|
Several mammals - including sheep mice,cows, goats, pigs, rabbits, cats(1). a mule(2), a horse(3) and a litter of thm rats(4)- have beencloned by transfer of a nucleus from a somatic cell into an egg cell(oocyte) that has had its nucleus removed. This technology has not so far been successful in dogs because of the dif.ficulty of maturing canine oocytes in vitro.Here we describe the cloning of two Afghan hounds by nuclear transfer from adult skin cells into oocytes that had matured in vivo.Together with detailed sequence information generated by the canine-genome project(5,6), the ability to clone dogs by somatic-cell nudes,transfer should help to determine genetic and environmental contributions to the diverse biological and behavioural traits associated with the many different canine breeds(7,8).
Successful somatic-cell nuclear transfer(SCNT) depends on the quality, availability and maturation of the animals unfertilized oocytes. Unlike other mammals, dogs ovulate at first meiotic prophase. and their oocytes mature for 2 to 3 days in the oviduct's distal regions. Previously, intra- and interspecific canine embryos have been constructed by canine SCNT into canine and bovine oocytes,respectively, but this did not result in viable offspring(9).
We collected oocytes matured in vivo at metaphase II about 72 hours after ovulatim by flushing the oviducts. (For details of methods,see supplementary information.) Donor fihro blasts were obtained from an ear-slin biopsy of & male Afghan hound and cultured for two to five passages (in which fully grown cells are transferred to a new culture dish). For SCNf,the chromosomes of the unfertilized canine oocytes were removed by micrornanipulation.and a single donor cell was transferred into each enucleated oocyte. The couplets were fused and only successfully fused couplets (75%) were activated. The activated oocytes were then transferred into the oviducts or uterine horns of recipient dogs at times appro.priate to the embryos' developmental stages.We collected an average of 12 oocytes from each female, and a total of 1,095 reconstructed canine embryos were transferred into 123 recipients.
Three pregnancies were confirmed by dtra sound scan at 22 days(2) gestation in recipients after transfer of constructs- Pregnancy was established only after embryo transfer of very early-stage nuclear-transfer constructs (that is,less than 4 hour after oocyte activation). Transfer of early-stage embryos is a crucial factor in successful assisted reproductive technology for dogs One fetus miscarried and two others were carried to term
We named the first cloned dog Snuppy (forSeoul National University puppy); it is shown in Fig. la with the male Afghan fibroblast donor. Snuppy was delivered by caesarian sec-tion after 60 days (full term) from a yellow Labrador surrogate mother (Fig. lb); his birth weight was 530 g. The second SCNT dog.NT-2, was carried by a mixed-breed surrogate,and was also delivered at 60 days, weighing 550 g (normal range for Afghans in a litter.482-680 g). He experienced neonatal respin-tory distress during the first week, seemed to recover, but died on day 22 as a result of aspiration pneumonia; no major anatomical anomalies were evident post mortem.
We tested whether the cloned dogs were genetically identical by microsatellite analysis of genomic DNA from the donor Afghan, the doned dogs and the surrogates (see supple-mentary information). Analysis of eight canine-specific microsatellite loci confirmed that the Boned dogs were genetically identical to their donor dog. However, the efficiency of cloning is still very low (2 dogs from 123 recip-ients, or 1.6%) compared with the rates forcats' and horses(3).
In addition to the benefits that cloning tech-nology may generally provide (the preserva-tion of rare species and therapeutic cloning - once canine embryonic stem cells become available), this technology could become a useful research tool for studying the genetics of outcrossed populations.
Byeong Chun Lee*, Min Ky. Kim*, Goo Jana*,Hyun Ju Oh*, Fibrianto Yuda*, Hye An Kim*,M. Hossein ShamMim*, lung lu Kim*,Sung Keun Kang*, Gerald Schaftent,Wan Suk Hwang* *Department of Theriogenology and Biotechnology. College of Veterinary Medicine,Seoul National University, Gwanak-gu,Seoul 151-74Z South Korea
e-mail: hwangws@snuackr
tPittsburgh Development Censer, Magee-Womens Research Institute. Departments of Obstetrics-Gynecology-Reproductive Silences and Cel Biology-Physiology, University of Pittsburgh School of Medicine. Pittsburgh,
Pennsylvania 15213. USA
1、shine.t.ef Nature 415,859(2002).
2、 Woods,G.Let al Science 301,1063(2003)
3 、GALLi, C .et.al Nature 424, 635(200.3).
4 、Zhou Q.et.al Science 302,1179(2003).
5 、Ostranter,E.A & Giniger,E.Am Hum Geret.61,475-480(1997)
6、.Sutter,N.B.& Ostrander,E.A.Nature Rev Genet 5.900-910(2004)
7、 lohi,H.et.al.Science 30;81(2005)
8、 Modiana,J.F et al Caner Res,65 5654-5661(2005)
9、Westhusin,M.E.et.al.I Report. Fert Support 57,287-293(2001)
|
