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Researchers in Japan have produced 26 successful generations of cloned mice from a single individual. That's a total of 598 mice, all of whom are essentially genetic duplicates. The achievement was made possible by a new cloning technique that allowed researchers to overcome genetic degradation problems characteristic of generational re-cloning. The breakthrough shows that mammalian cloning lines can be extended and reproduced without limit.
Indeed, animal re-cloning (i.e. cloning a clone) works great, but up to a point. Eventually, over the course of several generations, a clonal line will ultimately fail, the result of accumulated lethal genetic and epigenetic abnormalities. But the Japanese researchers devised a crafty biohack that appears to remedy this problem.
The new technique, developed by Teruhiko Wakayama of the RIKEN Center for Developmental Biology in Kobe, Japan, was so successful that it resulted in well over two dozen generations of re-cloned mice. Moreover, the cloning efficiency did not decrease over the course of those generations, and the project was allowed to continue indefinitely (and in fact, the project is still going!). In all, nearly 600 viable offspring were produced from a single donor mouse. The experiment started seven years ago and it is considered the largest cloning project using a mammal to date.
Wakayama and his team achieved this by using the standard cloning technique, somatic cell nuclear transfer (SCNT), and adding a histone deacetylase inhibitor (trichostatin), and other chemicals to the process.
In SCNT, the nucleus of a somatic cell is transferred to the cytoplasm of an egg that has had its nucleus removed (an enucleated egg). Once inside the egg, the somatic nucleus is reprogrammed to become a zygote nucleus, what is really a fertilized egg.
But as noted, this can’t be done indefinitely, as genetic problems start to creep in over successive generations. But adding the HDI to the mix seemed to do the trick. It's a class of compounds that interfere with the function of histone deacetylase, a class of enzymes that allow histones (proteins that package and order the DNA into nucleosomes) to wrap DNA more tightly. They can also be used to alter gene expression.
According to the researchers, the cloned mice had normal biological features, including regular lifespans and reproductive capabilities. That said, genetic analysis did show some minor abnormalities, including an oversized placenta. But none of these characteristics had a detrimental impact on the line’s clonal health. The researchers noted that “serially recloned mice have the same characteristics as standard clones.”
Their results show that repeated iterative re-cloning is possible. The researchers wrote that “with adequately efficient techniques, it may be possible to re-clone animals indefinitely.”
Once refined, the technique could result in the large-scale production of cloned animals for farming or conservation purposes. Moreover, animals can continue to be cloned long after the source individual has died.
For the first time scientists have deciphered the DNA of flat worms, which may reveal new therapeutic targets for future drugs. The genome is a new resource and the path to faster development of new drugs are urgently needed - flat worms cause two of the seventeen "neglected" tropical diseases listed by the World Health Organization - echinococcosis and cysticercosis.
The research team determined the DNA sequence of the four types of tapeworms to better study the biology and genetics of intestinal parasites. In most species, adults cause few complaints while in the intestines. The larvae, however, can cause serious medical complications in moving their body. They form cysts in the bodies of humans and animals, which can lead to complications such as blindness or epilepsy.
According to Dr. Matthew Beriman of the Wellcome Trust Sanger Institute, parasitosis of flat worms are widespread. Their global burden is comparable to that of multiple sclerosis and melanoma.
Typically, the researchers analyzed the DNA of pathogens and compare it with that of man, and thus identify potential targets for future drugs. In this study, however, researchers are mainly interested in the similarities in the DNA of human intestinal parasites. This is because unlike most pathogens such as bacteria and viruses, flat worms are eukaryotic organisms like humans. Flatworms much more like a human structure and physiology of any bacteria or virus.
Furthermore, by analyzing the similarities between the genomes, researchers discovered which of already existing drugs might be effective against parasites. This can save hundreds of decades of work and millions of dollars in investments.
It turns out that some tapeworms are sensitive to the drugs currently used to treat cancer. Another potential solution is cholesterol lowering medication. In the course of evolution, flat worms have lost the ability to synthesize their own cholesterol, which obtain at the expense of the host. Promising target for new drugs are proteins, through which the larvae absorb cholesterol from the intestine. If the function of these proteins has been crossed, the larvae will stop development and will die.
