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Cloning and Telomeres


I read that the length of telomeres in Dolly was shorter, because the DNA was taken from an old sheep. Shorter telomeres may lead to early aging. What is happening now with cloning? Do we use a telomerase to restore the length in a donor cell?


No, it doesn't seem to be common practice. A telomerase treatment would probably do more harm than good, and may be completely unnecessary in the first place.

Betts and coworkers published a study in which they examined telomere length in cloned cattle. They found that while telomere length in cloned embryos does start out somewhat shorter than in naturally produced embryos, by the time that the normal processes of embryonic development were in full swing telomere length in cloned and natural embryos was equivalent.

Betts' results make sense in light of the fact that telomerase activity (and the activity of many, many related proteins) is normally upregulated in embryonic cells.

As for Dolly, while it is true that her telomeres were shorter than those of an average age-matched sheep at the time of her death, it's far from clear that those short telomeres had anything to do with her decline. Also keep in mind that she didn't just keel over suddenly in the night, she was euthanized. A subsequent autopsy showed that she had a form of lung cancer caused by a retrovirus common in sheep.

Edit: why were Dolly's telomeres shortened?

This is a good question, as the findings of shortened telomeres in Dolly's case do contradict the findings in the Betts paper. Clearly we need more citations:

-"Analysis of telomere lengths in cloned sheep." Shiels et al, 1999

This is the original citation for the claim that Dolly's telomeres were shortened. There isn't too much extra information to be had as it's a very short paper (1 page letter). They did look at other cloned sheep created from types of cells other than skin cells (as Dolly was) and saw the same kind of telomere shortening relative to controls. They also make a point of mentioning that Dolly's telomere length may be within the normal variation for sheep.

-"Remarkable differences in telomere lengths among cloned cattle derived from different cell types." Miyashita et al, 2002

The investigators found that telomere length in cloned cattle was strongly dependent on the type of cell from which the clone was derived. For example, clones made from mammary cells tended to have shortened telomeres, whereas those derived from muscle cells had normal length telomeres.

-"The analysis of telomere length and telomerase activity in cloned pigs and cows.", Jeon, 2005

This group found that cloned cattle had normal length telomeres, whereas cloned pigs had elongated telomeres. By way of explaining these results, they found that telomerase activity was upregulated in cloned pigs relative to control pigs, whereas telomerase activity in cloned cattle matched that of control cattle.

How should we take all of this information? At the very least we can say that telomere length in clones is highly dependent on species and on source tissue, and that telomere length can increase, decrease, or stay the same in cloned animals relative to control animals. Clearly telomere length in cloned animals is a fiddly property. It is very likely that it also depends on the exact details of the cloning protocol as it was carried out (i.e. how much did the experimenter's hands shake when they inserted the needle to suck out the nucleus?, etc.).

So I would say not to read too much into Dolly's shortened telomeres. They may have been a result of her being a clone, or they may have been a result of natural variation. In either case the shortened telomeres were not linked to her ill-health, and it's likely that small tweaks to the cloning protocol would eliminate any telomere length discrepancies in future clones.


[Cloning and analysis of the telomeres of five Streptomyces linear plasmids]

Objective: Streptomyces strains were isolated from soil samples of Tibet, five small linear plasmids were detected by pulsed-field gel electrophoresis.

Objective: Cloning, sequencing and analysis of telomeres of these plasmids.

Methods: The telomeres were cloned by a modified procedure--"alkaline treatment and enzyme digestion in gels".

Results: Telomeres of five linear plasmids were cloned and sequenced. Compared with the typical Streptomyces telomeres, the newly identified telomeres contained multiple palindromes, but some could not "fold-back" of their first conserved palindrome I with the internal palindromes to form a "super-hairpin", and palindromes of some telomeres did not contain the 3-nt "loop".

Conclusion: New telomere sequences were cloned by a modified procedure. Both folding-back of the telomere palindromes and 3-nt loop of palindromes varied among telomeres.


Blackburn, E. H. & Szostak, J. W. A. Rev. Biochem. 53, 163–194 (1984).

Shampay, J., Szostak, J. W. & Blackburn, E. H. Nature 310, 154–157 (1984).

Walmsley, R. W., Chan, C. S. M., Tye, B-K. & Petes, T. D. Nature 310, 157–160 (1984).

Sugawara, N. & Szostak, J. W. Yeast 2 (Suppl.) 373 (1986).

Blackburn, E. H. & Gall, J. G. J. molec. Biol. 120, 33–53 (1978).

Klobutcher, L. A., Swanton, M. A., Donini, P. & Prestcott, D. M. Proc. natn. Acad. Sci. U.S.A. 78, 3015–3019 (1981).

