The manipulation of cells took five years to complete and cost $1.5 million
SCIENTISTS in Singapore have become the first to 'semi-clone' an animal by fertilising an egg with an embryonic stem cell that mimics sperm.
Holly, a 4cm-long medaka fish, is now 15 months old and a great-grandmother, with a family of more than 100 fish which are able to reproduce normally and healthily.The success by the scientists from the National University of Singapore may spell promise in future for infertile couples.
The key to the discovery is that scientists managed to generate a haploid DNA cell which mimics sperm. The isolation and manipulation of these cells took five years to complete, and cost $1.5 million.
The work by Associate Professor Hong Yunhan of the National University of Singapore's Department of Biological Sciences came through persistence - he continued his work to create haploid cells even when Nobel Prize winners had given up.
In essence, what the scientists did was to take eggs from one fish, and sperm from another.
The sperm cells were then zapped with UV rays to strip them of their DNA code, and these were then used to 'fertilise' the eggs.
As only one set of DNA was contained in the eggs, the resulting division created haploid cells. These cells were then combined with eggs from another fish, and Holly was born.
This method opens up the possibility of obtaining a haploid cell from a man, enabling him to pass on his DNA even if he is infertile.
That will be some time in the future, however, as further tests will still need to be carried out.
There will also be ethical hurdles to cross: Scientists and others have long debated the issue of whether it is right to clone humans.
For now, though, scientists here are celebrating what they say is akin to turning science fiction into reality.
'Eight years ago, semi-cloning was science fiction. Our work with the fish as a first model has revealed the possibility of carrying it out on vertebrates, to which humans belong,' said Prof Hong.
Holly is quite different from animals like Dolly, the world's first cloned sheep, and the myriad of others that came after her.
Instead of a clone, which is an exact genetic replica of an organism, Holly is a semi-clone with a unpredictable genetic code - similar to how it occurs in nature.
Dr Alan Colman, a principal investigator at the Agency for Science, Technology and Research's Institute of Medical Biology, executive director of the Singapore Stem Cell Consortium, as well as one of the creators of Dolly, said the isolation of a haploid cell was interesting and valuable to science.
He said: 'The reason haploid cells are desirable is that all of us have two copies of each gene.'
He explained that being able to look at a human haploid cell, if one day possible, will allow scientists to study and treat genes that cause diseases such as cancer, Alzheimer's and Parkinson's.
The next step for Prof Hong's team is to seek international collaboration to try and carry out its work on mice. Such a study will take about five years and, if successful, will proceed to trials with monkeys and, finally, humans.
Dr Benjamin Capps of the Centre for Biomedical Ethics at NUS' Yong Loo Lin School of Medicine said: 'The results of this study are closer to IVF - replacing the role of sperm with that of an artificially created haploid cell - and so raise little immediate ethical concern as a technique.'
Prof Hong, however, noted: 'It will be interesting to see whether human society would accept these semi-cloned children once the technology is established in humans.'
A fish called Holly
Singapore the birthplace of the world's first semi-cloned animal
Ong Dai Lin, Today Online 16 Oct 09;
SINGAPORE - A fish named Holly may have placed Singapore on the world map for being the birthplace of the very first semi-cloned animal.
By combining the stem cell of an embryo - one which is created to contain only one set of chromosomes - with an egg which provides the other set, three National University of Singapore (NUS) researchers were able to create a fish that is not an exact clone.
In traditional cloning, a baby is formed from a stem cell with two sets of chromosomes from one parent, making it an exact duplicate - and spawning great debate about the ethics of cloning since the world's first animal, Dolly the sheep, was cloned from an adult stem cell in 1996.
Now, the successful birth of the first semi-cloned animal, Holly the fish, means researchers can possibly use semi-cloning as a method to treat infertile couples.
Associate professor Hong Yunhan, who led the research team from the National University of Singapore Department of Biological Sciences, said semi-cloning is better than cloning, as it produces babies in an "unpredictable" way.
"We can't even predict whether the offspring is a male or female," said assoc prof Hong.
What is needed, for instance, could be for a man without mature sperm to use stem cells - which can later develop into all other kinds of cells - from another part of his body, to fuse with his wife's egg.
But it may take as long as 10 years to get to the stage where the technique can be researched in humans, said assoc prof Hong, who added that the semi-cloning technique can also be used to study diseases by analysing gene mutations.
His five-year research programme, funded by NUS, the Agency for Science, Technology & Research (A*Star) and the Ministry of Education, has so far cost $1.5 million. Assoc prof Hong estimates he needs between $5 and $10 million more to further the work.
She's a mother of 308, grandmother of 977
Ong Dai Lin, Today Online 16 Oct 09;
SINGAPORE - She is 15 months old and she already has 308 children and 977 grandchildren.
Holly, the world's first semi-cloned fish, has a friendly nature and likes to play with other fish.
Every day, Professor Hong Yunhan presents a male medaka fish to Holly as her playmate to encourage the fish to breed.
The history-making fish has quirky eating habits, too. As a baby, Holly's favourite food was green algae and pet fish food. Now that she is an adult, Holly prefers to eat living brine shrimp embryos.
Contrary to the perception that cloned specimens have health problems, Professor Hong says that Holly is very healthy.
