Conceived and designed the experiments: YH KS TO AI KS. Performed the experiments: YH NH ST NK KS. Wrote the paper: YH KS.
The authors have declared that no competing interests exist.
Frozen animal tissues without cryoprotectant have been thought to be inappropriate for use as a nuclear donor for somatic cell nuclear transfer (SCNT). We report the cloning of a bull using cells retrieved from testicles that had been taken from a dead animal and frozen without cryoprotectant in a −80°C freezer for 10 years. We obtained live cells from defrosted pieces of the spermatic cords of frozen testicles. The cells proliferated actively in culture and were apparently normal. We transferred 16 SCNT embryos from these cells into 16 synchronized recipient animals. We obtained five pregnancies and four cloned calves developed to term. Our results indicate that complete genome sets are maintained in mammalian organs even after long-term frozen-storage without cryoprotectant, and that live clones can be produced from the recovered cells.
In several mammalian species, cloned animals have been successfully produced by somatic cell nuclear transfer (SCNT)
A bull named Yasufuku was one of the most important and famous sires in the history of breeding Wagyu cattle due to his contributions to the improvement of the quality of marbling, which is a major characteristic of Wagyu beef. Yasufuku died of senility at an age of 13.5 years in September 1993. His testicles were collected 12 hours after his death, then wrapped in aluminum foil and placed in a −80°C freezer without cryoprotectant for 10 years. The testicles were then transferred to liquid nitrogen without cryoprotectant for another 3 years. We examined whether intact and culturable somatic cells could be retrieved from the testicles and whether the nuclei from such cells could contribute to the development of viable offspring after SCNT with enucleated oocytes. In this study, we succeeded in obtaining four live cloned calves from these frozen organ cells. Three calves are healthy though one died two days after birth. To our knowledge, this is the first report of the resurrection of a dead elite livestock specimen from a non-cryoprotected frozen organ by cloning.
Before experimenting with tissues from Yasufuku, we conducted some preliminary experiments with fresh frozen testicles. We collected testicles from three 12- to 15-month-old bulls and froze them at −80°C without any special treatment in a freezer for one to four months. We then dissected the frozen testicles into different parts, caput epididymis, cauda epididymis, spermatic cords and testes. The parts were thawed, minced and digested with collagenase and dispase and then cultured. In our preliminary experiments, we used Dulbecco's modified Eagle's medium (DMEM) or α-minimum essential medium (α-MEM) to obtain primary cultures from the frozen testicles. However, no cells grew from the thawed tissue. It was possible that cells in the thawed tissue might be with quite low proliferating activity even when they were alive. Therefore we selected MF-start™ that was developed to readily obtain initial outgrowth of cells with low proliferating activity at primary culture. We obtained live and culturable cells from both the caput epididymis and the spermatic cords but not from the cauda epididymis or the testes. Most of the culturable cells proliferated actively and populations expanded, suggesting that the cells were in normal condition. We used these cells to produce SCNT embryos by electrofusion with enucleated oocytes. Fibroblasts taken from bovine ear tissue were used as controls. The SCNT embryos were then cultured for 168 hours
We isolated the caput epididymis and the spermatic cords from Yasufuku's frozen testicles and cut them into several small pieces (
The testicle was stored in a −80°C freezer for 10 years and then transferred to liquid nitrogen for 3 years. (A) Yasufuku's frozen testicle. (B) Part of the caput epididymis (arrow). (C) Spermatic cords that had been cut into three pieces. Scale bars represent 2 cm.
Cell lines A and B were used for SCNT after primary culture, and cell lines C and D were cryopreserved and then subcultured with five passages. (A) Cell line A, consisting of fibroblast-like cells. (B) Cell line B, consisting of epithelial-like cells. (C) Cell line C, consisting of fibroblast-like cells. (D) Cell line D, consisting of epithelial-like cells. Pregnancies were obtained from SCNT embryos that had been cloned from cells of line A (A) and line D (D). Scale bars represent 100 µm.
We produced SCNT embryos by electrofusion of cells of three different cell lines (A, B and D) with enucleated bovine oocytes. SCNT blastocysts were produced from each cell line (
(A) A male calf derived from Yasufuku's testicles was born on 30 November 2007. Parturition was induced by injection of prostaglandin F2α after 287 days of gestation and the recipient animal delivered this calf two days after induction. The calf's birth weight was 18.5 kg and he remains healthy at the time of writing. (B) A male calf, derived from a vitrified SCNT embryo, that was delivered by Caesarean section on 5 March 2008, after 286 days of gestation. The calf's birth weight was 47.5 kg; he died two days after birth. (C) Male calves derived from vitrified SCNT embryos. The calf with an ear tag “c95” was born on 22 July 2008, at 287 days of gestation. Its birth weight was 32 kg. The calf with an ear tag “c66” was born on 31 July 2008, at 288 days of gestation. Its birth weight was 30 kg. Parturitions were induced as described above. Both remain healthy at the time of writing.