Ponzi, M., Pace, T., Dore, E. & Frontali, C. EMBO J. 4, 2991–2995 (1985).

Emery, H. S. & Weiner, A. M. Cell 26, 411–419 (1981).

Blackburn, E. H. & Challoner, P. B. Cell 36, 447–457 (1984).

Van der Ploeg, L. H. T., Liv, A. Y. C. & Borst, P. Cell 36, 459–468 (1984).

Forney, J., Henderson, E. R. & Blackburn, E. H. Nucleic Acids Res. 15, 9143–9152 (1987).

Cooke, H. J., Brown, W. R. A. & Rappold, G. A. Nature 317, 687–692 (1985).

Cooke, H. J. & Smith, B. A. Cold Spring Harbor Symp. quant. Biol. 51, 213–219 (1986).

Allshire, R. C. et al. Nature 332, 656–659 (1988).

Moyzis, R. K. et al. Proc. natn. Acad. Sci. U.S.A. 85, 6622–6626 (1988).

Richards, E. J. & Ausubel, F. M. Cell 53, 127–136 (1988).

Pluta, A. F., Dani, G. M., Spear, B. B. & Zakian, V. A. Proc. natn. Acad. Sci. U.S.A. 81, 1475–1479 (1984).

Cooke, H. J. & Cross, S. H. Nucleic Acids Res. 16 11817 (1988).

Burke, D. T., Carle, G. F. & Olsen, M. V. Science 236, 806–812 (1987).

Burgers, P. M. J. & Percivals, K. J. analyt. Biochem. 163, 391–397 (1987).

Murray, A. W., Schultes, N. P. & Szostak, J. W. Cell 45, 529–536 (1986).

Szostak, J. W. & Blackburn, E. H. Cell 29, 245–255 (1982).

Gottschling, D. E. & Zakian, V. A. Cell 47, 195–205 (1986).

Berman, J., Tachibana, C. Y. & Tye, B-K. Proc. natn. Acad. Sci. U.S.A. 83, 3713–3717 (1986).

Sen, D. & Gilbert, W. Nature 334, 364–366 (1988).

Lathe, R., Kierny, M. P., Skory, S. & Lecocq, J. P. DNA 3, 173–182 (1984).

Bellis, M., Pages, M. & Roizes, G. Nucleic Acids Res. 15, 6747 (1987).

Nakaseko, Y., Adachi, Y., Funahashi, S., Niwa, O. & Yanagida, M. EMBO J. 5, 1011–1021 (1986).


Author information

Lin Liu: Lin Liu, received Doctorate degree in Reproductive Biology and Biotechnology from Beijing Agricultural University. He was a visiting scholar at the BBSRC Babraham Institute, Cambridge, UK, and then had two year post-doctorate training in Cornell University and University of Connecticut, USA. He worked as an Investigator and Assistant Professor (Research) at Women & Infants Hospital/Brown Medical School. He is Professor in Nankai University, Adjunct Assistant Scientist at Marine Biological Laboratory, Visiting Assistant Professor at the University of South Florida College of Medicine, and Member of H Lee Moffitt Cancer Center & Research Institute. He has been a member of American Association for the Advancement of Science (AAAS), Society for the Study of Reproduction (SSR), International Embryo Transfer Society (IETS), New York Academy of Sciences (NYAS), and council member of Reproductive Biology under Zoological Society China. Dr Liu's research focuses on reproductive aging and egg senescence, molecular mechanisms underlying early embryo development and differentiation, and differentiation of adult stem cells. He is also interested in derivation and understanding of patient-specific stem cells that can be effectively used for regenerative medicine, particularly for treatment of reproductive aging and associated diseases.

Affiliations

College of Life Sciences, Nankai University, Tianjin, China

Department of Obstetrics and Gynecology, University of South Florida College of Medicine, Tampa, Florida, USA

Lin Liu, Maja Okuka & David L Keefe

College of Life Sciences, Sun Yat-Sen University, Guangzhou, China

Lin Liu, Chao Li, Lingjun Zhou & Chao Wu

Center for Regenerative Biology and Department of Animal Science, University of Connecticut, Storrs, CT, USA


DNA CLONING VECTORS

Plasmids are the most commonly used cloning vectors. Plasmids are popular because they allow cloning and manipulation of small pieces of DNA thereby being helpful in molecular biology techniques. One of the first widely used plasmid DNA vectors, called pBR322 was designed to have genes for ampicillin and tetracycline resistance and several useful restriction sites.

FEATURES OF DNA CLONING VECTORS

Modern plasmid DNA cloning vectors usually include the features.

