Dolly is dead...now what will the ill informed public think?

[Mike C]

What will be the ill informed public´s reaction, in your opinion, to Dolly the sheeps "pre-mature" death? Is cloning again going to be restamped as dangerous, unthinkable, or down right evil? The way the press is reporting this story, with it´s 15 to 30 second news reports, I have a bad feeling that the public will be even more brainwashed into thinking that cloning=bad and cloning=unsafe. CR is going to have an even tougher time pushing the therapeutic cloning agenda, I fear.

[antiquity]

The Dolly article can be read here:

http://carecure.org/forum/showthread.php?t=2078

[Wise Young]

Cloning stem cells is really quite different from cloning an organism. To get the latter, you need to have all the cells functioning and developing with respect to each other. To get cloned stem cells, you just need to select those cells that are healthy and robust for transplantation. Wise.

[dogger]

Dolly had a pretty fair innings . if the economic and natural climate are on my side i try and keep all of my flock under 5 yr. old . lately there have been a few falling off the perch at less than that due to the drought . sheep breeders have a saying '' a sheep's object in life is to try and commit suicide as quickly and originally as possible : our object is to do our best to ensure they fail in their attempts ''

thank you
dogger

every day i wake up is a good one .

[vic]

yeah dogger, who wants to eat 6 year old sheep anyways :-)

[Wise Young]

In retrospect, Dolly was a very successful case of cloning. Not only was she the first and oldest cloned mammal and she survived 6 years, she was fertile (actually producing four lambs). It is true that she became obese, developed arthritis earlier than other sheep, and had progressive lung diseases. Hundred of animals have been cloned around the world. The experience around the world suggest that many cloned animals suffer from similar problems.

1. Galli C, Lagutina I, Vassiliev I, Duchi R and Lazzari G (2002). "Comparison of microinjection (piezo-electric) and cell fusion for nuclear transfer success with different cell types in cattle." Cloning Stem Cells 4(3): 189-96. Contact: Laboratorio di Tecnologie della Reproduzione, CIZ srl, Cremona, Italy. cgalli@tin.it.
Amongst the many variables that can determine success of cloning, the source of nuclei, the procedure used for nuclear transfer, and the activation of the reconstructed embryo are very important aspects. In this study, we have compared the two most common procedures for transferring nuclei to enucleated oocytes--cell fusion (CF) and piezoelectric microinjection (PEM) using different somatic cells--and we have investigated the effect of different activation procedures. Granulosa cells and fibroblasts were grown to confluency or in low serum to induce a quiescent state, while lymphocytes were thawed immediately prior to use. Enucleated oocytes were reconstructed either with CF or PME by 21-23 h postmaturation. For cell fusion, one pulse of 1 kVolt/cm for 30 microsec was used; for PEM, the cell membrane was broken by repeated pipetting and transferred in a 12% PVP solution to facilitate injection. Manipulated oocytes were activated with ionomycin and cycloheximide (CHX) or 6-DMAP (DMAP) and cultured in microdrops of SOF-BSA-AA. On day 7 (day 0: nuclear transfer), embryo development was evaluated and embryos were either transferred fresh or were frozen. More embryos were successfully reconstructed with PEM than CF, but a higher number of reconstructed embryos by CF developed to blastocyst at D + 7. In addition, in both systems more embryos were obtained after activation with DMAP than with CHX. The transfer of 141 embryos to recipients resulted in a pregnancy rate of 50%, and no differences were observed between the source of donor cell, the reconstruction methods, or the activation protocol. Six calves were delivered at term, and four survived. High pregnancy losses were observed throughout the gestation period.

2. Yanagimachi R (2002). "Cloning: experience from the mouse and other animals." Mol Cell Endocrinol 187(1-2): 241-8. Contact: The Institute for Biogenesis Research, School of Medicine, University of Hawaii, Honolulu, HI 96822, USA. yana@hawaii.edu.
Cloning mammals has been successful for many years by splitting an early embryo or transferring embryonic cell nuclei into enucleated oocytes. Cloning is now possible with adult somatic cells. At present, cloning efficiency--as determined by the proportion of live offspring developed from all oocytes that received donor cell nuclei--is low regardless of the cell type (including, embryonic stem (ES) cells) and animal species used. In all animals, except of Japanese black beef cattle, the vast majority (>97%) of cloned embryos perish before reaching full term. Even in the Japanese cattle, less than 20% of cloned embryos reach the adulthood. This low efficiency of cloning seems to be due largely to faulty epigenetic reprogramming of donor cell nuclei after transfer into recipient oocytes. Cloned embryos with major epigenetic errors die before or soon after implantation. Those with relatively 'minor' epigenetic errors may survive birth and reach adulthood. We found that almost all fetuses of inbred mice die at birth from respiratory problems, while those of hybrid mice do not, suggesting that genomic heterogeneity masks-to some extent-faulty epigenetic errors. Thus far, the majority of cloned mice that survived birth, had a normal life span and were fertile. However, these animals may not be totally free of health problems. Postpubertal obesity in certain strains of mice is one example. A trial and error approach may discover better cells for cloning, but it would be wiser to understand the molecular mechanisms of epigenetic nuclear programming and reprogramming to find the way to make cloning safer and more efficient. The relatively high cloning success rate in the Japanese black cattle may provide us a clue of solving the problem of high mortality of cloned offspring.

