Science in Society Archive

Handmade Cloning More Efficient But Obsolete

The dangers of new animal cloning techniques are evading regulatory bureaucrats while safer more ethical options for tissue replacement are making them obsolete Prof. Joe Cummins and Dr. Mae-Wan Ho

The state of food animal cloning

Governments in North America and in the European Union (EU) have approved the production and sale of meat and milk products from cloned animals and they have not required that those items be identified and accounted for so it is not presently possible to state how much of our diet includes cloned animal products [1].

The United States Food and Drug Administration (FDA) approved the sale of unidentified cloned animal products in 2008 but there was ‘voluntary’ agreement to sell only the products of offspring of the cloned animals because the first clones are frequently unhealthy [2]. Unlabeled cloned meat and milk were approved by the EU in 2011 as food safety proposals were undemocratically ripped up [3] (see Cloned Meat & Milk Coming, Be Very Afraid,SiS50).  The FDA claims “Clones are really just genetic copies of the animals from which they are produced” [4] and that claim is parroted by bureaucrats in EU. However, that is patently false because the animals cloned by somatic cell nuclear transfer (SCNT) are not true clones but all bear a mixture of mitochondrial genes from the donor somatic cell and the egg cytoplasm, a condition referred to as heteroplasmy (see [5] (‘Cloned’ Food Animals Not True Clones,SiS48). In nature, only the maternal parent provides mitochondrial genes. SCNT is a process entirely new to nature, and also departs significantly from in vitro fertilization. Mitochondrial heteroplasmy, and ensuing mitochondrial depletion, has been implicated in diseases affecting the brain, the central nervous system and the heart.

When the cloned female is mated to a cloned male the offspring are all heteroplasmic and likewise, when the cloned female is mated with a normal male the offspring will always be heteroplasmic. However, when a cloned male is mated with a normal female the offspring will all be normal homoplasmic, but carrying the mitochondria of the female parent.  The regulatory regimes of USA and EU based their false claim that the cloned animals are true clones on the baseless assumption that the mitochondrial genomes do not count when in fact the mitochondria play crucial roles in in a number of diseases of the nervous system, in cell suicide and in aging.

Handmade Cloning - speedy somatic cell nuclear transfer

The first cloned animal Dolly the sheep was created in 1996 using expensive and time-consuming cell surgery with a micromanipulator. The manipulator is used to remove the nucleus from an egg which is then fused with a cell from a donor animal.

In 2002, a technique called handmade cloning was introduced, which is cheaper and simpler, without the need for micromanipulation and also works better. Handmade cloning is done with micro blades and free-hand cell surgery under the dissecting microscope. The receptor egg is split in half and the half containing the nucleus is discarded.  The half egg lacking a nucleus is fused with a donor somatic cell and the fused egg half is fused again with a second enucleate half egg before it is activated (with a mild electric shock) to produce an embryo that is then implanted in a surrogate  mother [6]. The speedy technique could be performed in a clean home kitchen and it has begun to dominate the production of animal ‘clones’ that are not true clones, but heteroplasmic for mitochondrial DNA as explained above [5].

In 2004, an apparently healthy heifer calf was obtained by handmade cloning. It was the sole survivor of five blastocysts (embryos) inserted into surrogate mothers [7].  In 2007, piglets were produced using handmade cloning.  Litters of 3 and 10 piglets were produced with a live birth per transferred embryo of 17.2 % [8]. Water buffalo embryos were produced using handmade cloning.  It was found that the success of the embryos depended on the cytoplasmic volume of the egg. One live calf was delivered but died 4 hours after birth [9]. A rosy review of handmade cloning in mammals suggested that the procedure will benefit humans through production of animals with superior genetic traits, contribute to rescue of endangered species and through transgenic animals, produce medicine for human diseases [10].