  1. SIZE – They should be small enough to be easily separated from chromosomal DNA of host bacteria.
  2. ORIGIN OF REPLICATION(ori) – The site for DNA replication that allows plasmids to replicate separately from the host cell’s chromosome. The number of plasmids in the cell is called the copy number. Most of the desirable cloning plasmids are known as high copy number plasmids because they can replicate to create hundreds or thousands of plasmid copies.
  3. MULTIPLE CLONING SITE – The MCS , also called a poly linker is a stretch of DNA with recognition sequences for many different restriction enzymes. A MCS provides a lot of opportunities for possible DNA inserts to be added.
  4. SELECTABLE MARKER GENES – These genes allow the selection and identification of bacteria that have been transformed with a recombinant plasmid compared to non transformed cells. Example, ampicillin resistance and tetracycline resistance and lacZ gene used for blue – white selection.
  5. RNA POLYMERASE PROMOTER SEQUENCE – These sequences are used for transcription of RNA in vivo and in vitro by RNA polymerase.
  6. DNA SEQUENCING PRIMER SEQUENCE – These sequences permit nucleotide sequencing of cloned DNA fragments that have been inserted into the plasmid.
TYPES OF VECTORS

Each vectors has its significant role in molecular biology techniques. Therefore, there are different cloning vectors.

DNA from bacteriophage lambda one of the first phage vectors used in cloning. The lambda chromosome is a linear 49 kb size structure. Cloned DNA is inserted into restriction sites in the centre of the lambda chromosome Recombinant chromosomes are hen packaged into viral particles in vitro, and these phages are used to infect E.coli growing as a lawn. At each end of the lambda chromosome are 12 nucleotide sequences called cohesive sites (COS) that can base pair with each other . When lambda infects E.coli as a host, the Lambda chromosome uses these COS sites to circularize and then replicate. A primary advantage of using these vectors is that they allow cloning of larger DNA fragments than plasmids.

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2. COSMID VECTORS

Cosmid vectors contain COS ends of lambda DNA, a plasmid origin of replication, and genes have been removed. DNA is cloned into a restriction site, and the cosmid is packaged into viral particle, a s is done with bacteriophage vectors. Bacterial colonies are formed on a plate, and recombinants are screened by antibiotic selection. A primary advantage of cosmids is that they allow for the cloning of DNA fragments in the 20- 45 kb range.

3. EXPRESSION VECTORS

Protein expression vectors allow high level expression of eukaryotic proteins with in the bacterial cells because they contain a prokaryotic promoter sequence adjacent to the site where DNA is inserted into the plasmid. Bacterial RNA polymerase can bind to the promoter and synthesize large amounts of RNA which is then translated into protein. Protein can then be isolated using biochemical techniques.

4. BACTERIAL ARTIFICIAL CHROMOSOMES

Bacterial artificial chromosome (BACs) are large low copy number plasmids, present as one or two copies in bacterial cells that contain genes encoding the F- factor. BACs can accept DNA inserts in the 100 – 300 kb range. BACs were widely used in the Human Genome Project to clone and sequence large pieces of human chromosomes.

5. YEAST ARTIFICIAL CHROMOSOMES

Yeast artificial chromosomes (YACs) are small plasmids grown in E.coli and introduced into yeast cells. A YAC is a miniature version of a eukaryotic chromosome. YACs contain an origin of replication, selectable markers, two telomeres, and a centromere that allows for replication of the YAC and segregation into daughter cells during cell division. Foreign DNA fragments are cloned into the restriction site into the center of the YAC. YACs are useful for cloning large fragments of DNA from 200 kb to approx. 2 megabases (mb= 1 million bases) in size. YACs have also been used in Human Genome Project.

6. Ti VECTORS

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These are naturally occurring plasmids isolated from the bacterium Agrobacterium tumefaciens. A soil borne Gram negative pathogen that infects dicot plants by causing crown gall disease. On entering a cell the A. tumefaciens inserts a apart of its DNA(T-DNA) from the Ti plasmid into the host chromosome. Therefore the plant geneticists exploited this property if the bacteria and used it to insert their gene of interest using A. tumefaciens as a vector into the host cell.

