Cloning by somatic cell nuclear transfer (SCNT) is a technique that produces embryos or animals that are genetically identical to the donor cells. Since the birth of Dolly the sheep derived from an adult mammary gland cell[1], SCNT has been heralded as a promising assisted reproductive technology with a broad range of applications[2], including conservation. The first SCNT mouse was reported in 1998 by Wakayama et al. at the University of Hawaii[3], and since then, the mouse has played a seminal role in the fundamental research on the low developmental efficiency of mammalian SCNT over the last 25 years. However, the broader applications of SCNT technology have been hampered by its extremely low birth rate. Typically, less than 5% of embryos transferred to pseudopregnant mothers result in live births[4].
Cloning in mouse is generally performed by removing the metaphase-II spindle (enucleation) followed by the injection of somatic cells using micromanipulator. The reconstructed embryos are further activated and reprogrammed in the activation and reprogramming medium before being cultured until the formation of blastocyst or being transferred into a surrogate mother at usually 2-cell stage.
Due to the different genetic background, clonable mouse strains have been limited to hybrid F1 strains, such as B6D2F1 and B6C3F1[5]. However, recent advancements have reported the success of clone from an outbreed mouse strain (ICR oocyte receiving ICR cumulus cells) with 30% Morula/Blastocyst rate and 0.9 - 4.5% number of live offsprings[5,6].
The following is one of my trial in doing cloning of ICR mouse using cumulus cell.
Photos are personal documentation.
References:
[1] Wilmut, I., Schnieke, A., McWhir, J. et al. Viable offspring derived from fetal and adult mammalian cells. Nature 385, 810–813 (1997). https://doi.org/10.1038/385810a0
[2] Kamimura S., Inoue, K., Mizutani, E. et al. Improved development of mouse somatic cell nuclear transfer embryos by chlamydocin analogues, class I and IIa histone deacetylase inhibitors. Biology of Reproduction. 105(2): 543–553, https://doi.org/10.1093/biolre/ioab096
[3] Wakayama, T., Perry, A., Zuccotti, M. et al. Full-term development of mice from enucleated oocytes injected with cumulus cell nuclei. Nature 394, 369–374 (1998). https://doi.org/10.1038/28615
[4] Inoue, K. Mouse somatic cell nuclear transfer: What has changed and remained unchanged in 25 years. Journal of Reproduction and Development. 69(3): 129-138. https://doi.org/10.1262/jrd.2022-105
[5] Kishigami, S., bui, H.T., Wakayama, S. et al. Successful Mouse Cloning of an Outbred Strain by Trichostatin A Treatment after Somatic Nuclear Transfer. Journal of Reproduction and Development, 2007, 53(1): 165-170
[6] Tanabe, Y., Kuwayama, H., Wakayama, S., et al. Production of cloned mice using oocytes derived from ICR-outbred strain. Reproduction, 2017.154(6), 859-866. https://doi.org/10.1530/REP-17-0372