The AccuPower DNA Ligation PreMix is a lyophilized master mix containing T4 DNA Ligase, ATP, reaction buffer, and patented stabilizer. This DNA ligation premix is conveniently aliquoted in strip-tubes for reactions; you need only add DNAs to be ligated and water. The reaction will work for DNA ligation for all applications: blunt cloning, sticky end cloning and TA cloning. The premix is stable up to four months at room temperature and for three years at -20°C.
Features and Benefits
|Ready to use premix:||Minimal set up time and handling required|
|Fast:||Only 5 minutes for cohesive-end ligation and 10 minutes for blunt-end ligation at room temperature|
|Stable:||Enzyme activity for up to four months at room temperature and for three years in the freezer|
Cloning into vectors, library construction, TA cloning, linker ligation, and re-circlization of linear DNA
Figure 1. Stability test of AccuPower DNA Ligation PreMix at room temperature.
Lane 1 – 12: Lambda DNA / Hind lll fragment (1 µg)
Lane 12 – 24: Lambda DNA / EcoR V fragment (1 µg)
Lane 2, 3, 14, 15: Ligation with AccuPower Ligation PreMix stored at room temperature for 1 month
Lane 5, 6, 17, 18: Ligation with AccuPower Ligation PreMix stored at room temperature for 2 months
Lane 8, 9, 20, 21: Ligation with AccuPower Ligation PreMix stored at room temperature for 3 months
Lane 11, 12, 23, 24: Ligation with AccuPower Ligation PreMix stored at room temperature for 4 months
Figure 2. DNA Ligation efficiency comparison between AccuPower DNA Ligation PreMix and other competitors’ products.
Lane 1, 9: Intact Lambda DNA (1 µg)
Lane 2 – 8: Lambda DNA / Hind lll fragment (1 µg)
Lane 10 – 16: Lambda DNA / EcoR V fragment (1 µg)
Lane 3, 4, 11, 12: Ligation with AccuPower Ligation PreMix
Lane 5, 13: Ligation with T4 DNA Ligase from company N
Lane 6, 14: Ligation with Quick Ligation Kit from company N
Lane 7, 15: Ligation with LigFast Rapid DNA Ligation System from company P
Lane 8, 16: Ligation with Ready-To-Go T4 DNA Ligase from company A
Up to 10 per cent of the active genes of an organism that has survived 80 million years without sex are foreign, a new study from the University of Cambridge and Imperial College London reveals. The asexual organism, the bdelloid rotifer, has acquired a tenth of its active genes from bacteria and other simple organisms like fungi and algae.
The findings were reported Nov. 15 in the journal PLoS Genetics.
Bdelloid rotifers are best known for going 80 million years without sex, as they have evolved to reproduce successfully without males. Many asexual creatures go extinct without the benefit of traditional genetic evolution. However, bdelloids have flourished by developing ingenious ways of overcoming the limitations of being asexual.
Bdelloids have also developed the fascinating ability to withstand almost complete desiccation when the freshwater pools they typically live in dry up. They can survive in the dry state for many years only to revive with no ill effect once water becomes available again.
"We were thrilled when we discovered that nearly 10 per cent of bdelloids' active genes are foreign, adding to the weirdness of an already odd little creature," said Professor Alan Tunnacliffe, lead author of the study from the University of Cambridge. "We don't know how the gene transfer occurs, but it almost certainly involves ingesting DNA in organic debris, which their environments are full of. Bdelloids will eat anything smaller than their heads!"
Because some of the foreign genes are activated when the bdelloids begin to dry out, the researchers believe that the genes play a role in bdelloids' ability to survive desiccation.
Professor Tunnacliffe added: "Other researchers have shown that bdelloids contain powerful antioxidants, which help protect them from the toxic oxidising agents that are the by-products of desiccation. These antioxidants have not yet been identified, but we think that some of them result from foreign genes."
For the study, the researchers extracted all of the messenger RNA (genetic code similar to DNA which provides a blueprint for the creation of proteins) from bdelloid rotifers and sequenced each message, creating a library of the animal's active coding information. Using a supercomputer, they then compared these messages with all other known sequences and found that in many cases similar sequences had been found in other organisms.
Strangely, however, these other organisms were often not animals, but simple microbes. This means that bdelloids have genes that are not present in other animals, but have been acquired from micro-organisms and adapted for use in the rotifer.
The research was funded by the Biotechnology and the Biological Sciences Research Council (BBSRC) and the European Research Council.