3. Niemann H and Rath D (2001). "Progress in reproductive biotechnology in swine." Theriogenology 56(8): 1291-304. Contact: Department of Biotechnology, Institute of Animal Science and Behavior, Mariensee (FAL), Neustadt, Germany.
This article summarizes recent progress in reproductive biotechnology in swine with special reference to in vitro production of embryos, generation of identical multiples, and transgenic pigs useful for xenotransplantation. In vitro production (in vitro maturation, in vitro fertilization, and in vitro culture) of viable porcine embryos is possible, although with much lower success rates than in cattle. The main problems are insufficient cytoplasmic maturation of porcine oocytes, a high proportion of polyspermic fertilization and a low proportion of blastocysts that, in addition, are characterized by a low number of cells, hampering their development in vivo upon transfer to recipients. Microsurgical bisection of morula and blastocyst stage embryos leads to a 2 to 3% monozygotic twinning rate of the transferred demiembryos, which is similar to that in rabbits and mice but considerably lower than in ruminants. It was found that with decreasing quality an increasing proportion of demi-embryos did not possess an inner cell mass. Porcine individual blastomeres derived from 4- and 8-cell embryos can be cultured in defined medium to the blastocyst stage. Leukemia inhibitory factor has been shown to be effective at defined embryonic stages and supports the formation of the inner cell mass in cultured isolated blastomeres in a concentration-dependent manner. For maintaining pregnancies with micromanipulated porcine embryos, it is not necessary to transfer extraordinarily high numbers of embryos. Porcine nuclear transfer is still struggling from the inefficiency of producing normally functioning blastocysts. Blastomeres, blastocyst-derived cells, fibroblasts and granulosa cells have been employed as donor cells in porcine nuclear transfer and have yielded blastocysts. Recently, the generation of the first piglets from somatic cell nuclear transfer has been achieved. DNA-microinjection into pronuclei of porcine zygotes has reliably resulted in the generation of transgenic pigs, which have special importance for the production of valuable pharmaceutical proteins in milk and xenotransplantation. It has been demonstrated that by expression of human complement regulatory proteins in transgenic pigs the hyperacute rejection response occurring after xenotransplantation can be overcome in a clinically relevant manner. Although biotechnological procedures in swine have recently undergone tremendous progress, the development is still lagging behind that in cattle and sheep. With regard to genetic engineering, considerable progress will originate from the possibility of employing homologous recombination in somatic cell lines and their subsequent use in nuclear transfer. In combination with the increasing knowledge in gene sequences this will allow in the foreseeable future widespread use in the pig industry either for agricultural or biomedical purposes.

4. Wakayama T and Yanagimachi R (2001). "Effect of cytokinesis inhibitors, DMSO and the timing of oocyte activation on mouse cloning using cumulus cell nuclei." Reproduction 122(1): 49-60. Contact: The Institute for Biogenesis Research, Department of Anatomy and Reproductive Biology, University of Hawaii School of Medicine, Honolulu, HI 96822, USA. teru@advancedcell.com.
Cloning methods are now well described and in almost routine use. However, the frequencies of production of live offspring from activated oocytes remain at < 3% and little is known about the factors that affect these frequencies. The effects of cytokinesis inhibitors, dimethylsulphoxide [DMSO) and the cell cycle of recipient cytoplasm on the cloning of mice were examined. Reconstructed oocytes, which were activated immediately after nucleus injection and cultured without cytochalasin B, developed into blastocysts at a frequency of 30--54% and into live cloned offspring at a frequency of 2--3%. Activated zygotes did not support development to full term after nuclear transfer. Reconstructed oocytes were activated 1--3 h after nuclear transfer and were exposed separately to three inhibitors of cytokinesis [cytochalasin B, cytochalasin D or nocodazole) to examine the toxicity of these inhibitors on cloning. All of the oocytes exposed to nocodazole-containing media formed many small pseudo-pronuclei, whereas with cytochalasin-containing media most of the activated oocytes formed only two pseudo-pronuclei. Despite such differences, 42--61% of reconstructed embryos developed to the morula-blastocyst stage and 1--3% developed to full term in all groups. Addition of 1% [v/v) DMSO to the activation medium significantly improved the frequency of development to the blastocyst stage and full term; however, this improvement did not lead to a higher success rate in the generation of live cloned offspring. These results show that activated mouse oocytes/zygotes are not effective cytoplasmic recipients with the methods described and that the lack of success of cloning is not due to inhibition of cytokinesis.