PROGRESS IN NATURAL SCIENCE Vol .14 , N o .1 , January 2004

REVIEW ARTICLE

Progress in interspecies cloning of mammals＊

WEN Duancheng , BI Chunming and CHEN Dayuan＊＊ (State Key Laboratory of Reproductive Biology , Institu te of Zoology , Chinese Academy of Sciences, Beijing 100080 , China)

Received May 7 , 2003

Abstract Interspecies mammalian cloning can be achieved by application of tw o key techniques, i.e.the technique of interspecies nuclear transfer and the technique of interspecies .The general principles, problem s and possible solutions, as w ell as the recent advances of interspecies mammalian cloning have been summarized in this review .

Keywords: interspecies cloning , interspecies nuclear transfer, interspecies pregnancy , mammals.

Intraspecies animal clo ning is the technique fo r plasmic hybrids;secondly , the interspecies pregnancy the production of clonally derived mammals .The pro- is essential for the success of IAC .The nucleocyto- cedure involves the transfer of a nucleus from a dono r plasmic constructed in vitro need to cell into an enucleated oocy te of the same species . be transferred back into the uterus of a surrogate ani- Live offsprings have been born after somatic nuclear mal to com plete its full term of development .As the transfer in sheep , bovine , mouse , goat , pig , rabbit nucleus and cytoplasm of the hybrid embryos are from and .As opposed to intraspecies anim al cloning , different species , the hybrid embryos are heteroge- interspecies animal cloning (IAC), the transfer of a neous in some deg ree to the surrogate animal regard- cell nucleus of one species into an enucleated oocyte of less of the surrog ate species . ano ther species , is the technique fo r cloning those an- imal species w hose oocytes are difficult to obtain .IAC 1 .1 Interspecies nuclear transfer in mam mals usually includes the technique of interspecies nuclear In 1938 , Spemann proposed a “thought experi- transfer and the technique of interspecies pregnancy . ment” .He w ondered w hat would happen if a nucleus As IAC breaks through the restriction of “reproduc- from a differentiated cell , even an adult cell , w ere tive isolation” among species and is involved in the in- transferred into an egg w hose nucleus had been re- teraction of different species in molecular , cellular and moved .Fourteen years past befo re his gedanken ex- phy siological level , it is an inv aluable tool for study- periment could be carried out in Amphibia .Brun in ing the nucleus-cy toplasmic interaction , gene ex pres- 1973 first reported interspecies nuclear transfer in sion and regulation , differentiation and evolution . mammals .He transferred several m ammalian som atic The technique of IAC also provides possible applica- cell nuclei into the Xenopus oocytes[ 1] .In 1977 , De tions in animal breeding and conservation ;mean- Roeper[ 2] found chrom atin dispersal and DNA sy nthe- w hile , it could become a hopeful approach to reviving those extinct species . sis after injecting the nuclei of Hela cells into the Xenopus eggs .The result suggested that factors to 1 Principles and recent advances of IAC reactivate mam malian nuclei exist in Xenopus oocy te cy toplasm .Professo r Tong Dizhou , a Chinese scien- IAC differs from intraspecies animal cloning in tist , is the pioneer of nuclear transfer in fish .He is tw o aspects .Firstly , it involves a technique of trans- also the first scientist w ho conducted interspecies nu- ferring a donor nucleus of one species into an enucle- clear transfer in fish and obtained interspecies cloned ated oocy te of another species to form a nucleocy to- fish .He successfully conducted the nuclear transfer in