Direct comparisons of the efficiency of cloning between handmade and micromanipulation are difficult to locate in the scientific literature.  A direct comparison of bovine handmade cloning (HMC) and in vitro fertilization (IVF) shows that the two have similar success in producing viable embryos (IVF27% HMC 31%) [11]. The health of surrogate mothers in cloning studies has not been reported.  In the case of transgenic clones, or the human or humanized animal  clones  the mother may continue to carry transgenic or human  cells from the  embryo. During pregnancy there is transplacental traffic of fetal cells into the maternal circulation. Remarkably, cells of fetal origin can then persist for decades in the mother and are detectable in the circulation and in a wide range of tissues. [12].

Interspecies handmade embryos

Handmade cloning has prompted exhaustive studies on development of SCNT embryos that employ the somatic cells of one species and the eggs of another. Interspecies somatic cell nuclear transfer (iSCNT) involves the transfer of a nucleus or cell from one species into the cytoplasm of an enucleated oocyte from another. Once activated, reconstructed oocytes can be cultured in vitro to the blastocyst stage, the final step of pre-implantation development. However, they often arrest during the early stages of pre-implantation development; fail to reprogram the somatic nucleus (a switch in nuclear gene expression of one kind of cell to that of an embryo or other cell type); and eliminate the accompanying donor cell's mitochondrial DNA (mtDNA) in favour of the recipient oocyte's genetically more divergent population. The incompatibility of mitochondria of the enucleate egg cell and that of the somatic cell nucleus donor appears to be a major factor.  Even though the mitochondrial DNA level was not changed in hybrids of pig-mouse embryos generated in one such experiment, the expression of a number of important mitochondrial genes was significantly reduced [13].  Mouse fetal fibroblasts in enucleated porcine oocytes, resulted in extremely low blastocyst rates (0.48%); and failure to replicate nuclear DNA and express Oct-4, the key marker of reprogramming.  When the pig eggs were depleted of mitochondrial DNA using a cancer drug, the HIV drug dideoxycytidine and reprogramming factors, the developmental outcomes of the iSCNT embryos were enhanced [14].

The possibility of producing interspecies handmade cloned embryos by nuclear transfer from donor cells of cattle, goat and rat using water buffalo oocytes as recipient enucleate eggs has also been explored. Interspecies blastocyst stage embryos can be produced using buffalo enucleate eggs and differentiated somatic cells from cattle and goat, with the source of donor nucleus affecting the developmental competence of interspecies embryos. However those embryos did not progress beyond the early stage embryos. Rat donor cells did not progress beyond the 32 cell stage while cattle donor cells developed as far as the 175-cell stage [15].  The reconstruction of embryos of the endangered Tibetan antelope using goat eggs met modest success.  Embryos were produced and morulas were formed but the embryos did not develop into blastocysts. Somatic cells of the antelope can be reprogrammed in the egg of the goat [16]. Interspecies hybrids have a long way to go before they can be used to preserve threatened species or to resurrect extinct species.

Handmade embryos to produce spare parts for transplant

For therapeutic transplants, a lot of work has been done on human embryonic stem (ES) cell line derivation, but immune rejection is a major problem for this cell based therapy. For a long time, deriving patient-matched stem cells has been the main approach. Oocytes after nuclear transfer are the most reliable source for patient-matched ES cell derivation for therapeutic use. In humans, using oocytes for stem cell research raises sensitive logistical and ethical questions on the participation of women as oocyte donors. Therapeutic cloning would lead to commercial exploitation of poor women. On the other hand, the therapeutic promise of embryonic stem cells is so huge that there is a strong incentive to find some alternate sources of human oocytes.  Would it be reasonable to use cross-species oocytes supplemented with human ES cellular extracts to establish patient-specific ES cell lines [17]?

Using handmade cloning, nuclear transplant derived embryonic stem cells were obtained from goat somatic cells and eggs [18]. In terms of therapeutic medicine, the goal is to produce patient-specific embryonic stem cells. However, the problem of mitochondrial heteroplasmy in the derived cell lines has not yet been resolved.  Mouse embryonic stem cells have been used to generate dorsal spinal cord neurons [19].  In such constructs mitochondrial heteroplasmy is a particular concern on account of the detrimental impact on oxidative metabolism.

