Indians find simpler way to make transgenic mice

September 21st, 2008 - 4:34 pm ICT by IANS  

Bangalore, Sep 21 (IANS) Indian scientists, traditionally known for their skill to find low-cost solutions to complex problems, have developed a technique that should potentially reduce the cost of drug development worldwide.Scientists at the National Institute of Immunology (NII) in New Delhi say their technique may enable biologists produce transgenic mice in their own labs instead of having to buy them at exorbitant prices.

As well as potentially reducing the cost of drug development, their “deathless” technique of generating transgenic mice should gladden animal lovers as it avoids needless sacrifice of animals, its developers claim.

A transgenic animal is one that carries a foreign gene that has been deliberately inserted into its genome. Since the first gene transfers into mice were successfully executed in 1980, transgenic mice (carrying human genes) have become models for studying human diseases.

They are one of the most sought-after tools in drug discovery because human diseases can be induced in such mice and experimental drugs tested on them.

But the current procedures for the production of transgenic mice are technically demanding, labour intensive and very cumbersome, requiring trained personnel and costly infrastructure.

For instance, to produce transgenic mice, genes responsible for particular traits or disease susceptibility are chosen and extracted. Next they are injected into fertilized mouse eggs removed from females that are later sacrificed.

The embryos are then implanted in the uterus of surrogate mothers. The selected genes will be expressed by some of the offspring. Success of such techniques is so low that several animals will be killed, without ever being used, because they do not have the required trans gene.

Because of the complex steps involved, transgenic mice are currently produced in central facilities or supplied by private companies mostly in the US and Europe. The high cost of importing transgenic animals is recognized as one of the factors that have retarded drug research in India.

The inexpensive and less time-consuming technique recently reported by NII’s Subeer Majumdar and his colleague Suveera Dhup in the journal Nature Methods should therefore be welcome news for the research community in general and for Indian scientists in particular.

“With this simple technique every biologist can generate his or her own transgenic animals at low cost and without the help of specialized labs or personnel,” Majumdar told IANS.

Instead of inserting the foreign gene into the fertilized egg, the NII team introduced it into the undifferentiated germ cells (spermatogonia) in the testis of males.

“Our reasoning was simple,” explains Majumdar. “The females produce a few eggs over a span of some days whereas the males produce about 100-200 million sperms in a day. If the passing of the foreign (human) genes in animals (from one generation to the next) is to be through sperms or eggs, it is better to pick the sperms because of their high numbers.”

The NII technique involves injecting the gene constructs at a specific site in the mice testis and coax them to enter the testicular germ cells (spermatogonia) by passing a mild current for a fraction of a second — a process called “in vivo electroporation”.

The process leads to integration of the gene permanently into the chromosomes of the male germ cells, says Majumdar. Such mice, called “founders”, can continuously generate transgenic pups for more than one year by natural mating, the researchers report.

In contrast, the existing technology, says Majumdar, “requires assisted reproductive techniques like preparation of surrogate mothers and transfer of manipulated embryos into their womb”.

To check the efficiency of their procedure, the NII scientists electroporated 17 mice, inserting four different gene constructs. All except one produced transgenic pups, indicating a success rate of about 94% which is 3-4 fold higher than that achieved by existing techniques.

The NII scientists report that, to their knowledge, “this is the first electroporation-mediated technique for transfection of undifferentiated spermatogonial cells in vivo that resulted in integration and long-term maintenance of the transgene in the germ cell and its transmission via mating.”

In the past, in vivo electroporation either did not result in generation of transgenic animals or required assisted reproductive to achieve a live birth of transgenic progeny, the authors said.

The technique is also claimed to be ethically superior to existing method of generating transgenic mice because of the massive reduction in the number of animals used and absolute avoidance of any killing to collect eggs.

“Limited use of animals and the potential to produce large numbers of transgenic animals in a short duration are the two major benefits underlying this technique,” says the report. For instance, first transgenic progeny can be generated within 60 days of electroporation as compared to 82-177 days in other methods.

Since the human genome is deciphered, the pace of basic as well as drug development research will see a phenomenal rise because this procedure will allow rapid generation of transgenic animals using various available genes, the authors say. According to Majumdar, the new procedure, if adopted, could drastically reduce Indian scientists’ dependence on the western world for the supply of transgenic animals.

Officials of India’s Department of Biotechnology (DBT) that funded the NII study say that this procedure can potentially open avenues for generating transgenic cattle expressing human proteins in their milk, and for creating models of difficult human diseases using monkeys.

Says Majumdar: “Understanding the biological basis of AIDS, mental retardation, Parkinson’s and Alzheimer’s disease requires monkey models and we also need transgenic cattle to study foot and mouth disease, mad cow disease etc.”

He says costly medicines like human gamma interferon, clot dissolving factors and similar proteins of human importance can be produced in huge quantities in the milk of transgenic cattle in what is known as “biopharming”.

The complexity of existing technology and need for several donor females have so far restricted generation of transgenic cattle and transgenic monkeys, adds Majumdar. “I hope this will change.”

Majumdar has now an Indo-US grant to make transgenic monkeys and the DBT has asked him to undertake a big project with more partners to make transgenic buffaloes.

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