＊ S upported by the Ministry of Science and Technology of China(Grant No .Climbing-special-08)and the Chinese Academy of S ciences(Gran t No . KSCX1-05-01). ＊＊ To w hom correspondence should be addressed .E-mail:chendy @panda.ioz.ac.cn 2 Progress in Natu ral Science Vol.14 No.1 2004 tw o subfamilies of goldfish (Carassius euratus and panda .At the same time , Dominko et al.[ 9] found Rhodeus sinensis)w ith his self-made micromanipula- that bovine oocyte cytoplasm supported development tor[ 3] .Thereafter , Professor Tong and his successo rs of the embryos produced by nuclear transfer of somat- obtained higher rates for interspecies nucle- ic cell nuclei from v arious mam malian species .They ar transfer embry os between species of families and used enucleated bovine oocytes as nucleus recipients, o rders , such as transferring the nuclei of goldfish into transferring somatic cell nuclei from sheep , pig (Sus the enucleated oocy tes of Param isgurnus dabr- scrofa), monkey (Macaca fascicularis ) and rat [ 4] ryanus .These results demonstrated that cy toplas- (Rattus rattus)into the enucleated bovine oocy tes. mic factors fo r dedifferentiating and reprogramming Those reconstructed embryos of sheep-bovine , pig- of differentiated cell nuclei ex ist in the oocy tes of bovine and monkey-bovine could develop to the blas- some low class animals , and these factors may not be tocy st stage after in v itro culture .Although no preg- species-specific . nancies had been carried to term after transfer of these interspecies cloned embryos into surrogate ani- The success of mammalian somatic cell clo ning mals , tw o surrog ate sheep receiving sheep-bovine em- shows that facto rs in mammalian oocy tes can make bryos show ed signs of early ;however , no somatic cell nuclei dedifferentiated and repro- heartbeats w ere detected by transvaginal ultrasono- g rammed .Are these factors not species-specific as graphy after 32 days of em bryo transfer ;the surro- those in Amphibia ? McGrath and Solter[ 5] exchanged gates hysterectomized displayed sacks of fluid and dis- pronuclei between rodents and found that the recon- tinctly developed caruncles within uterine horns ;no structed embryos could divide in vitro .In 1992 , Wolfe et al .[ 6] transferred the bovine (Bos Taurus) fetal membranes or fetal remnants were found .An- nuclei from blastomere of 16-32 stage embryos into other g roup of scientists in USA established pregnan- enucleated oocy tes of buffalo (Bubalus bubalis), cy after the transfer of nuclear transfer embryos pro- sheep (Ov is aries)and hamster (Mesocricetus aura- duced from the fusion of argali (Ovis ammon)nuclei tus);the reconstructed embry os of bovine-buffalo and into domestic sheep (Ov is aries)enucleated oocy tes. By transferring the argali som atic cell nuclei into the bovine-sheep could develop to the blastocy st stage af- [ 10] ter in v itro culture ;however , the reconstructed em- enucleated sheep oocy tes White et al . obtained bryos of bovine-hamster did not divide in his experi- 1 .56 % of blastocy st rate for these reconstructed em- ment.In 1993 , M ei et al .[ 7] transferred the mouse bryos.28 interspecies clo ned w ere trans- (Mus m usculus)nuclei of blastomere from the 8-cell ferred into 6 surrog ate sheep , and one surrogate was stage embryos into enucleated rabbit (Oryctolagus found preg nant with ultrasonography 49 day s after cuniculus)oocytes .Nuclear swollen and prem ature em bryo transfer .Unfo rtunately , the pregnancy was chromosome condensation (PCC )were observed in terminated 10 days after the ultrasonograph detec- the mouse-rabbit cloned embry os, 5 .4 % of the re- tion .Although no live offspring was obtained , these constructed embryos developed into the blastocy st studies have demonstrated that some mammalian stage , and karyotype analysis of the blastocysts con- oocytes (i .e .bovine , sheep and rabbit)are able to firmed that the chromosomes w ere from the mouse , accept , dedifferentiate and reprog ram the nuclei from not from the rabbit .These early studies showed that other species. some mammalian oocytes could accept and dedifferen- In January of 2001 , Advanced Cell Technology tiate the nuclei from o ther species . (AC T)announced[ 11] that an ordinary cow named In 1999 , Professo r Chen et al.