Better, safer, and more ethical options exist

Actually, better, safer options already exist that do not raise the same ethical concerns. There is no need of handmade cloning, nor indeed of humanized animal cells for tissue replacement.

The first option is to use the patient’s own stem cells, a practice that has been demonstrating clinical successes since the beginning of the present century, despite the fact that proponents for embryonic stem cells have done their best not to mention it (see [20, 21] Hushing Up Adult Stem Cells, SiS 13/14; Patient's Own Stem Cells Mend Heart, SiS 25).

The second option is to use induced pluripotent stem (IPS) cells [22] The Promise of Induced Pluripotent Stem Cells, SiS 51). Within the past 6 years, scientists have discovered various ways in which 0rdinary cells can be induced to become pluripotent stem cells that can develop into practically any differentiated tissue, making it unnecessary to use human embryos. However, as with conventional embryonic stem (ES) cell, there is a significant risk of tumour formation if iPS cells are to be used for transplant. A further concern is the accumulation of mutations and genomic rearrangements during the iPS cell generation process, especially in genes related to cancers. There is also evidence that IPS cells are subject to immune reaction, even if they are derived from the patient’s own cells [23]

A third, most promising option being developed is to stimulate the patient’s own adult stem cells in situ to carry out tissue repair, as was demonstrated for animal models of multiple sclerosis (see [24] Stem Cells Repair without Transplant, SiS 50).

To conclude

Handmade cloning is a significant technical advance, as SCNT cloning can now be done rapidly and inexpensively.  However, SCNT cloning will always give rise to heteroplasmic clones which contribute to disability and death of the embryos. Depleting the eggs’ mitochondrial population to replace it with the mitochondria from the donor of the somatic cell is presently rather toxic to the egg and embryo due to the drugs employed.  The use of iSCNT to produce patient-specific replacement parts poses interesting ethical questions.   

Most of all, SCNT cloning is rapidly becoming obsolete for tissue replacement, which had been the main reason for continuing with the technique, as other safer, more ethical options are becoming available.