Cell line | No. of experiments | No. of SCNT embryos | No. (%) of embryos fused |
No. of embryos cultured | No. (%) of blastocysts |
No. of embryos transferred |
No. (%) of pregnancies |
No. of live offspring |
A | 1 | 64 | 32 (50) | 21 | 9 (43) | 6 | 3 (50) |
2 |
B | 1 | 62 | 21 (34) | 21 | 7 (33) | 4 | 0 (0) | - |
D | 6 | 289 | 242 (84) | 162 | 27 (17) | 6 | 2 (33) |
2 |
Subtotal | - | 415 | 295 (71) | 204 | 43 (21) | 16 | 5 (31) | 4 |
Control | 6 | 289 | 226 (78) | 106 | 41 (39) | - | - | - |
Fusion was examined one hour after electrofusion of couplets of cells and enucleated oocytes. Percentage of the number of the couplets.
Blastocyst development was examined 168 h after nuclear transfer. Percentage of the number of cultured embryos.
Single blastocysts were transferred into single synchronized recipients. Several blastocysts were stored in liquid nitrogen by a vitrification technique. Ten vitrified and warmed embryos were transferred to recipients.
Pregnancy was examined by ultrasonography at 33 days after embryo transfer. Percentage of the number of transferred recipients.
One recipient delivered a healthy male calf on 30 November 2007. One delivered a mummified fetus on 27 August 2007. One recipient that received a vitrified embryo delivered a healthy male calf on 5 March 2008 but the calf died two days after birth.
Two recipients, which received vitrified embryos delivered two healthy male calves on 22 and 31 July 2008.
BM1824 | BM2113 | ETH10 | ETH225 | ETH3 | INRA023 | SPS115 | |
Yasufuku's frozen semen | 180,188 | 135,137 | 274,274 | 145,147 | 117,117 | 198,208 | 246,256 |
Frozen testicles | 180,188 | 135,137 | 274,274 | 145,147 | 117,117 | 198,208 | 246,256 |
Donor cultured cells | 180,188 | 135,137 | 274,274 | 145,147 | 117,117 | 198,208 | 246,256 |
Mummified fetus | 180,188 | 135,137 | 274,274 | 145,147 | 117,117 | 198,208 | 246,256 |
Recipient | 180, |
274,274 | 145, |
198,208 | 246,256 | ||
Cloned offspring |
180,188 | 135,137 | 274,274 | 145,147 | 117,117 | 198,208 | 246,256 |
Recipient |
180, |
274,274 | 145, |
117,117 | 198,208 | 246,256 | |
Cloned offspring |
180,188 | 135,137 | 274,274 | 145,147 | 117,117 | 198,208 | 246,256 |
Recipient |
180, |
145, |
117,117 | ||||
Cloned offspring |
180,188 | 135,137 | 274,274 | 145,147 | 117,117 | 198,208 | 246,256 |
Recipient |
180, |
135,137 | 274,274 | 145,147 | 117, |
198,208 | |
Cloned offspring |
180,188 | 135,137 | 274,274 | 145,147 | 117,117 | 198,208 | 246,256 |
Recipient |
115,117 | 198, |
TGLA122 | TGLA126 | TGLA227 | TGLA53 | DIK023 | DIK010 | |
Yasufuku's frozen semen | 148,150 | 111,117 | 77,93 | 157,167 | 90,100 | 185,195 |
Frozen testicles | 148,150 | 111,117 | 77,93 | 157,167 | 90,100 | 185,195 |
Donor cultured cells | 148,150 | 111,117 | 77,93 | 157,167 | 90,100 | 185,195 |
Mummified fetus | 148,150 | 111,117 | 77,93 | 157,167 | 90,100 | 185,195 |
Recipient | 148,150 | 111,117 | 77, |
90,100 | 185,195 | |
Cloned offspring |
148,150 | 111,117 | 77,93 | 157,167 | 90,100 | 185,195 |
Recipient |
148, |
77,93 | 167, |
185, |
||
Cloned offspring |
148,150 | 111,117 | 77,93 | 157,167 | 90,100 | 185,195 |
Recipient |
148,148 | 111,117 | 77,93 | 157, |
185, |
|
Cloned offspring |
148,150 | 111,117 | 77,93 | 157,167 | 90,100 | 185,195 |
Recipient |
148,150 | 111,117 | 77, |
167, |
100, |
|
Cloned offspring |
148,150 | 111,117 | 77,93 | 157,167 | 90,100 | 185,195 |
Recipient |
111,117 | 77,93 | 185,195 |
Polymorphism in 13 microsatellites was examined by the Maebashi Institute of Animal Science, Livestock Improvement Association of Japan, Inc (LIAJ).
Values different from those of donor cultured cells are indicated in boldface.