The team of researchers from the National University of Singapore practically treats Holly like family, celebrating each stage of her growth with a meal.
When Holly was born and started swimming, the team went for a meal at a restaurant in West Coast. When Holly produced eggs after mating, the team celebrated over a seafood Chinese dinner.
Holly belongs to a species of fish called the medaka fish or Japanese rice fish. They are a freshwater species and are indigenous to Japan, Korea, China and Vietnam where they inhabit slow-moving waters.
They are reared as aquarium fish and are also used extensively for scientific research.
A medaka fish can live for up to two years and grow to 4cm.
The making of Holly, a semi-cloned fish
Breakthrough by NUS team spells promise for infertile couples
Victoria Vaughan, Straits Times 17 Oct 09;
FOR 15 years, molecular biologist Hong Yunhan dreamt of making a breakthrough in a new technology that could help infertile couples have children.
Now, he and his team have come a step closer to doing so.
They have worked on isolating and maintaining haploid cells for five years. These are cells containing half the DNA of a normal cell and are found in eggs and sperm before fertilisation.
It did not matter to the team that 2007 Nobel Prize winner in medicine Martin Evans, with Dr Matthew Kaufman, had closed the door on isolating these cells 26 years ago, after their mouse study found them to be unstable and prone to mutation.
Associate Professor Hong and his team at the National University of Singapore's Department of Biological Sciences pressed on and have made remarkable inroads.
To prove the viability of their haploid cells, the team went on to harness them to create Holly, a medaka fish, the world's first semi-cloned vertebrate.
Holly is healthy and so are her offspring, numbering more than 600. And it has over 1,000 grandchildren.
Created from two haploid reproductive cells - one an egg cell, the other an embryonic stem cell manipulated to mimic sperm - Holly could hold promise for infertile couples much further down the road if scientists learn how to obtain stable human haploid cells.
Prof Hong, 53, said he was doing this research for two reasons: First, fewer people are having babies and, second, couples are having them later, a factor that is accompanied by a decline in the quality of the human genome.
'Child production is not only a medical problem, it's a social problem and has an impact on daily life as well as the country,' said the Chinese national, who moved here eight years ago with his wife and two daughters, and is applying for permanent residency.
'With medical technology, there is a greater possibility of people with ill health surviving and having children. But by doing so humans are passing on bad genes. Consequently, the human gene pool will become worse.'
The technique can theoretically allow scientists to study the genetic makeup of a haploid cell and correct any disease mutations - or discard non-viable cells - before it is used in fertilisation.
During fertilisation, two haploid cells are joined together. This process can allow weaker genes, which cause diseases like Alzheimer's, Parkinson's and cancer, to hide making it difficult for scientists to study and 'correct' them.
Prof Hong was helped by molecular biologist Yi Meisheng and PhD student Hong Ni.
Dr Yi, 41, took seven months to create a batch of stable haploid cells. He did this by zapping the fish sperm cells with UV rays to kill their DNA but without destroying their ability to swim.
A sperm is then used to 'fertilise' a fish egg. This sparked the 'embryo' into cell division, but with only the DNA of the egg cell, making it a haploid cell.
'When I had a medaka haploid cell, I kept it very carefully. When the cells were still growing well 20 days later, I told Prof Hong and he was happy. After 59 days, he bought me a beer,' said Dr Yi. The cells were cultured for 400 days to confirm they were stable.
Molecular biologist Hong, 29, was bought in to validate that the cells were good, while Dr Yi took a month to train his hands to be steady enough to inject a haploid cell into a 1mm egg.
After many attempts over two months, three embryos began to grow.
One became Holly, created in 2007. It was named by Prof Hong in tribute to Dolly the sheep, the world's first cloned animal, but with an 'h' for haploid.
Previous efforts
Straits Times 17 Oct 09;
# 1996, Edinburgh, Scotland: Dolly the sheep is created in the first successful cloning of a mammal from an adult cell.
# 2003: Dolly is euthanised because of an incurable lung disease. It is only six years old, while the average lifespan of a sheep is 12 years.
# February 2004, Seoul National University, South Korea: Professor Hwang Woo Suk's team claims it has created 30 cloned human embryos.
# May 2005: Prof Hwang's team says it has made stem cell lines from the skin cells of 11 people.
# August 2005: Prof Hwang announces the creation of the world's first cloned dog, an Afghan hound named Snuppy.
# November 2005: Prof Hwang apologises for using eggs from his own researchers.
# December 2005: A university panel finds Prof Hwang's cloning research to be fabricated and he steps down as a professor at the university.
# 2008, South Korea: RNL Bio arranges for the re-creation of Booger the pit bull terrier through its refrigerated ear tissue. Ms Bernann McKinney pays US$50,000 for the five puppies that are born.
# November 2008: Dr Teruhiko Wakayama of the Riken research institute in Yokohama, Japan, clones mice whose bodies had been frozen for as long as 16 years.
# April 2009, South Korea: Cloned pig Xeno is created by a team of scientists doing research on making pigs suitable for human transplants.
# June 2009, Harayana, India: Scientists clone buffalo Garima at the National Dairy Research Institute in Karnal, northern India. The institute had cloned the world's first buffalo four months earlier, but it died five days after its birth.