Article first published 26/03/12



References

  1. Fox News Offspring of Cloned Meat Making Its Way Into U.S. Food Supply, 13 Nov 2009 www.foxnews.com/story/0,2933,574867,00.html
  2. Poulter, S. Unlabelled clone meat allowed on shop shelves as food safety proposals are ripped up dailymail.co.uk, 26th May 2011  http://www.dailymail.co.uk/news/article-1390876/Unlabelled-clone-meat-allowed-shop-shelves-food-safety-proposals-ripped-up.html
  3. Ho MW. Cloned Meat & Milk Coming Be Very Afraid. Science in Society 50, 44-45, 2011
  4. United States Food and Drug Administration Animal and Veterinary Consumer Q&A Genetic Engineering, 2009 www.fda.gov/.../DevelopmentApprovalProcess/GeneticEngineering/GeneticallyEngineeredAnimals/ucm113672.htm
  5. Cummins J and Ho MW. ‘Cloned’ food animals not true clones.Science in Society 48, 48-50, 2010
  6. Westphal S. 'Handmade' cloning cheap and easy. New Scientist 14 August 2002 http://www.newscientist.com/article/dn2670-handmade-cloning-cheap-and-easy.html
  7. Vajta G, Bartels P, Joubert J, de la Rey M, Treadwell R, Callesen H. Production of a healthy calf by somatic cell nuclear transfer without micromanipulators and carbon dioxide incubators using the Handmade Cloning (HMC) and the Submarine Incubation System (SIS). Theriogenology 2004, 62, 1465-72.
  8. Du Y, Kragh PM, Zhang Y, Li J, Schmidt M, Bøgh IB, Zhang X, Purup S, Jørgensen AL, Pedersen AM, Villemoes K, Yang H, Bolund L, Vajta G. Piglets born from handmade cloning, an innovative cloning method without micromanipulation. Theriogenology 2007 ,68, 1104-10
  9. Panda SK, George A, Saha AP, Sharma R, Manik RS, Chauhan MS, Palta P, Singla SK.Effect of cytoplasmic volume on developmental competence of buffalo (Bubalus bubalis) embryos produced through hand-made cloning. Cell Reprogram 2011, 13, 257-62.
  10. Elsheikh, A. Handmade cloning of mammals. African Journal of Biotechnology 2007, 6, 1862-68.
  11. Mezzalira JC, Ohlweiler LU, da Costa Gerger RP, Casali R, Vieira FK, Ambrósio CE, Miglino MA, Rodrigues JL, Mezzalira A, Bertolini M. Production of bovine hand-made cloned embryos by zygote-oocyte cytoplasmic hemi-complementation. Cell Reprogram. 2011, 13(1), 65-76.
  12. Lissauer DM, Piper KP, Moss PA, Kilby MD Fetal microchimerism: the cellular and immunological legacy of pregnancy. Expert Rev Mol Med. 2009 Nov 12;11:e33.
  13. Amarnath D, Choi I, Moawad AR, Wakayama T, Campbell KH. Nuclear-cytoplasmic incompatibility and inefficient development of pig-mouse cytoplasmic hybrid embryos. Reproduction 2011, 142, 295-307.
  14. Jiang Y, Kelly R, Peters A, Fulka H, Dickinson A, Mitchell DA, St John JC. Interspecies somatic cell nuclear transfer is dependent on compatible mitochondrial DNA and reprogramming factors. PLoS One 2011, 6, e14805.
  15. Selokar NL, George A, Saha AP, Sharma R, Muzaffer M, Shah RA, Palta P, Chauhan MS, Manik RS, Singla SK. Production of interspecies handmade cloned embryos by nuclear transfer of cattle, goat and rat fibroblasts to buffalo (Bubalus bubalis) oocytes. Animal Reproduction Science 2011, 123, 279-82.
  16. Yu HH, Guo ZL, Guo SC, Qi DL, Yang XL, Yue PP, Cao H, Xie L, Zhao XQ. Interspecies embryo reconstruction in Tibetan antelope Pantholops hodgsonii by handmade cloning. African Journal of Biotechnology 2011, 21, 2360-265
  17. Riaz A, Javeed A, Zhou Q. Therapeutic cloning by xenotransplanted oocytes, supplemented with species specific reprogramming factors. Med Hypotheses. 2011, 76, 527-9.
  18. Dutta R, Malakar D, Khate K, Sahu S, Akshey Y, Mukesh M. A comparative study on efficiency of adult fibroblast, putative embryonic stem cell and lymphocyte as donor cells for production of handmade cloned embryos in goat and characterization of putative ntES cells obtained from these embryos. Theriogenology 2011, 76, 851-63
  19. Shan ZY, Liu F, Lei L, Li QM, Jin LH, Wu YS, Li X, Shen JL. Generation of dorsal spinal cord GABAergic neurons from mouse embryonic stem cells. Cell Reprogram 2011, 13, 85-91.
  20. Ho MW and Cummins J. Hushing up adult stem cells. i-sis news13/14, 6-7, 2002.
  21. Ho MW. Patient’s own stem cells mend heart. Science in Society 25, 38-39, 2005.
  22. Sirinathinghji E. The promise of induced pluripotent stem cells. Science in Society 51, 42-44.
  23. “Study finds therapies using induced pluripotent stem cells could encounter immune rejection problem”, Kim McDonald and Chris Palmer, UC San Diego Neww Center, 13 May 2011, http://ucsdnews.ucsd.edu/newsrel/science/5-13-11stemcells.asp
  24. Sirinathinghji E. Stem cells repair without transplant. Science in Society 50, 48-49, 2011.

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Todd Millions Comment left 28th March 2012 07:07:47
Thanx for this -decades ago I read of work showing'de-differlization of specialized cell,to ones that where'embrionic',and so could reform into any new tissue.The next few decades were-a slowly smoldering build up of cold rage that these leads were inconveinent too the medical mafia(along with other items) and their corprate pharma masters.Its good too see some signs of follow up being done on Dr Becker's observations.May I suggest a reprise of 'silver neddles-of both the healing and infection fighting polarities?

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