•
cloned by somatic cell
nuclear transfer
•
Adult nucleus put into
Go phase in culture, transplanted into enucleated egg
•
Nucleus from Finn Dorset
into egg from Scottish Blackface
•
made a viable lamb (1997)
(and many nonviable lambs and embryos)
•
Showed that irreversible
modifications do not always occur to the genome in somatic cells
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| Cloning various species June 2000 |
from Clones, a hard act to follow Science 288:1722 (2000) |
SCNT=somatic cell nuclear transfer |
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| Species Cell type | Nuclear transfers (% embryos formed) |
Live births/ Number transferred |
(%) | Transgenic donor? |
Cow |
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| Adult granulosa | 552 (69) | 10/100 | (10) | No |
| Fetal fibroblast | 276 (12) | 4/28 | (14.3) | Yes |
| Adult cumulus | 47 (38) | 5/6 | (83) | No |
| Adult oviduct epithelial | 94(21) | 3/4 | (75) | No |
| Fetal fibroblast | 174 (20) | 2/7 | (29) | No |
| Fetal germ cell | 85 (?) | 1 | No | |
| Adult fibroblast | ? (?) | 1 | No | |
| Adult fibroblast | 338 (30) | 6/54 | (11) | No |
| Adult muscle | 346 (21) | 4/26 | (15) | No |
Goat |
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| Fetal fibroblast | 71 (68) | 1/47 | (2.1) | Yes |
| Fetal fibroblast | 54 (76) | 2/38 | (5.3) | Yes |
Sheep |
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| Adult mammary gland |
227 (12) | 1/29 | (3.4) | No |
| Fetal fibroblast | 172 (27) | 3/40 | (7.5) | No |
| Embryonic epithelial-like |
385 (33) | 4/87 | (4.6) | No |
| Fetal fibroblast | 507 (13.6) | 6/62 | (9.7) | Yes |
| Embryonic epithelial-like |
128 (24.2) | 2/31 | (6.5) | No |
| Embryonic epithelial-like |
258 (17) | 1/44 | (2.3) | No |
| Embryonic epithelial-like |
176 (14.8) | 4/26 | (15.4) | No |
| Embryonic epithelial-like |
68(11.7) | 1/8 | (12.5) | No |
Mice |
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| Adult cumulus | 2468 (56) | 16/1385 | (1.2) | No |
| Adult fibroblast | 250 (39) | 1/97 | (1.0) | No |
| Adult fibroblast | 467 (38) | 2/177 | (1.1) | No |
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Copy cat (Cc): Nuclear-donor cat (left), and cloned kitten with its surrogate mother. from Nature 415:859
(2002) Shin et. al. |
| a, Adult female cat that supplied cumulus cells for nuclear transplantation. The cells were cultured in DMEM/F12 medium for 5 days at 37 °C until confluent; micromanipulation was used to enucleate ova obtained after routine ovariohysterectomy and to transfer the donor's cumulus cells into the perivitelline space. Enucleated ova and cumulus cells were fused by electrofusion; a second electropulse was applied to activate the oocytes. b, The surrogate mother, a synchronized recipient of three cloned embryos, produced one cloned kitten, which was delivered by caesarian section 66 days after embryonic transfer. | |
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| ES cell derived mice at term (right) |  |
| The pup and placenta on the left are derived from tetraploid embryo complementation and appear grossly normal. In contrast, the pup on the right, derived by nuclear transfer of the same ES cell line, shows a dramatic example of the commonly observed overgrowth phenotype seen in cloned mice. Extensive fetal and placental overgrowth are observed accompanied by edema, and this animal did not survive. Scale bars, 1 cm. [from Eggan et al. (55); copyright 2001 National Academy of Sciences, U.S.A.] | |
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. from Lanza RP et al Science 288:665 (2000) |
| Normal heifers cloned from senescent somatic cells. (A) CL53-8, CL53-9, CL53-10, CL53-11, and CL53-12 (nicknamed Lily, Daffodil, Crocus, Forsythia, and Rose, respectively) at 5 months of age and (B) CL53-1 (Persephone) at 10 months of age. Fig. 2 | |
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Ability of nuclear transfer to restore the proliferative life-span of senescent donor cells. (A) The growth curve of the original BFF cell strain (green) is compared with that of cells derived from the fetus (ACT99-002) (black) that was cloned from late-passage BFF cells (CL53 cells). (B) The growth curve of the CL53 donor cells demonstrating that the cultures had about two population doublings (PDs) remaining. (C) Late-passage CL53 cells (n = 97) were seeded at clonal density, and the proliferative capacity after 1 month was determined. (D) Single-cell clones from early-passage BFF cultures (original) and early-passage ACT99-002 (clone) showed a capacity for extended proliferation. Fig. 3 from Lanza RP et al Science 288:665 (2000) |
 Fig.