[ 8] repo rted that Bessie g ave birth to a remarkable baby bull , Noah . they obtained panda (Ailuropoda Melanoleuca)em- The 80-pound new born gaur , an endangered ty pe of bryos , a highly endangered animal, by using the w ild ox from India , Indochina and Southeast Asia , technique of interspecies nuclear transfer . They w as the first cloned mam mals obtained by using the transferred the panda somatic cell nuclei from uterus technique of IAC .The gaur and the cow are classified epithelia , skeleton muscle and mammary gland ep- as tw o different species , for the karotype of gaur is 58 ithelia into enucleated rabbit oocytes , resulting in (2 n =58)and the cow (Bos Taurus)is 54 (2n = 9 .9 %, 6 .8 % and 11 .7 % of blastocy st rates from 54).Noah w as produced by introducing nucleus from these reconstructed embryos , respectively .Analyses the skin fibroblast of an adult m ale gaur into enucleat- of karyo type , mtDNA and nDNA confirmed that ed oocyte of the cow , and transferred back into the these reconstructed embryos w ere embryos of giant uterus of a surrogate cow after the reconstructed em- Progress in Natural Science Vol.14 No .1 2004 3 bryo developed to the blastocyst stage in vitro .Un- produced by transferring fibroblasts from ear skin of a fortunately , Noah succumbed to com mo n dysentery tiger (panthra tigiris altaica)into enucleated bovine w ithin 48 hours after his birth , but his sho rt life indi- oocytes, one tiger and 3 lion surrogate mothers re- cated that the somatic nucleus of the gaur could be ceived the tiger-bovine embryos , however , no preg- dedifferentiated and reprogram med in the enucleated nancy w as reported . oocy te of the cow and suppo rt the full term develop- ment of the interspecies cloned .Shortly after In our laborato ry , w e have produced various in- the birth of Noah , another endangered animal , mou- terspecies cloned embryos using enucleated rabbit flon (Ov is orientalis musimon)w as also cloned using oocytes as nucleus recipients .We cloned panda em- the technique of IAC .Loi et al .[ 12] used post-mortem bryos by transferring panda fibroblasts into enucleated somatic cells from a mouflon as donor cells and enu- rabbit oocy tes, these panda-rabbit cloned embry os at cleated sheep oocy tes as recipients to produce cross- 2 ～ 4-cell stage w ere transferred into the oviduct of 21 species nuclear transfer embryos, a total of 23 recon- cat recipients , 19 recipients returned estrus and the other tw o recipients died of pneumonia .One of them structed embryos were produced and 7 of them devel- w as found pregnant w ith 6 early fetuses after autop- oped to the blastocy st stage .Transferring these 7 blastocy sts into 4 surrog ate sheep resulted in 2 preg- sy .M icrosatellite DNA analysis of these early fetuses confirmed that tw o of them were from giant panda- nancies , one surrogate abo rted 40 ～ 50 days after em- [ 17] bryo transfer , the other surrogate carried preg nancy rabbit cloned embryos .Cat-rabbit clo ned embryos to term .The success of interspecies cloning of g aur were also produced by transferring cat fibroblasts into enucleated rabbit oocytes , 14 .5 %of the reconstruct- and mouflon demonstrated that the technique of IAC ed embry os developed to the blastocy st stage after in is practical , and it is another milestone in animal v itro culture .Comparing the developmental capacity cloning after Dolly the lamb . and timing of embry ogenesis of the cat-rabbit cloned M any o ther results have been obtained using the em bryos with that of cat-cat cloned embryos , no sig- bovine oocy tes as recipients since Dominko et al . nificant difference of developmental capacity w as ob- demonstrated that enucleated bovine oocy tes could served between cat-rabbit and cat-cat cloned embry os. support the development of interspecies cloned em- The timing of the first three cleavages for the cat-rab- bryos from various mammalian species .Lanza et bit embryos was similar to that of the rabbit-rabbit al .