Cloned calves born on 30 November 2007, 5 March, 22 and 31 July 2008, respectively.
The probability that the genotype of a non-cloned animal would completely match the donor cells at these 13 loci is less than 10−12.
DIK069 | DIK024 | DIK102 | DIK097 | DIK106 | DIK068 | DIK039 | DIK096 | BM6026 | |
Yasufuku's frozen semen | 163,163 | 239,245 | 135,135 | 190,190 | 109,109 | 152,160 | 195,195 | 256,258 | 167,167 |
Donor cultured cells | 163,163 | 239,245 | 135,135 | 190,190 | 109,109 | 152,160 | 195,195 | 256,258 | 167,167 |
Mummified fetus | 163,163 | 239,245 | 135,135 | 190,190 | 109,109 | 152,160 | 195,195 | 256,258 | 167,167 |
Cloned offspring |
163,163 | 239,245 | 135,135 | 190,190 | 109,109 | 152,160 | 195,195 | 256,258 | 167,167 |
Cloned offspring |
163,163 | 239,245 | 135,135 | 190,190 | 109,109 | 152,160 | 195,195 | 256,258 | 167,167 |
Cloned offspring |
163,163 | 239,245 | 135,135 | 190,190 | 109,109 | 152,160 | 195,195 | 256,258 | 167,167 |
Cloned offspring |
163,163 | 239,245 | 135,135 | 190,190 | 109,109 | 152,160 | 195,195 | 256,258 | 167,167 |
ETH185 | ILSTS6 | RM041 | CYP21 | BM4505 | INRA130 | BM103 | ILSTS93 | DIK093 | |
Yasufuku's frozen semen | 235,237 | 287,289 | 135,135 | 195,202 | 244,248 | 112,112 | 148,150 | 186,186 | 239,243 |
Donor cultured cells | 235,237 | 287,289 | 135,135 | 195,202 | 244,248 | 112,112 | 148,150 | 186,186 | 239,243 |
Mummified fetus | 235,237 | 287,289 | 135,135 | 195,202 | 244,248 | 112,112 | 148,150 | 186,186 | 239,243 |
Cloned offspring |
235,237 | 287,289 | 135,135 | 195,202 | 244,248 | 112,112 | 148,150 | 186,186 | 239,243 |
Cloned offspring |
235,237 | 287,289 | 135,135 | 195,202 | 244,248 | 112,112 | 148,150 | 186,186 | 239,243 |
Cloned offspring |
235,237 | 287,289 | 135,135 | 195,202 | 244,248 | 112,112 | 148,150 | 186,186 | 239,243 |
Cloned offspring |
235,237 | 287,289 | 135,135 | 195,202 | 244,248 | 112,112 | 148,150 | 186,186 | 239,243 |
Polymorphism in 18 microsatellites were examined by our laboratory.
Cloned calves born on 30 November 2007, 5 March, 22 and 31 July 2008, respectively.
In this study, we demonstrated that normal and actively proliferating cells can be retrieved from mammalian organs that have been frozen and stored without cryoprotectant for more than a decade and that normal healthy offspring can be produced from such cells by the nuclear transfer technique. To our knowledge, this is the first report of the cloning of a dead livestock specimen from unprotected frozen tissue. SCNT offspring have been produced from dead
Gametes, zygotes, embryos and/or cell cultures derived from elite livestock or endangered animals have often been cryopreserved in ‘Gene Banks’
Testicles after castration were immediately wrapped in aluminum foil, and then frozen in a −80°C freezer without cryoprotectant. The testicles were stored in the freezer for one to four months. Yasufuku's testicles were taken from his scrota 12 hours after his death, then wrapped in aluminum foil, frozen in a −80°C freezer without cryoprotectant, Ten years later, his testicles were plunged into liquid nitrogen and stored for 3 years.
Primary cultures of cells from thawed tissue were generated as described earlier
SCNT by electrofusion of somatic cells with enucleated bovine oocytes was performed as described earlier
Some cloned embryos were vitrified prior to transfer to recipient animals as described elsewhere
Cloned blastocysts at 7 days post fusion were transferred into synchronized recipient animals non-surgically (one embryo per recipient) on day 7 or day 8 of the estrous cycle (estrus = Day 0) . All animal procedures in the present study were approved by the Committee for Experimental Animals of Gifu Prefectural Livestock Research Institute.
Microsatellite analysis was performed, for parentage testing, for 13 microsatellites by the Maebashi Institute of Animal Science, Livestock Improvement Association of Japan, Inc. (LIAJ: Maebashi, Japan) as described elsewhere
Japanese Translation of the Full Text by YH
(0.07 MB DOC)
We thank Mr. D. Sipp (RIKEN, Center for Developmental Biology, Kobe Japan) for critical and useful comments on the manuscript. A translation of the article into Japanese is available as Alternative Language