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Arnold C, Hodgson IJ (1991) Vectorette PCR: A novel approach to genomic walking. PCR Meth Appl 1:39–42

Belostotsky DA, Ananiev EV (1990) Characterization of relic DNA from barley genome. Theor Appl Genet 80:374–380

Bennet MD, Smith JB (1976) Nuclear DNA amounts in angiosperms. Phil Trans R Soc London (Biol) 274:227–274

Blackburn EH (1991 a) Structure and function of telomeres. Nature 350:569–573

Blackburn EH (1991 b) Telomeres. Trends Biochem Sci 16:378–381

Broun P, Ganal MW, Tanksley SD (1992) Telomeric arrays display high levels of heritable polymorphism among closely related plant varieties. Proc Natl Acad Sci USA 89:1354–1357

Chan CSM, Tye B-K (1983) Organization of DNA sequences and replication origins at yeast telomeres. Cell 33:563–573

Cooke HJ, Brown WRA, Rappold GA (1985) Hypervariable telomeric sequences from the human sex chromosomes are pseudoautosomal. Nature 317:687–692

Corcoran LM, Thompson JK, Walliker D, Kemp DJ (1988) Homologous recombination within subtelomeric repeat sequences generates chromosome size polymorphisms in P. falciparum. Cell 53:807–813

Cross SH, Allshire RC, McKay SJ, McGill NI, Cooke HJ (1989) Cloning of human telomeres by complementation in yeast. Nature 338:771–774

Devereux J, Haeberli P, Smithies O (1984) A comprehensive set of sequence analysis programs for the VAX. Nucleic Acids Res 12:387–395

Ganal MW, Lapitan NLW, Tanksley SD (1991) A molecular and cytogenetic survey of major repeated DNA sequences in tomato (Lycopersion esculentum). Plant Cell 3:87–94

Kleinhofs A (1992) The NABGMP mapping progress report. Barley Genet Newsletter (in press)

Kleinhofs A, Chao S, Sharp PJ (1988) Mapping of nitrate reductase genes in barley and wheat. In: Miller TE, Koebner RMD (eds) Proc 7th lnt Wheat Genet Symp Vol 1, Bath Press, Bath, pp 541–546

Kleinhofs A, Kilian A (1992) RFLP maps of barley. In: Phillips RL, Vasil IK (eds) DNA-based markers in plants. Advances in cellular and molecular biology of plants, vol. 1, Kluwer Academic Publishers, Dordrecht, (in press)

Lander ES, Green P, Abrahamson J, Barlow A, Daly MJ, Lincoln SE, Newburg L (1987) MAPMAKER: an interactive computer package for constructing primary genetic linkage maps of experimental and natural populations. Genomics 1:174–181

de Lange T, Shiue L, Myers RM, Cox DR, Naylor SL, Killery AM, Varmus HE (1990) Structure and variability of human chromosome ends. Mol Cell Biol 10:518–527

Murray V (1989) Improved double-stranded DNA sequencing using the linear polymerase chain reaction. Nucleic Acids Res 17:8889

Petracek ME, Lefebvre PA, Silflow CD, Berman J (1990) Chlamydomonas telomere sequences are A + T-rich but contain three consecutive G · C base pairs. Proc Natl Acad Sci USA 87:8222–8226

Richards EJ, Ausubel FM (1988) Isolation of a higher eukaryotic telomere from Arabidopsis thaliana. Cell 53:127–136

Riley J, Butler R, Ogilvie D, Finniear R, Jenner D, Powell S, Anand R, Smith JC, Markham AF (1990) A novel, rapid method for the isolation of terminal sequences from yeast artificial chromosome (YAC) clones. Nucleic Acids Res 18:2887–2890

Sambrook J, Fritsch EF, Maniatis T (1989) Molecular cloning a laboratory manual. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York

Schwarzacher T, Heslop-Harrison JS (1991) In situ hybridization to plant telomeres using synthetic oligomers. Genome 34:317–323

Weber B, Collins C, Robbins C, Magenis RE, Delaney AD, Gray JW, Hayden MR (1990) Characterization and organization of DNA sequences adjacent to the human telomere-associated repeat (TTAGGG)n. Nucleic Acids Res 18:3353–3361

Zakian AW (1989) Structure and function of telomeres. Annu Rev Genet 23:579–604


Resources

BOOKS

Brown, Terry. Gene Cloning and DNA Analysis. Boston: Blackwell Publishing, 2006.

Caplan, Arthur and Glenn McGee, eds. The Human Cloning Debate. Berkeley: Berkeley Hills Books, 2006. McGee, Glenn. The Human Cloning Debate. Berkeley: Berkeley Hills Books, 2002.

Scientific American. Understanding Cloning. New York: Warner Books, 2002.

PERIODICALS

Loi, Pasqualino, Grazyna Ptak, Barbara Barboni, Josef Fulka Jr., Pietro Cappai, and Michael Clinton, “ Genetic Rescue of an Endangered Mammal by Cross-species Nuclear Transfer Using Post-mortem Somatic Cells. ” Nature Biotechnology (October 2001):962 – 964

Ogonuki, Narumi, et al. “ Early Death of Mice Cloned from Somatic Cells. ” Nature Genetics (11 February 2002):253-254


Interactive resources for schools

Transcription factors

Transcription factors are proteins which bind to the nuclear DNA, regulating the transcription of the genetic material and sometimes changing the mRNA which is formed as a result.