[ 13] cloned gaur embryos by electrofusion of g aur cloned embryos , but sig nificantly faster than that of fibroblasts w ith enucleated oocy tes from a domestic the cat-cat embry os, w hile the time to fo rm blasto- cow .Tw elve percent of the reconstructed embryos cysts w as similar to that of cat-cat embryos , but sig- developed to the blastocy st stage , and 18 % of these nificantly slow er than that of the rabbit-rabbit em- embryos developed to the fetal stage w hen transferred bryos.The result indicated that the timing of em- to surrogate mothers .Three of the five pregnant sur- bryogenesis for interspecies cloned embryos is recipi- rogates were removed at day s 46 to 54 of gestation fo r ent oocy te specific in early cleavage divisions , and is o ther studies , and one surrogate aborted on day 201 nucleus specific after the blastocyst stage (our unpub- of gestation , the abortive fetus w eighed 10 .7 kg with lished data).The monkey cloned embryos also ob- the same karyoty pe and phenoty pe as the gaur , the tained in our labo ratory by transferring somatic mon- o ther surrogate developed no rmally to term .Ki- key cell into the enucleated rabbit oocy tes , these tiyanant et al .[ 14] transferred fetal buffalo fibroblasts monkey-rabbit cloned embryos could develop into the into enucleated bovine or buffalo oocy tes , and no dif- blastocyst stage after in v itro culture , analysis of m tDNA found that the mtDNA from both monkey ference of the blastocy st rates for buffalo-buffalo and [ 18] buffalo-bovine embryos w as observed . In 2001 , and rabbit coexist before the blastocyst stage . [ 15] M eirelles et al . electrofused the blastomere of early 1 .2 Interspecies preg nancy in m ammals Bos indicus embry os with enucleated bovine oocy tes and obtained Bos indicus interspecies cloned em- Preg nancy between different species of mam- bryos , five bovine surrogates received blastocy sts of mals , namely , interspecies pregnancy , is rarely suc- the cloned embryos and two of them became preg- cessful in nature , and it is prevented by the mecha- nant , finally , one live cloned offspring of Bos indicus nisms of reproductive isolation at different level , such w as bo rn .In the same year , Hw ang et al .[ 16] in as genetic , cellular and phy siological isolations .How- South Ko rea reported that tiger-bovine embryos were ever , the reproductive isolation in mammals is not 4 Progress in Natu ral Science Vol.14 No.1 2004 stringent , in some of the cases, interspecies preg nan- failure of pregnancy has been demonstrated by suc- cies of hy brids from different species do occur in na- cessful gestation of chimaeras, composed of xenogene- ture .Interspecies pregnancies can be classified as ic embryonic cells protected by , species- three categories :(1)the pregnancies of hybrids of homologous to the em bryo transfer recipient[ 23, 27 , 28] . different species ;(2)the pregnancies of chimeras ; In the murine model of interspecies preg nancy , in- and (3) the preg nancies of embryos from different volving Mus caroli and Mus musculus , fully xeno- species via .Three different models of geneic Mus caroli w ere viable in Mus m usculus fol- interspecies pregnancies have been established , the low ing transfer of chimeric blastocysts com posed of a murine model using Mus musculus and M .caroli ; Mus caroli w ithin a Mus m usculus the equine model involving primarily the domestic trophoblast vesicle .Non-manipulated Mus caroli em- and donkey , and the bovid model of domestic bryos gestating in the Mus m usculus uterus failed to sheep and goat[ 19] .Studying these models revealed develop to term and most of the embryos died and re- that tw o main reasons attributed to the failure of in- sorbed during the second half of pregnancy[ 23, 27] . terspecies pregnancies :(1)the failure of tissue inter- action betw een the trophectoderm of embryos and the The success or failure of establishing interspecies uterus of the surrog ate mother[ 20] ;(2)the immuno- preg nancy involves various facto rs , such as the genet- logical rejection of the surrogate mother to the fetuses ic relationship , gestation period , body tem perature after embryo implantation , this rejection prevents the and physiological structure of the uterus betw een the formation of placenta[ 19 , 21] . species of the embryos and the surrogate mother .Es- tablishment of interspecies preg nancy between tw o Embryo attachment and implantation to the genetic closer species should increase the possibility of uterus , occurring after the blastocy st stage , symbol- success;this speculatio n has been supported by the ize the beginning of pregnancy .The processes of at- facts of the successful cloning of gaur and mou- tachment and implantation between mother and em- flon[ 11 , 12] and the successful gestation of Indian desert bryos are complex and numerous among species .In cat (Felis sy lvestris ornate)in uterus of the domestic most of the m ammals, embryos are able to attach and cat[ 29] .Although it is unlikely to establish inter- implant to the uterus after the blastocyst stage , at the species pregnancy betw een any two species of mam- same time , the uterine wall of the mother develops to mals, it is possible to achieve interspecies pregnancy a certain state w hich allow s the embryos to attach and betw een tw o carefully selected species w ith consider- implant, the period of this state , specifically , “im- ing the differences of the genetic relatio nship , repro- plantation w indow ” , is transient .Only the comple- ductive phy siolog y and anatomic structure after the tion of embry o implantation in the period of window proper remolding the embryos . can pregnancy be normally established .In the delayed implantatio n mammals , the time of embryo implanta- 2 Problems and possible solutions in IAC tion is determined by the state of the mother .In most of these mammals , embryos arrest at the blastocy st The technique of somatic anim al cloning has stage until the “im plantation w indow” occurs and the solved the problem of nucleus dedifferentiation and process of im plantation is triggered .Interestingly , reprog ramming .At the basis of som atic nuclear these arrested embryos do not degrade and apoptosis transfer , the technique of IAC needs further to solve for days o r months according to different species in the problem s of nucleo-cytoplasmic compatibility and vivo[ 22] .Establishing a biological structure such as the problems of interspecies pregnancy between placenta for interactions between mo ther and fetus is species . crucial for the success of mamm alian pregnancy .Tro- phoblast cells play pivo tal role in the establishment of 2 .1 Problem s of nucleo-cy toplasmic compatibility in interactions between mother and fetus[ 23 , 24] .The IAC placenta , mainly derived from trophoblast cells, has Nucleo-cytoplasmic compatibility includes several been show n to secrete hormones and g row th factors , and is considered to be an immunological barrier be- aspects , i .e .w hether the nucleus and cytoplasm sup- tween the mo ther and her immunogenic concep- port each other structurally and functionally , whether tus[ 25, 26] . the sig nal passage can be correctly established be- tw een nucleus and cy toplasm , ect .The problems of That trophoblast alone can control the success o r nucleo-cy toplasmic compatibility in IAC mainly in- Progress in Natural Science Vol.14 No .1 2004 5 volve w hether the cy toplasmic facto rs in the oocy tes It remains largely unknow n to these cy toplasmic fac- of one species can support the dedifferentiatio n and to rs for their com position , characters and functions. reprogramming of the nucleus of the other species and To clarify these factors and to specify their roles in w hether the nucleus and mitochondria can support the nucleus dedifferentiation and reprogram ming is each other structurally and functionally . helpful to understand the mechanisms of animal cloning . 