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Nuclear transplantation in bovine embryos
THERIOGENOLOGY
J.
NUCLEAR TRANSPLANTATIGN IN BOVINE EMBRYOS M. Robl, R. Prather, W. Eyestone, F. Barnes, D. Northey, B. Gill Igan and N. L. First Department of Meat and Animal Science Unlverslty of Wisconsin 537 06 Mad ison. W lscons in
C I on lng of bov ine embryos wou I d be of great importance for svaluatIon and selection of genotypical ly superior animals. Cloning by nuclear transpl antation, al though not yet feasib I e, offers the potential of producing an unlimlted number of clones. We report here a method of transp I ant ing nut I ei in bov ine embryos that is based on a procedure Embryos developed by McGrath and Sol ter (Science 220:1300) for mice. were treated with the cytoskeletal inhibitors cytcchalason B (Sigma; 7.5 pg/ml 1 and demicolcine (Sigma; 0.1 ug/ml) approxlmatel y 10 mTn before Pronuclei were micromanipulation and during micromanipulation. visual ized using the centrifugatlon method of Wal I et al. (Blol. Reprod. 32:645). A beve I I ed, sharpened mlcrop i pet of 25 to 30 Mm (G.D.1 was Inserted through the zona pellucida, moved adjacent to the nut I eus, and with gentle aspiration the nucleus was pul led into the plpet without rupturing the plasma membrane. The membrane p inched off as the p 1 pet was pul led away from the embryo. The plasma membrane bounded nucleus was inserted Into an enucleated embryo by the die1 ectrophoretIc method of ccl I fusion. Fusion occured at a higher rate in the low ionic strength Zimmerman cell fusion medium@ (8/9; 89%) than in a Hepes buffered Tyrodes medium (l/9; 11%). Embryos fused at a higher rate when the fusion membranes were paral I el to the el ectrodes (22/24; 92%) versus random al ignment (13/19; 68%). The duration of a 100 volt electrical pulse also affected the rate of fusion with 14/18 (78%) embryos fusing at 40usecs, 15/19 (79%) fusing at 2Ousecs and 2/10 (20%) fusing at 10 usecs. When pronuc I el were removed and reinserted In bov lne embryos 9/29 (31%) developed to 7 to 12 ccl Is in vitro. In a dlfferent group of bov ine embryos, pronuc I e I were removed, reinserted and then immediately transfered to sheep oviducts for five days. Of the intact embryos recovered, 5/29 (17%) deve I oped to moru I a or b I astocysts compared to 1 l/30 (37%) nonman ipu I ated embryos. Two nuclear transfer embryos were One transfered to a recipient cow fol I ow Ing culture in a sheep oviduct. of the embryos developed at I east 43 days. ultrasound to have a beating heart and resu I t-s demonstrate that pronucl ei can be ear I y bov I ne embryos.
JANUARY 1986 VOL. 25 NO. 1
The embryo was observed by These was normal in size. successfu I I y transp I anted In
189
J.
NUCLEAR TRANSPLANTATIGN IN BOVINE EMBRYOS M. Robl, R. Prather, W. Eyestone, F. Barnes, D. Northey, B. Gill Igan and N. L. First Department of Meat and Animal Science Unlverslty of Wisconsin 537 06 Mad ison. W lscons in
C I on lng of bov ine embryos wou I d be of great importance for svaluatIon and selection of genotypical ly superior animals. Cloning by nuclear transpl antation, al though not yet feasib I e, offers the potential of producing an unlimlted number of clones. We report here a method of transp I ant ing nut I ei in bov ine embryos that is based on a procedure Embryos developed by McGrath and Sol ter (Science 220:1300) for mice. were treated with the cytoskeletal inhibitors cytcchalason B (Sigma; 7.5 pg/ml 1 and demicolcine (Sigma; 0.1 ug/ml) approxlmatel y 10 mTn before Pronuclei were micromanipulation and during micromanipulation. visual ized using the centrifugatlon method of Wal I et al. (Blol. Reprod. 32:645). A beve I I ed, sharpened mlcrop i pet of 25 to 30 Mm (G.D.1 was Inserted through the zona pellucida, moved adjacent to the nut I eus, and with gentle aspiration the nucleus was pul led into the plpet without rupturing the plasma membrane. The membrane p inched off as the p 1 pet was pul led away from the embryo. The plasma membrane bounded nucleus was inserted Into an enucleated embryo by the die1 ectrophoretIc method of ccl I fusion. Fusion occured at a higher rate in the low ionic strength Zimmerman cell fusion medium@ (8/9; 89%) than in a Hepes buffered Tyrodes medium (l/9; 11%). Embryos fused at a higher rate when the fusion membranes were paral I el to the el ectrodes (22/24; 92%) versus random al ignment (13/19; 68%). The duration of a 100 volt electrical pulse also affected the rate of fusion with 14/18 (78%) embryos fusing at 40usecs, 15/19 (79%) fusing at 2Ousecs and 2/10 (20%) fusing at 10 usecs. When pronuc I el were removed and reinserted In bov lne embryos 9/29 (31%) developed to 7 to 12 ccl Is in vitro. In a dlfferent group of bov ine embryos, pronuc I e I were removed, reinserted and then immediately transfered to sheep oviducts for five days. Of the intact embryos recovered, 5/29 (17%) deve I oped to moru I a or b I astocysts compared to 1 l/30 (37%) nonman ipu I ated embryos. Two nuclear transfer embryos were One transfered to a recipient cow fol I ow Ing culture in a sheep oviduct. of the embryos developed at I east 43 days. ultrasound to have a beating heart and resu I t-s demonstrate that pronucl ei can be ear I y bov I ne embryos.
JANUARY 1986 VOL. 25 NO. 1
The embryo was observed by These was normal in size. successfu I I y transp I anted In
189