17 January 2024

China has successfully cloned a rhesus monkey

Chinese researchers have improved somatic cell nuclear transfer technology and successfully used it to clone a rhesus macaque monkey (Macaca mulatta). Previously, it was not possible to obtain a viable monkey of this species by cloning, but now three clones have lived for more than two years. The report on the work was published in the journal Nature Communications.

Somatic cell nuclear transfer technology (SCNT) is widely used to clone various species of mammals: mice, rabbits, pigs, cattle, pigs and dogs. However, the efficiency of this approach is extremely low - it leads to the birth of a live animal only in 1-3 percent of cases (5-20 percent in cattle) and is often accompanied by developmental abnormalities.

Zhen Liu (Zhen Liu) and Qiang Sun (Qiang Sun) with colleagues at the Institute of Neuroscience of the Chinese Academy of Sciences optimized the SCNT protocol, applied epigenetic regulators and thus managed to clone conventional and genetically modified macaque crab-eating macaques (Macaca fascicularis) in 2018-2019. Using the histone demethylase Kdm4d and the histone deacetylation inhibitor trichostatin A (TSA) in these experiments, live birth rates of 2.5 and 1.5 percent were achieved, respectively. Cloning rhesus macaques has proved more difficult: although reports of such attempts first appeared as early as 1997, no viable individuals have been obtained. The only clone of a monkey of this species, born alive by caesarean section, lived only 12 hours and died due to multi-organ dysplasia as a result of intrauterine anoxia, a typical problem associated with placental developmental defects in SCNT.

In another series of rhesus SCNT experiments, Liu and Sun's team used Kdm4d and TSA. The rate of blastocyst formation (47.6 percent) was comparable to a control group that underwent fertilization by intracytoplasmic sperm injection (ICSI, ICSI). The implantation rate of clones was half as high (35 of 484 vs. 74 of 499), only one clone was born alive and lived for 23 hours. Moreover, most miscarriages with SCNT occurred around day 60 and with ICSI around day 130 of gestation. Taken together, this suggests that the major problems in the clones manifest in the implantation and peri-implantation stages.

Molecular analyses showed that embryos after ICSI and SCNT have significantly different levels and patterns of DNA methylation. At the same time, epigenetic reprogramming at ICSI occurs in an orderly and separate manner in parental genomes, while at SCNT somatic epigenetic modifications can persist up to the blastocyst stage despite the general demethylation of Kdm4d and TSA DNA, which leads to a large number of differentially hypo- and hypermethylated sites in the DNA of cloned embryos.

Aberrant maternal imprinting of four genes, THAP3, DNMT1, SIAH1 and RHOBTB3, was also observed in cloned blastocysts. Of these, they were confirmed as differentially expressed genes with normalized expression level score (FPKM) greater than one. This loss of imprinting was also observed in postimplantation embryos cultured in vitro on day 17 postfertilization and in tissues of the formed placenta, and their total DNA methylation levels were significantly higher than in ICSI. Ultrasound, morphologic, and histologic examinations of placentas revealed their hyperplasia and calcification at SCNT, which were not observed at ICSI.

Previous experiments with mice, rabbits, pigs and cows have shown that tetraploid complementation can reduce placental insufficiency and increase live birth rates in SCNT. The authors created tetraploid rhesus macaque embryos by electrospinning at the two-cell stage and observed inner cell mass (embryoblast) formation at the blastocyst stage. 49 such embryos were transferred to 16 females, resulting in the development of eight pregnancies with 16 implantations. Of these 16 implanted embryos, half developed into a fetal egg only; the rest formed fetuses. Three of them were born, one died after one year, the remaining two were still alive at the time of writing (after two years of follow-up). Their chromosome number was normal.

Given the limited success of tetraploid complementation in monkey embryos, the researchers developed a technology of trophoblast replacement (SCNT-TR, somatic cell nuclear transfer with trophoblast replacement). For this purpose, they took blastocysts obtained by ICSI, removed their embryoblast, leaving a blastocele (trophoblast with a cavity) and replaced it with an embryoblast from SCNT-blastocysts. This technique was supplemented by the use of Kdm4d and TSA. 11 such embryos were transferred to seven females, two of them became pregnant, one of them with twins. The latter miscarried on day 106 of gestation. The singleton pregnancy ended with the birth of a healthy male on the 157th day, at the time of writing, he lived well over two years. His nuclear genome was completely inherited from a fibroblast donor and his mitochondrial genome from an egg donor. No chimerism was observed in him. Additional study of the placentas showed that SCNT-TR prevented differential DNA methylation, inbreeding defects, hyperplasia and calcification, meaning that this technology greatly improves all aspects of monkey cloning.

In 2023, the same research team reported other advances. First, the Chinese researchers collected from stem cells of macaque crab-eater embryos, grew them to 17 days of development, and they were even implanted in the uterus of surrogate mothers, but did not develop further. Secondly, scientists managed to get a clone of a monkey of the same species with 90 percent of chimeric cells.

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