The first cohort of five gene-edited monkey clones made from fibroblasts of a monkey with disease phenotypes were born recently at the Institute of Neuroscience (ION) of the Chinese Academy of Sciences (CAS) in Shanghai.
The expression of BMAL1, a core circadian regulatory transcription factor, was knocked out in the donor monkey using CRISPR/Cas9-mediated gene editing at the embryo stage, and the fibroblasts of the donor monkey were used to clone five monkeys using somatic cell nuclear transfer, the same method that generated Zhong Zhong and Hua Hua, the first two cloned monkeys.
This major advance, reported in two articles published in the journal National Science Review on January 24, demonstrates that a population of customized gene-edited macaque monkeys with uniform genetic background will soon be available for biomedical research.
One of the articles described the generation of gene-edited donor monkeys, using the CRISPR-Cas9 method to edit the BMAL1 gene of in vitro fertilized monkey embryos. These monkeys exhibited a wide-range of circadian disorder phenotypes, including reduced sleep time, elevated night-time locomotive activities, dampened circadian cycling of blood hormones, increased anxiety and depression, as well as schizophrenia-like behaviors.
"Disorders of circadian rhythm are associated with many human diseases, including sleep disorders, diabetes mellitus, cancer, and neurodegenerative diseases. Our BMAL1-knockout monkeys thus could be used to study the disease pathogenesis as well as therapeutic treatments,” said CHANG Hungchun from ION.
The other article described the cloning of macaque monkeys from the fibroblast of a BMAL1-knockout monkey using somatic cell nuclear transfer. Researchers removed the nucleus from a monkey oocyte (egg cell) and replaced it with another nucleus from a fibroblast, a differentiated somatic (body) cell. This reconstructed egg then developed into an embryo that carries the genes of the replacement nucleus. The embryo was then transferred to the womb of a surrogate female monkey that later gave birth to the cloned monkey.
In the previous study, Zhong Zhong and Hua Hua were generated by using fibroblasts from an aborted fetus. The present study succeeded in using fibroblasts from a young adult gene-edited donor monkey with disease phenotypes.
"Our approach is to perform gene-editing in fertilized embryos to first generate a group of gene-edited monkeys, and then select one monkey that exhibits correct gene editing and the most severe disease phenotypes as the donor monkey for cloning by somatic cell nuclear transfer,” said SUN Qiang, Head of the Nonhuman Primate Research Facility, ION. "We believe this approach of cloning gene-edited monkeys could be used to generate a variety of monkey models for gene-based diseases, including many brain diseases, as well as immune and metabolic disorders and cancer."
Researchers plan to continue improving the technique in order to increase the efficiency of cloning.
ION is following strict international guidelines for animal research. “The research will help to reduce the amount of macaque monkeys currently used in biomedical research around the world. Without the interference of genetic background, a much smaller number of cloned monkeys carrying disease phenotypes may be sufficient for preclinical tests of the efficacy of therapeutics," said POO Muming, who helped supervise the project and is co-author on both studies.
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