2 .1 .1 Nucleo-cy toplasmic compatibility and selec- tion of recipient oocy tes Early embry o develop- 2 .1 .2 The fate of mitochondria in IAC The ment is controlled by maternally inherited RNA and fate of mitochondria is one of the most concerned is- proteins and little or no transcription is detectable sues in IAC .Mitochondria , the “pow er plants” and from zygo tic nucleus .At a particular stage of devel- the energy supplier for mammalian cell activities , are opment , w hich is species-dependent , a switch to zy- organelles that occupy a substantial portion of cell cy- go tic control occurs .This transition , namely , mater- toplasm .Mammalian mitochondrion consists of a nal to embryonic (or zygotic)transition (MET), is double-stranded circular DNA of about 16 .5 kb , the characterized by a large increase in detectable tran- genetics of which differs from that of nuclear scription .The zy gotic nucleus must then control de- genome .The mitochondrial DNA (mtDNA)carries velopment in a spatial and tem poral manner and result genes for 13 pro teins , 22 tRNAs and two rRNAs in fo rmation of specific differentiated cell types[ 30] . w hich are essential com ponents of o xidative phospho- For successful development of nuclear transfer em- rylation and electron transfer .Up to 95 %of proteins bryos , the transferred nucleus must therefo re first involved in biogenesis and functions of mitochondria abolish transcription and then reestablish the tempo- are encoded by the nucleus[ 38, 39] .The number of mi- ral, spatial , and quantitative patterns of gene ex pres- tochondria in a typical somatic cell is about 2 ×103 , sion associated with normal development[ 30 , 31] .This w hile the number of mitochondria in a single oocyte is process of reestablishment in nuclear transfer embryos about 2 ×105 [ 40] .In normal fertilized embryos , the is the like of MET in normal embryos[ 32] .The cor- mitochondria from the oocy tes are multiplied , and the rect MET of the interspecies nuclear transfer embryos mitochondria from the sperm are eliminated by an un- is crucial fo r the embryo development .The develop- known mechanism during the embryo development . ment can be continued only w hen the M ET of nuclear In the process of nuclear transfer , mitochondria of the transfer embryos occurs befo re maternal RNA and dono r cell , together with the nucleus , are transferred proteins are used up , or the embryo development ar- to the recipient oocy te .Thus , the cloned em bryo rest .The abilities to accept nuclei from other species should harbor mitochondria from both the dono r cell for oocy tes are different among mamm alian species . and recipient oocy te .What are the fates of mitochon- Interspecies nuclear transfer between mouse and rat dria from the do nors and recipients in IAC ? Three found that embryos produced by transferring rat nu- different patterns of mitochondria transfo rmation in clei into enucleated mouse oocytes were arrested at the IAC have been observed :(1)mitochondria from the 1-cell or 2-cell stage , and embryos produced by trans- dono r cell are g radually eliminated , w hile the mito- ferring mouse nuclei into enucleated rat oocy tes could chondria from the recipient oocy te multiply to domi- only develop to the 5 ～ 8 cell stage[ 33] ;however , em- nate in the embryo during development ;(2)mito- bryos produced by transferring nuclei from various chondria from the recipient oocy te are eliminated , mamm alian species into enucleated oocy tes of bovine , w hile the mitochondria from the dono r cell multiply sheep and rabbit can develop readily to the blastocy st to dominate in the embryo , and finally mitochondria stage[ 8 ～ 10 , 12, 13 , 17] .The MET of normal bovine em- from the dono r cell substitute for those from the re- bryos occurs at the 8-cell stage[ 34] , that of sheep and cipient oocy te completely ;(3) mitochondria from rabbit embryos occurs at the 8 ～ 16-cell stage[ 35, 36] , both the dono r cell and the recipient oocy te coexist all w hereas the MET of mouse embry os occurs at 1 ～ 2- the time during embryo development . cell stage[ 37] .From facts of these , w e may propose that the ability to accept nuclei from o ther species fo r If mitochondria multiply without any selective mamm alian oocy tes is related to the time of M ET oc- pressure , mitochondria from both the donor cell and curring in the species . the recipient oocyte will increase geometrically after the blastocyst stage .How ever , the number of mito- Nucleo-cy toplasmic compatibility involves the in- chondria from the recipient oocyte w ill dominate , and teractions of nucleus and various cytoplasmic factors . finally substitute for that from the donor cell after 6 Progress in Natu ral Science Vol.14 No.1 2004 many rounds of multiplication , since the number of somatic nuclear dono r cell mtDNA , but a mechanism mitochondria from the recipient oocy te is mo re than of neutral seg regatio n of mitochondria from both the 10 times of that from the dono r cell in the recon- dono r cells and recipient oocy tes in cloned animals . structed embryo .Lanza et al .