4 from Lanza RP et al Science 288:665 (2000) |
| Telomere length analysis. (A) Nucleated blood cells. Peripheral blood samples from cloned and control Holsteins were analyzed by flow FISH (34) in two separate blinded experiments. Duplicate samples (red and blue bars) of nucleated cells obtained after osmotic lysis of red cells with ammonium chloride were analyzed by flow FISH as described (33). The average telomere fluorescence of gated single cells was calculated by subtracting the mean background fluorescence from the mean fluorescence obtained with the FITC-labeled telomere probe. (B) Telomere lengths in nucleated blood cells of 25 normal Holsteins ranging from <2 weeks to 6 years of age, showing the decline in mean telomere lengths against age. (C) Elongation of telomeres in cells upon nuclear transfer. Terminal restriction fragment (TRF) analysis of DNA fragments obtained after digestion with Hinf I-Rsa I was performed on a 0.5% agarose gel run for 28 hours, as described (Telomere Length Assay Kit; Pharmingen, San Diego, California). Lanes 1 and 4, genomic DNA isolated from control cells (pretransfection BFF bovine fibroblasts) (mean TRF length = 18.3 kb); lanes 2 and 5, senescent CL53 cells (mean TRF length = 15.2 kb); lanes 3 and 6, fibroblasts from a 7-week-old cloned fetus (ACT99-002) obtained by nuclear transfer with senescent CL53 cells (mean TRF length = 20.1 kb) (lanes 4 to 6 are longer exposures of lanes 1 to 3); lane 7, senescent donor fibroblast clone 22-1 (mean TRF length = 14.4 kb); lane 8, nuclear transfer fetal fibroblasts obtained with senescent 22-1 cells (mean TRF length = 16.4 kb); lane 9, senescent fibroblast clone 25-1 (mean TRF length = 12.1 kb); and lane 10, nuclear transfer fetal fibroblasts obtained with senescent 25-1 cells (mean TRF length = 16.1 kb). |
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Telomerase is expressed in reconstructed embryos but not in donor bovine fibroblasts. Telomerase activity was measured with a TRAP assay kit (Pharmingen, San Diego, California). Lysates from adult donor senescent (CL53) fibroblasts and day 7 reconstructed bovine embryos (n = 15) were obtained and used in the TRAP assay. Lane 1, extract from 4000 K562 human erythroleukemia cell line cells; lane 2, 20-base pair ladder; lane 3, no cell extract; lane 4, heat-treated embryo (n = 1) extract; lane 5, embryo extract (n = 10); lane 6, n = 1; lane 7, n = 0.1; lane 8, n = 0.01; and lane 9, extract from 4000 donor CL53 fibroblasts. All lanes contain the internal control TRAP reaction (36 base pairs, arrow) Fig. 5 from Lanza RP et al Science 288:665 (2000) |
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From Nature Genetics 30:253 (2002) Ogonuki N … Ogura, Atsuo, |
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| From Nature Genetics 30:253 (2002) Ogonuki N … Ogura, Atsuo, | |
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| From Nature Genetics 30:253 (2002) Ogonuki N … Ogura, Atsuo, | |
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Hochedlinger and Jaenisch Nature 415:1035 (2002) |
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Hochedlinger and Jaenisch Nature 415:1035 (2002) |
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