Differentiation

The genetically-controlled process by which an unspecialised stem cell becomes a cell with specialised structures which carries out a particular function

Non-coding RNA

About 98% of the transcribed RNA thought to regulate transcription of the DNA directly, or by histone modification, or to modify the products of transcription.

Epigenetics

Epigenetics is the study of external factors which affect gene expression and differentiation, often by switching particular genes on or off, so changing the proteins made by a DNA sequence.

Pluripotent

capable of differentiating to form almost any type of cell in the body except those of the placenta and amniotic membranes.

Cloning problems

Although cloning has huge potential, the problems associated with it prevent it from being widely used because cloning isn't widely used, scientists are not easily able to investigate these problems and improve their techniques to overcome them. Cloning is inefficient because of problems associated with nuclear transfer - but these can be improved with practice. There are also certain genetic problems associated with cloning - these lead to some health problems, and also problems with appearance. None of the problems associated with cloning are exclusive to it: they are also seen in normally and artificially reproduced animals.

Epigenetics

One of the main problems with cloning is due to a naturally occurring complex process called epigenetics. Epigenetics controls differentiation, it results in changes in the genes expressed (producing proteins) in a cell or organism. Of the 20-25,000 genes that we possess, only 10-20,000 are expressed at any one time. Also as a result of epigenetics, we can produce far more proteins than we have genes. The most common way in which gene expression is controlled is by switching the transcription of specific genes on or off in the nucleus. This is brought about by transcription factors – proteins which bind to the DNA in the nucleus and affect the process of transcribing the genetic material. Some transcription factors bind to the DNA in a promotor region and simply start the transcription of the gene. Others make a particular gene more or less available for transcription, effectively switching genes on or off. The DNA sequence itself is not altered by this process – it occurs because specific compounds become attached to a section of DNA thereby ‘silencing’ or ‘activating’ it. The DNA may be methylated or demethylated directly, or the coiling of the histones (part of the chromosome structure) may be changed by having acetyl or methyl groups attached. Much of the RNA produced when the DNA code is transcribed is non-coding – it doesn’t code for a specific protein. But this non-coding RNA (ncRNA) affects the proteins which are made. When changes are made to the ncRNA, the proteins produced are also modified. At least some of these changes can be reversed – but as they are complicated and not fully understood, it is hard for scientists to do this.

SCNT in reproductive and therapeutic cloning involves using a nucleus that has undergone epigenetic-controlled differentiation. If these epigenetic changes cannot be properly reversed then the cell cannot be ‘reprogrammed’ or undifferentiated and cloning is likely to fail altogether or result in an organism with health problems. Reproductive cloning results in the birth of an organism that develops into an adult: this process has many steps where something can go wrong and therefore is likely to emphasise any epigenetic problems more than therapeutic cloning does. Embryonic cell nuclear transfer and artificial twinning are much less likely to have epigenetic problems because they do not use a previously fully differentiated nucleus.

The first cloned cat to be produced was called Carbon Copy, or CC for short. Even though she had exactly the same genetic material as her genetic mother (called Rainbow), she looked different because of a process called X-inactivation, a type of epigenetic control that occurs in female mammals. Female mammals have two X chromosomes, but they only need the genetic material from one of these. One entire X chromosome is switched off in each cell by ncRNA, which coats one of the X chromosomes and deactivates it. This switching off is entirely random so that different X chromosomes are switched off in different cells.

This inactivation occurs fairly early in development, and so cells with the same inactivation pattern are often found clustered together in adults because they are descended from one already X-inactivated cell. In cats, some coat colour genes are located on the X chromosomes. Rainbow and CC both had the same genetic material which coded for different coat colours on each of their X chromosomes. CC was cloned from a cell in which the X chromosome carrying the gene for orange colouring was switched off and it appears it was not switched on again.

Problems with nuclear transfer

Nuclear transfer is a very inefficient process: the donor egg nucleus must be removed without damaging the rest of the cell, and the donor nucleus removed from this cell and transferred into the egg without damaging it, which is not easy. It is even more inefficient if the embryo is then implanted into a surrogate mother as in many instances the embryo does not implant or miscarries. The fact that Dolly the sheep was even born was an achievement: 277 cells were taken from Dolly’s ‘genetic mother’, which were fused with 277 eggs. Only 29 of these formed viable reconstructed embryos which could be implanted into surrogate mothers. Of those 29 early embryos, only one developed into a sheep: Dolly.