[ 13] reported that the mitochondria in 11 tissues from 3 cloned gaur fetuses 2 .2 Possible techniques fo r mammalian interspecies produced by transferring gaur som atic nuclei into the preg nancy enucleated oocyte of the domestic cow were exclusive- Interspecies pregnancy , an inevitable process in ly from the oocy te of the domestic cow , and mito- IAC , is one of the key factors to confine the success chondria DNA of the donor g aur w as undetectable in of interspecies mammalian cloning .Although the the cloned fetuses .Loi et al.[ 12] cloned mouflon with study of interspecies mam malian pregnancy is limited the oocy tes of domestic sheep ;mitochondrial DNA of so far , it w ould be possible to achieve interspecies the recipient oocy te could be detected in the cloned preg nancy with the help of the follow ing techniques : mouflon, while mitocho ndrial DNA of the dono r mouflon w as undetectable . (1)Interspecies preg nancy by the help of con- If the nucleus from the donor cell does not sup- specific embryos to the surrogate .Co-transferring the po rt the biogenesis of mitochondria from the recipient conspecific embryos and interspecies cloned embryos into the surrogate mother would achieve interspecies oocy te , the interspecies cloned embryo w ill selectively preg nancy .In our laboratory , w e co-transferred the multiply mitochondria from the donor cell during em- panda-rabbit cloned embryos at the 2 ～ 4 cell stage bryo development .Althoug h the number of mito- chondria from recipient oocy te is 10 times of that w ith the cat-rabbit cloned embryos into the surrogate from the do nor cell in the early cloned embryo , mito- female .One pregnant surrogate cat carried 6 fe- chondria from the dono r cell will dominate those from tuses after 21 days of embryo transfer .Of the 6 fe- tuses, tw o w ere from the panda-rabbit cloned em- the recipient oocyte after the blastocy st stage .The [ 17] results from our laboratory have showed that the mi- bryos as confirmed by the satellite DNA analy sis . tochondria from the dono r panda cells and those from The giant panda is a delayed implantatio n animal, the recipient rabbit oocy tes co-exist in embryos before w hose gestation period varies from 80 ～ 150 days .We implantatio n;w hereas the mitochondria from dono r hy pothesize that the giant panda em bryos play an in- panda cells remain detectable , mitochondria from the active role during implantation fo r their delayed im- recipient rabbit oocytes are eliminated after implanta- plantation characteristics;the cat-rabbit embryos may tion[ 17] . act as implantation “inducers” o r “helpers” fo r the panda-rabbit cloned embry os, w hich may help im- If the nucleus from the dono r cell supports the plantation of panda-rabbit embryos in cat uterus. biogenesis of mitochondria from both the dono r cell During embryo implantation , the signals from em- and the recipient oocy te , however , the nucleus has a bryos to recipient uterus for implantation are con- preference fo r multiplying mitochondria from the trolled by the nucleus and these sig nals may be donor cell , in this case , mitochondria in the cloned species-specific .The cat-rabbit cloned embryos may animals will be at various levels of heteroplasmy . give implantation signals to the recipient cats , and [ 41] S teinbo rn et al . found that mitochondria in 4 of trigger the process of implantation fo r both the cat- the 11 cloned Bos indicus calves are heteroplasmy . rabbit and the panda-rabbit cloned embryos . Recently , Takeda et al.