Cloning of Monkeys by Somatic Cell Nuclear Transfer
Somatic cell nuclear transfer (SCNT) is a promising method to establish genetically modified monkeys with identical genetic background as models in biomedical research. We have recently cloned monkeys by optimization of the SCNT protocols and inclusion of
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Introduction Owing to their high similarity in physiology and behavior, monkeys are widely used as a model to study human biology and diseases. With the development of gene-editing technologies, monkey models will play increasingly important roles in the future. Lentiviral cell transduction and nuclease-mediated gene-editing in early embryos are the two main methods to generate genetically modified monkey models. CRISPR/Cas represents the latest progress for the nuclease-mediated gene-editing in animal embryos. However, there are still several limitations for these two methods. For example, there are multiple and random integrations of transgenes in the lentivirus-based transgenic monkeys, and mosaicisms in the CRISPR/Cas-edited monkeys, resulting in various genotypes for each individual founder monkeys. Another limitation is the inconsistency in genetic backgrounds among the transgenic monkeys, and this may lead to variations in experimental results among different monkeys. Cloning of macaque monkeys from a geneedited somatic cell by somatic cell nuclear transfer (SCNT) allows the generation of genetically identical monkeys without mosaicisms, and thus it has been considered as an ideal method to generate gene-modified monkey models. Somatic cell nuclear transfer (SCNT), also known as cloning, can produce animals through transfering of a single somatic nucleus
Kejin Hu (ed.), Nuclear Reprogramming: Methods and Protocols, Methods in Molecular Biology, vol. 2239, https://doi.org/10.1007/978-1-0716-1084-8_2, © Springer Science+Business Media, LLC, part of Springer Nature 2021
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into an enucleated oocyte, and has been intensively studied since John Gurdon successfully cloned frogs using nuclei from the intestinal epithelium cells of tadpoles [1]. After the successful cloning of Dolly the sheep in 1996 using SCNT [2], mammalian cloning has been achieved using similar methods in more than 20 different species including cows, mice, pigs [3–5], and others. The first cloning of cynomolgus monkeys was achieved by our group in 2017 using the fetal fibroblasts as nucleus donors [6]. Recently, we isolated the fibroblasts from the monkeys whose circadian transcription factor BMAL1 has been knocked out and used these fibroblasts as nucleus donors to generate five monkeys with both alleles harboring the BMAL1 mutation without mosaicisms [7]. This chapter provides the related protocols for cloning of monkeys using the key technology of somatic cell nuclear transfer. Since animal cloning relies on several techniques, we will describe the step-by-step procedures for the following integral experiments about monkey cloning: monkey superovulation (Subheading 3.1), oocyte collection (Subheading 3.2), preparation of monkey fibroblasts (Subheading 3.3), donor cell preparation (Subheading 3.4), somatic cell nuclear transfer (Subheading 3.5), monkey embryo transfer (Subheading 3.6), pregnancy test and monitoring (Subheading 3.7), as well as STR and SNP analyses (Subheading 3.8).
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Materials Prepare all the so
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