As reproductive cloning becomes more common, scientists are able to practice their techniques and so cloning is becoming more efficient. The first attempt to clone the endangered grey wolf at Seoul National University in South Korea lead to the birth of just two live females called Snuwolf and Snuwolffy in 2005. Once they had established the method to use, the scientists were able to more efficiently produce a further three wolves just three years later.

In the UK, it is illegal to place a human embryo, which hasn’t been created by fertilisation or has had its DNA altered, in a woman – with the exception of mitochondrial donation. At the moment, no artificial womb exists that is capable of supporting a developing embryo for nine months from its creation – so it is effectively illegal in the UK to create a human reproductive clone.

Reproductive cloning in humans would not be very efficient or effective if it were carried out. It can be seen from Dolly the sheep how many eggs were required to generate one healthy adult sheep. The number of human eggs wasted, the probability of the embryo not properly implanting in the uterus, miscarriage, and the possibility of babies being born with genetic abnormalities are so high that this work is prohibited by good scientific judgement as well as by law.

Telomere length

Many people noted that Dolly died when she was six years old. This is half the average lifespan for a sheep, but is not particularly unusual because Dolly had a severe lung problems caused by a virus. What was unusual were some of Dolly’s health problems (such as arthritis) which aren’t usually seen in sheep – especially of her age. Dolly’s genetic mother was six years old herself when her somatic cells from which Dolly was cloned were removed: does this mean Dolly was effectively 12 years old (a good age for a sheep) when she died?

As you age, your DNA is replicated each time your cells divide. The full length of the chromosome is replicated but the primer at the very end of the section-copied strand can’t be correctly joined up to the rest of the strand, so it is degraded and the DNA is shortened. This generally isn’t a problem because the section at the end of a DNA strand, called a telomere, doesn’t contain any genes. Over the many cell divisions that take throughout your lifetime, however, the telomere can shorten so much it disappears altogether and so sections of DNA containing important genes are lost. Telomere shortening is thought to be linked to ageing once the telomeres are lost altogether, important genetic material is damaged and this might be the reason for some of the severe health problems that are seen in older people.

Dolly’s telomeres were examined and found to be much shorter than those of ordinary sheep of a similar age. Therefore it is possible that Dolly’s telomeres were already half the length they should be (the same length as a six year old sheep) when she was born if her telomeres kept shortening at the same rate, she would have the telomere length of a 12 year old sheep at the age of six.

However, this isn’t a problem that is seen in all cloned animals – in fact, Dolly seems to be the exception, so perhaps this phenomenon wasn’t a result of cloning after all. CC, the first cloned cat, is still alive at 16 years old and has been normal and healthy throughout. After SCNT, the telomeres of most cloned cells are restored to their full, or near their full, length – in the very few cases where the telomeres are not sufficiently restored, the animal does show health problems or may even die before it is born. This problem only really affects reproductive cloning by SCNT: reproductive cloning using ECNT and artificial twinning both use ‘young’ cells where the telomeres are nearly full length and so it doesn’t matter if they are not fully restored. In therapeutic cloning using SCNT, the cloned cells are not kept long enough to divide many times, so it does not matter if the telomeres are not restored because they will not be shortened by much more.

Large offspring syndrome

Large Offspring Syndrome (LOS) is a condition which can occur in mammals at any time. It is more common, however, in animals produced by assisted reproduction - a term which includes techniques such as embryo transfer, artificial insemination and IVF as well as reproductive cloning. In this disorder, the cloned foetus carried by the surrogate mother is unusually large, and may have some additional health problems such as breathing and circulation difficulties caused by abnormally large organs. This not only affects the health of the clone - the large size of the foetus can cause problems in late pregnancy and birth for the surrogate mother.

Any surrogate carrying a clone is monitored so medical help (such as caesarean sections) is on hand to overcome these problems. Although an animal can have severely affected health at birth, in most cases it is of normal size and healthy within a few months its own offspring (if normally reproduced) are no more likely to have LOS than 'ordinary' animals. No one knows exactly why LOS occurs – apart from the fact that manipulation of the embryo makes it more likely – but there are several plausible theories.

It has been noticed that the different culture conditions the early embryo is placed in before it is implanted into the surrogate are associated with different rates of LOS. However, the ‘good’ and ‘bad’ culture conditions seem to vary from species to species, and rates of LOS can even vary widely when exactly the same scientific protocol is used in the same location for the same species at different times.

The conditions an embryo is exposed to once it has been transferred into the surrogate mother can also result in LOS: the environment in the womb changes frequently so the embryo's rate of development changes according to whether the surrogate is synchronised with the embryo age.

