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These glitches explain why it’s tough to clone cows

It’s been 20 years since scientists in Scotland cloned Dolly the sheep, but cloning mammals remains a challenge. A new study on gene expression in developing clones shows why most cloned embryos likely fail.

Dolly was cloned using the technique of “somatic cell nuclear transfer,” when a nucleus from an adult cell is transferred into an unfertilized egg that has had its nucleus removed, and is then shocked with electricity to start cell growth. Embryos are then transferred to recipient mothers who carry the clones to birth.

Cloning cattle is an agriculturally important technology and can be used to study mammalian development, but the success rate remains low, with typically fewer than 10 percent of the cloned animals surviving to birth. The majority of losses are due to embryonic death, a failure during the implantation process, or the development of a defective placenta.

cloned calves
The first cloned calves born in the Biology of Development and Reproduction Unit in Jouy-en-Josas, France. (Credit: Gérard Paillard/INRA)

In the study, published in the Proceedings of the National Academy of Sciences, Harris Lewin, professor in the University of California, Davis, department of evolution and ecology, and colleagues used RNA sequencing to look at gene expression in cloned cows during implantation in order to get a better understanding of the molecular mechanisms that lead to a high rate of pregnancy failure for clones.

“Our work tackled fundamental questions relating to the cloning process,” says Lewin. “The study has resulted in the redefinition of our understanding of how nuclear reprogramming affects gene expression in extra-embryonic tissues of cloned cattle embryos, and the exquisite communication between clones and their recipient mothers.”

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“The large amount of data our collaboration has generated sheds light on mechanisms that account for embryonic losses at implantation,” says Olivier Sandra, team leader for the study at the Institut National de la Recherche Agronomique in France. “They also provide new insights on how events taking place at implantation drive the progression of pregnancy and shape the post-natal phenotype of the progeny, in cattle as well as in other mammalian species.”

Anomalies in 5,000 genes

The researchers studied tissue from cloned cow embryos—all derived from the same cell line—at 18 and 34 days of development, as well as the corresponding endometrial lining of the pregnant cows. They also looked at noncloned cows conceived using artificial insemination.

Using RNA sequencing, the researchers found multiple genes whose abnormal expression could lead to the high rate of death for cloned embryos, including failure to implant in the uterus and failure to develop a normal placenta. Looking at the extra-embryonic tissue of the cloned cows at day 18, the researchers found anomalies in expression of more than 5,000 genes.

When they compared the results to the Mouse Genomic Informatics Knockout database they found 123 genes that corresponded with functional annotation of abnormal extra-embryonic tissue morphology, 121 associated with embryonic lethality, and 14 with abnormal embryo implantation.

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By day 34 of development, however, the pattern of gene expression was much more similar to control cows derived from artificial insemination, suggesting that these surviving clones were able to implant in the uterus and begin to form a placenta. These results indicate that the large losses of cloned cows before implantation probably result from problems with critical developmental genes in the extra-embryonic tissue.

The study also reveals other points of potential failure for the clones, including problems with hormonal signaling between the developing cloned embryo and the pregnant cow. For example, the study found down-regulation of genes involved in interferon tau, the major signal of pregnancy recognition. The clones also appeared to have an effect on the gene expression of the pregnant cows themselves; on day 34, some uterus tissue showed grossly different gene expression, which could affect the placenta.

Strict ban on human cloning

“Our data confirm that the interactions between the uterus and the extra-embryonic tissues is critical during implantation, making this step a major hurdle for the progression of pregnancy,” says Sandra.

“We now understand why clones fail, which can lead to improvements in the process of cloning of animals,” says Lewin. But, he cautions, “Our discoveries also reinforce the need for a strict ban on human cloning for any purposes.”

“It’s amazing that the process works at all, demonstrating the great plasticity that developing animals have to adapt to extreme conditions,” Lewin says.

A US Department of Agriculture–Agricultural Research Service Grant, a European Program SABRE Grant, and French National Research Agency Grants supported the work. The pregnancies were initiated at the experimental farm of the Institut National de la Recherche Agronomique in France and at the University of Illinois at Urbana–Champaign. The experiment took place in accordance with the rules and regulations of the European Convention on Animal Experimentation.

Additional coauthors contributed from institutions in the US and France.

Source: UC Davis

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