[ 42] used PCR-mediated sin- g le-strand conformation poly morphism (PCR-SSCP) (2)Replacement of inner cell mass .The tro- to analy ze the mitochondria of the cloned calves , and phoblast alone can control the success or failure of the found that 3 of the 9 cloned calves exhibited hetero- mammalian preg nancy[ 25] . Interspecies pregnancy plasmy w ith donor cell mtDNA populations ranging might be established with the composing of inter- from 6 % to 40 %.They contended about a significant species chimeric blastocysts , which can be produced replicative advantage of donor m tDNAs to recipient by transferring the inner cell mass of one species into mtDNAs during the course of embryogenesis in cloned the blastocysts, w ith the inner cell mass being re- calves .Hiendleder et al .[ 43] also observed varying de- moved , of the species to the surrog ate mother .Viable g rees of mitochondrial DNA heteroplasmy in nuclear offspring of Mus caroli preg nant in surrogate mo ther transfer embryos, fetuses , and offsprings ;however , of Mus m usculus have been obtained by transferring their results did not suggest a replicative advantage of the inner cell mass of Mus caroli into trophoblast Progress in Natural Science Vol.14 No .1 2004 7 vesicles (blastocy sts w ith inner cell mass being re- 2 De Roeper, A.Chromatin dispersal and DNA synthesis in G1 and [ 23] G2 Hela cell nuclei injected into Xenopus eggs.Nature , 1977 , moved)of Mus m usculus . 265 :469 . 3 Tong, D.Z .et al.Transplantation of cell nuclei.Acta Zool Sini- (3)Diploid/tetraploid (2 n/4 n)chimeras .2 n/ ca, 1963 , 15 :151 . 4n chimeras are the chimeras produced by agg regat- 4 Tong , D.Z .et al.Nuclear transfer betw een sub-families of fishes. ing the diploid embryos o r ES cells w ith the tetraploid Acta Zool Sinica , 1973 , 19 :201 . embryos .Mouse 2n/4n chimeras display nonrandom 5 McGrath , J.D.et al.Inability of mouse blastomere nuclei trans- ferred to enucleated zygotes to support development in vitro .Sci- distributive characteristics, w hich tetraploid cells ence, 1984 , 226 :1317 . mainly contribute to the extraembryonic tissues and 6 Wolfe, B .A.et al.Methods in bovine nuclear transfer.Theri- diploid cells form the conceptuses during embryo de- ogenology , 1992 , 37 :5 . velopment[ 44～ 46] .Agg regating the inner cell mass o r 7 Mei, Q .et al.Studies on the early development of nucleo-cytoplas- mic hybrid embryos betw een mouse and rabbit by nuclear transplan- ES cells of chinchilla 129/Sv mouse with the te- tation .Acta Exp .Biol.S inica, 1993 , 26 :389 . traploid embryos of albino CD1 mouse , chimeras em- 8 Chen , D .Y .et al.The giant panda (Ailuropoda melanoleuca) bryos were transferred into the surrogate mother ; somatic nucleus can dedifferentiate in rabbit ooplasm and support most of the newbo rns w ere derived from the inner cell early development of the reconstructed egg .Science in C hina (Se- mass or ES cells , and only a minor contribution ries C), 1999 , 42 :346 . 9 Dominko, T .et al.Bovine oocyte cytoplasm support development (<2 %)from tetraploid cells could be detected in of embryos p roduced by nuclear transfer of somatic cell nuclei from [ 44 , 45] some of the new bo rns .Making use of the char- various mammalian species.Biol.Reprod ., 1999 , 60 :1496 . acteristic of 2n/4n chimeras , w e may com pose inter- 10 White, K.L .et al.Establishment of pregnancy after the transfer species 2n/4n chimeras , of w hich the tetraploid em- embryos produced from the fusion of Argali(Ovis a mmon)nuclei into domestic sheep (Ovis aries) enucleated oocytes.Cloning , bryos are from the species of the surrog ate mother , 2000 , 1 :47 . and the diploid embryos are from other species . 11 Lanza, R.P.et al.Cloning Noah' s ark .Sci.Am .2000 , 283 : Transferring these interspecies 2n/4n chimeras into 84 . the surrogate mother , tetraploid cells , which is the 12 Loi, P.et al.Genetic rescue of an endangered mammal by cross- same species as the surrogate mother , will be dis- species nuclear transfer using post-mortem somatic cells.Nat . 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