There is also a correlation between foetuses with LOS and abnormal placentas – and it appears that the placenta becomes abnormal before LOS develops. It is thought that this placental abnormality could be caused by epigenetic defects: the embryo develops from pluripotent stem cells, but the placenta develops from totipotent stem cells which are even less differentiated than pluripotent cells. Perhaps after SCNT, epigenetic controls on nuclear material are reversed so that the cells are fully pluripotent but are not quite reversed enough for the cells to be fully totipotent. This would explain the difficulty in producing a normal placenta.


How A Sheep Revolutionized Cloning

Gone are the days where cloning is a far-fetched dream a scientist grinning in the glowing green light of his evil creation. Gone are the days where the concept of cloning is merely that now, it is reality.

Not many people know about Dolly the sheep the first mammal cloned from an adult somatic cell. Dolly the sheep isn’t a product of modern technology either she was “born” in 1996 in a laboratory in Scotland after 277 previous failed attempts. That was over 20 years ago.

Dolly was crea t ed through nuclear fission. Scientists took the DNA of a body cell of one sheep and fused it with the egg cell (with its nucleus removed) of another sheep. The resulting embryo was placed in a surrogate sheep and thus, Dolly was born.

Dolly’s impact was huge. The world was shocked that a sheep was cloned. A frog had been cloned in the 60’s but mammals were seen as too complicated to clone until now. Dolly brought up the discussion of cloning humans and other species. Since Dolly, 20 species have been cloned.

Over the years, Dolly had 6 children and lived the life of a normal sheep. Towards the end of her life, she developed arthritis and contracted a virus that caused her to develop lung cancer. She was euthanized to save her from any pain in 2003 at the age of 6.

According to “The Life of Dolly”, Dolly’s telomeres’ “were shorter than would be expected for a normal sheep of the same age. As an animal or person ages, their telomeres become progressively shorter, exposing the DNA to more damage. It was thought that, because Dolly’s DNA came from an adult sheep, her telomeres had not been fully renewed during her development. This could have meant that Dolly was biologically older than her actual age.”

While Dolly was not found to have any premature aging conditions, it brings up an interesting question: is cloning ethical?

In the United Kingdom, there is a law for the mother cow to be under anesthesia during the embryo transfer process due to the stress it causes the cows. And, according to PETA, cloned animals are also more likely to have faulty immune systems.

Autumn Fiester writes about Dolly and other cloned animals in his essay, “Ethical Issues in Animal Cloning”, writing, “There is a large body of literature citing high rates of miscarriage, stillbirth, early death, genetic abnormalities, and chronic diseases among cloned animals.”(331)

Dolly isn’t the only clone, however, to spark the conversation of bioethics, cloning and mammal-editing.

In 2018, a man by the name of He Jianku announced to the world that he had made the world’s first genetically modified/edited twins. They are also known as the world’s first CRISPR twins. The twins go by the pseudonyms Lulu and Nana. According to this article, there is a third baby as well. He, the first scientist, has been reportedly jailed for three years for “illegally carrying out human embryo gene-editing intended for reproduction, in which three genetically edited babies were born”.

The terrifying thing about Lulu and Nana is just how much is unknown about them. He has claimed that he will publish data regarding the twins, but he has yet to. He has injected the twins with a gene that is meant to protect the twins from HIV but scientists aren’t so sure that's all he did.

Dr. Kiran Musunuru, professor of cardiovascular medicine and genetics at University of Pennsylvania Perelman School of Medicine, says, “These babies were treated as subjects in a grand medical experiment, and we have to believe that they will be studied for the rest of their lives it’s sad actually.”

The notion of editing babies has been a concern of scientists for decades. Autumn Fiester of “Ethical Issues in Animal Cloning” writes, “What makes reproductive cloning morally troubling is that its primary purpose is to create children of a certain kind”. This could further perpuate toxic beauty standards and create hierarchies based on appearance even more than what is already present in society. This could lead to less diversity which could thus lead to less tolerance and acceptance.

“What makes reproductive cloning morally troubling is that its primary purpose is to create children of a certain kind”.

Cloned individuals may have trouble with genetic identity an incredibly important domain of life. Patricia A. Baird writes about this often looked over idea in her brilliant article “Cloning Of Animals And Humans: What Should The Policy Response Be?”. Baird writes about how individuals have certain attributes from both parents while still maintaining a unique identity and how similarities between parents and offspring are an important part of “human identity” (187).

The similarities between parents and offspring is an eternal reminder of the relationship the two share and helps strengthen the bond between them. Sadly, cloned individuals are incapable of experiencing that aspect of “human identity”.

Furthermore, Baird discusses that many children who are adopted show a need to know who their birth parents are to complete a part of their identity. Baird writes that cloned children will “have no chance of having this dual genetic origin” (187). Unfortunately, if cloned individuals do not have friends or people for support they essentially have no one.

