through more than 110
cell cycles had the competence to direct the formation of
tadpoles (Hoffner Orr et al. 1986). It is likely that somatic
nuclei are "rejuvenated"to some large extent in oocytes
because oocytes contain a large store of molecular substances that support nuclear replicationand mitosis. Further studies may revealthe mechanism of cellularrejuvenation. Telomeres,which are normally reduced in length
during the aging process (Greider and Blackburn 1996,
Shay 1997), may also be shortenedin animal clones. Dolly
and two other cloned sheep were reportedto have telomeres that were shorterthan those of age-matchedcontrols
(Shiels et al. 1999). Despite their shortenedtelomeres,the
cloned sheep were vigorous and healthy.It remains to be
seen if the reduced telomere lengths will have an effect
during the lifetime of the sheep.
Genomic imprinting. Nuclear transferand molecular
studies in mice elucidated genomic imprinting, a genetic
mechanismthat controlsthe differentialexpressionof certain pairs of autosomal alleles.Mouse nuclear transplants
constructedof two maternalor two paternalpronucleifail
to develop;only nucleartransplantscomprisingbiparental
nuclei form normal offspring (McGrathand Solter 1984,
Surani et al. 1984). Molecularanalysesof mouse embryos
revealed that certain autosomal genes are differentially
expressedfrom maternaland paternalgenomes at specific
times in development (Latham 1995). Thus, an embryo
with two inactive maternal (or paternal) genes fails to
transcribea necessary gene product. It is for this reason
that the mammalian embryo requiresa set of genes from
both the father and the mother for normal development.
The experimentalresultsin mice led to the clarificationof
the basis of several inherited human disorders, such as
PraderWilli and Angelmansyndromes,which presentdifferent phenotypes but are both due to deletions in different homologues of chromosome 15: paternaldeletions in
PraderWilli patients and maternaldeletions in Angelman
patients (Driscoll 1994).
Epilogue
Becauseof their fundamentalnature,scientificdiscoveries
in the basic sciences (e.g., anesthesia, atomic energy,
recombinant DNA) occasionally lead to unanticipated
deleteriousapplications.Knowledgein itself is amoral,but
the choices for its applicationsreside in the ethical decisions of humans. Cloning, like other developments in
basic science, was initiated to seek new fundamental
knowledge.In addition to yielding information about the
role of the nucleus during cell differentiation,the procedure also provided insight into basic aspectsof modifying
the cancer phenotype, rejuvenatingaged nuclei by oocyte
cytoplasm, nuclear reprogramming, and genomic
imprinting. It will continue to yield new knowledge in
Articles
these and other basic subjects.As scientistswho have
workedin frogcloning,we aregratifiedto see decadesof
basic researchculminatingin the productionof cloned
mammalsthatwillproducehumanproteinsforthe alleviation of humandisease.Cloningwill likelyresultin the
of livestock,the productionof anigeneticimprovement
mal models to study and treat human diseases,and
sourcesof animaltissuesandorgansforxenotransplantation to humans.Weconsidertheseapplications
of cloning
Indeed,they arethe reasonswhy Dollywas
appropriate.
produced.
OnceDollyappeared,
however,the newsmediabecame
consumedwith the ideaof cloninghumanadults,which
immediatelystimulateda worldwidedebateamongethicists, theologians,clerics, lawyers,legislators,and, of
course,laypeople.We do not intend to summarizethe
many proposalsand convictionsof others;rather,we
delineatebrieflythe reasonsfor our belief that human
and ethicallyunsound.We define
cloningis scientifically
humancloningasthe attemptto producea humanorganismby anycloningprocedure:
blastomereisolation,bisection (splitting)of preimplantation
embryos,and nuclear
transplantation.
Nucleartransplantation
of embryonic,fetal,or adult
cellsfromall speciesresultsin abnormalanimalsat a frequencythatincreaseswith the ageof the donorcell.The
failuresresultfrom incompletenuclearreprogramming
andfailedcellcyclematching,althoughothercausesmay
in some casesbe responsible.Abnormalnucleartransatvarious
plantsfromalldonorstagesarrestdevelopment
nuclear
transfer
and
activation,cleavage,organostages:
genesis, tadpole, and juvenile. In viviparousspecies,
abnormaltransplantembryosmay fail to implant,and
thosethatdo implantmayabortatvariousembryonicand
fetalstages.Finally,thosenucleartransplants
thatareborn
may die soon afterbirth or survivewith birth defects.
Analysesof abnormalnucleartransplantembryosfrom
frogs,mice,cattle,rabbits,andpigsrevealedchromosomal
thatcouldaccountforthe
and/ormolecularabnormalities
morphologicalaberrations(reviewedin Di Berardino
even some normalnucleartrans1997a).Furthermore,
plant frog blastulaederivedfrom embryonicor adult
in someof their
nucleicontainedabnormalchromosomes
cells,whereasthe chromosomesin othercellsof the same
blastulaeappearednormal. Such cases would pose confusion in monitoring normal-appearingnuclear transplants
for furthercultureand development.For these reasons,we
considerthe use of anycell type (not just those from adults)
for human cloning scientificallyand ethicallyunsound.
In the case of Dolly, she was the only successfulcase out
of 434 attempted fusions of oocytes and donor cells that
were taken from cultures of mammary gland. Even as
cloning of adult nuclei becomes much more efficient,
there will still be hazards to humans. For example, the
donor cells could suffer mutations in situ from radiation,
chemicals,and/or aging during the lifetime of the donor.
Mutationscouldalso arisein the donorcellsduringcell
culture,an eventthatis not unusual.Therearestillother
scientificconcerns.Willtelomereshorteningin the donor
celllimitthelife spanof the clone?Willstoredgeneproducts (RNAsandproteins)in oocytesfromforeigndonors
alwaysbe compatiblewiththe donornucleus?Finally,it is
importantto considerthatmeiosis,whichprecedessexual
reproduction,affordshumansanotheropportunityfor
DNArepairandthereforeshouldnot be avoidedin favor
of asexual(somatic)reproduction.
Forallof thesereasons,
we opposehumancloning.
Acknowledgments
Debra L. Carlson,Departmentof Biology,Augustana
College,SiouxFalls,SouthDakota,readandprovidedcritical commentson an earlyversionof this paper.MarkR.
Krampf,Departmentof Biochemistry,Universityof
Minnesota,providedvaluableassistancein preparingseveralof the illustrations
forpublication.Becauseof bibliothe
graphic restrictions,
primary referencesof some
authorswerenot cited,andwe apologizefor theseomissions. R. G. M:.'sresearchon nucleartransplantation
of
cancercells was supportedby Grant2675BR1from the
Councilfor TobaccoResearch-U.S.A.,
Inc. M. A. Di B.'s
research
on
the
cloning
genomicpotentialof frogcellswas
from
the United StatesNational
supportedby grants
Institutesof Healthandthe NationalScienceFoundation.
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