Funnily enough, this idea is presented in Frankenstein. In the novel, the monster has no friends, no acquaintances, and no family. His creator, Victor, shuns the monster which deteriorates the monster’s mental state. The monster feels like an outsider compared to society and promises revenge on all humans. When Victor dies, the monster has truly no one and no reason to live.

Now, as science advances and the world holds its breath, we can only watch to see where cloning takes us. For now, we can only contemplate the bioethics of cloning.


Discussion

We find that in the model species S. mediterranea, asexual animals demonstrate the potential to maintain telomere length during regeneration. Sexual animals appear to only lengthen their telomeres through the sexual reproduction process. This finding suggests that asexual individuals will be able to avoid senescence over evolutionary timescales using telomerase, a prerequisite for the formation of an evolutionarily stable fissionating asexual lineage. We did not observe any adverse affects of telomere shortening through Smed-tert(RNAi) or serial regeneration. The difference we observe between asexual and sexual animals is surprising, given that sexual animals also appear to have an indefinite regenerative capacity. We conclude that either they would eventually show effects of telomere shortening or that they are able to use another chromosome end-maintenance mechanism not involving telomerase.

In most species, telomeres erode in the absence of telomerase until a senescent phenotype is seen. In mice, effects of telomere loss are observed by generation 4 and certainly by generation 6 (23, 24). In Arabidopsis, cell cultures can be maintained for a number of population doublings before senescence, whereas sexual generations exhibit effects after six or seven generations (25, 26). In yeast grown asexually, cells senesce after ∼70 generations (27). Trypanosoma brucei appears to escape senescence entirely, with telomere length stabilizing at a shorter length, before senescence, by an unknown mechanism (28).

It is possible that the erosion of telomeres we observe or induce by RNAi is counteracted by mechanisms that give rise to alternative lengthening of telomeres (ALT) not requiring TERT (29, 30). ALT is characterized by an abrupt change in telomere length (29, 30). An ALT mechanism is responsible for telomere elongation in blastomeres of the early mouse embryo (31). This abrupt change in telomere length has not been observed in adult Smed-tert(RNAi) animals or adult sexual animals with declining telomere length. This suggests that the shortened telomeres achieved by RNAi are not short enough to trigger any ALT mechanism, or that any ALT mechanism does not use the telomere repeat for elongation.

Telomere maintenance mechanisms show adaptation in asexuals is achieved at the level of Smed-tert expression. First, PCR and in situ hybridization expression data in the context of irradiated animals suggest that Smed-tert is expressed in irradiated pASCs, and this is also supported by TRAP assay data from intact and irradiated worms (Figs. 1F, 2B, and 3). However, the majority of Smed-tert transcripts present in the pASCs of intact asexual and germ-line cells of sexual animals are missing the TRBD required for binding the telomerase RNA component (Fig. 4). By analogy with all other species, these would not be able to engage in telomere extension. Alternate splicing has also been implicated in the generation of dominant-negative and inactive forms of TERT in vertebrate cells, and may be a common mechanism for differential control of telomerase activity in different animal cell types (32 ⇓ –34). Furthermore, we are able to show that during normal homeostatic turnover of planarian tissues telomerase activity is not sufficient to maintain telomere lengths. During regeneration and fission, asexual animals are able to increase telomere length by producing seven- to eightfold more TRBD-containing transcripts. Direct testing of the roles of the alternate transcripts awaits the development of transgenic techniques in S. mediterranea and/or the ability to use siRNA technology to target splice junctions and stretches of small sequences that are isoform-specific.

Previous work on colonial ascidians (35) and oligochaete worms (36) that have asexual life-history phases and reproduce by fission suggests that passage through a sexual reproductive cycle is required to avoid telomere depletion (35) and senescence (35, 36). In both cases, these animals have the natural option of a sexually reproductive cycle. In the urochordate Botryllus schlosseri that also propagates sexually and asexually, telomerase activity appears to be up-regulated in asexually formed buds (37), although these animals also eventually undergo senescence (38). Longevity experiments to investigate senescence in Hydra that reproduced asexually suggest that they are immortal (4, 39), whereas a sexually reproducing species showed clear signs of degeneration and mortality (40). Although immortal Hydra also appears to share the (TTAGGG)n telomere repeat, there is as yet no data on how or whether they avoid chromosome end depletion (41). These data suggest the possibility that senescence or death of asexual individuals and colonies may in part result from a failure to maintain chromosome ends that are restored by going through a sexually reproductive cycle (35). In the case of “effectively immortal” and obligate asexual S. mediterranea, the end-replication problem in somatic stem cells has been solved by the simple evolutionary changes we have characterized. These allow increased telomerase activity in somatic pASCs, allowing them to be an effective cellular unit of inheritance.